TW200530399A - Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof - Google Patents

Abstract

The present invention provides recombinant nucleic acid molecules, expression cassettes, and vectors useful for expression of polypeptides, including heterologous polypeptides, such as antigens, in bacteria. Some of the recombinant nucleic acid molecules, expression cassettes and vectors comprise codon-optimized sequences encoding the polypeptides and/or signal peptides. Some of the recombinant nucleic acid molecules, expression cassettes, and expression vectors comprise sequences encoding non-Listerial and/or non-secA1 signal peptides for secretion of the polypeptides. The invention also provides bacteria comprising the nucleic acid molecules, expression cassettes, and expression vectors, as well as compositions such as vaccines comprising the bacteria. Methods of making and using the bacteria, recombinant nucleic acid molecules, and expression cassettes are also provided.

Description

200530399 9. Description of the invention: [Technical field to which the invention belongs] The field of the present invention is generally related to novel polynucleotides and expression cassettes used to express polypeptides in recombinant bacteria, including heterologous polypeptides. In particular, the present invention relates to recombinant bacteria, including novel expression cassettes and / or nucleic acid molecules, which can be used in vaccine compositions. [Prior art] Methods have been developed for use as vaccines for delivery of heterologous antigens. The delivery of heterologous antigens by microorganisms has been modified for certain nucleic acid sequences encoding proteins or antigens originating from different species. Heterologous antigen delivery is particularly advantageous for treating or preventing a disease or condition that results primarily from a toxic or lethal source, such as cancer or a pathogen (such as HIV or Hepatitis B), where injection of a natural infectious agent or cancer cell is beneficial to the receiving organism. It may be harmful, and administration of attenuated or killed formulations or cells has proven to be unsuccessful in eliciting an effective immune response, or where there is no assurance that sufficient attenuation of infectious agents or cancer cells is acceptable. Recently, certain bacterial lines have been developed into recombinant vaccines. For example, modified oral vaccines for attenuated Salmonella have been shown to express circumsporozite antigens in piasmodium berghei and protect mice against malaria (Aggarwal et al. 1990, j. Exp Med. 172: 1083). Listeria monocytogenes (Listeria monocytogenes) (Listeria) is a Gram-positive facultative intracellular bacteria that is being developed for use in antigen-specific vaccines It, because it provides pathways via class Z and class II antigens, provides the ability to effectively (: 〇4 + / 〇:] 〇8 + 1 ^ cell mediate inverse 98561.doc 200530399. See 'for example, US Patent No. No. 6,051,237, 6,565,852, and 5,830,702. Listeria has been studied for many years in a model that stimulates both innate and adaptive T cell-dependent antibacterial immunity. Listeria is effective in stimulating cellular immunity The ability is based on its intracellular life cycle. When infected, the cells are rapidly ingested by the cells, including giant cells and dendritic cells (DC). Part of the bacteria. In the swallowing body of infected APCs, peptides caused by proteolytic degradation of pathogens are directly loaded on MHC class II molecules and pass through the intracystic pathway of class I to provide antigens. Cell surface Processed antigens, and these MHCII-peptide complexes activate CD4 + facilitators, T cells, which stimulate the production of antibodies. In the acidic compartment, certain bacterial genes are activated, including cholesterol-dependent lysing cells LLO, which can degrade the bacteriolytic digestion, releases pupae into the cytosolic compartment of the host cell, where the surviving Listeria genus multiplies. To effectively provide heterologous antigens via the MHC Class I pathway, it is required The endogenous protein is re-expressed by Listeria. In the cytoplasm of the antigen-providing cells (APC), samples of the protein synthesized and secreted by Listeria 'were sampled and degraded by proteosome. By TAP protein The resulting peptide was shuttled in the endoplasmic reticulum and loaded on MHC class I molecules. The MHC I-peptide complex was delivered to the cell surface, mixed with sufficient co-stimulation (signal 2), activated and stimulated with Cytotoxic T lymphocytes (CTLs) of the same family of τ cell receptors were expanded and subsequently recognized as MHC I-peptide complexes displayed on, for example, tumor cells. In the appropriate microenvironment, activated τ fines Aim and kill cancer cells. 98561.doc 200530399 Based on the mechanism provided by Listeria, write a plan to provide heterologous antigens via MHC class 1 pathway, express heterologous genes and newly synthesized proteins from bacteria The potency of both secreted into the cytoplasm of infected (providing antigen) cells is directly related to the potential for CD8 + T cell triggering and / or activation. Because the degree of Ag-specific T cell triggering is directly related to vaccine efficacy, The efficacy of heterologous protein expression and secretion is directly related to the potential of the vaccine. Therefore, 'new techniques are needed to optimize the performance of heterologous protein expression and secretion in order to enable Listeria-based vaccines and other bacteria The effectiveness of the base-based vaccine is maximized. It would also be beneficial to optimize the effectiveness of heterologous protein expression and secretion in a bacterial host-expression system, where it is desirable to have a large number of heterologous protein expression and secretion. [Summary of the Invention] The present invention generally provides novel polynucleotides, including novel recombinant nucleic acid molecules, performance cassettes and vectors, for expression and / or secretion of polypeptides (eg, heterologous polypeptides) in bacteria, especially Listeria spp. . In some embodiments, these polynucleotides provide enhanced expression and / or secretion of polypeptides in bacteria. The present invention also generally provides bacteria ' including recombinant nucleic acid molecules, cassettes or vectors, and pharmaceutical, immunogen, and vaccine compositions including the bacteria. These bacteria and compositions can be used to induce an immune response and treat and / or prevent a variety of diseases or other conditions, including cancer, infections, and autoimmunity. One aspect of the present invention provides a recombinant nucleic acid molecule, including a first polynucleotide encoding a signal peptide, wherein the first polynucleotide is codon-optimized for expression in bacteria, and encodes a polypeptide (eg, an antigen) The second polynucleotide, wherein the second polynucleotide has the same translation editing architecture as the first polynucleotide 98561.doc 200530399, and wherein the recombinant nucleic acid molecule encodes a fusion protein, which includes a signal peptide And the polypeptide. In some embodiments, the second polynucleotide codon is also-optimized for expression in bacteria. The invention also provides a performance cassette comprising the recombinant nucleic acid molecule, and further comprises a promoter gene operably linked to the recombinant nucleic acid molecule. Vectors and bacteria including recombinant nucleic acid molecules and / or expression cassettes are also provided, as are pharmaceutical compositions, immunogenic compositions and vaccines including the bacteria. Methods of using the bacterium or a composition comprising the bacterium in a host to induce an immune response and / or prevent or treat a condition, such as a disease, are also provided. In another aspect, the invention provides a recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a signal peptide that is natural to bacteria, wherein the first polynucleotide codon-most Optimization, and (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein, including a signal peptide And the polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the polypeptide encoded by the second polynucleotide is foreign to the bacteria. The invention also provides a performance cassette including the recombinant nucleic acid molecule, and further includes a promoter gene operatively linked to the recombinant nucleic acid molecule. Vectors and bacteria including recombinant nucleic acid molecules and / or expression cassettes are also provided, as are pharmaceutical compositions, immunogenic compositions, and vaccines including the bacteria. Methods of using the bacterium or a pharmaceutical composition comprising the bacterium in a host to induce an immune response and / or prevent or treat a condition (such as a disease) are also provided. 98561.doc 200530399 In another aspect, the present invention provides a recombinant Listeria bacterium that includes a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes the first multinucleate gene encoding a signal peptide. The performance is to optimize the first polynucleic acid codon 'and the second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing structure as the first polynucleotide, where the recombinant nucleic acid The molecule encodes a fusion protein, including a signal and a peptide. In some embodiments, the polypeptide encoded by the second polynucleic acid is heterologous to the signal peptide. In some embodiments, the signal peptide is natural to Listeria. Methods of using Listeria (or a composition comprising Listeria) in a host to induce an immune response and / or prevent or treat a condition (eg, a disease) are also provided. In another aspect, the present invention provides a recombinant nucleic acid molecule, including a first polynucleic acid encoding a non-secAu «bacteria signal peptide, and a second polyacid encoding a polypeptide (such as an antigen). Scholate is the same translation editing framework as the first polynucleic acid, in which the recombinant nucleic acid molecule encodes a fusion protein, including a signal peptide and the polypeptide. In some embodiments, the first and / or second polynucleotide codons are optimized for performance in bacteria such as Listeria. The invention also provides a performance cassette comprising the recombinant nucleic acid molecule, and further comprises a promoter gene operably linked to the recombinant nucleic acid molecule. Vectors and bacteria including recombinant nucleic acid molecules and / or expression cassettes are also provided, as are pharmaceutical compositions, immunogenic compositions, and vaccines including the bacteria. Methods of using the bacterium or a composition comprising the bacterium in a host to induce an immune response and / or to treat a condition such as a disease are also provided. 98561.doc -10- 200530399 — In another aspect, the present invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises a first multinucleate ㈣ encoding a non-commercial ... bacterial signal peptide, and ( b) The second polynucleic acid encoding a signal peptide that is heterologous or paired: is a foreign polypeptide, and the second polynucleic acid in the salamander is the same translation editing structure as the first polynucleic acid, where the weight is: The nucleic acid molecule encodes a fusion protein, including a signal peptide and the polypeptide. In one, ', body & example towel' by the second multi-nucleus g "encoded polypeptide is signal to two kinds or to bacteria is foreign (that is, to fine ® is heterogeneous), or to both Yes. Pharmaceutical compositions, immunogenic compositions and vaccines including Listeria are also provided. Methods of using the Listeria (or a composition comprising the Listeria) in a host to induce an immune response and / or prevent or treat a condition (eg, a disease) are also provided. In one aspect, the present invention provides a recombinant nucleic acid molecule comprising a polynucleic acid encoding a polypeptide that is foreign to the genus Listeria (eg, a cancer or non-listeria infectious disease antigen), wherein Expressed in Yitella genus, the polynucleotide codon encoding the foreign polypeptide is maximized. In some embodiments, the recombinant nucleic acid molecule further includes a polynucleus encoding a polypeptide that is foreign to Listeria spp. Polynuclear picric acid encoding a signal peptide encoding the same translational editing architecture as picric acid. In some embodiments, the signal peptide is natural to Listeria bacteria. In other examples, the 4 signal peptide is foreign to Listeria spp. In some specific embodiments, in order to be expressed in Listeria spp. : ^ G ㈣ code optimization. Also provides Listeria spp. Which includes the heavy d core molecule. Also provides pharmaceuticals including Listeria s. 98561.doc 200530399 compositions, immunogenic compositions and vaccines. In addition, the present invention Methods of using the recombinant Listeria spp. In a host to induce an immune response and / or prevent or treat a condition (such as, but not limited to, a disease) are also provided. In another aspect, the invention provides recombinant Listeria spp.囷 'wherein the performance card E includes a polynucleic acid encoding a polypeptide that is foreign to Listeria (such as a cancer or non-Listeria infectious disease antigen), and in order to be expressed in Listeria, the encoding Polynucleotide codons of foreign polypeptides · Optimization and operably linked promoter genes encoding polymorphisms of foreign polymorphisms. In some embodiments, the performance cassette further includes The same translation and editing architecture as that encoding a polynucleic acid that is a foreign polypeptide to Listeria, a polynucleotide encoding a signal peptide (a signal peptide that is natural or foreign to Listeria bacteria), and The method of operation is linked with the promoter gene, and the expression cassette is expressed by making a fusion protein including a signal peptide and a foreign polypeptide. In some specific embodiments, the signal will be encoded for expression in Listeria spp. Polynucleotide codons for peptides-optimization. Pharmaceutical compositions, immunogenic compositions, and vaccines including the Listeria spp. Are also provided. In addition, the present invention provides the use of the recombinant Listeria spp. In a host to induce an immune response And / or a method for preventing or treating a condition, such as a disease. In another aspect, the invention provides a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes (a) a first multinucleus encoding a non-Listeria signal peptide And (b) a second polynucleotide encoding a polypeptide with the same translation editing architecture as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a A fusion protein of non-Listeria spp. Peptide and the polypeptide. The present invention also provides 98561.doc 200530399 for the performance card E including the recombinant nucleic acid molecule, in which the performance card is further improved and includes an operable manner with the The first and second polynucleic acid-linked promoter genes of the recombinant nucleic acid molecule. Vectors including the recombinant nucleic acid molecule and / or expression cassette are also provided. In addition, Lees including the recombinant nucleic acid molecule and / or expression cassette are also provided. Special bacteria. Also provided are pharmaceutical compositions, immunogenic compositions, and vaccines including the Listeria bacterium. The present invention provides the use of the Listeria bacterium (or a combination comprising the Listeria bacterium) in a host Method) to induce an immune response and / or prevent or treat a condition (eg, disease). In another aspect, the present invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises a non-Listeria Is the first polynuclear acid of the signal peptide, and (b) encodes the second polynuclear picric acid of the polypeptide with the same translational editing architecture as the first polynucleate, in which The recombinant nucleic acid molecule encodes a fusion protein including a non-Listeria spp. Signal peptide and the polypeptide. Pharmaceutical compositions, immunogenic compositions and vaccines comprising the Listeria genus are also provided. Methods of using the Listeria (or a composition comprising the Listeria) in a host to induce an immune response and / or prevent or treat a condition (e.g., a disease) are also provided. In another aspect, the present invention provides a Listeria bacterium including a manifestation card E (e.g., from a species of Listeria monocytogenes) that includes a first multinucleus encoding a non-Listeria signal peptide Glycylic acid, with the same translation and editing architecture as the first polynucleotide, a second polynucleotide * encoding a polypeptide (such as an antigen), and an operable approach to both the first and second polypicric acid The flail starts the base s. This expression encodes a fusion protein of the B. spp. Liszt 98561.doc -13 · 200530399 Mycobacterium spp. Peptide and the polypeptide Λ ^, human, and both. In some specific embodiments: the expression in Listeria spp. Is optimized for the first and / or the second multi-core common code. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising the Listeria spp. In addition, the present invention also provides a method for using the recombinant Liszt gene in a host to induce an immune response and / or prevent or treat a condition (e.g., a disease). The present invention also provides a recombinant nucleic acid molecule comprising a first polynucleic acid encoding a bacterial autolysin or a catalytically active fragment or a catalytically active variant, and (b) a second encoding a polypeptide Two polynucleotides, wherein the first nucleotide is the same translation and editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a polypeptide including a second polynucleotide encoding Lysin, or a catalytically active fragment or a catalytically chimeric protein chimera thereof, wherein in the protein f chimera, the polypeptide and autolysin or a catalytically active fragment or The variants are fused, or located within. Vectors and bacteria including the recombinant nucleic acid molecule and / or expression cassette are also provided, as are pharmaceutical compositions, immunogenic compositions, and vaccines including the bacteria. Methods of using the bacteria or composition in a host to induce an immune response and / or treat a condition, such as a disease, are also provided. In another aspect, the invention provides a recombinant nucleic acid molecule, wherein the nucleic acid molecule encodes at least two isolated non- Listeria polypeptides. The present invention further provides a performance cassette ' which includes a recombinant nucleic acid molecule, and further includes a promoter gene ' wherein the promoter gene is operatively linked to the recombinant nucleic acid molecule. Further provided is a vector comprising the recombinant nucleic acid molecule and / or a performance cassette. In addition, 98561.doc -14- 200530399-Lichenella bacteria including the recombinant nucleic acid molecule (and / or performance cassette) are also provided. Pharmaceutical combinations including the Listeria spp., Immunogen compositions, and vaccines are also provided. Methods of using the Listeria (or a composition comprising the Listeria) to induce an immune response and / or prevent or treat a condition (eg, a disease) in a host are also provided. In another aspect, the present invention provides a recombinant Listeria bacterium comprising a polycistronic expression cassette, wherein the polycistronic expression kalma includes at least two non-Listeria polypeptides. Medical music compositions, immunogenic compositions and vaccines including the Listeria spp. Are also provided. Methods of using the Listeria (or a composition comprising the Listeria) to induce an immune response and / or prevent or treat a condition (eg, a disease) in a host are also provided. Aspect n provides a recombinant nucleic acid molecule, which includes a first polynucleotide that encodes a signal, a first polynucleic acid that encodes a secreted protein or a fragment thereof, wherein the second polynucleotide is the same as the first The same translation and editing architecture of polynucleotide, and a third polynucleotide encoding a polypeptide heterologous to the secreted protein or fragment thereof, wherein the third polynucleotide is the same as the first and second The same translation and editing architecture of polynucleotides' wherein the recombinant nucleic acid molecule encodes a protein chimera including a signal peptide, a polypeptide encoded by a third polynucleotide, and a secreted protein or a fragment thereof, and in the protein chimera Wherein the polypeptide encoded by the third polynucleotide is fused to the secreted protein or a fragment thereof, or is located within the secreted protein or a fragment thereof. A performance cassette is also provided that includes a recombinant nucleic acid molecule and further advances include a promoter gene operably linked to the first, first, and second polynucleotides of the recombinant nucleic acid molecule. Vectors and bacteria including recombinant nucleic acid molecules and / or expression cassettes are also provided, as are 98561.doc -15 · 200530399 including pharmaceutical compositions, immunogenic compositions and vaccines including the bacteria. Methods of using the bacterium or a composition comprising the bacterium to induce an immune response and / or prevent or treat a condition are also provided. In some embodiments, a method of inducing an immune response to an antigen in a host 'includes administering to the host an effective amount of a composition comprising a recombinant bacterium described herein (e.g., in any of the views above, Or in the methods or examples below), wherein the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or carrier in bacteria includes the antigen. In some specific embodiments, a method of preventing or treating a condition, such as a disease, in a host includes administering to the host an effective amount of a composition, including a recombinant bacterium described herein. The invention further provides the use of a recombinant bacterium described herein (for example, in any of the points above, or in the methods or examples below) for the manufacture of a medicament that induces an immune response to an antigen in a host, Wherein the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette and / or carrier in bacteria includes the antigen. In some embodiments, the antigen is a heterologous antigen. The invention also provides the use of a recombinant bacterium described herein for the manufacture of a medicament for the prevention or treatment of a condition (e.g. cancer or infectious disease) in a host. The invention further provides a recombinant bacterium described herein that can be used to induce an immune response to an antigen in a host, wherein the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or vector in the bacterium includes the antigen. The invention further provides recombinant bacteria described herein for use in the prevention or treatment of conditions (e.g., diseases) in a host. In more aspects, the invention provides improved methods for the expression and screening of heterologous 98561.doc -16-200530399 proteins in host bacteria. Manufacture also includes each of the above recombinant nucleic acid molecules and performance cards! Of bacteria. Methods of using the bacteria to produce vaccines are also provided. The present invention further provides various polynucleic acids encoding signal peptides and / or antigens, including polynucleotides that have been optimized by dense stone horses for expression in Listeria monocytogenes. [Embodiments] I. Introduction The present invention provides various polynucleotides, including recombinant nucleic acid molecules, expression cassettes, and polypeptides for expression and / or secretion in bacteria, such as Listeria, including heterologous polypeptides ( Such as antigens and / or mammalian proteins). In some embodiments, these polynucleotides can be used to improve the expression and / or secretion of polypeptides in bacteria. Some performance cards include codon-optimized codon sequences for peptides and / or signal peptides. In addition, some performance cards g ' used on bacteria contain signal peptide sequences derived from other bacterial sources and / or derived from a variety of different secretory pathways. Bacteria are also provided that include the epi cassette, just like compositions containing the bacteria, such as vaccines. Methods are also provided for using the polynuclear picric acid, bacteria, and compositions in a host to induce an immune response and / or prevent or treat a condition, such as a disease (eg, cancer). : This month is based on the codon optimization of signal-like sequences in the performance card g. The southern peptide was obtained from a recombinant bacterium (especially the most efficient combination with the dense polypeptide of a heterologous polypeptide) ( (Eg, antigen) expression and / or secretion based on discovery 'even when the signal sequence is natural to bacteria (see, eg, Examples 19 and 27 below). In addition, it has been found that 98561.doc -17- 200530399 can also be used to obtain signal peptide sequences derived from non-secAl secretion pathways and / or signal peptide sequences derived from non-Listeria bacteria Effective expression and / or secretion of genus heterologous polypeptides (see, eg, Examples 19, 27, and 30 below). A part of the present invention is also based on the codon optimization of the codon sequence of the heterologous polypeptide, which improves the extra discovery of the heteropolypeptide in Listeria spp. And / or secretion (see, eg, Example 19 below). It has also been shown that improved expression and / or secretion of heterologous proteins obtained through optimization of the performance cassette results in improved immunogenicity of bacteria including the optimized performance cassette (see, eg, Example 2 below). ). In addition, it has also been shown that the performance card E encoding a protein chimera including a heterologous antigen embedded in autolysin can be used in Listeria for efficient expression and secretion of a heterologous antigen (see, eg, Example 29 below) ). Autolysin protein chimeras have also been shown to be immunogenic (see, e.g., Example 3ia below). In addition, Listeria spp. That includes the codon-optimized performance card g and / or performance carduns that include non-Listeria spp. Signal peptides are immunogenic and low tumor volume in mouse mode And increase survival (see, eg, Example 31B-E below). Therefore, 'on the one hand, the present invention provides Panyu Rizhan, a host-provided recombinant nucleic acid molecule, including a first polynucleotide encoding a signal peptide', wherein the first polynucleic acid codon_ Optimization, and a second polynucleic acid encoding a polypeptide (such as an antigen) with the same translation structure as the first polynucleic acid, and wherein the recombinant nucleic acid molecule is incorporated into the fusion of the peptide and the polypeptide protein. In some specific examples, the 7 gs also optimizes the second polynucleotide codon (usually in Yuwang in the same bacteria as the first polynucleic acid 98985.doc • 18-200530399 which performed). In some embodiments, in order to be used in Listeria, Bacillus, Yersinia pesds, Salmonella, Shigella, Brucella ( Brucella), Mycobacterium or E. coli, the first polynucleotide or the first and second polynucleotide codons are optimized. In some specific embodiments, the polynucleotide (s) codon is optimized for performance in Listeria, such as Listeria monocytogenes. In some embodiments, the polypeptide encoded by the second polynucleotide is (or includes) an antigen, and in some cases, it may be a non-bacterial antigen. For example, in some embodiments, the antigen is or is derived from a tumor-associated antigen. For example, in some specific embodiments, the antigen is 1 to §, H Ras N-Ras, 12-K-Ras, mesotheHn, pSCA, NY ESO-1, WT-1, survivin ), Gpl〇〇, Protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or derived from K-Ras, H-Ras, N-Ras, mRas, mesothelin, pscA, ny -eso], WT], survival protein, gpi〇pAp, protease], SPAS-1, SP-17, PAGE-4, TARP or CEA. For example, in some specific examples, the antigen is mesothelin, an antigenic fragment or an antigenic variant of mesothelin.纟 In some other specific embodiments, the antigen is an antigenic fragment or an antigenic variant of NY-ESCM 'or ny-esqj. In one example, the antigen is an infectious disease antigen, or derived from an infectious disease antigen. In some embodiments, the signal peptide is bacterial (Listeria = non-Listeria). In some embodiments, the signal peptide encoded by the codon-optimized first polynucleotide is natural to bacteria. In another specific embodiment, the first codon-optimized 98561.doc -19-200530399 polynucleotide-encoded signal peptide ' is foreign to bacteria. In a specific embodiment, the signal peptide is a secAl signal peptide, such as the LLO signal peptide obtained from Listeria monocytogenes, the UsP45 signal peptide obtained from Lactococcus lactis, Or a protective antigen signal peptide from BaciUusanthracis. In some embodiments, the signal peptide is a secA2 signal peptide. For example, the signal peptide may be a p 6 0 signal peptide obtained from Listeria monocytogenes. In addition, the recombinant nucleic acid molecule may optionally include a third polynucleotide sequence encoding P60 or a fragment thereof in the same translation and editing framework as the first and second polynucleotides, wherein the second polynucleotide is Located within the third polynucleotide, or between the first and third polynucleotides. In another specific embodiment, the signal peptide is a Tat signal peptide, such as a Bacillus subtilis Tat signal peptide (e.g., PhoD). The invention also provides a performance cassette comprising a recombinant nucleic acid molecule, and further includes operably interacting with the recombinant nucleic acid molecule (eg, with a first and second polynucleotide (if present with a third polynucleotide) ) Linked promoter genes. Performance vectors and recombinant bacteria (such as Listeria) including the performance cassette are also provided, as are pharmaceutical compositions, immunogenic compositions, and vaccines that include the bacteria. Methods are also provided for inducing an immune response and / or preventing or treating a condition, such as a disease, using the bacterium or a composition comprising the bacterium. It is also provided to use the bacterium in a host to manufacture a pharmaceutical product that induces an immune response against the antigen, wherein the polypeptide encoded by the second polynucleotide includes the antigen. In a secondary aspect, the present invention provides a recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a signal peptide that is natural to bacteria, wherein for performance in bacteria, the first polynucleotide Codon optimization, and (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is 98561.doc -20- 200530399 the same translation editing structure as the first polynucleotide, Wherein the recombinant molecule encodes a fusion protein including a signal peptide and the polypeptide. In some embodiments, the polypeptide encoded by the first polyacid is heterologous to the signal peptide. In this case, it can be distinguished as follows: In the case of dioctahedron, the second polynucleotide, nucleoside, is heterologous. In some embodiments, the peptide is foreign to the bacteria, but the signal peptide is: two to the bacteria. In some embodiments, ' is encoded by a second polynucleotide as much: heterologous to the T signal peptide, foreign to the bacteria, or both. In some specific embodiments, the bacteria from which the signal-like cells are derived are bacteria within the cell. In some embodiments, the bacterium is selected from the group consisting of Listeria bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacterium or E. coli Group of people. In some embodiments, the bacteria are bacteria of the genus Listeria (such as Listeria monocytogenes). In some embodiments, the second polynucleotide codon is optimized for performance in bacteria. In some specific examples, codon-optimization of the second polynucleotide and / or the second polynucleotide improves the performance in or secretes from bacteria encoding fusion proteins (as opposed to non-codons. Optimized sequence). In some embodiments, the polypeptide encoded by the second polynucleic acid comprises an antigen. The polypeptide encoded by the second polynucleic acid is the antigen. In some embodiments, the antigen is a non-bacterial antigen. In some embodiments, the antigen is a tumor-associated antigen, including antigens derived from tumor-associated antigens. In some embodiments, the antigen is selected from K_Ras, H_Ras, N_Ras, 12-K-Ras, mesothelin, PSCA, NY_ES01, survival protein, gP 100, PAP, protease 3, SPAS_i, sp ] 7, pAGE 4, TArp 98561.doc -21-200530399 or a group of CEA, or derived from the group selected from K_Ras, H_Ras, N-Ras 12-K-Ras, mesothelin, psca, NY-ESO-1, Antigens consisting of WT-1, survival protein, gplOO, PAP, protease 3, SPAS ", spi7, pAGE4, TArp or CEA. For example, in some embodiments, the antigen is mesothelin 'or an antigenic fragment or variant thereof, or NY ESO 1 or an antigenic fragment or variant thereof. In some alternate specific examples, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the signal peptide is a secA1 signal peptide (e.g., the LLO signal peptide from Listeria monocytogenes). In some embodiments, the signal peptide is a secA2 signal peptide (e.g., a P60 signal peptide from Listeria monocytogenes). A performance cassette comprising a recombinant nucleic acid molecule is also provided, and further comprises a promoter gene operably linked to the first and second polybasic acids of the recombinant nucleic acid molecule. A performance carrier including the performance card E is also provided. Also provided is a recombination comprising the recombinant nucleic acid molecule, "Agrobacterium" wherein the first polynucleotide codon is optimized for performance in the recombinant bacterium. In some embodiments, the recombinant bacterium is an intracellular bacterium In some embodiments, the recombinant bacteria is selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, and groups. In some embodiments, the bacteria are recombinant Listeria bacteria (eg 1 ·

Wherein, the second polynucleotide encodes Shigella spp., Brucella, Mycobacterium, or E. coli, and the number of codes is as large as the method; and the method of polypeptides that group the recombinant bacteria is an antigen. . It also extracts the compound, which is from Chu Chu-Jia Zhiji and 98561.doc -22- 200530399; the bacterium is used in the manufacture of medical products that induce immune responses to antigens in the host = of which is the second polynucleic acid The polymorphism of the code includes the following aspects: The present invention provides a recombinant Liszt gene (eg, Listeria monocytogenes) comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid is a A polynucleotide, in order to express in the bacteria of the Listeria genus, the first polynucleotide codon-optimum ^, and (b), the second polynucleotide of the 4-coded polypeptide, The second polynucleotide is the same translation editing structure as the first polynucleotide, wherein the heavy and nuclear & knife encoding includes a signal peptide and the polymorphic fusion protein. In the integrated example The polypeptide encoded by the second polynucleic acid is heterologous to the signal peptide. In some specific embodiments, the recombinant nucleic acid molecule is a part of the performance card S, which further includes being operable with First and first polynucleic acid linked initiation In other words, in some embodiments, 'the recombinant Listeria spp. Includes a performance card E' which includes a recombinant nucleic acid molecule, wherein the performance card is further enhanced-further including in an operable manner A promoter gene linked to both the first and second 核 nucleotides of the recombinant nucleic acid molecule. In some specific embodiments, the performance card E 疋 polycistronic performance card is concealed. In some specific embodiments, To express in the fine g of Listeria 'optimize the second polynucleic acid codon. In some embodiments, the codon-optimization effect of the first and / or second polynucleic acid To improve the performance of the edible fusion protein in Listeria bacteria and / or to secrete it (relative to the non-codon · optimized sequence). In some specific embodiments, the second Polynucleotide-encoded polypeptides are foreign to the bacteria of the genus Listeria (ie, heterologous to the bacteria of the genus Listeria 98561.doc -23- 200530399). In some specific embodiments, the The polypeptide encoded by the second polynucleotide includes an antibody (Eg, non-Listeria or non-bacterial antigens). In some embodiments, the polypeptide encoded by the second polynucleic acid is the antigen. In some embodiments, the antigen is related to a tumor Related antigens or derived from tumor-related antigens. In some specific implementations of mesothelin, PSCA, NY-ESO-1, WT-1, survival proteins, gpl〇〇, PAP, protease 3, SPAS], sp_17, pAGE 4. The group consisting of Ding Ba Chu and ⑶, or derived from K_Ras, H_Ras, N_Ras, 12_K-Ras, mesothelin, PSCA, NY-ESO-1, WT_1, survival protein, gpl〇〇, PAP, protease 3. Antigens of a group consisting of SPAS-1, SP-17, pAGE-4, TArp and CEA. For example, in some embodiments, the antigen is mesothelin or an antigenic fragment or antigenic variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is human mesothelin with its signal peptide and GPI cross-linker functional site deleted. In some alternate embodiments, the antigen is NY_ES0_ 丨, or an antigenic fragment or antigenic variant thereof. In some alternate embodiments, the antigen is an infectious disease antigen, or an antigen derived from an infectious disease antigen. In some specific examples, the signal peptide is non-Listeria. In some embodiments, the # peptide is bacterial. In some embodiments, the signal peptide is foreign to Listeria. In other specific embodiments, the # peptide is natural to Listeria. In some embodiments, the signal peptide is a secAl signal peptide (for example, the LLO signal peptide obtained from Listeria monocytogenes, the Usp45 signal peptide obtained from Lactococcus lactis, and 98561.doc -24-200530399 and Protective antigen signal peptide from Anthrax rod &). In some implementations, the h 5 tiger-like signal secA2 (for example, p 6 (Ms peptide) from Listeria monocytogenes). In some embodiments, the signal is a Tat signal. Peptides (such as the PhoD signal peptide from Bacillus subtilis). In some specific embodiments, the Listeria bacterium is attenuated. For example, cell-to-cell spread can be used to enter non-pupid cells, Or proliferation, attenuating Listeria. In some embodiments, the recombinant Listeria bacterium is defective for ActA, internaiin B, or both ActA and surface protein b (eg, ΔΜΐΑΔίπΙΒ dual Deleted mutants In this specific example, the recombinant Listeria bacterium deleted both the functional ActA, surface protein B4ActA, and surface protein B. In some specific examples, 'already by (Such as psoralen (ps0 oil η) compounds) to modify the nucleic acid of the recombinant bacteria. The present invention also provides a recombinant Listeria bacterium and a pharmaceutical composition, to An immunogen comprising a recombinant Listeria bacterium, wherein the polymorphism encoded by the second polynucleic acid is an antigen. The present invention also provides a vaccine including a recombinant Listeria bacterium. Also provided in a host, A method for inducing an immune response to an antigen, comprising administering to the host an effective amount of a composition including a recombinant bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen. It also provides prevention or treatment of a condition in the host. (E.g., a disease such as cancer or an infectious disease), comprising administering to the host an effective composition comprising a recombinant Listeria bacterium. The bacterium is also provided to induce an immune response to the antigen in the host. For use in pharmaceuticals, wherein the polypeptide encoded by the second multinucleate is an antigen. 98561.doc • 25-200530399 In a fourth aspect, the present invention provides a recombinant nucleic acid molecule comprising a non-c A1 ,,, The first polynucleotide of the Lactobacillus peptide L and the second polynucleic acid encoding a polypeptide (such as an antigen), wherein the second polynucleic acid is the same as the first polynuclear acid Translation and editing frame Xiao 'and wherein the recombinant nucleic acid molecule encodes a fusion protein including a signal peptide and the polypeptide. In some specific embodiments, in order to be expressed in a particular type of bacteria, the first polynuclear acid and / or— Second polynuclear ⑽ codon optimization. In some embodiments = codons of 7th or 2nd polynucleotide-optimization improves the expression and / or secretion of the fusion protein in the bacterium (Vs. non-dense 2 sub-optimized sequences). In some specific embodiments, in order to be in the Listeria: genus, Bacillus, Yersinia pestis, Salmonella, Zhibei , Brucella, Mycobacterium, or E. coli, optimize the first polynucleotide and / or the second polynucleotide codon_. In a real β case, in order to express in Listeria, such as Listeria monocytogenes, the polynucleic acid codons were optimized. In the one- and two-body example, the L-bleptide ' encoded by the first codon-optimized polynucleotide is natural to the bacterium and is codonized for this purpose. In this and one embodiment, the first polynucleotide encodes the

The peptide is heterologous to the second polynucleotide. In some specific implementations, the ancient female. The polypeptide encoded by Gu Xi nucleotide is an alien to the signal peptide. In some specific embodiments, the original encoded by the second polynucleic acid is in some specific In the embodiment, the polypeptide rabbit encoded by the second polynuclear acid is an antigen, which may be a non-bacterial antigen in some cases. ^ 'One here *' Bai-qu / ϊλ- > In one embodiment, the antigen is a tumor-associated antigen, 9856l.doc 200530399 or derived from such a tumor-associated antigen. For example, in some specific implementations In the example, 'the antigens are K-Ras, H-Ras, N_Ras, 12_K_Ras, mesothelin, PSCA, NY-ESO], WT-i, survival protein, 、 100, pA proteinase 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, Mesothelin, psCA, NY-ESO-1 'WT-1, Survival Protein, gplOO, pAp, protease 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For example, in some embodiments, the antigen is mesothelin, or an antigenic fragment or antigen thereof Variants. In some other specific embodiments, the antigen is NY_ES0_1, or an antigenic fragment or antigenic variant of NY-ESO-1. In some embodiments, the antigen is an infectious disease antigen Or derived from an infectious disease antigen. In some embodiments, the signal peptide of Listeria monocytogenes encoded by the first polynucleotide of the recombinant nucleic acid molecule. In other In specific embodiments, the signal peptide is non-Listeria. In some embodiments, the signal peptide is derived from Gram-positive bacteria. In some embodiments, the signal peptide is derived from a spore. A bacterium of the genus Bacillus, Staphylococcus, or Lactococcus. In some specific embodiments, the signal peptide is a secA2 signal peptide. For example, the 'δHk' peptide may be a signal pattern obtained from Listeria monocytogenes In addition, 'the recombinant nucleic acid molecule may optionally include a third polynucleotide sequence encoding ρ60 or a fragment thereof, which is located in the third polynucleotide, or in the first and third polynucleotides. The same translation and editing architecture of the second polynucleotide between the two. In another specific embodiment, the signal peptide is a Tat signal peptide, such as the Tat signal peptide of Bacillus subtilis (for example, the P. subtilis signal peptide) ° The invention also provides a performance cassette comprising the recombinant nucleic acid molecule, 98561.doc -27- 200530399 which further includes a primer operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule Therefore, expression vectors and bacteria including expression cassettes and / or recombinant nucleic acid molecules are also provided, as are pharmaceutical compositions, immunogenic compositions, and vaccines including the bacteria. In some embodiments, expression cassettes or recombinants are included The recombinant bacteria of the nucleic acid molecule are intracellular bacteria. In some specific embodiments, the bacteria is selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Cloth A group of bacteria of the genus Mycobacterium, Mycobacterium, or E. coli. In some embodiments, the bacterium is a bacterium of the genus Listeria (e.g., a member of the species Listeria monocytogenes). In some embodiments, the polypeptide encoded by the first polynucleotide is foreign to the bacterium (p is heterologous to the bacterium). Methods of using the bacterium or a composition comprising the bacterium in a host to induce an immune response and / or prevent or treat a condition (e.g., a disease) are also provided. In some embodiments, the condition is cancer. In 1 his specific implementation shot, the condition was an infectious disease. Also provided is the use of the bacterium for the manufacture of a medicament for inducing a response to an antigen in a wounded subject, wherein the polypeptide encoded by the second polynucleotide includes an antigen. f In another aspect, the present invention provides a recombinant Lisztium bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises: the first polynuclear picric acid which encodes a non-descriptive peptide; and the second polynuclear polypeptide which encodes a polypeptide. The second polynucleic acid is the same as the first polynucleic acid, and it is a wooden structure, in which the recombinant nucleic acid molecule encodes a fusion protein including a signal peptide and a nuclear-shaped protein. In some specific embodiments, the polypeptide encoded by the second oxalic acid is heterologous to the signal peptide or to the bacterium 98561.doc -28- 200530399

It's foreign, or both. In some embodiments, the Listeria genus belongs to the species Listeria monocytogenes. In some embodiments, the recombinant nucleic acid molecule is part of a performance card, which further includes a promoter gene operably linked to both the first and second polynucleic acids. In other words, in some specific embodiments, the recombinant Listeria bacterium includes a performance card g, which includes a recombinant nucleic acid molecule, wherein the performance card further includes a operably linked A promoter linked to both the first and second polynuclear picric acid. In some embodiments, the performance card is a multi-cistronic performance card E. In some specific embodiments, in order to express in Listeria bacteria (for example, monocytogenes), the first polynucleotide and the second polynuclear bud Codon optimization for acid or both first and second polynucleic acids. In some embodiments, the 'codon optimization of the first and / or the second polyphosphonate · improves the expression and / or secretion from the bacteria (compared to the non- Optimized sequence). Some specific implementation

In the example, the ' βHadi- and second polynucleotides are heterologous to each other. In some specific examples, the polypeptide encoded by the second polynucleic acid and the signal state are heterologous to each other. In some embodiments, the polypeptide encoded by the second polynucleic acid is foreign to the Listeria bacterium (eg, heterologous to the Listeria bacterium. In some embodiments, The polypeptide encoded by the second polynucleotide includes an antigen. In some specific cases, the polypeptide encoded by the second polynuclear acid is an antigen (such as a non-Listeria or non-bacterial antigen) ). In some specific implementations, the antigen is a tumor-associated antigen 'or derived from a tumor-associated antigen. Here 98561.doc -29- 200530399 In specific embodiments, the antigen is selected from K-Ras , H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO_l, WT-1, survival protein, gplOO 'PAP, protease 3, spAs ", SP-17, pAGE-4, The group consisting of TARp and CEA, or derived from K_Ras, H_Ras, N_Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survival protein, gplOO, PAP, protease 3, SPAS ", sp-17, pAGE4, TArp and CEA antigens. For example, in some embodiments, the antigen is mesothelial Or its antigenic fragments or antigenic variants. In some specific embodiments, the antigen is human mesothelin. In some specific embodiments, the 'delta' antigen deletes its signal peptide and gpi crosslinker functional sites. Human mesothelin. In some alternate embodiments, the antigen is an infectious disease antigen, or a bamboo-derived infectious disease antigen. In some embodiments, the signal peptide is non-Listeria. In some embodiments In some embodiments, the non-secA1 signal peptide is a Listeria spp. Signal peptide. In other specific embodiments, the non-secAl signal peptide is a non-Listeria spp. Signal peptide. In some embodiments, the k-shaped Is a secA2 signal (eg, a p60 signal peptide from Listeria monocytogenes). In some embodiments, the recombinant nucleic acid molecule includes a secA2 signal peptide, and further includes an encoding secA2 autolysin (eg, p60 or N-acetylcellulose enzyme) 'or a fragment thereof (e.g., a fragment having catalytic activity), a third polynucleotide with the same translation editing structure as the first and second polynucleic acids, Wherein the second polynucleotide is located within the third polynucleotide of the recombinant nucleic acid molecule, or between the first and third polynucleotides. In some embodiments, the second polynucleotide The nucleotide is located in the third polynucleotide. In some embodiments, the signal peptide is 98561.doc -30 · 200530399

Tat signal peptide. In some embodiments, the signal peptide is a Tat signal peptide derived from Bacillus subtilis (e.g., a pOD signal peptide obtained from Bacillus subtilis). In some embodiments, the Listeria bacterium is attenuated. For example, cell-to-cell spread, into non-phagocytic cells, or proliferation can be used to attenuate Listeria. In some embodiments, the recombinant Listeria pallidum is defective for both ActA, surface protein ActA, and surface protein B (e.g., a double deleted AactAAinlB mutant). In some embodiments, the recombinant Listeria bacterium deletes functional ActA, surface protein B, or both ActA and surface protein B. In some embodiments, the nucleic acid of a recombinant bacterium has been modified by reacting with a nucleic acid of a quasi-compound (such as a psoralen compound). The invention also provides a pharmaceutical composition comprising a recombinant Listeria bacterium and a pharmaceutically acceptable carrier. The invention also provides an immunogen composition comprising a recombinant bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen. The present invention also provides a vaccine comprising a recombinant Listeria® cellulite. A method of inducing an immune response to an antigen in a host is also provided, comprising administering to the host an effective amount of a composition comprising the recombinant bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen. Also provided is a method for preventing or treating a condition (e.g., a disease, such as cancer or wood-borne disease) in a host, including administering to the host an effective amount of a composition including the recombinant Listeria bacterium. The use of the bacterium for the manufacture of a medicament for inducing an immune response to an antigen in a host is also provided, wherein the polypeptide encoded by the second polynucleotide includes an antigen. In other aspects, the present invention provides a recombinant nucleic acid molecule, which includes a polypeptide (such as an antigen, such as antigens, such as anti-inflammatory disease 98561.doc 31 200530399) that is expected to be Listeria®. Antigen), and the polynucleotide codon was optimized for expression in Listeria. In some embodiments, the codon-optimization effect of the polynucleotide improves the expression and / or secretion of the polypeptide in Listeria bacteria (versus the non-codon-optimized sequence). ). In some embodiments, the foreign polypeptide includes an antigen. In some embodiments, the foreign polypeptide is an antigen. In some embodiments, the antigen is a non-bacterial antigen. For example, in some embodiments, the antigen is or is derived from a tumor-associated antigen. For example, in some embodiments, the polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1 'WT-1, survival protein, gpl 〇〇, PAP, protease 3, SPAS], SP-17, PAGE-4, TARP or CEA, or derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, wt-1, survival protein, gpl00, PAp, protease 3, SpAS1, sp i7, PAGE-4, TARP or CEA. In some embodiments, the antigen is mesothelin 'or an antigenic fragment or antigenic variant thereof. In some other specific embodiments, the antigen is NY-ESO-1, or an antigenic fragment or variant of NY-ESO-1. In some other specific embodiments, the antigen is an infectious disease antigen ' or derived from an infectious disease antigen. In some specific embodiments, the heavy nucleic acid molecule further includes a polynucleotide encoding a signal peptide in the same translation editing framework as the foreign polypeptide, so that the recombinant nucleic acid molecule can be edited to include the k-number peptide and the foreign Polypeptide fusion protein. In some embodiments, 'for expression in Listeria monocytogenes, a polynuclear 98561.doc 200530399 encoding a signal-like signal (which may or may not be natural to Listeria monocytogenes) will be encoded Sub-optimization. The invention further provides a performance cassette comprising the recombinant nucleic acid molecule, which further comprises a promoter gene operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule. A vector (e.g., a performance vector) comprising the recombinant nucleic acid molecule and / or a performance cassette is also provided. The present invention also provides a recombinant Listeria bacterium comprising the recombinant nucleic acid molecule and / or a performance cassette. In some embodiments, the Listeria bacterium belongs to the species Listeria monocytogenes. Also provided are pharmaceutical compositions, immunogenic compositions, and vaccines including the Listeria bacterium. The invention further provides a method for inducing an immune response to an antigen in a host, comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium, wherein the polypeptide is an antigen. In addition, the present invention also provides a method for inducing an immune response to an antigen and / or preventing or treating a condition (e.g., a disease) in a host using the Listeria bacterium. Also provided is the use of the bacterium for the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the foreign polypeptide includes an antigen. In other aspects, the present invention provides a recombinant Listeria bacterium including the expression of Carcun, the expression card E comprises encoding a polypeptide that is foreign to Listeria bacterium (such as an antigen such as a cancer antigen or non-Lister. Bacterium bacteria = antigen) of polynucleic acid 'wherein the polynuclear codon is optimized for expression in Listeria genus' and a promoter gene operably linked to a polynucleic acid encoding the foreign polypeptide. In some embodiments, the Listeria monocytogenes belongs to the species Listeria monocytogenes. In some specific examples, the codon optimization effect of the multinucleate cymbal enhances the performance of the polypeptide in the Liszt g gene and / or secretes it from 98561.doc 200530399 (compared to the non-codon Optimized sequence). In some embodiments, the foreign polypeptide includes an antigen. In some embodiments, the foreign polypeptide is an antigen, and in some cases it may be a non-bacterial antigen. For example, in some embodiments, the antigen is a tumor-associated antigen, or is derived from such a tumor-associated antigen. For example, in some specific embodiments, the polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survival protein, gpi 〇〇, pap, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA , NY-ESO-1, WT-1, survival protein, gplOO, pAp, protease 3, SPAS-1, SP-17, PAGE-4, D-ARP or CEA. In some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant thereof. In some other specific embodiments, the antigen is NY-ESH, or an antigenic fragment or antigenic variant of NY ESO-1. In some other specific embodiments, the antigen is an infectious disease antigen, or is derived from an infectious disease antigen. In some specific embodiments, the performance cassette further comprises: encoding a signal-like polynucleic acid with the same translation editing structure as the foreign peptide, which can be connected with the promoter gene in a manner of, This performance card B can be encoded, peptide and. Fusion of foreign peptides. In some embodiments, in order to be expressed in Listeria monocytogenes, a multi-core encoding signal peptide (which may or may not be natural to Listeria S) may be multi-core =,: sub-optimized. Also provided is a medical instrument including the recombinant Listeria bacterium, and a method for inducing immunological anti-cardiologists in an immunogen composition and epidemic host, for the host ... to include effective administration of 98561 to the host. doc-34. 200530399 A composition comprising a recombinant Listeria bacterium. In addition, the present invention also provides a method for using the recombinant Listeria to specifically fix the host tissue to the antigen and / or prevent or treat a condition (such as a disease). The bacteria can be used to induce an immune response to the antigen in the host. Use of Pharmaceuticals I 'wherein the foreign polypeptide includes an antigen.

In other aspects, the present invention provides a recombinant Listeria bacterium (eg, Listeria monocytogenes) comprising a recombinant nucleic acid molecule, wherein the recombinant nuclear cultivar molecule comprises a signal encoding a non-Listeria bacterium The first multi-core peptide of the peptide and the second multi-core peptide encoding the polypeptide, wherein the second multi-core peptide is the same translation editing framework as the first multi-core peptide, where The recombinant nucleic acid molecule encodes a fusion protein including a signal peptide of a non-Listeria bacterium and the polypeptide. In some embodiments, the recombinant nucleic acid molecule is located in a performance card, which further includes a promoter gene operably linked to both the first and second polynucleic acid. therefore,

In some specific embodiments, the recombinant Listeria bacterium includes a performance card ε ′ which includes a recombinant nucleic acid molecule, wherein the performance card further includes a first and A second polynucleotide-linked promoter gene. In some specific embodiments, the performance cassette is a multi-cistronic performance cassette (eg, a bicistronic performance card E). In some embodiments, in order to Cells, Listeria monocytogenes), the first polynucleotide, the second polynucleotide, or both the first and the second polynucleotide are codon-optimized. In one or two specific examples, the codon-optimization effect of the first and / or the second polynucleotide improves the performance of the fusion protein in the bacteria, and / or from it 98561.doc -35- 200530399 Synthetic protein secretion (relative to 1 and for non-codon-optimized sequences). In the example of a physical yoke, the first and second polynucleotides are seeded with each other. In some embodiments, the polypeptide depleted by the second polyacid and the signal peptide are heterologous to each other. In some embodiments, the polypeptide encoded by the first polynuclear #acid is foreign to the Listeria bacterium (ie, heterologous to the Listeria bacterium). In some specific embodiments, the polypeptide encoded by the second multi-core: y: ^ t / ^ nuclear glycine includes antigens (such as non-Listeria antigens). In some embodiments, the polypeptide re-encoded by the second polynucleoside S is an antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. In some specific embodiments, the antigen is selected from the group consisting of K_Ras, H_Ras, n_Rm, 12-K-Ras, mesothelin, PSCA, Nγβ01, WT1, survival protein, gplOO, PAP, protease 3, SpAS] , Sp_17, pAGE 4, TArp and CEA, or derived from the group selected from K_Ras, H_Ras, N-Ras, 12-K-Ras, Mesothelin, PSCA, NY-ESO-1, WT-1, survival protein , GplOO, PAP, protease 3, SPAS-1, SIM7, pAGE 4, TArp and CEA. For example, in some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen 疋 deletes human mesothelin from its signal peptide and GPI cross-linker functional site. In some embodiments of the parent, the antigen is an infectious disease antigen, or is derived from an infectious disease antigen. In some embodiments, the signal peptide is bacterial. In some embodiments, the signal peptide is derived from an intracellular bacterium. In some embodiments, the signal peptide is derived from Gram 98561.doc -36-200530399 positive bacteria. In some embodiments, the signal peptide is obtained from a bacterium belonging to the genus Bacillus, Staphylococcus or Lactococcus (e.g., Charcoal, Bacillus subtilis, Staphylococcus aureus or Lactococcus lactis). In some embodiments, the signal peptide is a secA 1 signal peptide (e.g., the Usp45 signal peptide from Lactococcus lactis or the protective antigen signal peptide from Bacillus anthracis). In some embodiments, the signal peptide is a secA2 signal peptide. In some embodiments, the signal peptide is a Tat signal peptide (e.g., a pDO signal peptide from Bacillus subtilis). In some embodiments, the Listeria bacterium is attenuated. For example, in some embodiments, Listeria can be attenuated by cell-to-cell spread, into non-phagocytic cells, or proliferation. In some embodiments, the recombinant Listeria bacterium is defective for ActA, surface protein B, or both ActA and surface protein B (eg, Δ & (^ ΑΔίη1Β double deleted mutant). In some specific implementations In the example, a recombinant Listeria bacterium deleted functional ActA, surface protein B, or both ActA and surface protein B. In some embodiments, a compound (such as a psoralen compound) has been used to target The nucleic acid reacts to modify the nucleic acid of the recombinant bacteria. The present invention also provides a pharmaceutical composition including a recombinant Listeria bacterium and a pharmaceutically acceptable carrier. The present invention further provides an immunogen combination including the recombinant bacteria The polypeptide encoded by the second polynucleotide is an antigen. The present invention also provides a vaccine including a recombinant Listeria bacterium. A method for inducing an immune response to the antigen in a host is also provided, including the host. An effective amount of a composition comprising the recombinant Listeria bacterium is administered, wherein the polypeptide composed of the second polynuclear picolinic acid is an antigen. Formula for preventing or treating a condition in a host, such as a disease such as cancer or infectious disease 98561.doc -37- 200530399 The method includes administering to the host an effective amount of a composition including the recombinant Listeria bacterium. Also provided Use of the bacterium for the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleic acid includes an antigen.-In other aspects, the invention provides a recombinant Lisz Agrobacterium (e.g., from the species Listeria monocytogenes) comprising a first polynucleotide encoding a non-Listeria signal peptide, the same as the first t-nucleotide Translation and editing architecture, a second polynuclear bitter-encoded polypeptide and a promoter gene operably linked to both the first and second polynucleic acid. The performance cassette encoding includes non-Listeria A fusion protein of both the signal peptide and the remote polypeptide. In some specific embodiments,-field cell pairs-cell proliferation, into human non-living cells, or proliferation, can attenuate Listeria bacteria. In some cases In the present embodiment, in order to optimize the first polynucleotide, the second polynucleotide, or the first and second and the first and the second codons in Listeria monocytogenes, the codons are optimized. In some specific The codon-optimization effect of the second and / or second polynucleotides in the examples improves the performance of the encoded fusion protein in the bacterium, and / or divides it from 苴: (vs. non-codon_ Optimized sequences). In some specific examples, the performance of the first polynuclear shoots and / or the first polynucleic acid codon-optimized is shown in Listeria monocytogenes. In some specific embodiments, the polypeptide encoded by the second polynuclear acid includes an antigen. In some specific examples, the polymorphism encoded by the second polynucleic acid is an antigen, and in A-case, it can Is a non-bacterial antigen. For example, in some specific known examples, the antigen is an antigen associated with cancer 'or derived from such an antigen associated with cancer 98561.doc -38-200530399. For example, in some specific embodiments, the antigen is K-Ras, H-Ras, N_Ras, 12_K_Ras, mesothelin, psCA, NY-ESO-1, WT-1, survival protein, gplOO, pAp, protease 3. SPAS-1, SP-17, PAGE_4, TAR] ^ cea, or derived from K Ras, H-Ras, N-Ras, 12-K-Ras, Mesothelin, PSCA, NY_ES〇 !!, WT- 1. Survivin, gp100, pAp, protease 3, spas-i, SP-17, PAGE-4, TARP or CEA. For example, in some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant thereof. In some other specific embodiments, the antigen is an antigenic fragment or an antigenic variant of NY_ESO_B or. In some embodiments, the antigen is an infectious disease antigen ' or is derived from #affected disease antigen. In a preferred embodiment, the signal peptide is bacterial. In some embodiments, the signal peptide is obtained from a bacterium belonging to the genus Bacillus, Staphylococcus, or Lactococcus. For example, in some embodiments, the signal peptide is obtained from Bacillus anthracis, Bacillus subtilis, Staphylococcus aureus®, or Lactococcus lactis. In some specific embodiments, the signal peptide is a SeeA1 peptide, such as a protective antigen signal peptide obtained from Lactococcus lactis-M signaling or Bacillus anthracis. In some specific examples, the 'secA2' signal peptide. In more specific embodiments, the peptide is a Tat signal peptide, such as a Bacillus subtilis plus a signal peptide (eg, pHOD). Also mentioned are pharmaceutical compositions, immunogen compositions and vaccines of recombinant Listeria bacteria described herein. In addition, the present invention also provides a method for inducing an immune response using the Listeria bacterium and / or preventing or treating a condition such as a disease. It is also used for the purpose of manufacturing a quilted mouth for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynuclear 98561.doc 200530399 glycoside includes an antigen . The present invention further provides a recombinant nucleic acid molecule 'comprising (a) a first polynucleotide encoding a bacterial autolysin, or a fragment or a catalytically active variant thereof; and (b) a second encoding a polypeptide Polynucleotides, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a polypeptide and an autolysin encoded by the second polynucleotide Or a protein chimera having a catalytically active fragment or a catalytically active variant thereof, wherein in the protein chimera, the polypeptide is fused to an autolysin, or a catalytically active fragment or a catalytically active variant thereof , Or located in autolysin, or its catalytically active fragment or catalytically active variant. In some embodiments, the first polynucleotide encodes a bacterial autolysin. In some embodiments, the protein chimera is as catalytically active as autolysin. In some specific embodiments, the bacterial autolysin is derived from a bacteria in a cell (e.g., Listeria). In some embodiments, the bacterial autolysin is an autolysin of the genus Listeria. In some specific embodiments, the second polynucleic acid encoding the polypeptide is located in the first polynucleotide encoding autolysin, or a catalytically active fragment or a catalytically active variant thereof, and the The recombinant nucleic acid molecule encodes a protein chimera, wherein the polypeptide is located in an autolysin, or a catalytically active fragment or a catalytically active variant thereof (that is, the polymorphic is buried in autolysin 'or has catalytic activity). Fragments or catalytically active variants in vivo). In some alternative embodiments, the second polynucleotide is located outside the first polynucleotide encoding autolysin, or a catalytically active fragment or a catalytically active variant thereof, and the recombination Nucleic acid points 98561.doc -40- 200530399

The daughter encodes a protein chimera in which the polypeptide is fused to autolysin, or a fragment having catalytic activity or a variant having catalytic activity. In some embodiments, the polypeptide is heterologous to autolysin. In some embodiments, the first polynucleotide and the second polynucleotide are heterologous to each other. In some specific embodiments, the recombinant nucleic acid molecule further includes (c) a third polynucleotide encoding a signal peptide with the same translation editing architecture as the first and second polynucleotides, wherein the recombinant nucleic acid The molecular code includes a signal peptide, a polypeptide encoded by the second polynucleotide, and an autolysin, or a catalytically chimeric fragment or a protein chimeric variant thereof. In some embodiments, the signal peptide is a secA2 signal peptide (eg, p60). In some specific embodiments, the signal peptide is an autolysin-related signal peptide in nature (e.g., the signal peptide is p60 and the autolysin is p60). In some embodiments, the autolysin is a secA2-dependent autolysin. In some embodiments, the autolysin is a peptidoglycan hydrolase (e.g., N-acetamomerase or p60). In some embodiments, the polypeptide encoded by the second polynucleotide includes an antigen. In some embodiments, the polypeptide is an antigen (for example, an antigen associated with a tumor, an antigen derived from an antigen associated with a tumor, an infectious disease antigen, or an antigen derived from an infectious disease antigen). In some embodiments, the antigen is selected from the group consisting of K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, pscA, NY-ESO-1, WT-1, survival protein, gplOO , PAP, protease 3, SPAS-Bu SP-17, PAGE-4, TARP and CEA, or derived from the group selected from K_Ras, H-Ras, N-Ras, 12-K_Ras, mesothelin, pSCA, NY -ESO-1, WT-1, survival protein, gplOO, PAP, protease 3, SPAS-b SP-17, PAGE-4, TARP 98561.doc -41-200530399 and CEA antigens. For example, in some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant thereof. In some specific embodiments, the antigen is human mesothelin. In some embodiments, the antigen does not delete its 间 § peptide and Gpi-determined human mesothelin. The present invention also provides a performance cassette comprising a recombinant nucleic acid molecule, further comprising a promoter gene operatively linked to the first and second polynucleotides of the recombinant nucleic acid molecule, and including the performance card. Carrier of performance. The present invention further provides a recombinant bacterium including the recombinant nucleic acid molecule. In some embodiments, the recombinant bacteria are intracellular bacteria, such as bacteria of the genus Listeria (e.g., Listeria monocytogenes). In some embodiments, the polypeptide encoded by the second polynucleotide is foreign to the recombinant bacteria. Also provided are pharmaceutical compositions comprising (a) the recombinant bacteria and (1)) a pharmaceutically acceptable carrier. In addition, an immunogen composition comprising the recombinant bacteria is also provided, wherein the polypeptide encoded by the second polynucleotide is an antigen. A vaccine comprising the recombinant bacterium is also provided, wherein the polypeptide encoded by the second polynucleotide is an antigen. The invention also provides a method for inducing an immune response to an antigen in a host, comprising administering to the host an effective amount of a composition comprising the recombinant bacteria, wherein the polypeptide encoded by the second polynucleotide is an antigen. Methods of preventing or treating conditions in a host are also provided, comprising administering to the host an effective amount of a composition comprising the recombinant bacteria. The use of the bacteria in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host is also provided, wherein the polypeptide encoded by the second polynucleotide includes an antigen. In another aspect, the present invention provides a recombinant Listeria bacterium comprising a polycistronic expression cassette, wherein the polycistronic expression cassette encodes at least two 98561.doc -42- 200530399 non- Listeria polypeptides. For example, in some specific embodiments, the cash card includes a first polynucleic acid encoding the first non-Listeria polypeptide, and a second polymorphism encoding the second non-Listeria polypeptide. Glycoside s 夂 α and a promoter gene operably linked to the first and second polynucleotides. In some embodiments, the performance card further includes intergenic sequences between the first and second polynucleotides. In some examples, the polycistronic expression card E is a bicistronic expression cassette that encodes two isolated non-Listeria polypeptides. In some specific embodiments, the recombinant Listeria bacterium belonging to the genus 30 belongs to the species Listeria monocytogenes. In some embodiments, at least one non-Listeria polypeptide encoded by a polycistronic expression cassette includes an antigen. In some embodiments, at least two non-Listeria polypeptides each include a fragment of the same antigen. In some embodiments, the antigen is a tumor-associated antigen ' or derived from a tumor-associated antigen. For example, in some embodiments, the antigen is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY_ESO], WT-1, survival protein, gPl. 〇, PAP, protease 3, spaS-1, SP-17, PAGE-4, TARP and CEA antigens, or derived from K-Ras, H-Ras, N-Ras, 12-K-Ras , Mesothelin, PSCA, nY-ESO-1, WT-1, Survivin, gplOO, PAP, Protease 3, SPAS-1, SP-17, PAGE-4, TARP and CEA. In some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is human mesothelin with its signal peptide and GPI anchored. In some specific 98561.doc -43- 200530399 embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the at least two non-Listeria polypeptides encoded by the polycistronic performance card E include a signal peptide (signal of Listeria or signal peptide of non-Listeria ). In some embodiments, the signal peptide is a secAl signal peptide. In some embodiments, the signal peptide is a "M" signal peptide. In other embodiments, the signal peptide is a Tat signal peptide. In some embodiments, the performance card includes a multi-core encoding signal. Picric acid, in which the codon for the polynucleotide encoding the signal is optimized for expression in Listeria. The invention also provides a pharmaceutical composition comprising: (a) a recombinant Listeria bacterium, and (B) A pharmaceutically acceptable carrier. An immunogen composition including the recombinant Listeria bacterium is also provided. A vaccine including the recombinant Listeria bacterium is also provided. An inducement against the host is also provided. A method for an immune response to an antigen, comprising administering to the host an effective amount of a composition comprising the recombinant Listeria bacterium, wherein the at least one non-Listeria spp. Polypeptide is an antigen. Prevention or treatment is also provided in the host. A method for the condition, comprising administering to the host an effective amount of a composition comprising the recombinant Listeria bacterium. The bacterium is also provided to induce immune response to the antigen in the host in the manufacture thereof. For use in pharmaceuticals, one of which is a non-Listeria polypeptide encoded by a 5 cistronic expression caliper including antigens. In another aspect, the invention provides a recombinant nucleic acid molecule comprising (a) an encoding signal The first polynucleotide of the skin, (b) the first glycoside transcript encoding a secreted protein or fragment thereof, wherein the second polynucleotide is the same translation editing structure as the first polynucleoside transcript, And (c) a third multinucleus encoding a polypeptide that is heterologous to the secreted protein or its 98561.doc -44- 200530399 fragment, wherein the third nucleoside i 疋 and the first and second multinucleates The same translation and editing shelf of the same nucleotides-the heavy, and the nuclear molecule encoding includes a signal peptide, a key encoded by a third polynucleic acid, and a protein enemy complex that secretes a protein or a fragment thereof and wherein the protein interferes with the complex The polypeptide encoded by the third polynuclear picric acid is fused to the secreted protein or a fragment thereof, or is located in the secreted protein or eight pieces of I. In some specific embodiments, the secreted protein is a natural knife / must Protein Protein secreted from its natural cells. In some embodiments, the third polynuclear gene is located in a second polynuclear gene of the recombinant nucleic acid molecule. The polypeptide encoded by the third polynucleotide is located in a secreted protein or fragment thereof. In some embodiments, the third polynucleotide is located in the second polynucleotide of the recombinant nucleic acid molecule And in the protein chimera, the polypeptide encoded by the third polynucleotide is fused to a secreted protein or a fragment thereof. A performance caliper is also provided, which includes the recombinant nucleic acid molecule, and further includes operable Promoter genes linked to the first, second and third polynucleotides of the recombinant nucleic acid molecule. In some embodiments, the peptide encoded by the second polynucleotide includes an antigen. In some specific embodiments In an embodiment, the polypeptide encoded by the second polynucleotide is an antigen. For example, in some embodiments, the antigen is a tumor-associated antigen, or derived from a tumor-associated antigen (eg, selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, Antigen of the group consisting of dermatan, pSCA, NY-ESO-1, WT-1, survival protein, gpi〇〇, pAp, protease 3, SPAS-1, SP-17, PAGE-4, TARP and CEA, 98561 .doc -45- 200530399 or derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survival protein, gpi〇〇. , Pap, protein S # 3, SPAS-1, SP-17, PAGE-4, TARP and CEA antigens). In some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant thereof. For example, in some embodiments, the antigen is human mesothelin, or its signal peptide and GPI-anchored human mesothelin are deleted. In some embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. A performance carrier including the performance cassette is also provided. A recombinant bacterium comprising the recombinant nucleic acid molecule is also provided. Recombinant Listeria bacteria (such as Listeria monocytogenes) are also provided, and in some embodiments, the polypeptide encoded by the third polynucleotide is foreign to the Listeria bacteria . The invention also provides an immunogen composition comprising the recombinant bacterium, wherein the polypeptide encoded by the third polynucleotide is an antigen. A method for inducing an immune response to an antigen in a host is also provided, comprising administering to the host an effective amount of a composition comprising the recombinant bacteria, wherein the polypeptide encoded by the third polynucleotide is an antigen. Also provided are pharmaceutical compositions and epidemic fields that include the bacteria, as well as methods of using the recombinant bacteria or a composition comprising the bacteria in a host to prevent or treat a condition. The use of the bacterium for the manufacture of a medicament for inducing an immune response to an antigen in a host is also provided, wherein the polypeptide encoded by the third polynucleotide includes an antigen. In a further aspect, the present invention provides improved methods for the expression and secretion of heterologous proteins in host bacteria. The invention also provides methods for improving the expression and secretion of heterologous proteins in bacteria. The invention further provides methods of making the recombinant nucleic acid molecules, expression cards, expression vectors, and recombinant bacteria described herein. 98561.doc -46-200530399 It should be understood that in the Markush ethnic group, the bite passage, Shi Wu " Zhan and Zhan, in the scope of patent application, Yuan, and Er You b Lu, the specific embodiment stated, ordered妫, +, ^ indicates that they are not explicitly stated otherwise or by context, including each individual concrete example # / 丨 example only, any combination of each individual specific target case, and the present invention consisting of . “Individual specific examples and additional specific examples and views that provide more explanations of the above-mentioned viewpoints and specific examples are provided below. Σ Listeria recombinant nucleic acid molecules The present invention provides a variety of Listeria spp. Exhibits polynuclear picric acid, such as heteronuclear polynuclear polynuclear tadpoles. For example, a recombinant nucleic acid comprising a sequence encoding a signal peptide (or a polypeptide including a signal peptide) and a code sequence of the polypeptide, such as a novel combination of heterologous antigen Molecule. Provides a recombinant nucleic acid molecule that includes a codon-optimized polynucleotide sequence. In some embodiments, these recombinant nucleic acid molecules are heterogeneous because they include a polynucleotide (ie, a polynucleotide sequence) Is not naturally found in a combination that becomes part of the same nucleic acid molecule with each other. In some embodiments, the recombinant nucleic acid molecule is separate. In some embodiments, the recombinant nucleic acid knife is located on the performance card. , Expression vectors, plastid DNA in bacteria and / or even in genomic DNA of bacteria (after insertion) In some embodiments, the recombinant nucleic acid molecule provides improved expression and / or secretion of the polypeptide (eg, a heterologous polypeptide) in bacteria. In some embodiments, the recombinant nucleic acid molecule is DNA. In some specific embodiments In embodiments, the recombinant nucleic acid molecule is RNA. In some specific embodiments, the recombinant nucleic acid is single-stranded. In other specific embodiments 98561.doc -47- 200530399, the recombinant nucleic acid is double-stranded F. In some specific embodiments, the recombinant nucleic acid molecules described herein encode fusion proteins, such as fusion proteins including signal peptides and other polypeptides, such as heterologous polypeptides for the signal peptide. In some specific embodiments In the specific peptide, the signal peptide is a fine g. It is understood that the listed fusion custard polymorphous can be 'but not necessarily, directly fused with each other. In some embodiments, the polypeptide component of the fusion protein may be On the polypeptide sequence, it is separated by one or more intervening amino acid sequences. In some embodiments, there is another polymorphous non-bacterial, such as mammal or virus. According to an aspect of the present invention, there is provided a recombinant nucleic acid molecule comprising a first polynucleic acid encoding a signal peptide, wherein the first polynucleotide codon is optimized for performance in bacteria; and 0) encoding A second polynucleotide of a polypeptide (eg, an antigen), wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a Fusion protein. In additional specific embodiments, and also for bacterial performance, the second polynucleotide (polynucleotide encoding a polypeptide, such as an antigen) is codon-optimized. The first and The second polynucleotide codon-optimized bacterium should be a bacterium that attempts to place a 5-nucleotide recombinant nucleic acid molecule therein. In another aspect, the present invention provides a recombinant nucleic acid molecule comprising (a) an encoding The first polynucleotide that is a natural signal peptide to the bacterium, where the first polynucleotide is codon-optimized for performance in the bacterium; and (b) the second one encoding a polypeptide Polynucleoside Wherein the second polynucleotide and the first polynucleotide is the same translational reading compiled architecture, wherein the recombinant nucleic 98561.doc • 48 - 200530399 acid molecule encodes a fusion protein comprising the signal peptide and the polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the second polynucleotide is heterologous to the first polynucleotide. In some embodiments, the polypeptide is heterologous to a bacterium that is natural to the signal peptide (ie, foreign to the bacterium). In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide, foreign to the bacteria, or both. In some embodiments, the bacteria from which the signal peptide is derived are intracellular bacteria. In some embodiments, the fine line is selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacterium, and E. coli Group of people. In some embodiments, the signal peptide is natural to bacteria of the genus Listeria. In some embodiments, the peptide is natural to a Listeria bacterium belonging to the species Listeria monocytogenes. In some embodiments, the second polynucleotide codon is optimized for performance in bacteria. In another aspect, the present invention provides a recombined nucleic acid molecule, wherein the recombined nuclear knife comprises (a) a first polynucleotide encoding a signal peptide, wherein the first Multinucleotide ㈣ codon optimization 'and (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation and editing framework as the first polynucleotide 忒, where the 忒 recombinant nucleic acid molecule encodes A fusion protein of a signal peptide and the polypeptide. In some embodiments, the signal peptide is natural to Listeria®. In some embodiments, the signal peptide is foreign to Listeria ^ 98561.doc -49- 200530399. In some embodiments, the signal peptide is heterologous to the polypeptide encoded by the first polyacid, and among the polypeptides encoded by the second polynucleic acid, the bacteria against Listeria are Words are heterogeneous. In this and the first case, the Listeria bacterium belongs to the species Listeria monocytogenes. The present invention also provides a recombinant nucleic acid molecule comprising a polynucleotide encoding a polypeptide that is foreign to Listeria bacteria (e.g., cancer or non-Listeria infectious disease antigen). As shown in bacteria of the genus, the polynucleotide codons encoding the foreign polypeptide are optimized. In another aspect, the present invention provides a recombinant nucleic acid molecule comprising: a first polynuclear M encoding a non-secA1 bacterial signal peptide, and a second polynucleotide encoding a polypeptide such as an antigen, the second Polynucleotide is the same translation editing structure as the first polyacid. The recombinant nucleic acid molecule in # encodes a fusion protein including a signal peptide and the polypeptide. In some embodiments, the non-secA1 bacterial signal peptide is a secA2 signalling or signalling peptide. In some specific examples, 'A' is expressed in a bacterium (eg, Listeria) in which the recombinant nucleic acid molecule is intended to be placed. optimization. In some specific embodiments ... in the attempt to place the recombinant nucleic acid molecule in its second bacterium, the second polynucleotide acid codon encoding the polypeptide, such as an antigen, will be optimized. In some embodiments, the polypeptide encoded by the second polynuclear acid is heterologous to the signal peptide. In some embodiments, the polypeptide encoded by the second multinucleate is foreign to the bacteria into which the recombinant nucleic acid molecule is to be introduced or has been introduced. In some specific 98561.doc -50- 200530399 embodiments, the polypeptide encoded by the second polynucleic acid is foreign to the bacterium that is to be inserted into or has been introduced into the recombinant nucleic acid molecule, and The polypeptides encoded by the polynucleotides are also heterologous to the signal peptide. The present invention further provides a recombinant nucleic acid molecule, including a first polynucleic acid encoding a non-seeAi bacterial signal peptide, a second polynucleic acid encoding a polymorphism (such as a heterologous protein and / or antigen), and The same translation and editing architecture as the second polynuclear * acid, encoding a third polynuclear acid of SecA2 autolysin or a fragment thereof, and the second polynuclear cyanic acid is located in the third polynucleic acid ' Or between the-and the third multicore. In some embodiments ' the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide, a polypeptide, and an autolysin. In some embodiments, the autolysin fragment is as catalytically active as autolysin. In some embodiments, the autolysin is derived from bacteria in a cell. In some embodiments, the autolysin is a peptidoglycan hydrolase. In some embodiments, the bacterial autolysin is an autolysin of the genus Listeria. In some embodiments, the autolysin is P60. In some specific embodiments, the autolysin is a cell wall enzyme. The present invention also provides a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes (a) a first peptide encoding a non-Listeria signal peptide. A polynucleotide; and (b) a second polynucleotide encoding a polypeptide, which is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a gene comprising non-Listeria A fusion protein of both the signal peptide and the polypeptide. In some embodiments, the non-Listeria signal peptide is heterologous to a polypeptide encoded by a second polynucleic acid. In some embodiments, the first polynucleic acid, the second polynuclear 98561.doc 51-200530399 nucleotides, or both the first and second polynucleotides are expressed in order to express in Listeria bacteria. Codon-optimization. The invention also provides a recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a bacterial autolysin 'or a catalytically active fragment or a catalytically active variant thereof, and (b) a second encoding a polypeptide Polynucleotides, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a polypeptide and an autolysin encoded by the second polynucleotide Or a protein chimera having a catalytically active fragment or a catalytically active variant, wherein in the protein chimera, the polypeptide is fused to autolysin, or a fragment or a catalytically active variant thereof , Or located in autolysin, or its catalytically active fragment or catalytically active variant. In some specific embodiments, the second polynucleotide is located in the first polynucleotide, and the recombinant nucleic acid molecule encodes a protein chimera, and the polypeptide encoded by the second polynucleotide is located in Autolysin, or a catalytically active fragment or catalytically active variant thereof. In some specific embodiments, the second polynucleotide is located outside the first polynucleotide, and the recombinant nucleic acid molecule encodes a protein chimera, wherein the polypeptide encoded by the second polynucleotide is Lysin, or a catalytically active fragment or a catalytically active variant thereof is fused. In some specific embodiments, the unitary society ± t green μ ^

Or a catalytically active variant of a protein chimera 'or a catalytically active lean chimera. In some specific examples of 98561.doc -52- 200530399, the polypeptide encoded by the second polynucleotide is heterologous to the self-contained and the self-contained. In some embodiments, the autolysin fragment is at least about 30, at least about 40, at least about 50, or at least about 100 amino acids in length. In some embodiments, the autolysin is derived from bacteria in a cell. In some embodiments, the bacterial autolysin is an autolysin of the genus Listeria. Catalytically active variants of autolysins include variants that differ from the original autolysin by one or more substitutions, deletions, additions and / or insertions. In some embodiments, the autolysin is a peptidoglycan hydrolase. In some embodiments, the autolysin is p6O. In some specific embodiments, the autolysin is N-acetamylwallinase. Enzyme mapping, a technique known to the artist (see, for example,

Lenz et al. (2003) PrOc Natl Acad Sci. USA 10012 12432-12437) to identify additional autolysins and characterize them. Enzymes can also be used to determine whether specific fragments and / or variants of autolysins are catalytically active like autolysins. This technique can also be used to assess whether specific proteoglycans are as active as autolysins. In some embodiments, the catalytically active fragments and / or variants of autolysin have at least about 10%, at least about 30%, at least about, at least about 75%, at least about 5% or at least about Catalytic activity like natural autolysins. In some embodiments, the protein chimera has catalytic activity like autolysin. In some embodiments, the protein chimera is at least about 10%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or at least about 95% like a natural autolysin. Catalytic activity of 98561.doc -53- 200530399. Another option for the expression of heterologous proteins is to use a protein, a scaffold, in which the heterologous protein is inserted in a functional manner, in the framework. " In this combination, an intact gene or a gene component equivalent to, for example, MHC class I or ^ 11 (: epitope of type 11 is inserted and spread throughout the scaffold protein. The scaffold protein may be a highly expressed bacterial protein ( Such as Listeria proteins, such as LLO or P60), but in other specific embodiments, it can be a heterologous protein selected for its high performance, stability, secretion and / or (lack of) immunogenicity. Hawk Representative examples of self-producing eggs are chicken eggs or other human proteins, such as β-hemoglobin or albumin. The present invention also provides recombinant nucleic acid molecules, including the third polynucleotide encoding a signal peptide. b) editing the second polynuclear acid of the secreted protein or fragment thereof, wherein the second multinucleus is the same translation editing structure as that of the first multinucleus, and ⑷ encodes the secreted protein or the fragment thereof; Lu is a heterogeneous polymorphic ::: a mimicry shows a nucleotide, wherein the third polynucleotide is the same as the nucleotide of the first and third -Gannon gu 1 Translation and editing architecture The nucleic acid molecule encodes a protein chimera, including a L peptide, a polypeptide encoded by the second polynucleotide, and a secreted protein Zeng Formula 1 or a fragment thereof, and in the protein chimera, Xi Xiyi , W Haixi peptide is fused to the secreted protein or a fragment thereof, or is located in the negative sense eight tablets & of the secreted protein. In some specific embodiments, the second polymorphine lacks Λ and X glycosides. Encodes a secreted protein. In some specific embodiments, the secreted protein is a protein secreted from its natural cells. In some embodiments, the two polynucleotides of r are located in the recombinant nucleic acid molecule. The second polymorphism / all homology 'and in the protein chimera encoded by the recombinant nucleic acid molecule 98561.doc -54- 200530399, the third polymorphic polymorphism is located at the secretion Protein f or a fragment thereof. In some specific embodiments, in the nucleic acid molecule, the ancient female flute / m ^ μ two nucleoside 驮 system is located outside the second polynucleotide and is in the The third multicore An acid-encoded polypeptide is fused to the secreted protein f or a fragment thereof. In some embodiments, 'μ secretin f is albumin. In some embodiments, a truncated form of egg / month protein is used. In some specific embodiments, the secreted protein is P60. In some specific embodiments, the secreted protein is v · acetamycin. In some embodiments, the signal peptide is related to the normal secreted protein. Signal peptides. In some embodiments, the Lung peptide is heterologous to the secreted protein. In some implementations; the length of the cleavage / biprotein fragment is at least about 30, at least about 40. , At least about 50 or at least about 100 amino acids. In some specific embodiments, a recombinant nucleic acid molecule, a performance card E or a performance vector comprising a coding sequence of a polypeptide that is foreign to the bacterium is embedded in Within some or all of the codon sequences of highly expressed proteins in this bacterium. In some embodiments, the ' Xuan highly expressed sequences are natural to the bacteria in which they are to be expressed. In other embodiments, the highly expressed sequence is not natural to the bacteria in which it is to be expressed, but provides sufficient performance. In another aspect, the invention provides a recombinant nucleic acid molecule, wherein the nucleic acid molecule encodes at least two isolated non-Listeria polypeptides. In some embodiments, the polynucleotide codon-encoding the non-Listeria polypeptide is optimized for performance in Listeria bacteria. 98561.doc -55- 200530399 Methods for preparing recombinant nucleic acid molecules, including those described above, are well known to those skilled in the art. For example, a recombinant nucleic acid molecule can be prepared by synthesizing long oligonucleotides on a DNA synthesizer, which overlap each other, and then performing an extension reaction and / or PCR to produce a desired amount of double-stranded DNA. Restriction enzymes can be used to cleave the double-stranded DNA and insert it into the desired expression or selection vector. Sequences can be performed to confirm that the correct sequence has been obtained. Also via non-limiting examples, one or more parts of the recombinant nucleic acid molecule can be obtained separately from protein-containing plastids. The pcR of the plastid-related part, or restriction enzyme excision of the plastid-related part can be performed, followed by ligation and / or pCR, mixing the related polynucleic acid to generate the desired recombinant nucleic acid molecule. This type of technology: This technique is standard. Standard breeding techniques can also be used to insert the recombinant nucleic acid sequence into the plastid and replicate the recombinant nucleic acid in a host cell, such as a bacterium. The recombinant nucleic acid can then be isolated from the host cell. The invention also provides methods for producing recombinant bacteria (eg, recombinant Listeria bacteria) using any of the recombinant nucleic acid molecules described herein. In some embodiments, a method of making a recombinant bacterium using a recombinant nucleic acid molecule described herein includes introducing the recombinant nucleic acid molecule into a bacterium. In some embodiments, the recombinant nucleic acid molecule is integrated into the genome of a bacterium. In some other specific embodiments, the recombinant nucleic acid molecule is on a plastid incorporated into a bacterium. In some embodiments, the recombinant nucleic acid molecule is incorporated into a bacterium by conjugation. It can be incorporated into bacteria by any standard technique known in the art. For example, recombinant nuclei can be incorporated into bacteria by conjugation, transduction (metastatic infection), or transformation. 98561.doc -56- 200530399 ιπ · Signal peptide In some specific embodiments, the recombinant nucleic acid molecule, expression card, and / or vector of the present invention encodes a fusion protein or protein chimera including a signal peptide, and is suitable for host cells, As manifested in bacteria and secreted from them. Therefore, in some embodiments, the recombinant nucleic acid molecule, the expression card S and / or the vector of the present invention includes a polynucleotide encoding the signal peptide. The terms "signal peptide" and "signal sequence" are used interchangeably herein. In some embodiments, the signal peptide helps to make it easier for the polypeptide fused to the signal peptide to move across the cell membrane of a cell (eg, a bacterial cell), It is possible to secrete a polypeptide from the cell. In some specific embodiments, the signal peptide is " secretory signal peptide " or " secretory sequence ". In some specific embodiments, the signal peptide is located at the site to be secreted. In some embodiments, the sequence encoding the signal peptide in the recombinant nucleic acid molecule or expression cassette is located in the recombinant nucleic acid molecule or expression cassette, so that the encoded signal peptide will complete the sequence from A desired host cell (eg, a bacterium) secretes a polypeptide fused to it. In some embodiments, in the recombinant nucleic acid molecule or expression cassette, the polynucleotide encoding the signal peptide is located in the framework (directly or by The polynucleotide involved is separated) at the 5 'end of the polynucleotide encoding a polypeptide to be secreted (eg, a polypeptide including an antigen). In some embodiments, the letter A peptide is a part of a fusion protein or protein chimera encoded by a recombinant nucleic acid molecule, expression cassette and / or expression vector, which is heterologous to at least one other polypeptide sequence in the fusion protein and / or protein chimera In some specific embodiments, the signal encoded by the recombinant nucleic acid molecule, the expression card and / or the expression vector, 98561.doc -57- 200530399 shows the expression of the recombinant nucleic acid molecule to be introduced or has been introduced into it. The cassette and / or expression vector are heterologous (foreign). In some specific embodiments, the signal peptide is for a bacterium into which the recombinant nucleic acid molecule, cassette and / or expression vector is to be introduced. Natural. In some embodiments, the polynucleotide codon-encoding the signal peptide is optimized for performance in bacteria (eg, Listeria, such as Listeria monocytogenes). In some In specific embodiments, codon-optimized polynucleotides for a particular bacterium are foreign to the bacterium. In other specific embodiments, for special Codon-optimized polynucleotides are natural to the bacteria. Various signal peptides are known in the art. In addition, various algorithms and software programs can be used, such as the "SignalP" algorithm To predict signal peptide sequences that can be used in this technique. See, for example, Antelmann et al., Genome

Res., 11: 1484-502 (2001); Menne et al., Bioinformatics, 16: 741-2 (2000); Nielsen et al., Protein Eng., 10: 1_6 (1997); Zhang et al., Protein Sci ,, 13: 2819-24 (2004); Bendtsen et al., J. Mol. Biol., 340: 783-95 (2004) (About SignalP 3.0); Hiller et al., Nucleic Acids Res. 32: W375-9 (2004 ); Schneider et al., Proteomics 4: 1571-80 (2004); Chou, Curr. Protein Pept. Sci., 3: 615-22 (2002); Shah et al., Bioinformatics, 19: 1985-96 (2003); And Yuan et al., Biochem. Biophys. Res. Commun. 312: 1278-83 (2003). In some embodiments, the signal peptide is prokaryotic. In some alternate embodiments, the signal peptide is eukaryotic. At 98561.doc -58- 200530399

Humphreys et al., Protein Expression and PurificatiOn, 20: 252-264 (2000) describe the use of eukaryotic signal peptides in order to express proteins in, for example, E. coli. In some embodiments, the signal peptide is a bacterial signal peptide. In some embodiments, the signal peptide is a non-Listeria signal peptide. In some embodiments, the signal peptide is a signal peptide of Listeria. In some embodiments, the signal peptide is derived from a Gram-positive bacterium. In some embodiments, the signal peptide is derived from intracellular bacteria. In some embodiments, signal peptides (e.g., non-secA1 bacterial signal peptides) used in recombinant nucleic acid molecules, expression cards, and / or expression vectors are derived from Listeria. In some embodiments, the signal peptide is derived from Listeria monocytogenes. In some embodiments, the signal peptide is derived from a signal peptide of Listeria monocytogenes. In some specific examples, the signal peptide is not derived from Listeria, but instead is derived from bacteria other than Listeria. In some embodiments, the bacterial signal peptide is derived from Bacillus subtilis. In some specific examples, the '4 bacterial signal peptide is derived from a bacterium belonging to the genus Staphylococcus. What-in specific embodiments, the bacterial signal line is derived from Lactococcus. In some embodiments, the bacterial signal peptide is derived from a spore of a bacterium belonging to the genus Staphylococcus or Lactococcus. In some implementations, the number 7 is a bacterium derived from Bacillus, Staphylococcus, or Lactone ... In some embodiments, the bacterial signal is a peptide No. 仏 derived from A. anthracis, Bacillus subtilis, Staphylococcus aureus, or Lactococcus lactis 98561.doc -59- 200530399. In some embodiments, the bacterial signal peptide is a signal peptide obtained from Bacillus anthracis. In some embodiments, the bacterial signal peptide is a signal peptide obtained from subtilis g. In some embodiments, the bacterial signal peptide is a signal peptide obtained from Lactococcus lactis. In some embodiments, the bacterial signal peptide is a signal peptide obtained from S. aureus. In some specific embodiments of the polypeptides described herein, the signal peptide derived from an organism, such as a bacterium, is the same as the naturally occurring signal peptide sequence obtained from the organism. In other specific embodiments, the signal peptide sequence encoded by the recombinant nucleic acid molecule, expression card, and / or expression vector is derived from a naturally occurring signal peptide sequence, that is, a fragment and / or variant of a naturally occurring signal peptide sequence Where the fragment or variant still has the same function as the signal peptide. Bosomes include polypeptides that differ from the original sequence by only one or more substitutions, deletions, additions and / or insertions. For example, in some embodiments, the signal peptide encoded by the polynucleotide contains one or more retention mutations. The possible retentive amino acid changes are well known to those skilled in the art. See, for example, Chapter iv, detailed below, for additional information on changes in retentive amino acids. Signal peptides derived from other signal peptides (ie, fragments and / or variants of other signal peptides) are preferably substantially equivalent to the original signal peptide. For example, a signal peptide derived from another signal peptide has the ability to function like the signal peptide and should be substantially unaffected by changes (deletions, mutations, etc.) to the natural signal peptide sequence. In some embodiments, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the derived signal peptide can have a signal peptide function similar to a natural signal peptide sequence. In some specific embodiments, the signal peptide is on the amino acid sequence and has at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity to the original signal peptide. 98561.doc -60- 200530399 Sex. In some embodiments, the only change made in the signal peptide sequence is a retention amino acid substitution. The fragment of the signal peptide is preferably at least about 80% or at least about 90/0 of the original signal peptide. In some embodiments, the signal peptide encoded by the polynucleotide in the recombinant nucleic acid molecule, expression cardmaker and / or expression vector is a secAHf peptide, a secA2 signal peptide, or a bis-arginine translocation (Tat) signal Peptide. In some embodiments, the signal peptide is a secAl signal peptide. In some embodiments, the peptide is a non-secAl signal peptide. In some embodiments, the signal peptide is a secA2 signal peptide. In some embodiments, the signal peptide is a bis-arginine translocation (Tat) signal peptide. In some embodiments, these secAl, secA2 or Tat signal peptides are derived from Listeria. In some specific embodiments, these secAh secA2 or Tat signal peptides are non- Listeria. For example, in some embodiments, these secA1, secA2 *

The Tat signal peptide is derived from bacteria belonging to one of the following genera: Bacillus, Staphylococcus, or Lactococcus. Bacteria use multiple pathways for protein secretion, including secAb secA2 and double Arg translocation (Tat). The path used is determined mainly by the type of signal peptide 'located at the N-terminal of the pre-protein. Most secreted proteins make use of the Sec pathway, in which the protein is displaced in an unfolded configuration through the Sec pores of the protein embedded in the cell membrane. In contrast, proteins that make use of the Tat pathway are excreted in a dysmorphic configuration. A nucleotide sequence encoding a signal peptide that conforms to any of these protein secretion pathways can be genetically fused to the desired heterologous protein code sequence in architecture. The M-signal peptide preferably contains 98561.doc at its ㈣ terminal. 61- 200530399 Signal peptidase cleavage site to release a reliably desired protein into the extracellular environment (Sharkov and Cai. 2002 J. Biol. Chem. 277: 5796-5803; Nielsen et al. 1997 Protein Engineering 10: 1 6; and www.cbs.dtu.dk/services/SignalP/). The signal peptide used in the polynucleotide of the present invention can be derived not only from various secretory pathways' but also from various bacterial genera. Signal peptides usually have a common structural organization with a charged N · terminal (N_functional site), a water-repellent core region (Η-functional site), and a more polar c-terminal region ((> Month & site), however, they do not show sequence retention. In some embodiments, the C-terminal of the signal peptide carries a type I signal peptidase ^ 卩 enzyme cleavage position, a position relative to the cleavage position-丨 and _3 have a consensus sequence of A-χ_Α. Proteins secreted via the sec pathway have an average of 28 residues of signal peptides. The secA2 protein secretion pathway was first found in Listeria monocytogenes; by agar The crude colony phenotype on the medium, and the attenuating virulence phenotype in mice, identified the characteristics of mutants in the secA2 allogene (paralogue) (Lenz and pornoy, 2002 μ 〇1 · _ Microbiol. 45: 1043-1056; and Lenz et al. 2003 PNAS 100 ·· 12432-12437). The signal peptides related to the protein secreted by the Tat pathway have a three-part organization similar to the Sec signal peptide, but It is characterized by N_ functional sites The / η- functional site has a RR_ motif at the boundary, where # is a water-repellent residue). The bacterial Tat signal peptide has an average of 14 amino acids, which is longer than the sec signal peptide. The subtilis secretory (secreme) can contain up to 69 putative proteins that utilize the secretory pathway, 14 of which contain the spase I cleavage site (Jongbloed et al. 2002 J. Biol. Chem. 277: 44068- 98561.doc -62- 200530399 44078; Thalsma et al. 2000 Microbiol. Mol. Biol. Rev. 64: 515-547). Examples of non-limiting signal peptides shown in Table 1 below can be used in fusion combinations (including protein chimeric compositions) with selected other polypeptides, such as heterologous polypeptides, resulting in secretion from bacteria encoding the protein. Table 1 · Some representative signal peptide secretion pathways Signal peptide amino acid sequence (NHrC〇2) Signal peptidase position (represents the incision position) Gene genus / species secAl MKKIMLVFITLILVSLPIAQQ TEAKD (sequence number 45) TEA'KD ( (Sequence number 54) hly (LLO) Listeria monocytogenes MKKKIISAILMSTVILSAAAP LSGVYADT (sequence number 46) VYA'DT (sequence number 55) Usp45 Lactococcus lactis MKKRKVLIPLMALSTILVSS TGNLEVIQAEV (sequence number 47) IQA'EV ( (Sequence number 56) pag (protective antibody) Bacillus anthracis secA2 MNMKKATIAATAGIAVTAF AAPTIASAST (sequence number 48) ASWST (sequence number 57) iap invasion-associated protein p6〇 monocytes Listeria polymorphism MQKTRKERILEALALQEEKKN KKSKKFKTGATIATIAGVTAIAT SITVPGIEVIVSADE Sequence No. 49) VSA'DE (Sequence No. 58) NamA lmo2691 (Autolysin) Listeria monocytogenes MKKLKMASCALVAGLMFS GLTPNAFAED (Sequence No. 50) AFA'ED (Sequence No. 59) * BA 0281 ( NLP / P 60 homes i) Bacillus anthracis MAKKFNYKLPSMVALTLVG SAVTAHQVQAAE (sequence | J No. 51) VQA AE (Sequence No. 60) * atl (Autolysin) Staphylococcus aureus Tat MTDKKSENQTEKTETKENK GMTRREMLKLSAVAGTGIA VGATGLGTILNVVDQVDK LT (Sequence No. 52) DKA'LT (Sequence No. 61) lmo0367 Listeria monocytogenes SLDSTIAGLDGFQWQQKK (Sequence No. 53) VGA, FG (Sequence No. 62) PhoD (Alkaline Phosphatase) A bacterial autolysin secreted by the sec pathway from Bacillus thuringiensis (not determined as secAl or secA2) Therefore, in some specific embodiments Here, the sequence encoding the signal peptide encodes the secAl signal peptide. An example of a secAl signal peptide is the Listeriolysin O (LLO) signal -63- 98561.doc 200530399 peptide from Listeria monocytogenes. In some embodiments, the recombinant nucleic acid molecule, expression cassette, and / or expression vector includes a polynucleotide encoding an LLO signal peptide, and further includes a polynucleotide sequence encoding an LLO PEST sequence. Other examples of secAl signal peptides suitable for use in the present invention include signal peptides derived from the UsP45 gene in Lactococcus lactis (see Table 1 above, and Example 12 below) and Pag (protective from Bacillus anthracis) Antigen) gene. Therefore, in some embodiments, the signal peptide is a protective antigen signal peptide obtained from Bacillus anthracis. In some other specific embodiments, the signal peptide is a secA1 signal peptide other than the protective antigen signal peptide of Bacillus anthracis. Another example of a secA1 signal peptide is the SpsB signal peptide from S. aureus (Sharkov et al., J. of Biological Chemistry, 277: 5796-5803 (2002)). In some alternate embodiments, the heterologous code sequence is genetically fused to a signal peptide recognized by the secA2 pathway protein secretion complex. SecA transverse homologs (ParalOg) (SecA2), which has indeed been identified as a helper in 9 Gram-positive bacteria that cause severe or lethal human infectious diseases. SecA2g is necessary for the secretion of a subgroup of the export proteome (secretosomes) of Listeria, Mycobacterium, and Streptococcus (Braunstein et al., MoI Micr0bi 48: 453-64 (2003 ); Bensing et al., Mο1 · Microba1, 44: 1081-94 (2002) 'Lenz et al., Mol · Microbiol., 45: 1043-1056 (2000); and Braunstein et al., J Bacteri〇1 Gy, 183: 6979 699 (2001)). Confirmation of Listeria monocytogenes & 〇 八 2 through its association with smooth and rough changes in cells, and canine cytotoxicity reduction of secA2 of Listeria monocytogenes and Mycobacterium tuberculosis . For example, the protein P60 of Listeria is secreted via the secA2 pathway, 98561.doc _64-200530399 peptide polyauxin. For example, the secA2 signal peptide and the signal peptidase cleavage site obtained from p60 are linked to the amino terminal of the gene (such as an antigen) of the desired protein. In a specific example, the translation of the seeA2 signal peptide and the signal peptidase-antigen fusion allo-proteins from the card E in bacteria is carried via a Gram-positive cell wall, in which a reliable heterologous protein will be transported Released into the extracellular environment. Alternatively, the heterologous sequence can be incorporated into the framework " into p6〇, so that the heterologous protein f can be secreted in the form of a hybrid P60 · heterogeneous protein. A heterologous protein code sequence can be inserted into the framework within p6o For example, at the junction between the signal peptidase cleavage position and the protein-forming protein f. In this specific embodiment, the chimeric protein retains the appropriate “secretion signal, and its autolysin activity” means P6G secretes heterologous proteins in a f-free form. The heterologous antigen can be designed at any point to incorporate the structure within p6o, such as 0, which still retains the secretion and autolysin activities of the P60 protein. In Example 13 below Examples of partial expression cassettes suitable for inserting the coding sequence of a desired antigen or other heterologous polypeptide are described. In some embodiments, a fusion protein encoded by a recombinant nucleic acid molecule is a protein including a bacterial protein with special desired properties (except for Heterologous protein, such as a chimera other than an antigen). In some embodiments, the chimera includes a hydrolase. In some embodiments, the recombinant nucleic acid The sub-encoding includes an endopeptidase p60, a p60 chimera of a peptidoglycan hydrolase that degrades bacterial cell walls. In some embodiments, the fusion protein encoded by the recombinant nucleic acid molecule includes a monocytic polyclonal lister A bacterial hydrolase such as p60 (see, for example, Genbank accession number np_46411〇) ^ n 98561.doc -65- 200530399 Acetylpyramidin (NamA) (Genbank accession number 1 ^? 466213), both of which degrade the cell wall SecA2-dependent secreted proteins. This special combination of protein chimeras not only facilitates the molecular chaperone required for protein secretion, but also bacterial protein activity that can promote its secretion. Special protein chimeras include heterologous protein coding sequences and The correct configuration of Listeria monocytogenes hydrolase results in effective expression and secretion of heterologous proteins. (See, for example, the specific example of Example 29 below). Therefore, in some embodiments, 'encoded by the recombinant nucleic acid molecule The signal peptide that becomes part of the fusion protein ^ is the p60 signal peptide. In some specific embodiments, the recombinant core The signal peptide that is molecularly encoded and becomes part of the fusion protein is a NamA signal peptide. In some embodiments, the recombinant nucleic acid molecule includes a first polynucleotide encoding a p60 佗 5 tiger peptide and encodes another polypeptide (eg (Antigen), the same translation and editing framework for the second polynucleotide, encoding the third polynucleotide sequence of the p60 protein or a fragment thereof. Then, the recombinant nucleic acid molecule encodes a second polynucleotide comprising a signal peptide, A fusion protein of an encoded polypeptide (eg, an antigen) and a protein or a fragment thereof. In such specific embodiments, the second polynucleotide is preferably located in a third polynucleotide, or in a first and a third Between three polynucleotides. In some embodiments, the secA2 signal peptide is a secA2 signal peptide derived from Listeria. For example, in some embodiments, the signal peptide is a SecA2 signal peptide, such as a p60 signal peptide from Listeria monocytogenes or an N-acetamylar walloplastase (NamA) signal peptide. In addition, other Listeria monocytogenes proteins that have not been secreted in the absence of secA2 have been identified 98561.doc -66- 200530399 (Lenz et al., Mol. Microbiology 45: 1043-1056 (2002)) and can be used in some In specific embodiments, polynucleotides encoding signal peptides obtained from these proteins are used. Alternatively, a secA2 signal peptide obtained from a bacteria other than Listeria can be used to express and secrete heterologous proteins from recombinant Listeria or other bacteria. For example, by way of illustrative but non-limiting example, the secA2 signal peptide from Bacillus anthracis can be used in a recombinant nucleic acid molecule and / or a performance cassette. In other specific examples, a secA2 # peptide from golden yellow fe is used. See Table 1. Proteins that have been secreted via the secA2 pathway in other bacteria have also been identified (see, eg, Braunstein et al., Mol. Microbiol., 48: 453-64 (2003) and Bensing et al., Mol. Microbiol. 44: 1081-94 ( 2002)). Other proteins secreted through the secA2 pathway can be identified. SecA2 homologues have indeed been identified in many fine-grained species (see, for example, Lenz et al., Mol.

Microbiology 45: 1043-1056 (2002) and Braunstein et al., J. Bacteriology, 183: 6979-6990 (2001)). Additional sequence comparisons can be performed to identify additional secA2 homologues using techniques known to those skilled in the art. Once the homolog is confirmed, the homolog can be deleted from the bacterial organism, resulting in a Δ ^ οΑ2 mutant. Supernatant proteins from field and mutant bacterial cultures can be precipitated by TCA_ and analyzed by any proteomic technique known in the art 'to determine proteins secreted by field bacteria but not AsecA2 mutants. For example, secreted proteins can be analyzed via SDS-PAGE and silver plating staining. The resulting bands can be compared to confirm those proteins that are not secreted in the absence of secA2 (see 'e.g., Lenz et al., Mol. Microbiology 45: 1043-1056 (2002)). The n-terminal sequence 98561.doc -67- 200530399 of these proteins can then be analyzed (for example, using an algorithm to predict the signal peptide incision position) to determine the SecA2 signal peptide sequence used by the protein. N-terminal sequencing by automated Edman degradation can also be performed to confirm the sequence of the signal peptide. In a specific embodiment of the substitution, the polynucleotide encodes a polypeptide (e.g., a heteropolypeptide sequence) ' which is typically fused to a signal peptide that is recognized by a Tat pathway protein secretion complex. To secrete proteins that are folded in bacteria, the Tat secretion pathway is used by bacteria, including Listeria. For example, the Lista innocua protein YwbN has a putative Tat motif at its amine terminal and is therefore secreted using the Tat pathway (Genbank Accession No. NP-469731 [gi | l6799463 | ref | NP-469731 "丨The putative protein retained is' similar to the YwbN protein of Bacillus subtilis (Listeria harmless)], incorporated herein by reference). Other Tat signal peptide-containing proteins are YwbN proteins from Listeria monocytogenes strain EGD (e) (Genbank Accession No. NP-463 897 [gi | 16802412 | r * ef | NP-463897.11) , Similar to the Bacillus subtilis YwbN protein (Listeria monocytogenes EGD (e))]). For example, the YwbN signal peptide and signal peptidase cleavage site obtained from YwbN can be genetically linked to a gene encoding an amine terminal of a desired protein, such as an antigen. In this combination, the pre-protein includes the Tat signal peptide, and the signal peptidase_ antigen fusion is translated from the expression cassette in bacteria, transported via the Gram-positive cell wall, where the reliable heterologous protein is secreted into the cell Outside environment. The other protein predicted to be secreted from Listeria monocytogenes via the Tat pathway is 3-oxoxacyl-fluorenyl carrier protein synthetase (Genbank accession number NP-471636 [gi | 16801368 | ref | NP-471636 · 1 | Similar to 3 (Oxygen group (醯 98561.doc -68- 200530399 group (vehicle protein synthase (Listeria harmless))). Polynucleotides, performance cassettes and / or performance described in this article Polynucleic acids encoding k 5 tiger sequences derived from any of these proteins predicted to be secreted from Listeria via the Tat secretory pathway can be used in the vector. Tat signal sequences from other bacteria can also be used as signal peptides, including but Not limited to phoD from Bacillus subtilis. In Jongbloed et al., J. 0f Biological Chemistry, 277: 44068-44078 (2002); Jongbloed et al., J. of Biological Chemistry, 275: 41350-41357 (2000), Pop et al. Human, J. of Biological Chemistry, 277: 3268-3273 (2002); van Dijl et al., J. Biotechnology, 98: 243-254 (2002); and Tjalsma et al., Microbiology and Molecular Biology Reviews, 64: 5 15-547 (2000) Examples of Tat signal peptides from Bacillus subtilis are all incorporated herein by reference. Others confirmed in Bacillus subtilis that Tat proteins secreted through the Tat pathway have been predicted to include those sequences with the following Genbank / Embl accession numbers: 〇 八 315017 [^ | 263 5 523 | 61111) | 〇 八 815017.1 | Similar to two (component sensor histidine kinase (YtsA) (Bacillus subtilis)]; CAB12056 [gi | 2632548 | emb | CAB12056.1 | Phosphodiesterase / Alkaline Phosphatase D (Bacillus subtilis)]; CAB 12081 [gil26325 73 | emb | CAB 1208 1.1 丨 Similar to the putative protein (Bacillus subtilis)]; CAB 13278 [gi | 263 3776 | emb | CAB 13278.11 Similar to putative protein (Bacillus subtilis)]; CAB14172 [gi | 2634674 | emb | C ΑΒ14172 · 1 | menaqUinol: cytochrome c oxidoreductase (iron (sulfur subunit)) (subtilis Bacillus)]; CAB15089 [gi | 98561.doc -69- 200530399 2635595 | emb | CAB15089.1 | yUbF (Bacillus subtilis)]; and CAB15852 [gi | 2636361 | emb | CAB15852.1 | Alternating gene name: iPa ( 29d ~ similar to the putative protein) (Bacillus subtilis)], and its sequence is all cited Ways are incorporated herein. Therefore, in some embodiments, the signal peptide encoded by the polynucleic acid in the recombinant nucleic acid molecule and / or expression kakun is a Tat signal peptide derived from Bacillus subtilis. Information on the Tat signal peptide from Pseudomonas aeruginosa is provided in Ochsner et al., PNAs, 99: 8312-8317 (2002). Also described in Dnks et al., J. 〇f Bacteriology, 185: 1478-1483 (2003) and Eru et al., Dobson Microbiology, 35: 260-274 (2000) Bacterial Tat signal peptides, both of which are incorporated herein by reference in their entirety. An additional Tat signal peptide can be identified by its "bis-arginine," consistent motif, and distinguished from the Sec-type peptide # 5. As noted above, it is secreted by the Ding μ pathway The protein-related signal peptide has a three-part tissue, similar to the Seck peptide, but is characterized by an RR-motif (RR-X44, where # is a water-repellent residue) at the functional site boundary. Tat signal calls Peptides are generally longer than Sec-type signal peptides and less water-repellent. See, for example, Berks et al., Adv. Microb. Physiol., 47: 187-254 (2003) and Berks et al., Mol. Microbiol. 35 : 260-74 (2000). In addition, similar technologies to those described above for confirming novel proteins secreted by the SecA2 pathway and their corresponding SecA2 signal peptides can also be used to confirm novel proteins secreted via the Ding pathway and their Signal peptides. References

Jongbloed et al., J. Biotechnology chem., 277: 44068-44078 (2002) provides a protein that can be used to confirm the expression of a certain type of bacteria. It is bis-arginine via 98561.doc -70- 200530399 Technical examples of proteins secreted by translocation pathways. IV. Polypeptides Recombinant nucleic acid molecules described herein, as well as the performance cassettes or performance vectors described herein, can be used to encode any desired polypeptide. In particular, the recombinant nucleic acid molecule, expression cassette and expression vector can be used to express heterologous polypeptides in bacteria. In some embodiments (depending on the recombinant nucleic acid molecule, expression cassette or expression vector used), the poly-a encoded by the recombinant nucleic acid molecule, expression cassette and / or expression vector is fused with a signal peptide Part of the protein. In other specific embodiments, the encoded polymorphism is encoded by the recombinant nucleic acid molecule, expression card E, or expression vector in the form of an individual polypeptide. In another specific embodiment, the polypeptide ' encoded by the polynucleic acid of a recombinant nucleic acid molecule, expression cassette, or expression vector is encoded with a-portion of a fusion protein that does not include a signal peptide. In another specific embodiment, the polypeptide 'encoded by the multinucleic acid of the recombinant nucleic acid molecule, expression card E or expression vector of the present invention is encoded as part of a fusion protein (also referred to herein as a protein chimera), wherein the polypeptide Embedded in the sequence of other polypeptides. Therefore, as enumerated herein (below or elsewhere), each polypeptide encoded by the multinucleic acid of the recombinant nucleic acid molecule, expression card E or expression vector of the present invention can be fused with a protein (fused with a signal peptide and / or with other polypeptide Fusion or in it), or the expression of recombinant nucleic acid molecules, expression cards or expression vectors in the manner of individual polypeptides 1 will depend on the particular recombinant nucleic acid molecules, expression cards and expression vectors. For example, in some embodiments, a recombinant nucleic acid molecule comprising a polynucleic acid encoding the antigen CEA will encode cea in the form of a fusion protein with a 98561.doc -71-200530399 k-peptide. In some embodiments, the polypeptide is part of a fusion protein encoded by a recombinant nucleic acid molecule, expression cassette or expression vector, and is heterologous to the signal peptide of the fusion protein. In some embodiments, the polypeptide is located within another polypeptide sequence that is heterologous to it (e.g., a secreted protein or autolysin, or a fragment or variant thereof). In some embodiments, the polypeptide is bacterial (Listeria or non-Listeria). In some embodiments, the polypeptide is not bacterial. In some embodiments, the polypeptide encoded by the polynuclear acid is a mammalian polypeptide. For example, the polypeptide may correspond to a polypeptide sequence (ie, a human polypeptide) found in humans. In some embodiments, the polypeptide is of the genus Listeria. In some embodiments, the polypeptide is non-Listeria. In some embodiments, the polypeptide is not natural (i.e., foreign) to a bacterium in which the recombinant nucleic acid molecule, expression cassette, and / or expression vector is intended to be incorporated. In some embodiments, the polynucleotide codons encoding a polypeptide are optimized for performance in bacteria. In some embodiments, the polynucleotide encoding the polypeptide is fully codon-optimized for performance in bacteria. In some embodiments, the polypeptide encoded by the codon-optimized polynuclear picric acid is foreign to the bacterium (ie, heterologous to the bacterium. In this document, the term "polypeptide" may be used interchangeably. And "peptides" and "proteins", and are not limited to the length or size of the amino acid sequences contained therein. However, the polymorph will generally include at least about 6 amino acids. In some embodiments ^ at least About 20 of the polypeptides will include at least about 9, at least about 98561.doc-72 · 200530399, at least about 30, or at least about 50 amino acids. In some specific embodiments, the polypeptide includes at least Approximately 100 amino acids. In some embodiments, the polypeptide is a specific functional site of a protein (eg, an extracellular functional site, an intracellular functional site, a catalytic functional site, or a binding functional site). In some specific embodiments The polypeptide includes a complete (ie, full-length) protein. In some embodiments, a polynucleotide encoded by a recombinant nucleic acid molecule, a performance cassette, and / or a polynucleotide of a performance vector , Including antigens or proteins, which provide ameliorative treatment of the disease. In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, the expression cassette and / or the polynucleotide of the expression vector is an antigen or a protein, which provides the disease Relief treatment. In some specific embodiments, the encoded polypeptide is a therapeutic protein (or includes a therapeutic protein). In some specific embodiments, the recombinant nucleic acid molecule, expression cassette and / or expression vector polynucleoside Acid-encoded polypeptides include antigens (such as any of the antigens described herein). In some embodiments, the polypeptide encoded by the polynucleic acid of a recombinant nucleic acid molecule, expression cassette, and / or expression vector is an antigen. In some In specific embodiments, the antigen is a bacterial antigen. In some embodiments, the antigen is a non-Listeria bacterial antigen. However, in some embodiments, the antigen is a non-Listeria antigen. In specific embodiments, the antigen is a non-bacterial antigen. In some specific examples, the antigen is a mammalian antigen. In some embodiments, the antigen is a human antigen. In some embodiments, the scab is (or includes) an antigen that includes one or more immunogenic epitopes. In some 98561.doc 200530399 In specific embodiments, the antigen includes one or more bridging antigenic epitopes. In other specific embodiments, the antigen includes one or more mhc type π antigens. In some specific embodiments, the antigen The epitope is a CD4 + T-cell epitope. In other specific embodiments, the epitope is a CD8 + T-cell epitope. The antigen encoded by the polynucleotide is not limited to any precise nucleic acid sequence (such as Encoding a naturally-occurring, full-length antigen), but can be any sequence encoding a button, and the polypeptide is within a bacterium or composition of the invention when administered to an individual 'is sufficient to elicit the desired immune response. #When used in this document, the term antigen is also understood to include fragments of larger antigenic proteins, as long as the fragment is antigenic (ie, immunogenic). In addition, in some embodiments, the antigen encoded by the polynucleic acid of the recombinant nucleic acid molecule, expression card or expression vector may be a variant of a naturally occurring antigen sequence, like a polynucleotide encoding other non-antigen proteins, The sequence of a polynucleotide encoding a specific protein can be changed so long as the desired protein is expressed and the desired effect (such as a mitigating effect) is provided when administered to an individual). The antigens derived from other antigens include antigens that are antigenic (ie, immunogenic) fragments of other antigens, antigenic variants of other antigens, or antigenic variants of fragments of other antigens. Variants of an antigen include antigens that differ from the original antigen by one or more substitutions, deletions, additions and / or insertions. Antigenic fragments can be of any length, but most are usually at least about 6 amino acids, at least about 9 amino acids, at least about 12 amino acids, at least about 20 amino acids, at least about 30. Amino acids, at least about amino groups, or to; about 100 amino acids. Antigenic fragments of the antigen include 98561.doc • 74- 200530399 at least one epitope derived from the antigen. In some embodiments, the epitope is an MHC class I epitope. In other specific embodiments, the epitope is a MHC class II epitope. In some embodiments, the epitope is a CD4 + T-cell epitope. In other specific embodiments, the epitope is a CD8 + T-cell epitope. Skilled artisans can use a variety of algorithms and software packages to predict antigenic regions (including epitopes) within proteins. For example, algorithms that can be used to select epitopes that bind to MHC class I and II molecules are publicly available. For example, a publicly available " SYFPEITHI " algorithm can be used to predict peptides that bind to MHC (Rammensee et al. (1999) Immunogenetics 50: 213-9). For other examples of publicly available algorithms, see the following References: Parker et al. (1994) «Γ-ΐ mmunol 152: 163-75; Singh and Raghava (2001) Bioinformatics 17: 1236-1237; Singh and Raghava (2003) Bioinformatics 19: 1009-1014; Mallios (2001) Bioinformatics 17: 942-8; Nielsen et al. (2004) Bioinformatics 20: 1388-97; Donnes et al. (2002) BMC Bioinformatics 3:25; Bhasin et al. (2004) Vaccine 22: 3195-204; Guan et al. (2003) ) Nucleic Acids Res 3 1: 3621-4; Reche et al. (2002) Hum. Immunol. 63: 701-9; Schirle et al. (2001) J. Immunol Methods 257: 1-16; Nussbaum et al. (2001) Immunogenetics (2001) 53: 87-94; Lu et al. (2000) Cancer Res. 60: 5223-7. See also, for example, the Vector NTI® Suite (Informax, Inc., Bethesda, MD), GCG Wisconsin Suite Software 98561. doc -75- 200530399 (Accelrys, Inc., San Diego, CA), Welling et al. (1985) FE BS Lett. 188: 215-218, Parker et al. (1986) Biochemistry 25: 5425-5432, Van Regenmortel and Pellequer (1994) Pept. Res 7: 224-228, Hopp and Woods (1981) PNAS 78: 3824-3828 , And Hopp (1 993) Pept. Res. 6: 183_190. Some of the algorithms or software packages discussed in the references cited above in this paragraph are aimed at combining with MHC Class I and / or Class II For the prediction of peptides, the other is to confirm the incision position of protein disintegration, and the other is to predict the antigenicity based on hydrophilicity. Once it has been confirmed that it is believed to contain at least one candidate epitope with the desired property, the polynucleic acid sequence encoding the sequence can be incorporated into the expression cassette and introduced into a Listeria vaccine vector or other bacteria Inside the vaccine vector. The immunogenicity of the antigenic fragment can then be confirmed by assessing the immune response produced by Listeria or other bacteria expressing the fragment. Standard immunological assays can be used, such as EuspOT assay, cells

An internal cytokine staining (ICS) assay, a cytotoxicity / cell activity assay, or the like confirms that the selected antigen fragments still maintain the desired immunogenicity. Examples of these types of assays are provided in the examples below (see, e.g., Example 2D. In addition, the methods described in the examples below are also used (e.g., CT26 mouse colon cells expressing antigen fragments in famous mice, followed by candidate # Vaccinate mice and observe the effect on tumor size, metastasis, survival, etc. relative to the control group and / or full-length antigens, 9) 5Evaluate the resistance of Listeria and / or bacterial vaccines -Tumor potency. In addition, large databases containing epitopes and / or views (98561.doc -76- 200530399) for confirming antigenic fragments are publicly available. See, for example, Brusic et al. (1998 ) Nucleic Acids Res. 26: 368-371; Schonbach et al. (2002) Nucleic Acids Research 30: 226-9; and Bhasin et al. (2003) Bioinformatics 19: 665-666; and Rammensee et al. (1999) Immunogenetics 50: 213-9 o The amino acid sequence of the antigenic variant is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% identical to the original antigen. in In some embodiments, the antigenic variant is a retention variant that has at least about 80% identity to the original antigen, and the substitution between the antigenic variant and the sequence of the original antigen is a retention amine group Acid substitutions. The following substitutions are considered to be retention amino substitutions: • Replacement of alanine, isoleucine, or leucine with valine; substitution of arginine, glutamine, or aspartate with lysine Pyridoxine; Replace asparagine, histamine, lysine, or arginine with glutamine; replace aspartic acid with glutamic acid; replace cysteine with serine; replace with asparagus Amine substituted amidoamine; glutamine with aspartic acid; glycine with proline or alanine; histamine with asparagine, glutamine, lysine or arginine Acid; replace isoleucine with leucine, collarine, methionine, alanine, phenylalanine, or norleucine; replace with norleucine, isoalanine, leucine, Methionine, alanine, or phenylalanine replaces parabens, and arginine, glutamine, or aspartic acid replaces lysine; Replace methionine with phenylalanine or isoleucine; replace phenylalanine with leucine, lauryl, isoleucine, alanine, or melamine; replace proline with alanine; and threonine Substitute serine; replace threonine with serine 98561.doc -77- 200530399 Substitute threonine; replace tryptophan with tyrosine or phenylalanine; tryptophan, phenylalanine, threonine or serine Substituted complex amino acids; substituted complex amino acids with tryptophan, phenylalanine, threonine, or serine; replaced with isoleucine, leucine, sulfanilic acid, phenylalanine, alanine, or deselenium Acid replaces valine. In some embodiments, the antigenic variant is a retentive variant that is at least about 90% identical to the original antigen. In some embodiments, antigens derived from other antigens are substantially equivalent to other antigens. If the derived antigen is at least about 70% identical to the original antigen in the amino acid sequence and maintains at least about 70% of the immunogenicity of the original antigen, then the antigen derived from the other antigen is substantially equivalent to Derived from the original antigen. In some embodiments, the substantially equivalent antigen is at least about 80%, at least about 90%, at least about 95%, or at least about 98% identical in amino acid sequence to the original antigen. In some embodiments, the substantially equal antigens include only retention substitutions relative to the original antigen. In some embodiments, the substantially equal antigen retains at least about, at least about 90%, or at least about 95% of the original antigen's immunogenicity. To determine the immunogenicity of a specific derived antigen and compare it with the original antigen, determine whether the derived antigen is substantially equal to the original antigen. You can be familiar with any of the derivations in many immunogenicity assays known to this artist For example, Listeria monocytogenes and original antigens can be prepared as described herein to produce Listeria 1 expressing the original or derived antigen. Immunization against mice can be assessed by standard techniques such as EUSPOT assay, intracellular cytokine staining (Ics) assay, cytotoxic T 'cell activity, or the like, by immunizing mice with Listeria -78- 200530399 should be used to measure the ability of Z. spp. Which expresses different antigens to generate an immune response. Examples of this type of head measurement are provided in the examples below (see, for example, Example 21). In some embodiments, the polypeptide encoded by a polynucleotide comprising a double-stranded, 1, and a nucleic acid molecule, a performance cassette, and / or a vector includes an antigen. In some specific embodiments, the antigen is selected from the group of tumor-associated gamma antigens, related antigens, polypeptides derived from tumor-associated antigens, infectious disease antigens, and peptides derived from 彳 4 Wang Mufu's disease antigen Group of people. In some embodiments, the polypeptide encoded by a polynucleotide of a recombinant nucleic acid molecule, expression cassette and / or vector includes a tumor-associated antigen, or an antigen derived from a tumor-associated antigen. In some embodiments, the limulus polypeptide includes a tumor-associated antigen. In some embodiments, the encoded polypeptide includes more than one antigen, which is a tumor-associated antigen ' or an antigen derived from a tumor-associated antigen. For example, in some embodiments, 'the encoded polypeptide includes mesothelin (or an antigenic fragment or an antigenic variant thereof) and K-Ras, 12-K-Ras, or PSCA (or K_Ras, 12-K-Ras, or Antigenic fragments or antigenic variants of PSCA). In some embodiments, the antigen encoded by the polynucleic acid of the recombinant nucleic acid molecule, expression cassette and / or expression vector is a tumor-associated antigen or an antigen derived from a tumor-associated antigen. In some embodiments, the antigen is a tumor-associated antigen. In some embodiments, the polynucleotide encoding the antigen (or encoding a polypeptide comprising an antigen) in the recombinant nucleic acid molecule, expression cassette, and / or expression vector is 'not the same as an antigen associated with a tumor' but preferably is Antigen derived from 98561.doc -79- 200530399 tumor-associated antigen. For example, in some embodiments, an antigen encoded by a polynucleotide of a recombinant nucleic acid molecule, expression cassette, and / or expression vector may include a fragment of a tumor-associated antigen, a variant of a tumor-associated antigen, or a variant thereof Variants of fragments of tumor-associated antigens. In some cases, 'the antigen', such as a tumor antigen, can induce a more pronounced immune response in a vaccine when its amino acid sequence differs from the host's endogenous tasks. In other cases, a 'derived antigen induces a less pronounced immune response than the original antigen' but, for example, because of its smaller size, it is more advantageous for xenogeneic expression in a vaccine vector of Listeria. In some specific embodiments, the amino acid sequence of a variant of a tumor-associated antigen, or a variant of a fragment of a tumor-associated antigen, differs by one or more from the tumor-associated antigen or a corresponding fragment thereof. Multiple amino acids. Antigens derived from tumor-associated antigens will include at least one epitope sequence that, when expressed in a host, encodes a polynucleotide encoding the antigen, capable of inducing a desired immune response. Thus, in some embodiments, the polynucleotide in a recombinant nucleic acid molecule, expression cassette or vector encodes a polypeptide comprising an antigen derived from a tumor-associated antigen, wherein the antigen includes at least one tumor-associated antigen Antigenic fragment. In some embodiments, the polynucleotide in a recombinant nucleic acid molecule, expression cassette or vector encodes an antigen derived from a tumor-associated antigen, wherein the antigen includes at least one antigenic fragment of a tumor-associated antigen. The antigenic fragment includes at least one epitope of a tumor-associated antigen. In some embodiments, antigens derived from other antigens are antigenic (i.e. immunogenic) fragments or antigenic variants of other antigens. In some embodiments, the antigen is an anti-98561.doc -80- 200530399 primary fragment of another antigen. In some embodiments, the antigen is an antigenic variant of another antigen. Many tumor-associated antigens have been identified by τ cells (Renkvist et al. Cancer Immunol Innumother 50: 3-15 (2001)). These tumor-associated antigens can be differentiation antigens (such as PSMA, tyrosinase, gplOO), tissue-specific antigens (such as PAP, PSA), development antigens, tumor-associated viral antigens (such as HPV 16 E7), Cancer-testicle antigens (eg MAGE, BAGE, NY-ESO-1), embryonic antigens (eg CEA, α-fetal protein), tumor protein antigens (eg Ras, p53), over-expressed protein antigens (eg ErbB2 (Her2 / Neu), MUC1) or mutant protein antigens. Tumor-associated antigens that can be encoded by heterologous nucleic acid sequences, including but not limited to 707-AP, Annexin II, AFP, ART-4, BAGE, β-catenin / m, BCL-2, bcr-abl, bcr-ablpl90, bcr-ablp210, BRCA-1, BRCA-2, CAMEL, CAP], CASP-8, CDC27 / m, CDK-4 / m, CEA (Huang et al., Exper Rev. Vaccines ( (2002) 1: 49-63), CT9, CT10, Ctp-B, Dek-cain, DAM-6 (MAGE-B2), DAM-IO (MAGE-Bl), EphA2 (Zantek et al., Cell Growth Differ. 1999) 10: 629-38; Carles-Kinch et al. Cancer

Res. (2002) 62: 2840-7), ELF2M, EphA2 (Zantek et al., Cell Growth Differ. (1999) 10: 629-38; Carles-Kinch et al., Cancer Res. (2002) 62: 2840-7 ), ETV6-AML1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GnT-V, gplOO, HAGE, HER2 / neu, 98561.doc -81-200530399 HLA-A * 0201-R170I, HPV-E7, H-Ras, HSP70-2M, HST-2, hTERT, hTRT, iCE, inhibitors of cell death (such as survival proteins), KIAA0205, K-Ras, 12-K-Ras (K-Ras with codon 12 mutation), LAGE, LAGE small LDLR / FUT, MAGE small MAGE-2, MAGE-3, MAGE-6, MAGE-A1, MAGE -A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-B5,

MAGE-B6, MAGE-C2, MAGE-C3, MAGE-D, MART-1, MART-1 / Melan-A, MC1R, MDM-2, Mesothelin, Myosin / m, MUC1, MUC2, MUM- 1.MUM-2, MUM-3, New-Poly A polymerase, NA88-A, N-Ras, NY-ESO-1, NY_ESO-la (CAG-3), PAGE-4, PAP, Protease 3 (PR3 ) (Molldrem et al., Blood (1996) 8 8: 245 0-7; Molldrem et al., Blood (1997) 90: 2529-34), PI5, pl90, Pml / RARa, PRAME, PSA, PSM, PSMA, RAGE , RAS, RCAS, RIH, RU2, SAGE, SAR Ding Xiao SART-2, SART-3, SP17, SPAS-1, TEL / AML1, TPI / m, tyrosinase, TARP, TRP-1 (gp75), TRP-2, TRP-2 / INT2, WT-1, and the alternately translated NY-ESO-ORF2 and CAMEL proteins are derived from the NY-ESO-1 and LAGE-1 genes. In some embodiments, the antigen encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, and / or vector can include any tumor-associated antigen that can induce a tumor-specific immune response, including those that have been identified Of the antigen. In some embodiments, the polynucleotide in the recombinant nucleic acid molecule, expression cassette and / or vector encodes more than one tumor-associated antigen. In some embodiments, the antigen is mesothelin (Argani et al., Clin 98561.doc -82-200530399

Cancer Res. 7 (12): 3862-8 (2001)), Spl7 (Lim et al., Blood 97 (5): 1508-10 (2001)), gpl00 (Kawakami et al., Proc. Natl. Acad · Sci. USA 91: 6458 (1994)), PAGE-4 (Brinkmann et al., Cancer Res. 59 (7): 1445-8 (1999)), TARP (Wolfgang et al., Proc. Natl. Acad. Sci. USA 97 ( 17): 9437-42 (2000)), EphA2 (Tatsumi et al., Cancer Res. 63 (15): 4481-9 (2003)), PR3 (Muller-Berat et al., Clin Immunol Immunopath. 70 (1 ): 51-9 (1994)), prostate stem cell antigen (PSCA) (Reiter et al., PI · oc · Natl · Acad · Sci ·, 95: 1735-40 (1998); Kiessling et al., Int · J · Cancer , 102: 390-7 (2002)) or SPAS-1 (US Patent No. 2002/0 1 50588). In some embodiments of the invention, the antigen encoded by the recombinant nucleic acid molecule or expression cassette is CEA. In other specific embodiments, the antigen is an antigenic fragment and / or an antigenic variant of CEA. CEA is a 180-kDa membrane intercellular adhesion glycoprotein that is over-represented in a significant proportion of human tumors, including 90% of colorectal, gastric, and pancreatic cancers, 70% of non-small cell lung cancers, and 50% of breast cancers (Hammarstrom, Semin · Cancer Biol ·, 9: 67-81) 〇 Various immunotherapeutics mimicking CEA, such as anti-hereditary monoclonal antibodies (Foon et al., Clin · Cancer Res ·, 87__982-90 ( 1995), or immunization with a recombinant vaccine virus expressing CEA (Tsang et al., J. Natl. Cancer Inst., 87: 982-90 (1995)), but unfortunately has only limited success. Despite this, research The authors have identified HLA * 0201-restricted epitope, CAP-1 (CEA605-613), which was recognized by human T cell strains obtained from vaccinated patients. DC immunization with this epitope pulse Patients, unable to induce a clinical response (Morse et al., Clin · Cancer 98561.doc -83-200530399

Res., 5: 1331-8 (1999)). Recently, CEA605-613-like agonists have replaced asparagine (CAP1-6D) with irregularly changing aspartic acid at position 610. Although the amino acid substitution did not change the MHC binding affinity of the peptide, the use of the modified peptide ligand (APL) resulted in improved production of CEA-specific cytotoxic T lymphocytes (CTL) in vitro . CAP1-6D-specific CTLs maintain their ability to recognize and lyse tumor cells expressing native CEA (Zaremba et al. Cancer Res., 57: 4570-7 (1997); Salazar et al., Int. J. Cancer, 85 : 829-38 (2000)). Fong et al. Demonstrated that CEA-specific immunity was induced in colon cancer patients who were expanded with Flt3-ligand and immunized with the APL-cultured DC. It is encouraging that 2 of the 12 patients experienced dramatic tumor regression after vaccination, which was related to the induction of peptide-MHC tetramer + T cells (Fong et al., Proc. Natl. Acad Sci. USA., 98: 8809-14 (2001)). In other specific embodiments, the antigen is protease-3 or is derived from protease-3. For example, in a specific embodiment, the antigen includes the HLA-A2.1-restricted peptide PR1 (amino acids 169-177; VLQELNVTV (sequence number 63)). _ Information on protease-3 and / or PR1 epitopes can be obtained in the following references: US Patent No. 5,18,8,19, Molldrem et al., Blood, 90: 2529-2534 (1997 ); Molldrem et al., Cancer Research, 59: 2675-2681 (1999); Molldrem et al., Nature Medicine, 6: 1018-1023 (2000); and Molldrem et al., Oncogene, 21: 8668-8673 (2002). In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette and / or vector includes a polypeptide selected from K-Ras, H-Ras, 98561.doc -84- 200530399 N-Ras , 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survival protein, gplOO, PAP, protease 3, SPAS-B SP-17, PAGE-4 'TARP and CEA Group of antigens, or including antigens derived from the group selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survival proteins, gplOO, PAP, Antigens of a group of proteases 3, SPAS-1, SP-17, PAGE-4, TARP and CEA. In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette and / or expression vector includes a polypeptide selected from K-Ras, H-Ras, N-Ras, 12-K-Ras , Mesothelin, PSCA, NY-ESO-1, WT-B Survival Protein, gpl00, PAP, Protease 3, SPAS-B SP-17, PAGE-4, TARP or CEA. In some embodiments, the polypeptide includes K-Ras. In some embodiments, the polypeptide includes H-Ras. In some specific embodiments, the polypeptide includes N-Ras. In some embodiments, the polypeptide includes K-Ras. In some embodiments, the polypeptide includes mesothelin (e.g., human mesothelin). In some embodiments, the polypeptide includes PSCA. In some embodiments, the polypeptide includes NY-ESO-1. In some specific embodiments, the polypeptide includes WT-1. In some embodiments, the polypeptide includes a survival protein. In some embodiments, the polypeptide includes gp 100. In some embodiments, the polypeptide includes PAP. In some embodiments, the polypeptide includes protease 3. In some embodiments, the polypeptide includes SPAS-1. In some embodiments, the polypeptide includes SP-17. In some embodiments, the polypeptide includes PAGE-4. In some embodiments, the polypeptide includes TARP. In some specific examples of 98561.doc -85- 200530399, the polypeptide includes CEA. In some embodiments, the antigenic line encoded by the polynucleotide in the recombinant nucleic acid molecule, expression cassette and / or vector is selected from K-Ras, H_Ras, N-Ras, 12-K-Ras, mesothelin , PSCA, nY-ESO-1, WT-1, survival protein, gplOO, PAP, protease 3, SPAS-Bu SP-17, PAGE_4, TARP or CEA. In some embodiments, the antigen is K-Ras. In some embodiments, the antigen is H-Ras. In some specific embodiments, the antigen is N-Ras. In some embodiments, the antigen is K-Ras. In some embodiments, the antigen is mesothelin. In some embodiments, the antigen is P s CA. In some embodiments, the antigen is NY-ESO-1. In some embodiments, the antigen is WT-1. In some embodiments, the antigen is a survival protein. In some embodiments, the antigen is gp 1 0 0. In some embodiments, the antigen is PAP. In some embodiments, the antigen is protease 3. In some embodiments, the antigen is SPAS-1. In some embodiments, the antigen is SP-17. In some embodiments, the antigen is PAGE-4. In some embodiments, the antigen is TARP. In some specific embodiments, the antigen is CEA. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is mesothelin, SPAS-1, protease-3, EphA2, SP-17, gplOO, PAGE-4, TARP or CEA, or an antigen derived from one of those proteins. In some embodiments, the antigen is or is derived from mesothelin. In other specific embodiments, the antigen is EphA2 or an antigen derived from EphA2. In some specific implementations of 98561.doc-86-200530399, the antigen encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector described herein is not Epha2 (or an antigen derived from Epha2). In some embodiments, the antigen is a tumor-associated antigen other than Epha2. In some embodiments, the antigen is derived from a tumor-associated antigen other than Epha2. In some embodiments, the polypeptide encoded by a polynucleic acid in a recombinant nucleic acid molecule, expression cassette and / or expression vector includes an antigen other than Epha2. In some embodiments, the polypeptide encoded by polynuclear picric acid in a recombinant nucleic acid molecule, expression cassette, and / or expression vector includes Epha2 or an antigen other than an antigen derived from Epha2. In some embodiments, the polynucleotide encoding in the recombinant nucleic acid molecule, expression cassette and / or expression vector includes derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin , PSCA, NY-ESO-1, WT-1, surviving protein, gplOO, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA antigen peptide. In some embodiments, the polypeptide includes an antigen derived from K-Ras. In some embodiments, the polypeptide includes an antigen derived from H-Ras. In some specific embodiments, the polypeptide includes an antigen derived from N-Ras. In some embodiments, the polymorphism includes an antigen derived from 12-K-Ras. In some embodiments, the polymorphic envelope is derived from an antigen of mesothelin. In some embodiments, the polypeptide includes an antigen derived from PSCA. In some embodiments, the polymorphism includes an antigen derived from NY-ESO-1. In some embodiments, the polymorphic envelope is derived from an antigen of WT-1. In some embodiments, the polymorphism includes an antigen derived from a survival protein. In some embodiments, the polypeptide includes an antigen derived from gp 100. In some embodiments, the polymorphism is 98561.doc -87- 200530399 derived from an antigen of PAP. In some embodiments, the polypeptide includes an antigen derived from protease 3. In some embodiments, the polypeptide includes an antigen derived from SPAS-1. In some embodiments, the polypeptide includes an antigen derived from SP-17. In some embodiments, the polypeptide includes an antigen derived from PAGE-4. In some embodiments, the polypeptide includes an antigen derived from TARP. In some embodiments, the polypeptide includes an antigen derived from CEA. In some embodiments, the polynucleotide encoding in the recombinant nucleic acid molecule, expression card g, and / or expression vector is derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, Antigens of PSCA, NY-ESO-1, WT-1, Survivin, gp 100, PAP, Protease 3, SPAS_1, SP-17, PAGE-4, TARP or CEA. In some embodiments, the antigenic line is derived from K-Ras. In some embodiments, the antigenic line is derived from H-Ras. In some embodiments, the antigenic line is derived from N-Ras. In some embodiments, the antigenic line is derived from 12-K-Ras. In some embodiments, the antigen is an antigen derived from mesothelin. In some embodiments, the antigen is an antigen derived from PSCA. In some embodiments, the antigen is an antigen derived from NY-ESO-1. In some embodiments, the antigen is an antigen derived from WT-1. In some embodiments, the antigen is an antigen derived from a survival protein. In some embodiments, the antigen is an antigen derived from gp 100. In some embodiments, the antigen is an antigen derived from PAP. In some embodiments, the antigen is an antigen derived from protease 3. In some embodiments, the antigen is an antigen derived from SPAS-1. In some embodiments, the antigen is an anti-98561.doc-88-200530399 derived from SP-17. In some embodiments, the antigen is an antigen derived from page-4. In some embodiments, the antigen is an antigen derived from TArp. In some embodiments, the antigen is an antigen derived from CEA. In some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant thereof. Thus, in some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette and / or vector includes mesothelin ' or an antigenic fragment or antigenic variant thereof. In some embodiments, the polypeptide encoded by the polynucleotide is mesothelin, or an antigenic fragment or antigenic variant thereof. In some embodiments, the antigen is a mesothelin (e.g., human mesothelin) ' wherein the mesothelin signal peptide and / or Gpi (glycosylphospholipid inositol) anchor has been deleted. Thus, in some embodiments, the polypeptide encoded by the polynucleotide includes a mesothelin signal peptide and / or a GPI-anchored mesothelin. In some embodiments, the polypeptide encoded by the polynucleotide is a mesothelin signal peptide and / or GPI-anchored mesothelin has been deleted. In some embodiments, the polypeptide encoded by the polynucleotide is a human mesothelin in which the mesothelin signature and / or G PI have been deleted. In some specific embodiments, the polypeptide encoded by the polynucleotide is a human mesothelin in which both the mesothelin signal peptide and the GPI have been deleted. In some embodiments, the antigen is NY-eso- 丨, or an antigenic fragment or antigenic variant thereof. Therefore, in some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, and / or vector includes an antigen, which is NY-ESO-1 or an antigenic fragment or antigenic variant thereof . In some embodiments, the polypeptide is an antigen, which is NY-ESOq or 98561.doc -89-200530399 antigenic fragment or antigenic variant. In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette or vector includes at least one antigenic fragment of a tumor-associated antigen, such as a human prostate stem cell antigen (PSCA; GenBank Accession AF043498), human testicular antigen (NY-ESO-1; GenBank accession number NM-001327), human carcinoembryonic antigen (CEA; GenBank accession number M29540), human mesothelin (GenBank accession number U40434), human survival protein ( GenBank accession number U75285), human protease 3 (GenBank accession number X55668), human K-Ras (GenBank accession number M54969 & P01116), human H-Ras (GenBank accession number P01112), human N-Ras (GenBank accession number P01111) , And human 12-K-Ras (K-Ras includes Glyl2Asp mutation) (see, for example, GenBank Accession No. K00654). In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector includes an antigenic fragment of a tumor-associated antigen with an amino acid having at least one retention substitution. In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector includes an antigenic fragment having at least one amino acid residue deleted. In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette or expression vector includes a combination of antigenic sequences derived from more than one type of tumor-associated antigen, such as derived from A combination of antigenic fragments of both cortex and Ras. Representative regions of tumor antigens predicted to be antigenic include the following: Amino group 98561.doc of the PSCA amino acid sequence in GenBank accession number AF043498 • 90- 200530399 acids 25-35; 70-80 and 90-118; GenBank NY-ESO-1's amino acids 40-55, 75-85, 100-115 and 128-146 with accession number NM_001327; CEA amino acid sequence of GenBank accession number M29540 歹 J's amino acids 70-75, 1 50-1 55, 205-225, 330-340, and 510-520; GenBank accession number U40434 Amino acid 90-110, 140-150, 205-225, 280-3 10, 390 -410, 420-425, and 550-575; amino acid 12-20, 30-40, 45-55, 65-82, 90-95, 102-115, and 115-130 of the survival polypeptide sequence of GenBank accession number U75285 ; Amino acids 10-20, 30-35, 65-75, 110-120, and 160-170 of the amino acid sequence of protease 3 found in GenBank accession number X55668; GenBank accession number P01117 or M54968 (human K-Ras ) Amino acids 10-20, 30-50, 55-75, 85-110, 115-135, 145-155, and 160-185; GenBank accession number P01112 (human H-Ras) amino acids 10-20 , 25-30, 35-45, 50-70, 90_110, 115-1 35 and 145-175; Amino acids 10-20, 25-45, 50-75, 85-110, 115-135, 140-155, and 160-180 of GenBank Accession Number P01111 (Human N-Ras); and 12 -Top 25 amino acids of K-Ras (sequence disclosed in GenBank Accession No. K00654). These antigenic regions were predicted by Hopp-Woods and Welling antigenic mapping. In some embodiments, as an isolated polypeptide, or as a fusion protein with a selected signal peptide, or as a protein chimera in which the polypeptide has been inserted into other polypeptides, the polypeptides encoded by the polynucleotides of the present invention are all Is a polypeptide including one or more of the following peptides of human mesothelin: SLLFLLFSL (amino acid sequence 歹 ij 20-28; (sequence number 64)), 98561.doc -91 · 200530399 VLPLTVAEV (amino acid sequence 530 -538; (Sequence No. 65)), ELAVALAQK (amino acid sequence No. 83-92; (Sequence No. 66)), ALQGGGPPY (amino acid sequence 225-234; (Sequence No., J No. 67) )), FYPGYLCSL (amino acid sequence "M35-444; (sequence number 68)); and LYPKARLAF (amino acid sequence 475-484, (sequence number 69)). For example, in some embodiments, the antigen encoded by a polynucleotide of the invention is a (antigenic) fragment of human mesothelin that includes one or more of these peptides. Additional information on these mesothelin peptide sequences and their relationship to medically relevant immune responses can be found in PCT Publication WO 2004/006837. Alternatively, the polynucleotide in the recombinant nucleic acid molecule, expression cassette or expression vector may encode an autoimmune disease-specific antigen (or a polypeptide including an autoimmune disease-specific antigen). In autoimmune diseases mediated by T cells, T cells respond to their own antigens, resulting in autoimmune diseases. In order to use this type of antigen for the treatment of autoimmune diseases, the vaccine of the present invention can target specific T cells responsible for the autoimmune response. For example, the antigen may be part of a τ cell receptor that elicits an autoimmune response to a hereditary type specific to those T cells, wherein incorporating the antigen into a vaccine of the present invention will induce a response to those that cause an autoimmune response. T cell-specific immune response. Excluding those T cells would be a therapeutic mechanism for reducing autoimmune diseases. Other possibilities are the incorporation of a polynucleotide encoding an antigen into a recombinant nucleic acid molecule. The antigen result will produce antibodies aimed at self-antigens in autoimmune diseases, or specific B-cells that secrete the antibodies. Germline immune response. For example, 'a polynucleotide encoding a hereditary antigen can be incorporated into a recombinant nucleic acid molecule, which will lead to antibodies that fight this type of B cells and / or react with 98561.doc -92 · 200530399 autoantigens in autoimmune diseases Anti-hereditary immune response. Autoimmune diseases that can be treated with vaccines that include bacteria that include the expression cassettes and recombinant nucleic acid molecules of the present invention include, but are not limited to, rheumatoid arthritis, multiple sclerosis, Crohn's disease, lupus, Myasthenia gravis, leucorrhea, scleroderma, psoriasis, pneumonia vulgaris, fibromyalgia, colitis, and diabetes. A similar approach can be used to treat allergic reactions, in which antigens are incorporated into vaccine bacteria, targeting T cells, B cells, or antibodies effective in regulating allergic reactions. In some autoimmune diseases, such as psoriasis, the result of this disease is hyperproliferative cell growth, which can also target the manifestation of antigens. It is believed that such antigenic results will produce an immune response to hyperproliferative cells. In some embodiments, the antigen is an antigen that targets a protein structure associated with a unique disease. One example is the targeting of antibodies, B cells or τ cells using the hereditary type antigens discussed above. Other possibilities are targeting unique protein structures that result from specific diseases. One example is the incorporation of an antigen that will produce an immune response to a protein that has been observed to cause amyloid in diseases such as Alzheimer's disease, subacute spongiform encephalopathy (CJD), and bovine spongiform encephalopathy (BSE). spot. Although this method only provides a reduction in plaque formation, in the case of diseases like CJD, it may provide a vaccine for treatment. The disease is caused by an infectious form of prion protein. In some specific embodiments, the polynucleotide of the present invention encodes an antigen against an infectious form of the prion protein, so that the immune response generated by the vaccine can be eliminated and the infectious protein causing CJD can be reduced. In some embodiments, the polypeptide encoded by the polynucleotide of the recombinant nucleic acid molecule, expression cassette and / or 98561.doc • 93-200530399 expression vector includes an infectious disease antigen, or an antigen derived from an infectious disease antigen. In some embodiments, the polypeptide includes an infectious disease antigen. In some other specific embodiments, the polypeptide includes an antigen derived from an infectious disease antigen. In some embodiments, the polypeptide encoded by the polynucleotide of the recombinant nucleic acid molecule, expression cassette and / or expression vector is an infectious disease antigen, or an antigen derived from an infectious disease antigen. In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette and / or expression vector is an infectious disease antigen. In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule 'expression card and / or expression vector is derived from an infectious disease antigen. In other embodiments of the invention, the antigen is derived from a human or animal pathogen. The pathogen may be a virus, bacteria, fungus, or protozoa as needed. For example, 'the antigen may be an antigen of a virus or a fungus or a bacterium. In a specific embodiment, the antigen encoded by the pathogen-derived recombinant nucleic acid molecule, expression cassette and / or expression vector is a protein produced by a pathogen, or a protein derived from a free pathogen. For example, in some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette and / or expression vector is a fragment and / or variant of a protein produced by a pathogen. For example, in some embodiments, the antigenic line is derived from human immunodeficiency virus (such as gpl20, gpi60, gp41, gag antigens, such as p24gag and P55gag, and derived from HIVip01, env, tat, vif, rev, nef , Vpr, vpu, and LTR regions), feline immunodeficiency virus, or human or animal cancer repair virus. For example, in some embodiments, the antigen is gp 120. In a specific embodiment, the antigenic line is derived from herpes simplex virus (HSV) 98561.doc -94- 200530399 types 1 and 2 (such as gD, gB, gH, fast-acting early proteins, such as ICP27), obtained from Cytomegaloviruses (such as gB and gH), from metapneumovirus, from Eltonton virus, or from band cancer therapy viruses (such as gpl, II, or III). (See, for example, Chee et al. (1990) Cytomegaloviruses (edited by KK McDougall, Springer Verlag, pp. 125-169; McGeoch et al. (1988) J. Gen. Virol. 69: 153 1-1574; U.S. Pat. No. 5,171,568; Baer et al. (1984) Nature 3 10: 207-211; and Davison et al. (1986) J. Gen. Virol. 67: 1759-1816). In other specific embodiments, the antigenic line Derived from hepatitis viruses such as Hepatitis B virus (eg Hepatitis B surface antigen), Hepatitis A antigen, Hepatitis C virus, Hepatitis delta virus, Hepatitis E virus or Hepatitis G virus. See, for example, WO 89/04669 , WO 90/11089, and WO 90/14436. Hepatitis antigens can be surface, core, or other related antigens. The HCV genome encodes several viral proteins, including E1 and E2. See, for example, Houghton et al., Hepatology 14: 381-388 (1991) is an antigen of a viral antigen, derived from any of the following viruses, if necessary: from the family Picornaviridae (e.g., polioviruses, rhinoviruses, etc.); goblet Toxicidae (Caliciviridae); Togaviridae (such as wind treatment virus, dengue virus, etc.); Flaviviridae (Coronaviridae); Reoviridae (for example Rotavirus, etc.); Double RNA Viridae (Birnaviridae); Rhabdoviridae (eg, rabies virus, etc.); Orthomyxomyces 98561.doc -95- 200530399 Orthomyxoviridae (eg Influenza A, B, and C viruses, etc.); Filoviridae; Paramyxoviridae (e.g., poliovirus, measles virus, respiratory fusion virus, parainfluenza virus, etc.) Etc.); Bunyaviridae; Arenaviridae; Retiroviridae (e.g. HTLV-I, HTLV-11, HIV-1 (also known as HTLV_111, LAV, ARV , HTLR, etc.)), including but not limited to antigens obtained from isolates HIVIllb, HIVSF2, HTVLAV, HIVLAI, HIVMN); HIV-1CM23 5, HIV_1, HIV-2, etc .; Simian immunodeficiency virus (SIV)); Papilloma virus, Lice carried encephalitis virus; and its analogs. For a description of these and other viruses, see, for example, Virology, 3rd Edition (W. K. Joklik Edit 1988); Fundamental Virology, 3rd Edition (B. N. Fields, D. M. Knipe and P. M. Howley, Editor 1996). In a specific embodiment, the antigen is Flu-HA (Morgan et al., J. Immunol. 160: 643 (1998)). In some alternate embodiments, the antigen line is derived from a bacterial pathogen, such as a branch Bacillus, Bacillus, Yersinia, Salmonella, Neisseria, Borrelia (such as OspA or OspB or derivatives thereof), Chlamydia or Bordetella (eg, P.69, PT, and FHA), or derived from a parasite, such as Plasmodium or Toxoplasma. In a specific embodiment, the antigenic line is derived from Mycobacterium tuberculosis (eg, ESAT-6, 85A, 85B, 85C, 72F), Bacillus anthracis (eg, PA), or Yersinia pestis (eg, FI, V) . In addition, anti-98561.doc -96- 200530399 suitable for use in the present invention: laryngeal: \ or derived from a known cause of disease 'including but not limited to *, ri, tetanus, tuberculosis, fine g or fungal Pneumonia 'inner ear' gonorrhoea, cholera, typhoid fever, encephalitis, mononucleosis, murine-shell disease or salmonellosis, Legionnaires' disease, Lyme disease, leprosy, malaria hookworm, onchocerciasis , Live schistosomiasis, trypanosomiasis, Leishmania: dysentery moth, amoebiasis, filariasis, Borrelia and Trichinella. More antigens can be obtained or derived from non-traditional pathogens, such as Kourou disease, subacute spongiform encephalopathy (CJD), scrapie, infectious mink disease, and wasting disease Or from protein infectious particles, such as prions associated with rabies disease. In other specific embodiments, the antigen is derived from or derived from a gene involved in a neurodegenerative disease (such as Alzheimer's disease), a metabolic disease (such as type 1 diabetes), and a drug substance (such as nicotine addiction) Onset or progression of biological agents. Alternatively, the antigens encoded by heavy, " and nuclear " molecules are used for pain management, and the preparation is painful, or other preparations involving pain signal transmission. In some embodiments, the antigen is a human protein or is derived from a human protein. In other specific embodiments, the antigen is a non-human protein or is derived from a non-human protein (a fragment and / or variant thereof). In some embodiments, the antigenic portion of the fusion protein encoded by the recombinant cassette is a protein derived from a non-human animal, or a protein derived from a non-human animal. For example, 'even if the antigen to be expressed in a Listeria-based vaccine is intended for use in humans, in some embodiments, the antigen may be old mesothelin or derived from mouse mesothelin. V · codon-optimization 98561.doc -97- 200530399 In some specific embodiments, one or more polynucleotides (ie, polynucleotide sequences) within the recombinant nucleic acid molecule, expression cassette and / or expression vector ) Codon_optimization (relative to the natural codon sequence). In some embodiments' for expression in bacteria, the polynucleotide codons in a recombinant nucleic acid molecule encoding a signal peptide (and / or in a performance card E and / or a performance vector) described herein will be optimized . In some embodiments, the polynucleotide codons encoding polypeptides other than signal peptides, such as antigens or other therapeutic proteins, are optimized for performance in bacteria. In some embodiments, both the polynucleotide encoding the signal peptide and the polynucleotide encoding other polypeptides fused to the signal peptide are codon-optimized for expression in bacteria. In some specific embodiments, a polynucleotide encoding a secreted protein (or a fragment thereof) used as a scaffold, or a polynucleotide codon-optimizing an autolysin (or a fragment or a variant thereof) is used. If at least one codon of the polynucleotide's natural codon sequence has been replaced with a codon that is more commonly used by the organism in which it is expressed ("target organism") than the original codon of the natural codon sequence, then The polynucleotide includes a "codon-optimized" codon sequence. For example, if at least one of the codons obtained from a particular bacterial species in which a non-bacterial antigen is to be expressed is substituted for at least one derived from a natural bacterium The codons of the polynucleotide sequence will then optimize the polynucleotides and coders that encode non-bacterial antigens to be expressed in specific species of bacteria. For another example, if the The codons in the sequence are more commonly used by Listeria monocytogenes for amino acid codons, and replacing at least _ codons in the polynucleotide sequence will encode a recombinant mononuclear cell Liss Specialty bacteria 98561.doc -98- 200530399 Polynucleotide codons of human cancer antigens that represent part of the cassette_ Optimized. Similarly, if the The codons in the sequence are more often used by Listeria monocytogenes for amino acid codons, and replacing at least one codon in the polynucleotide sequence encoding the signal peptide will become a single in recombination. The part of the performance cassette encoding Listeria monocytogenes that contains fusion proteins including human cancer antigens is a natural signal peptide for Listeria monocytogenes (eg, from Listeria monocytogenes) Codon-optimization of the LLO signal peptide of Agrobacterium tumefaciens. In some embodiments, the most commonly used codon substitutions with the target amino acid encoding the same amino acid are replaced in the codon-optimized sequence In some embodiments, at least one natural codon sequence of the polynucleotide has been replaced with a codon 'that is more commonly used by the organism in which the codon sequence is to be expressed than the original codon of the natural codon sequence. Two codons. In some embodiments, the original codons of the natural codon sequence have been used more often than they are intended to express the codon sequence. Codons that are used at the same time, replace at least about 5 codons, at least about 10 codons, or at least about 20 codons in the natural codon sequence of the polynucleotide. In some embodiments, it has been used (more than the natural sequence The original dense stone horse) is more often (or most often) used by the target organism, replacing at least about 10% of the codons in the codon_optimized polynucleotide. In other specific embodiments' already used (More than the original codon of the natural sequence) more often (or most often) codons used by the standard organism, replacing at least about 25% of the codons in the codon_optimized polynucleic acid. In other specific implementations For example, 98561.doc -99 · 200530399 has used (more than the original codon of the natural sequence) the codons used more often (or most often) by the target organism, replacing at least the codon-optimized polynucleotide About 50% of the codons. In other specific embodiments, at least about 75 of the codon-optimized polynucleotides have been replaced with codons (or more often) used by the target organism (than the original codons of the native sequence). 〇 / 〇 codon. The codon advantage of different organisms has been extensively studied by those skilled in the art. For example, 'see Sharp et al., Nucleic Acids Res., 15: 1281-95 (1987) and Uchijima et al., The Journal of Immunology, 161: 5594-9 (1998). As a result, codon usage tables for various organisms are publicly available. For example, ‘codon usage tables for various organisms can be found on the Internet, at www.kazusa.or.jp/codon/, and other publicly available places. (See, eg, Nakamura et al. (2000) Nucleic Acids Research 28: 292). For convenience, a representative codon usage table obtained from www * kazusa.〇r.jP / c〇d〇n /, a codon usage table for Listeria monocytogenes (http: //www.kazusa.or.jp/codon/cgibin/showcodon.cgi7speci es = Listeria + monocytogenes + [gbbct]). Representatives of Bacillus anthracis, Mycobacterium tuberculosis, Salmonella typhimurium, Mycobacterium bovis BCG and Shigella flexneri are also provided in Tables 2B, 2D, 2E and 2F, respectively. Codon usage table. Table 2 A: Codon usage table of Listeria monocytogenes (available from www.kazusa.or.jp/codon/) ° Listeria monocytogenes: 3262 CDS, s (1029006 codes Sub) 98561.doc -100- 200530399 Scope: [Triad] [Frequency: per thousand] ([Number]) OUU 29.4 {30274) UUC 14.1 (1448β) UUh 36.8 (37821) UUG 12.3 (12704) UCU 13.2 (13586) UCC 6.5 (6714) UCA 10,4 (1D751) UCG 6.1 (6278) i 2 0 2 1 2 o

Ji ri /% i% 9 7 2 4 2 1 4 5 7 2 0 5 0 7 6 0 3 3 3 12 8 9 6 3 3 10 9 Be AG JQJGJO V uuu 3960) 1972) 583) 9580) CUU 21,0 -(21567) ecu 8.4 (8622) CAU 12.0 (12332) CGD 12.5 (12930) cue 5 Λ (S598) CCC l.? (1780) CAC 5 * 2 (5336) CGC 7.0 (7215) CUA 12.9 (13279) CCA 18.5 (18996;) CAA 29.9 (30719) CGA 5.6 (5732) OJG 5.0 (5120) CCG 7.0 (7219) CAG 5.1 (5234) CGG 2.8 (2384) AUO 49.3 (50692) ACU 17.1 (17614) AAU 33.0 (33908) AGU 14.1 (14534) AUC 18.4 (18894) ACC 6.9 (7089) AAC 15.3 (15790) AGC 8.8 (9031) AUh 9.4 (9642) ACA 2G.S (27318 »AAA SI · 6 (63379) AGA 6.9 (7111) AUG 25.9 (26 € 51) ACG 12.9 (13285) MG 10.4 (10734) AGG 1.2 (1254) GUU 2 ^ .4 (27202) GCU 24.3 (24978) GAU 39.8 (40953) GGU 24.2 (24871) GUC 8.7 (8990) GCC 8.4 (8512) GAC 14.3 (14751) GGC 14.2 (14581) QUA 21 ^ (22247) GCA 28 * 6 (29401 > GAA 60 * 4 (62167) ooa 19,1 (19 ^ 12) GUG 13.1 (13518) GCG 16.6 (17077) GAG 13.1 (13507) GGG 8.7 (9003) Table 2B: Codon usage table of Bacillus anthracis (available from www.kazusa.or.jp/codon/) . Bacillus anthracis [gbbet]: 312 CDS, (90023 codons) Range: [Triad] [Frequency: Every 1,000] ([Number])

UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG λι rv / \ / \ rf \ 4 4 7 4 4 7 4 4 2 0 3 1 4 3 2 4 3 1 4 X 1 1 2916) 934) 3931 ) 1024) 0 5 7 2 0 3 19 3 3 13 1 1 i

COT CCC CCA CCG 7 5 1 4 1 · 2 0 8 2 7 7 8 9 0 2 7 5 1 1

1547) UAU 453) UAC 1330) UAA 375) UAG 7 2 9 4 6 4 9 3 9 2 5 5 rf% .ft n ((/ 1% / lh / 1 5 9 7 3 3 6 4 8 4 1 2 3 0 4 6 0 4 12 2 2 2 1 4009) ACU 21 * 0 (1890) 1072) ACC 5.0 (453) 2042) ACA 2 ^ .8 (2414) 2098) ACG 9.4 (84 4 > 0 13 7 4 4 4 2 4 16 2 Is ffv / t \ / 1 »/ 1» 5 2 3 5 5 4 2 9 1 3 AUAC-AAAG c cac »l \ / 1/1 / \ 9 5 2 7 19 2 0 3 2876) UGU 853) UGC 199) UGA 66} UGG 1392) CGU 379) CGC 2 912) CGA 859) CGG 3959) AGO 1268) AGC 5786) AGA 2047} AGG 5X09 9 2 6 2 7 5 3 X 1 (/ 1 Jf \ /% / rv / V / 1 / ffv / 1 / IV fv / IN 18 5 3 8 5 3 0 4 2 7 1 5 4 7 5 5 6 4 3 4 1 8 3 3 9 9 8 2 6 7 8 2 5 1 1565 > 4 ^ 7) 1236) 368) LIT t /} irr 4 4 4 3 2X77 8 4 3 9 12

GCU GCC GCA GCG 3 15 9 7 4 3 7 1 2/1 · «fv i. R {. 3 0 9 9 99 3 7 3 SIi \ / \ / V / V / 8 2 7 9 9 7 10 5 3 17

3536) GGU 811) GGC 4855) GGA 1614) GOG 17.9 (1611) 5.8 (524) 24.5 (2203) 12.0 (X083) Password GC34.5 5% First letter GC44.99% Second letter GC33.16% Third letter GC25.51% 101-98561.doc 200530399 Table 2C: Codon usage table of Mycobacterium tuberculosis (available from www.kazusa.or.jp/codon/). Mycobacterium tuberculosis [gbbet]: 363 CDS's (131426 codons) Range: [Triad] [Frequency ·· per thousand] ([Number]) i IE ΙΙΞΙΙΙ / \ / \ / \ / 1/1/1/1 / \ · .Ri il / V / V / \ i \ /% 4 6 8 Θ 9 7 0 9 6 7 17 3 3 7 7 -7 45 72 2 9 824 5 1 4 3 1 3 3 s 5 1 4 2 1 709) 3359) 231) 1S45) 778) 2329) 521) 6032) 993) 4300) 282) 2591) UCU 2.0 (UCC 11.4 (UCA 4.3 (ITCG 19.2 (CCU 3.9 (CCC 18.3 (CCA 6.4 (CCG 33 * 2 (

10 7 4 * ·· * 4 6 4 6 3 1 IJCAG ACACIAC

1095) GCU 4249) GCC 622) GCA 4687) GCG 2 5 4 7 2X21 15 14 265) 1499) 571) 2522) 511) 2411) 843) 435S) S4S) 4735} 616} 2158} 1473) 6769) 1625} 5462 )

Ei slim 0 6 4 8 6 7 0 0 1 / mm. Ji. / I ί% 4 7 8 2 5 4 7 4 1 2

Jiw Ji / 1 yi d 3 8 5 * ♦ ♦ * 4 6 5 6 2 2 / \ (/ 1/1 6 6 8 8 »* -k» 5 4 6 5 14 13 2% / \ / AJr 8 7 2 3 8 0 5 0 7 3 1 711) 1928) 1030) 317β) vlr% /% / Ur 7 11s 3 5 6 8 6 4 7 4 3 3 2046) 5858) 2211) 4702)

ru-cAQ * ucag BBS port PCGUCG 2 5 7 1 #f \ / t \ / \ / \ 5.V0 5 9 e 8 1 2 2 3 0 5 3 7 2 3 2 /% / V / 1 c 0 7 8 1 8 6 5 1 2 2 1048) 3500) 7S4) 2772)

/ 1 / i / f% / Γ1 Q o 5 3 4 5 13 1 u CIAO GGGio AAAA t / Atf 1 / s / 16 2 9 3 7 9 2 5 9 14 5 3 3 5 5 8 8 1 4 3 12 2 6 12 7 6 0 9 **** 8 8 9 6 1 4 1i Password GC64.43% First letter GC65.27% Second letter GC48.28% Third letter GC79.75% Table 2D: Rat Codon usage table for Salmonella typhimurium (available from www.kazusa.or.jp/codon/) 〇 Salmonella typhimurium [gbbet]: 1322 OP84 (416065 codons) Range: [Triad] [Frequency : Per thousand] ([number]) UUU 21.7 (UUC 15.1 (UUA 13.6 (WJG 12.1 (CUU 12 * 1 (CUC 10.6 (CUA 4.7 (CUG 49.3 (\ / \ / \ / \ / 15 0 5 4 6 5 2 0 2 6 0 9 6 5 5 5038) 4396) 195Q) 20508)

ucuucc UCA UCG CCUccc CCA CCG 8 0 7 9 1 6 2 2 / V rf% / i / 1 / lm »f \ / 1. rf \ 5 6 9 4 9 0 5 7 8 0 6 4 13 8 2 5 4 2 9 3 4 3 3 3290) 2921) 2712) 9463)

Ei i / 1. Λι. / \ / V 5 6 8 3 6 110 11 3 6 11 5 2 3 2 8 8 7 1 e 4

UGU UGC UGA UGG ril /% rrv 6 111 4 6 14 1 / V ft Ji 2 2 8 8 2 9 2 0 HI X 3 5047) 3818) 5315) 12803) [/ \ / Λ J \ 18 12 8 0 4 7 12 u CAG8 888 1920) 2524) 465) S85I) 7542} 8659) 1695) 3004) 102 · 98561.doc 200530399 < rv / 1 yfi 2 0 0 6 3 4 8 3 2 1 3401) AAU 19.5 (8107) AGU 8,6 (3569) 9980) AAC 21.4 (8920) AGC 18.0 (7485} 3316) AAA 33,0 (13740) AGA 3.2 (1348) 7743) AAG 12,4 (5151) AGG 2.3 (959)

AUU 28.1 (11700) ACU AUC 23,9 (9941) ACC

ΆΙΓΑ 6 < 7 (2771) ACA

AUG 26 Λ (10842) ACG GUU ie.4 (6B21) GUC 17.7 (7367) GUA 11,9 (4935) GDG 24.3 (10092) GCU 14.4 (5985) GCC 27.5 (11462) GCA 14.8 (6156) GCG 37.0 (15387 ) GMJ 32.9 (13700) GJ ^ C 21-5 {8949) GAA 26.1 (15021) QAG 20.9 (8715) GGU 18. QGC 33.0 (GGA 5.1 (GGG 11 * 6 {10 8 4 4 3 8 3 5 7 7 8 7 3 3 4 1 Code GC52.45% First letter GC5 8.32% Second letter GC41.31% Third letter GC57.71% Table 2E · Codon usage table for Mycobacterium bovis BCG (from www.kazusa.or.jp/codon/) 〇 Mycobacterium bovis BCG [gbbct] ·· 51 CDS's (16528 codons) Range: [Triad] [Frequency: Every 1,000] ([Number]) ϋϋϋ 4 * 7 {UUC 27,4 (UUA 1.6 (UUG 14 * 7 (1 /% J-% J. \ Jr 7 3 6 3 7 5 2 4 4 2 / \ / \ / V / \ 9 4 B 8 114 0 1 2 p CAG accc ktf \ »r% /.% Mf 19 4 3 3 8 7 4 1 3 yi > / 1/1 (6 0 9 8 * 41« '«6 7 0 0 1 drawing lJAAl ϋ u ϋ ϋ 9 15 4 0 8 11 1 2 rrv / 11 / V / l \ 0 7 3 3 2 6 14 2 3 0 2 7 3 12 3 CUU 5,6 (92) CUC 14.8 (244) CUA S.1 ( 85) CUG 51.5 (852) / \ / \ / 1. 9 3 10 2 6 5 1 1 3 pc AG ac cccccc \ fr vlf i / \ / 8 0 4 2 4 7 8 1 2 5

AUU AUC AUA AUG / 1 / i / rl ft 1 β 2 2 6 9 2 0 3 2 0 4 7 4 0 5 3 3 1 6 3

_ί rv Λ \ / «ι. 8 1 _χ 6 7 0 4 7 3 3 uupmUG G δ G G 9 7 7 1 2 9 β 2 14 6

ACU ACC ACA ACG GCU GCC GCA GCG / \ / 4V / \ / \ 18 4 4 3 6 4 7 3 1 V /) i / i / 19 3 8 5 0 7 8 β 2 6 3 5 7 9 4 2 1 5 14 158) 89β) 206} 6Q9) / »ν Γι. Λν / V Γ \ / 1- / 1 · / 1/1 (. / 1 9 2 3 5 8 3 2 5 4 6 5 7« ， «'» · «» ♦ «*» 4 7 7 5 4 2 € 4 3 5 β 2 1 2 2 2 1 4 1 3 UAICI i 15 0 1 8 8 2 2 2 14 0 9 2 5 8 6 0 0 3 14 CGU 9.4 ( 155) CGC 33.8 (559) CGA 7.1 (118) CGG 26.7 (441) AGU 2.8 (46) AGC 14.5 (240) AGA 1.1 (19) AGG 3.8 (€ 2)

222) GGU 754) < 3GC 273) GGA 541) GGG

Ji / \ / \ / \ 9 6 3 7 6 2 7 6 1 4 1 0 4 0 6 8 0 2 7 2 7 12 The password is GC64.82. /. The first letter is GC65.3 6%, the second letter is GC48.07%, and the third letter is GC81.04%. Table 2F: Codon usage table of Shigella parasiticus (from www.kazusa.or.jp/codon/ ) 〇 Shigella parasitic dysentery [gbbet]: 706 CDS4 (180312 codons) -103-98561.doc 200530399 Range: [Triad] [Frequency: per thousand] ([Number]) 8 18 4 5 5 0 3 2 12 1 i

465a) UCU 2714) UCC 3756) UCA 2424) UCG 16.6 (2986) 9, S (1717) 15.6 (2821) 6.9 (1241) 5 2 2 1 4 9 6 9 9 9 3 3 1 9 0 0 5 II 2 Dii1

UGU UGC t / GA UGG 9 6 4 1-* _ «» 6 5 13 1 1252) 1011) 254) 2357) CUU 17.6 (3169) CUC 10.4 (1878) CUA 7.2 (1295) COG 33.5 (6045)

AUU AUC MJA AUG 7 8 2 8 110 9 4 0 4 1 5 3 3 4 / \ / 1 · / \ / \ 0 7 9 3 »« * »0 6 8 3 3 112 GOT 19.8 {3576) GUC 11.8 (2126 ) GUA 13,1 (2370) GUG 16.1 (2910)

ecuccc CCA CCQ ACU ACC ACA ACG GOT GCC GCA GCG

/ 1 / V / V / * \ 2 9 7 2 9 5 9 2 X / V rrlr ft ri 8 4 2 0 3 3 6 0 X 1 1 1/1 / V / \ / \ 6 5 2 2 9 8 2 5 112 1 2 2 2 2 1 \ / 11 / V / V »\ / \ / V / V / 6 2 4 0 ^ 0309 5 7 4 9 8 13 0 6 0 7 1 4 4 9 8 3527) 3338) 4009 ) 2732) CAUCACCAACAGIAACAAAI ΪΙ 12 9 6 5 8 5 3 1 12/1 Α \ / 1/1 5 S 6 4 + * «· 3 8 16 ^ 1 4 1 / V / 1- / \ / 1 0 3 5 7 4 6 7 1 3 13 2 2725) CGU 15.0 (2472} CCC 12.6 (2861} CGA 5.8 (4255) COG 9.0 (796 7 0 6 4 2 7 2 0 6 2 2 11

6044} AGU 3348) AGC 7507} AQA 2961) AGO 6123) GGU 2939) GGC 6763} GGA 3913) GGG

3 7 3 7 5 2 0 5 1 1 X 2764) 2281) 1865) 1029)

2 3 19 9 S 5 ο 1 1 1 X 8 4 7 0 6 5 2 7 4 7 7 9 3 2 2 1 Password GC44.63% First letter GC51.72% Second letter GC38.85% Third GC43.32% In some embodiments of the present invention, at least about 10%, at least about 25%, at least about 50%, or at least about 75% of the codons in the codon-optimized codon sequence, for The amino acid used in the target organism is the best codon. In other specific embodiments, 100% of the codons in the codon-optimized codon sequence are optimal codons for the amino acid in the target organism (i.e. the sequence is `` complete Codon-optimized. For example, in the examples shown below, all of the codons characterized by the codon-optimized sequence are the codons most commonly used by the target organism; however, any of these results can be Codon substitutions of codons that are more commonly used than the original (natural) sequence are considered π codons-optimized π. Table 3 below shows the optimal code for each amino acid in Listeria monocytogenes Utilization. -104- 98561.doc 200530399 Released in rabbits with mononuclear cells

In some embodiments, the codon-optimized polynucleotide encodes a signal peptide. In some embodiments, the signal peptide is foreign to the bacteria for which the sequence is codon-optimized. In other embodiments, the # peptide is natural to bacteria for which the sequence is codon-optimized. For example, in some embodiments, the codon-optimized polynucleotide encodes a signal peptide selected from Listeria monocytogenes, a Usp45 signal peptide obtained from Lactococcus lactis, and a signal peptide obtained from Bacillus anthracis Protective antigen signal peptide, a p60 signal peptide derived from Listeria monocytogenes, and a signal peptide derived from a group consisting of a PhoD signal peptide derived from Bacillus subtilis. In 98561.doc -105- 200530399, the codon_optimized polynuclear picric acid encodes a signal peptide other than the protective antigen signal peptide of Bacillus anthracis. In some specific embodiments, the polynucleotide codon_ encoding the signal peptide is optimized for expression in the monocytosis Lister g. In some embodiments, the codon. Optimized polynucleotide-encoded (non-signal peptide) protein has been optimized for the codon-optimized fine g foreign. In some embodiments, the codon-optimized polynucleotide encodes a polypeptide comprising an antigen. For example, in the specific example, the codon-optimized polynucleotide-encoded polypeptide includes an antigen, which is an antigen associated with a tumor or an antigen derived from an antigen associated with a tumor. In some specific embodiments, the codon-optimization effect of polynucleic acids encoding signal peptides and / or other polypeptides is improved compared to the corresponding polynucleus without codon-optimization, which includes the signal peptide and / Or other polypeptides (such as fusion proteins, protein chimeras, and / or foreign polypeptides encoded by recombinant nucleic acid molecules, expression cassettes, and / or expression vectors) in bacteria. In some embodiments, the codon-optimization effect of the multinucleate pupae improves performance by at least about 2 times, at least about 5 times, at least about 10 times, or at least about 20 "(compared with no codon-optimized Corresponding polynucleotide). In a specific embodiment, the coding-optimization effect of the polynuclear picric acid encoding the signal peptide and / or other polypeptides is compared to the corresponding multinucleus without codon-optimization. Homoacids, which increase the secretion of polypeptides (such as fusion proteins, protein disrupters, and / or foreign polypeptides) including the signal peptide and / or other polypeptides in bacteria. In some specific implementations, the codons of this multinucleated peptide are most Optimization effect, increase 98561.doc 200530399 at least about 2 times, at least about 5 times, at least about 10 times or at least about 20 times (relative to the codon-free optimization of the corresponding polynucleotide in the two specific implementation In the example, both the performance and the degree of secretion are improved. Standard techniques in this technique can be used, such as Western blots of various related bacterial cultures, to quickly assess the degree of performance and / or secretion. V I. Performance Cards! Performance cassettes are also provided by the present invention. For example, in some embodiments, the present invention provides performance cassettes including any of the recombinant nucleic acid molecules described herein, and further includes- A promoter gene linked to a codon sequence in the recombinant nucleic acid molecule (eg, a first polynucleotide encoding a signal and a second polynucleotide encoding another polypeptide). In some embodiments, this expression Card E is isolated. In some other specific embodiments, the performance card is contained in a human expression vector, which may be isolated or incorporated into bacteria. In a further specific embodiment, the performance card The E line is located in the chromosomal DNA of the bacteria. For example, in some specific embodiments, the performance card has been integrated into the genome of the bacteria: in one body; in the example, it is integrated into the genome of the bacteria The performance card includes-or more elements derived from genes, DNA. For example, in some specific embodiments; the word "recombinant nucleic acid molecule" is inserted into a certain position of the genome of the bacteria, such as By the integration of the specified position or the same kind of recombination), so that the recombinant nucleic acid molecule can be operatively linked with the starter gene that has already appeared in the genome ship ', thereby generating a new performance cassette integrated into the genome with A in- In other specific embodiments, the performance card is integrated into the genome 98561.doc 200530399 DNA (for example, integration through a specified location or homologous recombination) in a complete unit including both the promoter gene and the heavy and nuclear knife. Role). In some specific embodiments, the performance cassette is designed to express the polypeptide in bacteria. In some embodiments, the performance cassette is designed to express heterologous polypeptides, such as heterologous antigens, in bacteria. In some embodiments, the performance cassette provides improved performance and / or secretion of the polypeptide. Performance cassettes typically include the following sequence of elements: 0) a promoter gene, and (2) a polynucleotide encoding a polypeptide. In some embodiments, the performance cassette includes the following elements: (1) a promoter gene; (2) a polynucleotide encoding a signal peptide, and (3) a polynucleotide encoding a polypeptide (e.g., a heterologous protein). In other specific embodiments, the performance cassette includes the following elements: ("Prokaryotic start ℴ (2) Shine_Dalgarn 0 sequence 歹 4; (3) encoding a signal-like polynucleotide, and (4) A polynucleotide encoding a polypeptide (eg, a heterologous protein). In some embodiments, the performance cassette includes more than one promoter gene. In some embodiments, the performance card may also contain a relative to the heterologous Peptide, a transcription termination sequence inserted downstream of the terminal of the translation stop codon. For example, in some embodiments, a transcription termination sequence can be used in a construct for stable integration into a bacterial chromosome. Although not required, transcription The acceptor of the termination sequence becomes the last element to be seen in the heterogeneous gene expression card E to prevent the polar influence on the regulation of the expression of adjacent genes due to read transcription. Therefore, in some specific facts, it is possible to ^ § sequence elements known to the artisan to promote transcriptional termination that are dependent on or add to a heterogeneous protein expression cassette. Aspects of the present invention include the following performance cards E : ⑷ encodes the first polynucleotide of signal signal 98561.doc 200530399, Fuzhongduo # 舻-〃 in order to express in bacteria, the first nuclear nucleus is optimized. R亘 ”the second polynucleotide of the… code peptide, the first polynucleoside in BingTian County, Ganbiao 疋, and a polynucleotide-reading architecture; and 可 mowable m — Zeng Zhuanze The ligated starter gene was made to make the Fengfeng plant, Ganyuwen the polypeptide fusion protein / encoding including the signal and the two, the invention provides expression kaku, which includes the first encoding of the 细菌 ... A polynucleotide, in order to show up in bacteria: the first polynucleotide codons rhyme; (b) the brother of the encoding polypeptide-nucleoside acid, wherein the second polynucleoside is The first multinucleus translation and editing framework; and the promoter gene linked to the first and second polynucleotides of the expression cassette in an operable manner, wherein the beta recombination nuclear molecule coding includes the signal And polymorphic fusion proteins. In some embodiments, the second polynucleotide The coded polypeptide is a species of lLβ ^^^ ^-for this signal peptide. In some specific embodiments, the second polynucleic acid acts on the first mimicry & _ β cedarin & L 疋 heterogeneous. In some embodiments, the polypeptide is heterologous to the bacterium, while the signal peptide is natural to it (ie, foreign to the pupae). In some specific embodiments The bacteria from which the signal peptide is derived are intracellular bacteria. In some embodiments, the bacteria is selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shiga A group consisting of Bacillus, Brucella, Mycobacterium, and E. coli. In some embodiments, the bacteria are bacteria of the genus Listeria (eg, Listeria monocytogenes). In some specific examples, the second polynucleoside 98561.doc 200530399 was optimized for performance in bacteria. In another aspect, the present invention provides a performance cassette, wherein the performance cassette includes (a) a first polynucleotide encoding a signal peptide, wherein the first polynucleotide is coded for expression in a Listeria bacterium Sub-optimization, (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, and (c) is operable A promoter gene linked to the first and second polynucleotides of the expression cassette, wherein the recombinant nucleic acid molecule encodes a fusion protein including the signal peptide and the polypeptide. In some embodiments, the performance cassette is a polycistronic performance cassette. In some embodiments, this second polynucleotide codon is optimized for performance in Listeria bacteria. In some embodiments, the polypeptide encoded by the second polynucleotide is foreign to bacteria of the genus Listeria (ie, heterologous to bacteria of the genus Listeria). In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the performance card g includes more than one promoter gene. In another aspect, the present invention is for expression, including the first polynucleotide encoding a non-secAi bacterial "peptide; (b) the same translation and editing framework as the first polynucleotide, encoding the polypeptide A second polynucleotide; and 基因 a promoter gene operably linked to the first and second polynucleotides, allowing the performance card E to encode a fusion protein comprising the signal peptide and the polypeptide. In some specific In the examples, 'for expression in bacteria such as Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacterium ® or E. coli No.-9856l.doc • 110 · 200530399 polynucleotides and / or second polynucleotide codons _ optimized. In some specific embodiments, in order to be in Listeria, such as Listeria monocytogenes Codon-optimized the polynucleotide (s) in some bacteria. In some specific embodiments, the codon-optimized first polynucleotide encodes a codon-encoding peptide. Optimized bacteria are natural In some embodiments, the signal peptide encoded by the first polynucleotide is heterologous to the second polynucleotide. In some embodiments, the polypeptide encoded by the first polynucleotide The signal peptide is heterologous. In some embodiments, the performance cassette is a polycistronic performance cassette. In some embodiments, in order for Listeria bacteria (such as monocytes) Listeria monocytogenes), the first polynucleotide, the second polynucleotide, or both the first polynucleotide and the second polynucleotide codon _ optimized. In some specific embodiments Wherein the first and second polynucleotides are heterologous to each other. In some specific embodiments, the polypeptide and signal peptide encoded by the second polynucleotide are heterologous to each other. In some specific embodiments, The polypeptide encoded by the second polynucleotide is foreign to Listeria bacteria (ie, heterologous to Listeria bacteria). In some embodiments, the performance cassette includes more than one Promoter genes. Ming also provides performance cassettes that include the following (phantom encoding a polynucleotide that is a foreign polypeptide to Listeria spp .; and (1)) operably linked to a polynucleotide encoding the foreign polypeptide Promoter gene. In this specific embodiment, the polypeptide encoded by the expression cassette is an antigen (for example, see the description of some possible antigens above). In some embodiments, the expression cassette further includes an encoding Polynucleotide of signal peptide. The polynucleotide encoding the signal peptide 98561.doc 200530399 is also operably linked to the promoter gene, so that the expression cassette can express the fusion protein including the foreign polypeptide and the signal peptide. Suitable for use Polynucleotides encoding signal peptides in expression cassettes include, but are not limited to, those described above. In some specific embodiments, as described above, for bacteria such as Listeria (such as Listeria monocytogenes) Bacteria), will be included in the performance cassette, the polynucleotide codon encoding the signal peptide-optimization 0 The present invention also provides performance cassettes including the following a) the first polynucleotide encoding a non-Listeria signal peptide; ({3) the second polynucleotide encoding a polypeptide, which is the same translation editing structure as the first polynucleotide And a promoter gene operably linked to both the first and the second multinucleus, its performance card | encodes a fusion protein comprising both the non-Listeria monocytogenes peptide and the polypeptide . In some embodiments, the performance card E is a polycistronic performance card g. In some specific embodiments, in order to be expressed in Listeria (such as Listeria monocytogenes), the first polynuclear acid, the second polynuclear acid, or the first and second: Nucleic acid codon-optimization. In some embodiments, the first and second polyacids are heterogeneous to each other. In some embodiments, the polypeptide and signal peptide composed of the second polynucleic acid are heterologous to each other. In some specific examples, the peptide from the second polynuclear acid is foreign to the bacteria of the 4 Listeria species (i.e., heterologous to the Listeria species). In some embodiments, the expression includes more than one promoter gene. The present invention provides a performance card! Wherein, the performance card e includes (a) the first nucleus encoding the gene 98561.doc -112- 200530399, or a catalytically active fragment or a catalytically active variant thereof: an acid, (b) an encoded polypeptide The second polynuclear acid, of which the first nuclear bud is in translation with the first polynuclear acid, and (C) is operable with the first and second polynuclear acids. In the acid-linked ground-breaking factor, the performance cassette code includes the polypeptide and autolysin encoded by the second multinucleate bitter S-text, or a catalytically active fragment or a catalytically active variant of the protein ensemble In which the protein-inhibitor t β thia peptide is fused with autolysin or a fragment having catalytic activity or a variant having catalytic activity, or inserting the autolysin or a fragment having catalytic activity or a catalytic activity Variant. In some embodiments, the protein chimera is catalytically active like autolysin. In some embodiments, the peptides are heterologous to autolysins. In some embodiments, the bacterial autolysin is derived from an intracellular bacteria (e.g., Listeria). In some specific embodiments, the polypeptide encoded by the second polynucleotide is inserted into the autolysin encoded by the first polynucleotide or a catalytically active fragment or a catalytically active variant thereof, and the performance card The cassette encodes a protein chimera in which the polypeptide is inserted into an autolysin or a catalytically active fragment thereof or a catalytically active variant (that is, the polypeptide is embedded in an autolysin or a catalytically active fragment or a catalytically active fragment thereof). Within the body). In alternate embodiments, the second polynucleotide is located outside of the first polynucleic acid encoding an autolysin or a fragment thereof having catalytic activity or a catalytically active variant 'and the performance The cassette encodes a protein complex in which the polypeptide is fused to autolysin or a catalytically active fragment or a catalytically active variant thereof. In some embodiments, the polypeptide is heterologous to autolysin 98561.doc -113- 200530399. In some embodiments, the first polynucleotide and the second polynucleotide are heterologous to each other. In some embodiments, the autolysin is

SecA2-dependent autolysin. In some embodiments, the autolysin is a peptidoglycan hydrolase (e.g., N. acetamycin or p60). In some specific embodiments, the performance card E further includes a polynucleotide encoding a signal peptide (for example, a signal peptide normally bound to autolysin, or a signal peptide that is heterologous to the signal peptide). For example, in some embodiments, the performance cassette encodes a P60 signal peptide, a p60 protein (or a catalytically active fragment or a catalytically active variant thereof), and is buried in the p6o sequence as a heterologous p6o A protein chimera of a polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is a non-Listeria polypeptide. In another aspect, the present invention provides a performance cassette comprising (a) a first polynucleotide encoding a signal peptide, and (b) a second polynucleotide encoding a secreted protein or a fragment thereof, wherein the second polynucleotide Acid is the same translational editing framework as the first polynucleotide, encoding a heterologous secretory protein or fragment thereof

The third polynucleotide of the polypeptide, the third polynucleotide in # is a translation and editing framework corresponding to the first and second polynucleotides, and is operable with the first, second, and : A promoter gene linked to a third polynucleotide, wherein the recombinant nucleic acid molecule encodes a signal peptide, a polya encoded by the second polynuclear acid, and a protein chimera that secretes a protein or fragment thereof In the ensemble, the polypeptide encoded by the third polynucleic acid is fused to the secreted egg from f or a fragment thereof, or is located within the secreted protein or a fragment thereof. In some specific embodiments, the promoter gene in the performance card E (or the recombinant nucleic acid molecule described in this 98561.doc-114-200530399 text) described herein is a promoter gene of a prokaryote. For example, the prokaryotic promoter may be a promoter of Listeria. In some embodiments, the Listeria promoter is an hly promoter. In some embodiments, the promoter is a dependent promoter (e.g., an actA promoter). In some embodiments, the promoter is a constitutive promoter (e.g., a p60 promoter). In some embodiments, a performance cassette comprising a recombinant nucleic acid molecule described herein includes a hly 'actA or p60 promoter gene operably linked to a polynucleotide of the recombinant nucleic acid molecule. By attempting to use the performance card and the host bacteria in which the performance card will be placed, the skilled artisan will be able to quickly identify additional prokaryotes and / or Listeria promoter genes suitable for use in the performance card. For example, promoter genes for various mycobacteria suitable for use in recombinant expression cassettes in mycobacteria and other bacteria are known. These include the Mycobacterium bovis BCG promoter genes HSP60 and HSP70, and also include mycobactin promoter genes such as Mycobacterium tuberculosis and BCG, α-antigen promoter genes and 45 kDa antigen promoter genes, superoxide dismutase promoters Genes, MBP-70, asd promoters of mycobacterium halide, 14 kDa and 12 kDa antigen promoters of mycobacteria, mycobacteriophage promoters, such as Bxbl, Bxb2, and Bxb3 Promoters such as promoters, L1 and L5 promoters, D29 promoter, and TM4 promoter (see U.S. Patent No. 6,566,121). Promoter genes suitable for use in Bacillus anthracis include, but are not limited to, a pagA promoter, an alpha-amylase promoter (pamy), and

Pntr (see, for example, Gat et al., Infect. Immun., 71: 801-13 98561.doc -115- 200530399 (2003)). Promoters suitable for use in recombinant Salmonella expression cassettes and vaccines are known and include the nirB promoter, osmC promoter, P (pagC), and P (tac) (see, eg, Bumann, Infect. Immun ·, 69: 7493-500 (2001); Wang et al., Vaccine, 17: 1-12 (1999); McSorley et al., Infect. Immun 65: 1 71-8 (1997)). Various coliform promoter genes are also known to those skilled in the art. In some embodiments, the promoter genes used in the performance cassettes described herein are the constituent promoter genes. In other specific embodiments, the promoter genes used in the performance cassettes described herein are inducible promoter genes. Molecules (e.g., proteins) that are endogenous to the bacteria in which the expression cassette is to be used can induce inducible promoter genes. Alternatively, molecules that are heterologous to the bacteria in which the expression cassette is to be used (such as small molecules or proteins) can be used to induce an inducible starter gene. Various inducible promoter genes are well known to those skilled in the art. In some specific embodiments of the expression cassette, the 3'-end of the promoter gene is a poly-purine Shine-Dalgarno sequence, which makes the 30S ribosomal subunit (via 16S rRNA) coincide with the heterologous gene RNA transcript and starts Required components for rendering. The Shine-Dalgarno sequence usually has the following consensus sequence: 5'-NAGGAGGU-N5-10-AUG (start codon)-3 '(sequence number 85; sequence number 125-129). There are variants of the polypurine Shine-Dalgarno sequence. It is worth noting that the Listeria hly gene encoding Listerialysin O (LLO) has the following Shine-Dalgarno sequence: AAGGAGAGTGAAACCCATGf Sequence No. 70) (the Shine-Dalgarno sequence is underlined, and Bold indicates the translation start password 98561.doc • 116- 200530399). The construction of expression cassettes for use in bacteria, even the construction of expression cassettes specifically for use in recombinant bacterial vaccines, is known in the art. For example, instructions for the production and use of various bacterial expression cassettes and / or recombinant bacterial vaccines can be found in the following references, all incorporated herein by reference. Horwitz et al., Proc. Natl. Acad. Sci USA, 97: 13853-8 (2000); Garmory et al., J. Drug Target, 11: 471-9 (2003); Kang et al., FEMS Immunol Med. Microbiol., 37: 99-104 (2003) Garmory et al., Vaccine, 21: 3051-7 (2003); Kang et al., Infect. Immun., 1739-49 (2002); Russman et al., J. Immunol., 167: 357-65 (2001); Harth et al., Microbiology, 150: 2143-51 (2004); Varaldo et al., Infect. Immun., 72: 3336-43 (2004); Goonetilleke et al., J. Immunol., 171: 1602-9 (2003) Uno-Furuta et al., Vaccine, 21: 3149-56 (2003); Biet et al., Infect. Immun ·, 71: 2933-7 (2003); Bao et al., Infect. Immun ·, 71: 1656-61 (2003); Kawahara et al., Clin. Immunol., 105: 326-31 (2002); Anderson et al., Vaccine, 18: 2193-202 (2000); Bumann, Infect. Immun., 69: 7493-500 ( 2001); Wang et al., Vaccine, 17: 1-12 (1999); McSorley Human, Infect. Immun., 65: 171-8 (1997); Gat et al., Infect. Immun., 71: 801-13 (2003); US Patent No. 5,504,005; US Patent No. 5,830,702; US Patent No. 6,051 No. 237; U.S. Patent Publication No. 2002/0025323; U.S. Patent Publication No. 2003/0202985; WO 04/062597; U.S. Patent No. 6,566,121; and U.S. Patent No. 6,270,776. 98561.doc -117- 200530399 In some specific embodiments, it is desired to construct a performance cassette that uses the bicistronic and polycistronic (also known as polycistronic) performance of heterogeneous cipher sequences. Such performance cassettes can be utilized, for example, a single promoter gene operably linked to two or more independent password sequences. These code sequences may, for example, correspond to individual genes, or to the desired and / or selected sub-fragments of the entire designed gene. In the latter case, the gene may contain a sequence encoding a water-repellent penetrating membrane functional site, which has the potential to inhibit efficient secretion from Listeria. Therefore, you may want to divide the gene's code sequence into two sub-fragments from this water-repellent functional site; in this case, then express these two sub-fragments with bicistronic information. To take advantage of polycistronic performance, it takes 30s ribosomal subunits to retain polycistronic RNA information, followed by the termination of translation of the first codon sequence, and releases 50s ribosomal subunits, and then "read 1NA Information, to the next start codon, during which the 50s ribosome subunit is combined with the RNA_bound 30s ribosome subunit, and the translation function is initiated again.

Listeria monocytogenes, like other bacteria, takes advantage of its basic: polycistronic performance of group programs. For example, a sequence of the interstitial region of Listeria monocytogenes obtained from selected polycistronic information can be used to construct a polycistronic performance card E to represent selections from recombinant Listeria species Heterogeneous protein. For example, from the information of polycistronic, it appears from several prM reproductive agents of Listeria monocytogenes. For example, "Actis and PlcB proteins of Listeria monocytogenes are expressed in the form of bicistronic information". In the following, the sequence of Li A 98561.doc 200530399, which conforms to the mononuclear, cytoplasmic Listeria bacterium #acta_plcB intergenic sequence (Π), is shown below:

5f-TAAAAACACAGAGCGAAAGAAAAAGTGAGGTGAA TGA-3 '(Sequence 歹 ij No. 71) (The bold Shine-Dalgarno sequence showing the translation of plcB is shown. The first 3 nucleotides of the sequence are consistent with the Ochre stop codon). For a non-limiting example of a bicistronic expression vector, the bicistronic hEphA2 expression vector used in Listeria monocytogenes, see Example 28 below. Alternatively, other known intergenic or synthetic sequences can be used to construct polycistronic expression cassettes for use in Listeria or other bacteria. The construction of intergenic regions that guide the structure of parenchymal secondary RNA should be prevented, and unwanted transcription termination should be avoided by the rh0_ independence mechanism. It is important to 'if you want to secrete any or all of the translated proteins expressed from polycistronic messages, you must functionally link the signal peptides to each codon, and the specific implementations are different. These signal peptides are different from each other. The performance card g in the 0 genus or other thin halogens is 吝 値 m 疋 xi cistron (such as dicistronic). The two or more peptides' encoded by the bicistronic or polycistronic + zymotically, phylogenetic cassettes, and cassettes become isolated polypeptides. In this case, in the general, π-carcass embodiment, the bicistronic or polycistronic expression cassette includes its inter-gene sequence (eg, genes derived from bicistronic or polycistronic). ), Which is located between the code sequences of the two polypeptides. -In some 1-body embodiments, the intergenic sequence includes a ribosome entry

The sequence that initiated the translation. In an example of a solid yoke with a body ## η A cu., The intergenic sequence includes a Shlne-Dalgarno sequence. In the first example, in the case of the system, the intergenic 98561.doc -119- 200530399 column is the intergenic sequence of the Listeria prokaryotic actA_plcB gene. Generally, the intergenic sequence is located between the polynucleotide sequence encoding the first polypeptide (or the first fusion protein including the first polypeptide and the signal peptide) and the polynucleotide sequence (or the first Between two fusion proteins, including the second 7 peptide and the signal peptide). Accordingly, the present invention-aspect provides a performance cassette comprising the following: (i) a first polynucleic acid encoding a -peptide; (b) a polynucleic acid encoding a second polypeptide; (ii) a sacrifice position at the first and second Intergenic sequences between two polynuclear picric acids; and the expression card in a promoter gene operably linked to the first and second polynucleic acids, encoding the first and the second in the form of two separate polypeptides. The second peptide. In some embodiments, the first and second polynuclear are polynucleotides selected from any of the polypeptides described herein (e.g., in Chapter IV above). In some embodiments, at least one of the first and second nucleoside zepirin includes an antigen. In some embodiments, the first and second polynuclear acids respectively (different or identical) fragments of the same antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. The present invention further provides the following performance card E: ⑷ encoding the first polynucleotide of the first signal peptide; (b) encoding the second oligonucleotide r ′ 纟 of the first (non-signaling) polymorphism. The second polynuclear acid is the same translation editing structure as the first polynuclear acid; ⑷ a third polynuclear acid encoding a second signal peptide; ⑷ encodes a second (non-signaling) polypeptide The fourth polynuclear acid, where the fourth polynuclear acid is the same translation editing structure as the third polynuclear acid; the niche is between the second polynuclear acid and the third polynuclear acid 98561.doc -120- 200530399; and a promoter gene operably linked to the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide such that The performance cassette encodes both a first fusion protein including a first signal peptide and a first polypeptide, and a second fusion protein including a second signal peptide and a second polypeptide. In some embodiments, in order to be expressed in bacteria, one or more polynucleotide codons encoding a signal peptide are optimized. In some embodiments, in order to be expressed in bacteria, the third and / or fourth polynucleotide codons are optimized (preferably codons other than the polynucleotide encoding the signal peptide are optimized) outer). In some embodiments, the first and / or second signal peptide is a non-bacterial signal peptide. In some embodiments, the intergenic sequence is a Listeria monocytogenes actA-plcB intergenic sequence. In some specific embodiments, the second and third polypeptides are polypeptides selected from any of the polypeptides described herein (e.g., in Chapter IV above). In some specific embodiments, the first and second polypeptides are polypeptides selected from any of the polypeptides described herein (e.g., earlier in Section IV above). In some embodiments, at least one of the first or second polypeptides includes an antigen. In some embodiments, the first and first polynucleotides each include a fragment of the same antigen. In some embodiments, the antigen is a tumor-associated antigen or derived from a tumor-associated antigen. For example, to express heterologous polypeptides in the Listeria genus, the present invention provides a polycistronic performance cassette, wherein the performance cassette encodes at least two isolated non-Listeria polypeptides. In some specific embodiments, the polycistronic expression card ^ biscistronic expression card E encodes two separate non-Liss 98561.doc -121-200530399 aerobacillus polypeptides. In some specific embodiments, the performance cassette includes the following: (a) a first polynucleotide encoding a first non-Listeria polypeptide; (b) encoding a second non-Listeria A second polynucleotide of the polypeptide; (c) an intergenic sequence located between the first and second polynucleotides; and (a sentence operably linked to the first and second polynucleotides Promoter gene; wherein the expression cassette encodes the first and second polypeptides in two isolated polypeptides. If the expression cassette encodes three polypeptides in the cistronic expression cassette, the expression cassette The performance cassette will include a third polynucleotide operable to initiate the gene and a second intergenic sequence linkage between the second and third polynucleotides. In some embodiments, at least A non-Listeria spp. Polypeptide includes an antigen. In some embodiments, at least two non-Listeria spp. Polypeptides include fragments of the same antigen, respectively. In some embodiments, the performance cassette includes the following : (A) encoding the first signal peptide The first polynucleotide; (b) the second polynucleic acid encoding the first (non-signaling) non-Listeria spp. Peptide; wherein the second polynucleic acid is related to the first polynuclear acid ㈣the same translation and editing framework; ⑷the third polynucleotide encoding the first signal peptide; (d) the fourth polynucleotide encoding the second (non-signaling) non-Listerial polypeptide, The fourth polynucleotide is the same translation editing structure as the third polynucleotide; the intergenic sequence is niche between the second polynucleotide and the third polynucleotide; and (0 A promoter gene operably linked to the first polynucleotide, the second polynucleotide, the third polynucleotide #acid, and the fourth polyacid, enabling the Oral Performance Card E to include the first Signal peptide and the first polymorphic 98561.doc -122-200530399 The first fusion protein is ... λ-A and the younger brother including 4 signal peptides and a second polypeptide-a fusion protein-b A. In some specific embodiments, at least one non-Lisztia genus polymorph. Organic origin. In some specific embodiments, to At least two non-Listeria polypeptides are fragments of the same antigen, respectively. The present invention also provides any of the performance cards E described in this document, which produce heavy "and" "field fungus (such as recombinant Listeria bacteria ). In some embodiments, the 'method of manufacturing recombinant bacteria using the performance card E described herein' includes introducing the performance card into the bacteria. In some embodiments, the performance card! Integrated into the genome of the bacteria. In some other specific embodiments, the performance card is located on the plastid that is incorporated into the bacteria. In some embodiments, the performance card is 'E' by co-consumption Incorporation into bacteria. The introduction of performance cassettes into bacteria can be accomplished by any standard technique known in the art. For example, by conjugation, transduction (metastatic infection) or transformation, Introduction of expression cassettes into bacteria 0 VII. Vectors The present invention further provides vectors, such as expression vectors, which include any of the expression cassettes and / or recombinant nucleic acid molecules described herein. In some embodiments, the carrier is a plastid. In some embodiments, the carrier is linear. In some embodiments, the vector is circular. In some specific implementation examples, the 'remote vector' is an integrated or homologous recombinant vector. In some embodiments, the vector is pAM401. In some embodiments, the vector is pPL2. In some embodiments, the vector is isolated. As indicated above, in some specific embodiments, the performance card described in this article 98561.doc • 123 · 200530399 is incorporated into the performance vector. In some embodiments, the carrier is a plastid. In other embodiments, the carrier is linear. In alternate implementations, use this performance • Insert the performance card into the ϋ genomic DNA. In some specific embodiments, the telepresence carrier is linear. In other embodiments, the performance carrier is circular. Expression vectors suitable for use in bacteria 'such as Listeria are known to those skilled in the art. In order to assemble the performance card E ', there are various suitable carriers suitable as the main chain of the plastid construct. Select a special plastid construct backbone based on whether you want to express polynuclear * acids (ie, polynucleotides encoding heterologous antigens) from bacterial chromosomes or from staining: episomes. In some specific embodiments, an integration vector for the expression card E of Listeria phage ligase (ListedOphage) ligase integrated into a Listeria chromosome is contained in a specific sequence containing a catalytic vector, and the expression card K ( And / or recombinant nucleic acid molecules) and human monocyte multiplying Listeria g (Listeria) within the bacterial chromosome. For example, an integration vector called pPLw0pPL2 plans for the stable single-copy integration of heterologous protein shells in a harmless region of the pupae genome, and has been described in the literature (Lauer et al., 2002 J. Bactedol 184: 4177-4178; U.S. Patent Publication No. 20030023472). The integration vector stably becomes a plastid in E. coli, and is introduced into a desired Listeria spp. Via a conjugation. Each vector lacks the Liszt & genus of origin of replication and encodes a bacterial zygote ligase, making the vector stable only when integrated into the phage attachment site of the chromosome. Starting from the desired plastid construct, the process of producing a recombinant Listeria strain expressing the desired protein (s) 98561.doc -124- 200530399 took about i weeks. 1 and integration vectors are based on U1 53 and Listeria PSA phages, respectively. The pPL1 vector is integrated into the open editing framework of the comK gene, and ppL2 is integrated into the tRNAArg motif. In this way, the natural sequence of the gene is restored when it is successfully integrated, so it retains its natural expression function intact. The pPL1 * ppL2 integration vector contains multiple selection site sequences to facilitate the construction of plastids containing recombinant nucleic acid molecules or expression cassettes, such as heterologous protein expression cassettes. Specific examples of using the pPL2 integration vector are described in Examples 2 and 3 below. Alternatively, the expression cassette (and / or recombinant nucleic acid molecule) can be incorporated into the chromosomes of the Listeria spp. Via dual gene exchange methods known to those skilled in the art. In particular, a composition in which a gene encoding an antibiotic resistance protein is not to be incorporated as a part of a construct containing caliper is desired, and a method of dual gene replacement is desired. For example, the pKSV7 vector (CamiUi et al. Mier * Mier * obiol «(1993) 8, 143-157), which contains a temperature-sensitive Listeria gram-positive origin of replication, is used at unacceptable temperatures Let's choose the recombinant pure germline 'which represents the pKSV7 plastid recombined into the chromosome of Listeria. The pKSV7 dual gene exchange plastid vector contains multiple selection site sequences to facilitate the construction of plastids containing protein expression cassettes and aerobicin resistance genes. For insertion into the chromosome of Listeria, the expression cassette construct can be flanked by a chromosomal DNA sequence of approximately 1 kb, which is equivalent to the precise location where integration is desired. The pKSV7-Performance Cassette Protoplast can be incorporated into the desired bacterial strain by electropenetration according to the standard method of electropenetration of Gram-positive bacteria. A non-limiting example of performing a dual gene exchange method using the 98561.doc 200530399 PKSV7 vector is provided in Example 16 below. In other implementations, you may want to express peptides (including fusion proteins including peptides) from stable plastid episomes. By maintaining plastid episomes through multiple generations, a common expression of protein is required, which gives plastid-containing bacteria a selection advantage. As a non-limiting example, the protein f that is co-expressed with the polypeptide from the plastid may be an antibiotic-resistant protein, such as chloramphenicol, or may be a bacterial protein (in a field-type bacterium, from the chromosome) Performance), which can also give selection advantages. Non-limiting examples of bacterial egg quality include enzymes required for throat or amino acid biosynthesis (choose to use a defined medium lacking the relevant amino acid or other necessary precursor macromolecules) 'or appear in vitro or in Transcription factors required for in vivo donation and selection of dominant genes (Gunn et al. 2_J · 1 Sui delete 1. 167: 647 ^ 6479). As a non-limiting example, pAM is a plastid suitable for selecting epitope expression of polypeptides in a variety of Gram-positive cells (Wirth et al., M% Plant Bactenol 165: 83 1-836). For more explanation of typical uses of pAM401, see Examples 3 and 13 below. For example, the expression card g can be incorporated into the bacterial chromosome of Bacillus anthracis using an integration vector containing the expression caliper of phage ligase, which ligase catalyzes the integration of a specific sequence of the vector into the chromosome of Bacillus anthracis. Conjugating enzymes and attachment sites of P. anthracis can be used to derive conjugation vectors and incorporate key antigen expression cassettes into the vaccine composition. As a non-limiting example, a ligase and attachment site derived from the mild bacteriophage anthracis was used to derive an anthrax-specific integration vector (McCloy'EW · 1951. Study on the lysogenous Bacillus strain. Bacillus-specific phage 98561.doc -126- 200530399 (Studies on a lysogenic Bacillus stain. I. A bacteriophage specific for Bacillus anthracis ·) J. Hyg. 49: 114.125). Alternatively, antigen expression cassettes can be incorporated into the chromosome of Bacillus anthracis via dual gene exchange methods known to those skilled in the art. See, e.g., Infect. Imnum. 71: 801-13 (2003). In particular, a composition in which a gene encoding an antibiotic resistance protein is not intended to be part of a construct containing a cassette of expression is desired, and a method of dual gene exchange is desirable. For example, the PKSV7 vector (Camilli et al. Mo1 Microbi01 1993 8, M3-157), which contains a temperature-sensitive Listeria-derived gram-positive origin of replication, is used at non-permissive temperatures Recombinant pure germlines were selected to represent PKSV7 plastids recombined into the bacterial chromosome. 1) The £ 8 ¥ 7 dual gene exchange plastid vector contains multiple selection site sequences to make it easier to construct plastids containing expression cassettes and aerobicin resistance genes. Inserted into the chromosome of C. charcoal, the expression cassette construct can be located on the side of the chromosomal DNA sequence of approximately 1 kb, which is equivalent to the precise position to be integrated. The PKSV7-expression cassette can be introduced into a desired bacterial strain according to a standard method of electrical penetration of Gram-positive bacteria by electropenetration. Non-limiting examples of methods for performing dual gene exchange in Bacillus anthracis are provided in U.S. Patent Application No. 1 () / 883,599, all of which are incorporated herein by reference. For Xiao Xiao, the dual gene exchange using the PKSV7 vector can be used in the anthracnose strain to add the desired antigen expression caliper to any desired place in the bacterial chromosome. The described dual gene exchange method using PKSV7 can be widely applied to Gram-positive cells. In addition, various expression vectors useful in bacteria, 98561.doc -127- 200530399 include recombinant bacterial vectors, and are known to those skilled in the art. Examples include those vectors described in the following documents, each fully incorporated herein by reference: Horwitz et al., Proc. Natl. Acad. Sci. USA, 97: 13853 · 8 (2000); Garmory et al., J Drug Target, 11: 471-9 (2003); Kang et al., FEMS Immunol. Med. Microbiol., 37: 99-104 (2003); Garmory et al., Vaccine, 21: 3051-7 (2003); Kang Et al., Infect · Immun ·, 1739-49 (2002); Russman et al., J. Immunol ·, 167: 357-65 (2001); Harth et al., Microbiology, 150: 2143-51 (2004); Varaldo et al. Human, Infect. Immun., 72: 3336-43 (2004); Goonetilleke et al., J. Immunol., 171: 1602-9 (2003); Uno_Furuta et al., Vaccine, 21: 3149-56 (2003); Biet Et al., Infect · Immun ·, 71: 2933-7 (2003); Bao et al., Infect. Immun ·, 71: 1656-61 (2003); Kawahara et al., Clin. Immunol., 105: 326-31 ( 2002); Anderson et al., Vaccine, 18: 2193-202 (2000); Bumann et al., Infect. Immun., 69: 7493-500 (2001); Wang et al., Vaccine, 17: 1-12 (1999) McSorley et al., Infect. Immun., 65: 171-8 (1997); Gat et al. Infect. Immun., 71: 801-13 (2003); US Patent No. 5,504,005; US Patent No. 5,830,702; US Patent No. 6,051,237; US Patent Publication No. 2002/0025323; US Patent Publication No. 2003/0202985; WO 04/062597; US Patent No. 6,566,121; and US Patent No. 6,270,776. The invention further provides methods for producing recombinant bacteria (e.g., recombinant Listeria bacteria) using any of the expression vectors described herein. In some specific 98561.doc-128-200530399 Beta examples, the method of using the performance cassette described herein to make a recombinant bacterium includes introducing the performance card e into the bacteria. VIII. Bacteria and other host cells The present invention further provides recombinant nucleic acid molecules, expression cards, and / or vectors described herein (see, e.g., I, Pi, and Tozao, of the Summary and Embodiments above, And specific examples below). In some embodiments, the cells are prokaryotic. In some embodiments, the cell is eukaryotic. In some embodiments, the cells are mammalian. In some embodiments, the cells are antigen-providing cells, such as dendritic cells. In some embodiments, the cell is a bacterial cell. In some embodiments, the host cell is isolated. For example, the invention includes recombinant nucleic acid molecules, performance cassettes, and / or vectors described herein (see, for example, Chapters I, II, VI, and vπ of the Summary and Embodiments above, and specifics below). Example) of bacteria. Bacteria comprising these polynucleotides may be alternately referred to herein as, recombinant bacteria, and bacteria including the recombinant nucleic acid molecules, cassettes and / or vectors described herein may be alternately used herein Called "recombinant bacteria". In some embodiments, bacteria including recombinant nucleic acid molecules, expression cassettes and / or expression vectors are isolated. In some embodiments, a recombinant bacterium comprising a recombinant nucleic acid molecule, a performance cassette and / or a performance vector expresses a polypeptide or fusion protein encoded by the recombinant nucleic acid molecule contained therein, the performance card E and / or the performance vector. In some specific embodiments, the recombinant bacterium secretes a 98561.doc -129- 200530399 polypeptide or fusion protein encoded by a recombinant nucleic acid molecule contained therein, a performance card and / or a performance vector. In some embodiments, when the bacterium (or a composition comprising the bacterium) is administered to a host (eg, a human individual), the recombinant bacterium expresses and secretes a polypeptide and / or in an amount sufficient to generate an immune response in the host. Fusion protein. + In some embodiments, the bacteria is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, intracellular bacteria, and mycobacteria. In some embodiments, the bacteria are Gram-positive bacteria. In some specific embodiments of the present invention, the bacteria are intracellular bacteria (such as facultative intracellular bacteria). In some specific embodiments, the fines belong to the genus Listeria. In other specific implementations {Putida is a member of the Listeria monocytogenes species. In some other specific embodiments, the bacterium is a member of Listeria ivanovii, Listeria or Lisseehgen. In some embodiments, the bacterium is a member of the genus Tobacco. In another specific embodiment, the bacterium is Bacillus anthracis.纟 In another embodiment, the bacterium is Yersinia pestis. In other specific embodiments of the invention, the bacterium is Salmonella typhimurium. In some embodiments, the bacterial genus is at ... For example, in some embodiments ' the bacterium is a M. paralytica. In some embodiments, the bacterium is a member of the genus Brucella. In alternate embodiments, the bacterium is a Mycobacterium. Mycobacterium can be a member of the Mycobacterium tuberculosis species as needed. In some specific embodiments I ', the bacteria is a BCG m-body embodiment, the bacteria is a coliform such as E. coli which has been modified to express Listerialysin O (LLO). Therefore, in some specific embodiments, the bacterial line including the recombinant nucleic acid molecule, expression cassette and / or vector described herein in 98561.doc 200530399 is selected from the group consisting of Listeria, Anthracis, Yersinia pestis, A group of Salmonella and Mycobacterium. In some other specific embodiments, the bacterium that includes the recombinant nucleic acid molecules, expression cassettes, and / or vectors described herein is selected from the group consisting of Listeria, Anthracis, Yersinia pestis, and Salmonella , Shigella, Brucella, Mycobacterium and E. coli. In some embodiments, the recombinant nucleic acid molecules, expression cassettes, and / or vectors described herein are inserted (eg, see Chapters I, II, VI, and VII of the Summary and Embodiments above, and below Specific examples) to modify the polypeptide to express the polypeptide, and in at least some embodiments, the polypeptide-secreting bacteria are field-type Listeria bacteria, such as Listeria monocytogenes bacteria, including recombinant Nucleic acid molecules, performance calipers and / or vectors. However, in some embodiments of the present invention, the bacteria including the cassette and / or the carrier are mutant strains of bacteria. In some embodiments, this is attenuated. In some embodiments, the bacteria is an attenuated mutant strain of bacteria. In this article, the dog 'wheat species in which the gene "xyz " has been deleted is referred to as Axyz or xyz- or χντ: Λ1 私 织 炎 r ...

..., the eight major double ticks strains sometimes mean mutants or strains in which the XyZ gene has been deleted. , Sometimes will mean

The change can be any type of mutation. A mutation in the halberd mutant bacteria. For example, the mutation may be a point mutation, 98561.doc -131- 200530399 frameshift mutation 'insertion, deletion of some or all genes. In addition, in some embodiments of the modified strain, the bacterial base of the part has been replaced with a heteropolynuclear acid. In some embodiments, the mutation is naturally occurring. In other specific embodiments, the mutation is the result of human mutation stress. In another specific embodiment, the mutation in the bacterial genome is the result of genetic engineering. In some embodiments, for cell-to-cell transmission, entry into non-phagocytic cells, or proliferation (as opposed to field-type bacteria), the recombinant nucleic acid molecules, expression cards, and / or vectors described herein will be included Bacterial attenuation. Bacteria can be attenuated through canine changes or other modifications. In some embodiments, for cell-to-cell transmission (such as Listeria monocytogenes actA mutants), see any of the recombinant nucleic acid molecular tables described in this article for a card! And / or attenuating the bacteria expressing the vector. In some embodiments, for entry into non-phagocytic cells (eg, Listeria monocytogenes internalin mutants, such as iniB deletion mutants), any of the recombinant nucleic acids described herein will be included Attenuation of molecules, expression cassettes and / or bacterial expression vectors. In some embodiments, for proliferation, bacteria that include any of the recombinant nucleic acid molecules, expression cards, and / or expression vectors described herein will be attenuated. In some embodiments, the bacteria are attenuated for cell-to-cell transmission and for entry into non-phagocytic cells. In some embodiments, for cell-to-cell transmission, bacterial attenuation including the expression cassette and / or the expression vector described herein will be included. In some embodiments, the bacterium is defective in ActA (as opposed to non-mutant or wild type bacteria) or its equivalent (depending on the organism). In this 98561.doc -132- 200530399 embodiment, the bacterium includes one or more mutations in actA. For example, bacteria (such as Listeria) can be actA deletion mutants. ActA is an actin polymerase encoded by the actA gene (Kocks et al., CeU, 68: 521-531 (1992); Genbank accession number AL591974, nucleotides 9456-113 89). The actin polymerase protein is involved in the recruitment and polymerization of host F_actin in one of the listeria bacteria. The subsequent polymerization and lysis of actin caused Listeria to advance through the cytosol and enter adjacent cells. This mobility enables bacteria to spread directly from cell-to-cell without further exposure to the extracellular environment, thus escaping host defenses such as antibody development. In some specific embodiments, the attenuated Listeria spp. May optionally include mutations in surface protein genes, such as inlB, and in actA. In order to enter non-phagocytic cells, and for cell-to-cell transmission, the Listeria strain of this embodiment of the present invention is attenuated. In some embodiments, the bacteria) reduce the cell-to-cell transmission capacity of attenuated bacteria by at least about 10%, at least about 25%, at least about 50%, at least about 75 °, or at least about 90%. In some embodiments, the ability of cell-to-cell transmission of attenuated bacteria is reduced to approximately 25 relative to non-attenuated; transformed bacteria. /. . In some embodiments, the ability of attenuated bacteria to reduce cell-to-cell transmission is reduced by at least 50% relative to those without attenuating mutations. % _ The in vitro measurement of attenuated bamboo, stalk, and field stalks, such as Listeria bacteria, is well known. For example, 98561.doc • 133-200530399 measures the diameter of plaques formed over time after infection with a monolayer of selected cultured cells. Defective for intracellular growth and cell-to-cell transmission, as previously described in Sun, A., A. Camilli, and DA Portnoy. 1990, the isolation of small-lysoplaque mutants of Listeria monocytogenes (Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread.) Infect. Immun. 58: 3770-3778, as described by Skoble, J., DA Portnoy and MD Welch. 2000. Three regions within ActA promote Arp2 / 3 complex-mediated nucleation of Arp2 / 3 complex-actin actin and Listeria monocytogenes mobility. The description of mediated actin nucleation and Listeria monocytogenes motility.) J. Cell Biol. 150: 527-538 Modify the measurement method to perform plaque assays in L2 cell monolayers. Briefly, L2 cells were grown to confluence on a 6-well tissue culture dish and then infected with bacteria for 1 hour. After infection, cells are covered with a medium warmed to 40 ° C, which includes DME containing 0.8% agar, fetal bovine serum (eg, 2%), and gentamicin at the desired concentration. The concentration of gentamicin in the culture medium dramatically affected the plaque size and was a measure of the ability of selected Listeria strains to affect cell-to-cell transmission (Glomski, I.J., M.M. Gedde, AW Tsang, JA Swanson and DA Portnoy. 2002, J. Cell Biol. 156: 1029-1038). For example, 3 days after infection with a monolayer, when covered with a medium containing gentamicin at a concentration of 50 μg / ml, compared with field-type Listeria, it has a phenotype of cell-to-cell transmission defects. The bacterial plaque size of the Agrobacterium strain was reduced by at least 50%. On the other hand, when the infected monolayer was covered with 98561.doc -134- 200530399 medium + agarose containing only 5 μg / ml gentamicin, Liss had a phenotype of cell-to-cell transmission defect. The lytic plaque size was similar between the Agrobacterium strains and the field-type Listeria spp. Therefore, by selecting the concentration of gentamicin in the agarose-containing medium, it can be judged that the selected strain, relative to the field type Listeria, affects the cell-to-cell transmission in the monolayer of infected cells. Relative ability. If necessary, by adding a medium containing neutral red (GIBC0 BRL; diluted 1: 250 with DME + agarose medium) 48 hours after infection, the coloration and measurement of plaque diameter can be made easier. In addition, plaque assays can be performed in monolayers derived from other primordial or continuous cells. For example, HepG2 cells, a hepatocyte-derived cell line, or primitive human hepatocytes can be used to evaluate the ability of selected Listeria mutants to affect cell-to-cell transmission compared to field-type Listeria. In some embodiments, including Listeria spp.-mutated or other modified Listeria spp. Which attenuates Listeria spp., At high concentrations of gentamicin (approximately 50 μg / ml), it produces "Needle tip" plaque.

In some embodiments, the bacteria including the expression cassettes and / or the expression vectors described herein are mutant bacteria that attenuate nucleic acid repair (as opposed to wild type ' such as bacteria that do not attenuate genetic mutations). For example, in some embodiments, bacteria are defective in at least one DNA repair enzyme (e.g., Listeria monocytogenes uvrAB mutant). In some specific targets, the bacteria are defective in PhrB, UvrA, UvrB, UvrC, UvrD, and / or one of their equivalents (depending on the genus and species of the bacteria). In some embodiments, the bacteria are defective on UvrA, UvrB, and / or uVrC 98561.doc -135- 200530399. In some embodiments, the bacterium includes an attenuating mutation in the phrB, uvrA, uvrB, uvrC, uvrD, and / or recA genes. In some embodiments, the bacterium includes one or more mutations in the uvrA, uvrB, and / or uvrC genes. In some embodiments, the bacteria deletes UvrA, UvrB, and / or UvrC functions. In some embodiments, the bacteria delete functional UvrA and UvrB. In some embodiments, the bacterium is a uvrAB deletion mutant. In some embodiments, the bacterium is an AuvrAB AactA mutant. In some embodiments, the nucleic acid of a bacterium that attenuates nucleic acid repair and / or is defective in at least one DNA repair enzyme is modified by reacting with a nucleic acid-targeted compound (see below). U.S. Patent Publication No. 2004/0197343 describes in detail nucleic acid repair mutants, such as AuvrAB monocytogenes Listeria mutants, and methods of making the mutants (see, e.g., U.S. Patent Publication No. 2004 / Example 7 of 0197343). In some embodiments, the ability of attenuated bacteria to reduce nucleic acid repair is at least about 10%, at least about 25%, at least about 50%, at least about 75% relative to bacteria without attenuating mutations (such as field-type bacteria). Or at least about 90%. In some embodiments, the ability of attenuated bacteria to repair nucleic acids is reduced by at least about 25% relative to bacteria without attenuated mutations. In some embodiments, the ability of attenuated bacteria to repair nucleic acids is reduced by at least about 50% relative to bacteria without attenuated mutations. Specific mutations that occur in bacterial strains can be confirmed by various methods known to those skilled in the art. For example, the relevant part of the line's genome can be selected and sequenced. Alternatively, a pair of primers encoding regions adjacent to the deletion or other 98561.doc -136- 200530399 mutations can be used to confirm specific mutations via PCR. The soil station method can also be used to loosen changes in the bacterial genome. It is also possible to use the standard technique of this technique, > Western blotting method, to analyze whether the particular protein shell is expressed by this strain. It can also be confirmed by comparing the phenotype of the line with the previously reported phenotype 'that the line contains a mutation in the desired gene. For example, it is possible to measure the measurement of the nucleic acid force of a 2 test cell using a nucleotide excision repair (ner) mechanism and compare this ability with a field-type bacterium to assess nucleotide excision and make up for major changes, such as uvrAB deletion The presence. Such functions are known in the art. For example, the cyclobutane dimer excision or υν · induced (64) product excision can be measured to determine the enzyme defect in the mutant species (see, for example, Franklin et al., P. 21 3824 (1984)). Alternatively, a survival assay can be performed to assess the deficiency in nucleic acid repair. For example, the bacteria can be subjected to a psoralen guess A treatment, and then compared to the field type to evaluate its ability to proliferate and / or survive. In some embodiments, the bacteria are attenuated (as opposed to non-mutant or field-type bacteria) in order to enter non-phagocytic cells. In some embodiments, bacteria (e.g., Listeria) are deficient in one or more surface proteins (or equivalents). In some embodiments, the bacteria are defective on surface protein A. In the JSlfe dioctahedron example, the bacterium is deficient in surface protein B. In some embodiments, the bacterium includes a mutation in the tadpole. In some embodiments, the bacterium includes a mutation in in1B. ▲ In some embodiments, the bacterium includes mutations in both magnetic and continuous. In this case, the bacteria deleted functional ActA and surface protein B. In some specific examples, the bacterium is a 98561.doc -137- 200530399 double deletion mutant. In some embodiments, the bacterium is defective in both cognition and surface protein B. In some embodiments, the ability of attenuated bacteria to enter non-phagocytic cells is reduced by at least 10% compared to bacteria without attenuated mutations (such as field-type bacteria). , At least about 25%, at least about 50. / ❶, at least about 75. / 〇 or at least about 90%. In some embodiments, the ability of attenuated bacteria to enter non-phagocytic cells is reduced by at least about 25% relative to bacteria without attenuated mutations. In some embodiments, the ability of attenuated bacteria to enter non-phagocytic cells is reduced by at least about 50% relative to bacteria without attenuated mutations. In some embodiments, the ability of attenuated bacteria to enter non-phagocytic cells is reduced by at least about 75% relative to bacteria without attenuated mutations. In some specific examples, the attenuated bacteria, such as the Listeria strain, may be attenuated in order to enter more than one type of non-phagocytic cells. For example, attenuated drugs can be used to attenuate liver cells, but not to enter epithelial cells. As another example, the attenuated drug line can be attenuated for access to epithelial cells, but not liver cells. It is also known that attenuating bacteria that are specially modified in order to enter non-phagocytic cells is the result of mutations in designed genes, such as deletion of mutations, encoding invasive proteins called proteins, which react with special cell receptors and become Helps infect non-phagocytic cells. For example, & enter attenuated Listeria 4AinlB mutant strains into hepatocellular growth factor receptor (non-witch cells, including hepatocyte strains such as HepG2) and primitive human liver cells. f In some specific examples, although the larvae (such as the mutant Listeria s major Dante) are reduced in order to enter the non-swallowing cells, the Listeria can still be 98561.doc 200530399 Uptake by phagocytes, such as at least dendritic cells and / or macrophages. In a specific embodiment, the ability of attenuated bacteria to enter the phagocytic cells was not reduced by modification of the strain, such as modification of invasive proteins (ie, about 95% or higher was still measured after modification The strain's ability to be taken up by phagocytes). In other embodiments, the ability of the attenuated bacteria to enter phagocytes is reduced by no more than about 10% and no more than about 25%. , No more than about 50%, or no more than about 75%. In some embodiments of the present invention, the amount of bacteria (e.g., Listeria bacteria) is reduced in their ability to enter non-phagocytic cells, ranging from a 2-fold reduction to a considerable degree of attenuation. In some embodiments, the weakened ability of bacteria to enter non-phagocytic cells is at least about 0.3 log, about 1 log, about 2 log, about 3 log, about 4 log, about 5 log, or at least Approximately 6 logarithmic. In some embodiments, the attenuation is between about 0.3 to > 8 log, about 2 to > 8 log, about 4 to > 8 log, about 6 to > 8 log, about 0.3-8 log, There are also about 0.3-7 logs, about 0.3-6 logs, about 0.3-5 logs, about 0.3-4 logs, about 0.3-3 logs, and about 0.3_2 Logarithmic, and approximately ο] "logarithmic. In some embodiments, the attenuation is between approximately 1 to> 8 logarithms, 1 to 7 logarithms, 1-6 logarithms, approximately 2-6 logarithms, approximately 2-5 logarithms, and approximately 3-5 logarithms. In the range. Many surface proteins have been identified in Listeria monocytogenes (Boland et al., CHnicai Microbiogy Reviews, 2000 ^ 14: 584_640). These surface proteins include, but are not limited to, InlA, InlB, lnic, InlC2, InlD, InlE, InlF, 1111 (), and InlH (Dramsi et al.-98561.doc -139- 200530399 and Immunity, 65: 1615_1625 (1997); Raffelsbauer Et al., Mol. Gen. Genet. 260: 144-158 (1988)). Gene sequences encoding these proteins have been previously reported. For example, they have been reported in Gaillard et al., Cell, 65: 1127-1141 (1991). The sequences of both inlA and inlB are as in GenBank Accession No. M67471. In Table 2 of the web material of the appendix of Glaser et al., Science, 294: 849-852 (2001), it is confirmed that Genes of other members, (ww w. Sciencemag.org/cgi/content/ful 1/294/5 543/8 4 9 / DC1), including lmo0327, lmo033 1, lmo05 14, lmo0610, lmo0732, lmo 1136, lmo 1289 , Lmo2396, lmo0171, lmo0333, lmo0801, lmo 1290, lmo2026, and lmo2821. (Available in the Listeria monocytogenes EGD genome, GenBank accession number AL 5918 ^ and / or Listeria monocytogenes EGD -e genome, GenBank accession number NC The sequence of each member of the surface protein-related protein family is found in _003210. The positions of various surface proteins and related genes are indicated in Glaser * et al.).

InlA (Surface Protein A) (Gaillard et al., Cell, 65: 1127-1141 (1991); Genbank Accession No. NC_003210) directs Listeria to be taken up by epithelial cells, such as those in the small intestine.

InlB (Surface Protein B) (Gaillard et al., Cell, 65: 1127-1141 (1991); Genbank Accession No. AL591975 (Listeria monocytogenes strain EGD, complete genome, fragment 3/12, inlB gene region : Nucleotide 97008-98963); and Genbank Accession No. NC-003210 (Listeria monocytogenes EGD, complete genome, inlB gene region: nuclear 98561.doc -140- 200530399 nucleoside 45 7008-45 8963 ), Which are all incorporated herein by reference), instructing Listeria to be taken up by hepatocytes or endothelial cells, such as vascular endothelial cells of the cerebral microvascular system (microvasculature), including blood-brain disorders. (For more explanation of surface protein B, see ireton et al., J. 〇f

Biological Chemistry, 274: 17025-17032 (1999); Dramsi et al., Molecular Microbiology 16: 251-261 (1995); Mansell et al., J. of Biological Chemistry, 276: 43597-43603 (2001); and Bierne et al. 'J. Cell Science 1 15: 3357_3367 (2002), all incorporated herein by reference). In some embodiments, the bacterium is defective in Act A, surface protein b, or both Act A and surface protein B. In some embodiments, the bacteria deletes functional ActA, surface protein B, or both ActA and surface protein B. In some embodiments, the bacteria delete functional ActA. In some embodiments, the bacteria deletes functional surface protein B. In some embodiments, the bacteria deletes functional ActA and surface protein B. It is known to those skilled in the art to determine in vitro whether attenuating bacteria (such as the mutant strain of Listeria spp.) In order to enter non-swallowing cells. For example, Dramsi et al., Molecular Micr0bi0gy 16: 251-261 (l995) # 〇GaiUard et al., Cell65: U27 ii4i (i99i) Both describe the screening of mutant Listeria monocytogenes The ability to enter certain cell lines. For example, to determine whether to enter a special type of human: non-phagocytic cells with attenuated Listeria bacteria with special modifications, determine attenuated Listeria bacteria into this special type of non-swallowing 98561. doc 200530399 The ability to phagocytic cells is compared to the ability of the same Listeria bacteria to enter non-phagocytic cells without modification. Similarly, to determine whether a Listeria strain with a special mutation was attenuated to enter a particular type of non-phagocytic cells, determine whether a mutant Listeria strain has entered the special type of non-phagocytic cells And compared with the ability of non-mutated Listeria strains to enter non-phagocytic cells. In addition, by comparing the phenotype of this strain with the previously reported phenotypes of surface protein B mutants, it was confirmed that the strain was defective in surface protein B. In some embodiments, the attenuation of bacteria can be measured from the standpoint of the biological effects of Listeria on the host. The pathogenicity of bacterial strains can be assessed by measuring LDw in mice or other vertebrates. ld50s The amount or dose of Listeria spp. that will inject 50% of vertebrate deaths into vertebrates. The LDm value of bacteria with a special modification (such as a mutation) against bacteria without that special modification can be compared as a measure of the degree of attenuation. For example, if a bacterial strain without a special mutation has L050 of 103 bacteria and a bacterial strain with a special mutation has LD50 of 105 bacteria, then the strain has been attenuated 'so that LD50 increases. 100 times or 2 logarithmic. + Alternatively, the degree of attenuation of the ability of bacteria (such as Listeria spp.) To infect non-phagocytic cells can be assessed more directly in vitro. For example, you can compare the ability of a Listeria bacterium to infect non-phagocytic cells, such as liver cells, with the ability to swallow a non-modified Listeria spp. Or a field Listeria spp. . In such an assay, normally modified non-modified Listeria monocytogenes is added to non-phagocytic cells in vitro, for a limited period of time (for example, 1 hour), and then The solution rinses 9856l.doc • 142- 200530399 cells to kill any extracellular bacteria, lyse the cells, and then tile to evaluate the force value. An example of such an assay is found in U.S. Patent Publication No. 2004/0228877. For example, other biological effects can also be used to quantitatively measure the degree of attenuation 'such as histopathology or serum liver enzyme content. In clinical laboratories, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, and bilirubin were determined to be present in the serum of mice injected with Listeria (or other bacteria) . Comparing these effects in mice or other vertebrates that may be caused by Listeria with and without special modifications / mutations becomes a method to assess the attenuation of Listeria. Histopathology can also be used to measure the attenuation of Listeria. By comparing these values in injected mutants to non-mutant Listeria vertebrates, Listeria spp. Can be recovered from various tissues of infected vertebrates such as the liver, spleen and nervous system. Miles can also be used as a degree of attenuation. For example, by comparing these values in mice injected with mutant versus non-mutant Listeria, a function of time can be used to recover the number of Listeria from infected tissues such as the liver or spleen. As a measure of attenuation. Therefore, T k is known in the old-fashioned nature as a caterpillar of Listeria spp .: the viewpoint of bacterial load in selected organs; the amount of Listeria spp. Can be reduced by, for example, H2O + g.2% NP40 1 qualitative dilution) was plated on the random fat culture medium to count the colony counts (Miluo formation unit'.CFU), and the attenuation of Listeria was measured. For example, you can measure the liver or spleen by any route ’including intravenous, intraperitoneal, intramuscular, and subcutaneous, with modified Listeria monocytogenes time, measuring liver or spleen 98561.doc -143- 200530399 dirty pools. In addition, 'It is also possible to measure Listeria spp. In the liver or spleen (or any other organ of choice) in accordance with the instructions and for a period of time after the administration of the drug. Compare Listeria (or any other selected Listeria strain). The extent to which attenuated bacteria are taken up by non-phagocytic cells need not be absolutely attenuated 'in order to provide a safe and effective vaccine. In some embodiments, the degree of attenuating is to provide a reduction in toxicity that is sufficient to prevent or reduce symptoms of toxicity to a level that is not life threatening. In some embodiments of the present invention, the bacteria including the recombinant nucleic acid molecule, the expression card, and / or the expression vector described herein are mutant strains of the genus Listeria. In a further specific embodiment, the bacterium is an attenuated mutant strain of Listeria monocytogenes. In U.S. Patent Publication No. 60 / 446,051 (filed on February 6, 2003), 嶋 49,153 (filed on February 21, 2003), 60 / 511,719 (October 2003) On the 15th, Guangxi California will apply on January 15th), 60 / 5u (application on October 15, 2003), 60 / 541,515 (February 2, 2004 Applications) and 10 / 883,599 (June 30, 2004), and various representative mutant strains of the Listeria genus attenuated in U.S. Patent Publications 2004/0197343 and 2004/0228877 , Which are all incorporated herein by reference. The mutant strains of the genus Listeria are also described in International Patent Application No. PCT / US2004 / 23881, filed July 23, 2004, which is incorporated herein by reference in its entirety. For example, a bacterium including a performance card and / or a carrier may be a mutant strain of Listeria monocytogenes as required, which is 98561.doc on ActA or surface protein B, or both ActA and surface protein B. 144- 200530399 Beta bacteria are defective in monocyte proliferation. In some specific embodiments, this, ...

+ (E.g. DP-L4029 (in mutant strains of Listeria monocytogenes, which is described in actA < 97.537 (2000), Skoble et al., J. of Cell Biology, 150: i)) In the DP-L3078 strain, the entire description is described in US Patent Publication (Lauer et al., J. Bacteriol. 184: 4177 (2. Excluding its protoplasts), No. 2003/0203472) The internationally recognized actA-inlB- of the bismuth level | brake procedure (for example, DP-L4029inlB, in the Budapest Treaty of the Microbial Deposit for the purpose of # 广 卞, October 3, 2003, the Seven Hearts (ATCC), 10801 storage Collection of typical cultures in the United States f. 2101-20-22, United

University Blvd ·, Manassas, Virginia

named agu pTA-5562), States of America, and registered 5 tigers P actA-uvrAB_ (for example, DP_L4〇29uvrAB, under the provisions of the Budapest Treaty on the deposit of internationally recognized microorganisms for the purpose of patent private order) 'in 2003 Stored at the American Type Culture Collection (ATCC) October 3, '10801

University Blvd ·, Manassas, Virginia 20110-2209, United

States of America, named after registration number PTA-5563) ’or actA-inlB, vrAB ·. In some embodiments, the attenuated Listeria bacteria (e.g., Listeria monocytogenes) are deltaactAAinlB double deletion mutants. Mutation of bacteria can be accomplished through traditional mutagenesis methods, such as mutagenic chemicals or radiation, followed by selection of mutant species. It is also possible to accomplish the mutation effect of bacteria by the skilled artist through recombinant DNA technology. For example, the dual gene exchange method using the pKSV7 vector described in Cami lli et al., Molecular Micro. 8: 143-157 (1993), and the above-mentioned introduction of heterologous table 98561.doc -145- 200530399 into bacteria Intragenomic methods are suitable for producing mutants, including deleting mutants. (Camilli et al. (1993) are incorporated herein by reference in their entirety). A representative example of the use of the pKSV7 vector to generate Listeria monocytogenes surface protein B mutants is provided in Example 24 below. Alternatively, the gene replacement draft described in Biswas et al., 3 \ 6 Mad (^ 614〇1.175: 3628-3635 (1993)) may be used. Other similar methods are known to those skilled in the art. U.S. Patent Application The construction of various bacterial mutants is described in Case No. 10 / 883,599, U.S. Patent Publication No. 2004/0 197343, and U.S. Patent Publication No. 2004/0228877, each of which is incorporated herein by reference in its entirety. In some specific embodiments of the present invention, the bacterium including the recombinant nucleic acid molecule, the expression cassette and / or the expression vector is Bacillus anthracis. In some specific embodiments, the bacterium is a Sterne strain. In some specific implementations In an example, the bacterium is an Ames strain. In some specific embodiments, the Bacillus anthracis is a uvrAB mutant. In some specific embodiments, the Bacillus anthracis is a uvrC mutant. In some specific embodiments The Bacillus anthracis is a recA mutant. In some embodiments, the bacterium is an AuvrAB mutant of Bacillus anthracis (eg, the AuvrAB mutant of Bacillus anthracis Steiner, in the country for patent procedures). Under the provisions of the Budapest Treaty on the Accreditation of Accredited Microorganisms, it was deposited at the American Type Culture Collection (ATCC) on February 20, 2004, 10801 1 University Blvd., Manassas, Virginia 20110-2209, United States of America, to The registration number is named PTA-5825). 98561.doc • 146- 200530399 The method of altering the genome of Bacillus anthracis is known to those skilled in the art. The method of generating mutations in Bacillus anthracis is through dual gene exchange. A dual gene exchange vector known to this artist is used. A representative dual gene exchange plastid is pKSV7 described in Camilli et al., Molecular Microbiogy, 8: 143 ^ 47 (1993). The first step in the production of mutant bacillus bacillus is to expand the region of the genome to be deleted or otherwise mutated by PCR_, and about 1000 base pairs upstream and downstream of the anthracis genome, and then cloned to 1) Within 1 ^ 8 ¥ 7 plastid vector (or similar vector). (The Listeria ts replicon, which can appear in the PKSV7 dual gene exchange plastid vector, replaces the Bacillus-specific or Bacillus anthracis-specific temperature (ts) replicon). A restriction endonuclease recognition position in a region to be deleted or mutated may be used to delete a desired portion of a target gene in the region. Alternatively, a part of the target gene in the region may be removed and replaced with a sequence containing a desired mutation or other change. For example, restriction enzymes or a combination of restriction enzymes and synthetic gene sequences can be used to alter the enlarged T. anthracis gene tissue region before or after breeding into a dual gene-swap body. In some embodiments, the sequence can be changed into large regions and then cloned into PKSV7. In alternate embodiments, the enlarged region is first inserted into other plastids, then altered, incised, and inserted into ApKsv7. Alternatively, the PCR amplification region can be inserted directly into the PKSV7 plastid and then changed using a convenient restriction enzyme. The pKSV7 plastid containing the altered sequence was then introduced into Bacillus anthracis. This can be done via electrical penetration. Bacteria are then selected on the culture medium in the presence of aerobicin at a permissible temperature. Then, selection was made for a single cross-integration into the bacterial chromosome by substituting multiple generations at the temperature of 98561.doc -147- 200530399 in the presence of amycin. Finally, at a permissible temperature, the colonies are passed through multiple generations in a medium without antibiotics to achieve plastid deletion and correction (double crossover). U.S. Provisional Applications Nos. 60 /: 584,886 and 60 / 599,522, and U.S. Patent Publication No. 2004/0197343, all incorporated herein by reference, provide additional information regarding the construction of different types of anthracnose mutants Instructions. The bacteria of the present card E and / or expression vector in the present invention are those which have been modified to attenuate the bacteria (as opposed to unmodified bacteria). Preferably, the modified fine g maintains a sufficient degree of gene expression regardless of the modification. For example, in some embodiments, the degree of the 'gene expression is substantially unaffected by the modification, so that when the bacteria expressing the antigen is administered to the host, the antigen can be expressed to a degree sufficient to stimulate the immune response to the antigen. In some embodiments, the nucleic acid of a bacterium has been modified by reacting with a nucleic acid-targeted compound. In some specific embodiments, the fish: «is the target compound (which directly reacts with nucleic acids) to modify the nucleic acid of the bacteria that has passed through the text, thereby reducing the proliferation of the bacteria. : The compound of quasi-nucleic acid is nucleic acid / (） I group), 2_ [bis (2-Gaethyl) amino] ethyl vinegar. It is solid and solid: 一 :: =: Psoralen has been used to treat bacteria For example, bisphospholipids such as amines, trimethyl psoralen ("Heart,") and UVA light treatment to 98561.doc 200530399 modify bacteria. In some embodiments, the nucleic acids of bacteria have been modified by treatment with psoralen compounds and UVA radiation. A description of methods for modifying bacteria to reduce their proliferation using nucleic acid-targeted compounds is described in the following U.S. patent applications or publications, each of which is incorporated herein by reference in its entirety: No. 60 / 446,051 (2003 Application on February 6), No. 60 / 449,153 (application on February 21, 2003), No. 60 / 49〇, 〇89 (Shenzhen on July 24, 2003), brother 60 / 511,869 (filed on October 15, 2003), 60 / 541,515 (filed on February 2, 2004), ι0 / 883,599 (centered on June 30, 2004 ) And US 2004/0 197343. Modified bacteria and their uses are also described in International Application No. PCT / US 2004/23881, filed July 23, 2004, which is incorporated herein by reference in its entirety. For example, with regard to the treatment of Listeria monocytogenes AactA ^ uvrAB, in some specific embodiments, S-59 psoralen can be added to a logarithmic phase of (approximately) 0d6 () g = 0.05. The lipoprotein was reduced to 2000 nM, followed by inactivation with UVA light of 6 Joules per square meter, at which time the culture reached its optical density. By changing the concentration of S-59, the dose of UVA, the exposure time of s_59 before UVA treatment, and the growth during the growth of bacteria of the Listeria actA / uvrAB strain i: treatment time 'maximized the inactivation conditions. A Listeria strain of the Shaw parent was used as a control group. The inactivation of Listeria was determined by the inability of bacteria to form colonies on BHI (brain heart infusion) agar plates (log-kill). In addition, using pulsed chase experiments to confirm the virulence of heterologous proteins and S-59 / UVA-inactivated Listeria spp., Such as the performance of ll0 and p6〇, to determine the newly expressed protein inactivation in S_59 / UVA Synthesis and secretion after action. The 35s_metabolism marker can be used to routinely determine the performance of 110 and p6098561.doc-149-200530399. In the presence of 35s-methionine, S_59 / UVA-inactivated Listeria actA / uvrAB can be cultured for 1 hour. Both the lysate of the whole cell and the TCA precipitate of the bacterial culture broth can be judged for the antigen expression and the secretion of heterologous protein 'endogenous LLO and p60. Immunoprecipitation can be performed using LLO-, p60-, and heterologous shellfish-specific monoclonal antibodies, confirming continuous expression and secretion from recombinant Listeria after inactivation. In some embodiments, bacteria that have been attenuated in terms of proliferation can also be attenuated for nucleic acid repair and / or defects associated with at least one DNA repair enzyme. For example, in some embodiments, bacteria in which a nucleic acid has been used as a target compound, such as psoralen (used with UVA treatment) to modify nucleic acid, are uvrAB deletion mutants. In some embodiments, attenuating the proliferation of bacteria is at least about 0.3 logarithm, and at least about 1 log, about 2 log, about 3 log, about 4 log, about 6 log, or at least about 8 log. In other specific embodiments, the proliferation of attenuated bacteria is at least about 0.3 to > 10 log, about 2 to > 1 () log, about 4 to > 10 log, and about 6 to > ι log. , About 0.3-8 log, about 0.3-6 log, about 0.3-3 log, about 1-5 log, or about 2-5 log. In some embodiments, the antigen expressed by the bacterium is at least about 100% of the antigen expressed by a bacterium in which the bacterial nucleic acid is not modified. , About 25%, about 50%, about 75%, or at least about 90%. In some embodiments, the nucleic acid of the bacterium has not been modified by reaction with a nucleic acid-targeted compound. In some embodiments, the recombinant bacteria have not been attenuated in terms of proliferation. In some embodiments, the recombinant bacteria are not attenuated in their ability to repair nucleic acids. In some embodiments, 98561.doc -150- 200530399 recombinant nucleic acid is not defective in at least one DNA repair enzyme. In some embodiments, the number encoded by the first polynucleotide in the recombinant nucleic acid molecule, expression cassette and / or expression vector contained in the recombinant bacteria is natural to the recombinant bacteria. In some embodiments ' for performance in a recombinant bacterium, the polynucleotide codons encoding a signal peptide that is natural to the recombinant bacterium have been optimized. In some embodiments, the polynucleotide is fully codon-optimized. In some embodiments, the signal peptide ' encoded by the first polynucleotide of the recombinant nucleic acid molecule, expression cassette and / or expression vector contained in the recombinant bacteria is foreign to the host recombinant bacteria. In some embodiments ' for performance in recombinant bacteria, polynucleotide codons encoding signal peptides that are foreign to the recombinant bacteria have been optimized. In some embodiments, the second polynucleotide codon-optimizing the recombinant nucleic acid molecule, expression cassette and / or expression vector contained in the recombinant bacteria has been optimized for expression in the recombinant bacteria. In some embodiments, the second polynucleotide has been fully codon-optimized for performance in recombinant bacteria. In some embodiments, both codons of the first and second polynucleotides in the recombinant bacteria have been optimized for performance in the recombinant bacteria. In some embodiments, in order to perform in a recombinant bacterium, both the first and second polynucleosides in the recombinant bacterium have been fully codon optimized. One aspect of the present invention provides a bacterium including a performance card, wherein the performance cassette includes (a) the first polynucleoside bean encoding a signal peptide " / " Codon. Optimization; (b) a second polynucleotide encoding polymorphic 98561.doc -151-200530399, wherein the second polynucleotide is the same translation editing structure as the first polynucleotide; And (c) a promoter gene operably linked to the first and second polynuclear acids, wherein the expression cardon encodes a fusion protein including a signal peptide and a polypeptide. As described herein, for example, in Section III In the chapter, in some specific embodiments, the signal peptide encoded by it is derived from bacteria. In some embodiments, the bacterial signal peptide encoded by expression card is derived from the same bacteria as the expression cassette And / or species of bacteria. In some embodiments, the signal peptide is natural to the host recombinant bacteria. In other embodiments, the bacterial signal peptide line encoded by the expression cassette is derived from and includes The performance cassette Bacteria of different genus and / or species. In some embodiments, the signal peptide is foreign to the host recombinant bacteria. In some embodiments, the signal peptide is a secAl, secA2, or Tat signal peptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the bacterium (ie, in another aspect, the present invention provides a bacterium comprising a recombinant nucleic acid molecule, including (a) encoding the bacterium and The first polynucleotide is a natural signal peptide, in which the first polynucleotide is codon-optimized for performance in bacteria, and (b) the second polynucleotide encoding a polypeptide, wherein The second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein including a signal peptide and a polypeptide. In some embodiments, the bacterium is intracellular Bacteria. In some embodiments, the recombinant nucleic acid molecule is part of an expression cassette, which further includes operably linked to both the first and second polynucleotides. Genes. In some specific embodiments, the bacterium is selected from the group consisting of Listeria 98561.doc -152- 200530399 genera: Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella Shrimp from Mycobacterium, Mycobacterium and E. coli

In some embodiments, the bacterium is a Listeria spp. (E.g., Listeria monocytogenes). I In another aspect, the present invention provides a recombinant Listeria monocytogenes (eg, Listeria monocytogenes) comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes (a) a first polynucleoside encoding a signal peptide Acid, in which the _ polynucleotide codon is optimized for expression in Listeria spp., And (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is The first translational editing architecture of a polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein including a signal peptide and a polypeptide. In some embodiments, the recombinant nucleic acid molecule is part of an expression card, which further includes a first gene in a manipulable manner and a second polybasic acid linked promoter gene. In some embodiments, this second polynucleotide codon is optimized for performance in Listeria bacteria. In two examples, the polypeptide encoded by the second polynucleotide is foreign to Listeria bacterium (ie, heterologous to Listeria bacterium). In some embodiments, the Listeria fine g is attenuated. For cell-to-cell transmission, entry into non-phagocytic cells, or proliferation, the Listeria can be attenuated. In some specific embodiments, the recombinant Listeria bacterium is defective in ActA, surface protein B, or women and surface proteins (for example, the ΔactAMWB double deletion mutant). In some embodiments, the nucleic acid of the recombinant bacterium has been modified by reacting with a nucleic acid-targeted compound (such as a psoralen compound). 98561.doc -153- 200530399 In another aspect, the present invention provides a bacterium including a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes a first polynucleic acid encoding a non-secA 丨 bacterial signal peptide, and an encoded polypeptide (eg, an antigen) A second polynucleotide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, and wherein the recombinant nucleic acid molecule encodes a fusion comprising a signal peptide and a polypeptide

In some embodiments, the protein is heterologous to Peptide No. 4 encoded by the second polynucleic acid. In some embodiments, the recombinant nucleic acid molecule is part of a performance cassette, which further includes a promoter gene operably linked to both the first and second polynucleotides. In some specific embodiments, the bacterium is selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella Coronatus, and E. coli Groups of bacteria. In some embodiments, the polypeptide encoded by the second polynucleotide is foreign to the bacterium (i.e., heterologous to the bacterium).

In another aspect, the present invention provides a bacterium including a performance cassette, wherein the current cassette includes a first multi-core saccharin that encodes a non-secA1 bacterial signal peptide with the same translation editing structure as the first multi-core , A second polynucleic acid encoding a peptide (eg, a heterologous polypeptide to the bacterium) and ⑷ are operably linked to both the first and second polynucleotides 纟 a starter gene 'wherein the performance card The cassette encodes a fusion protein including a signal peptide and a polypeptide. As described herein, for example, in Chapter m above, in the first example, the non-seeA1 bacterial signal peptide is Kaba 2xin ^. In some other specific embodiments, the non-bacterial signal is a peptide. In some specific examples, the expression card Kunkun code white 98561.doc -154- 200530399 bacterial peptides derived from the same genera and / subspecies as including the performance card-ΛΛ, engineering <,,, and field halogen Tian In other specific embodiments, the secondary cassette is coded, and the Tianhao pseudo-peptide is derived from bacteria including the performance cassette and / or species. (,, different genus Tianhan and + on the other hand, the present invention provides a recombinant Listeria bacterium including a recombinant nucleic acid molecule, wherein the 4 sets of nucleic acid molecules include a first polynucleotide that is a non-bacterial signal, And (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion comprising a signal peptide and a polypeptide Protein. In some embodiments, the recombinant nucleic acid molecule is part of a performance cassette, which further includes a promoter gene operably linked to both the = and the first polynucleotide. In some implementations In some examples, the Listeria bacterium is attenuated. In some embodiments, the 'Listeria bacterium is Listeria monocytogenes. For example, it can be used for cell-to-cell transmission and non- Phagocytosis or proliferation in cells, attenuating the Listeria spp. In some embodiments, the recombinant Listeria spp. Are in Act A, surface protein B, or both Act A and surface protein b Is defective (such as AactAAinlB double deletion mutant). In one or two specific embodiments, the nucleic acid of the recombinant bacterium has been modified by reacting with a nucleic acid-targeted compound (such as a psoralen compound). In one aspect, the invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises a polynucleotide encoding a polypeptide that is foreign to the Listeria bacterium, wherein Codon-optimization of the polynucleotide. 98561.doc -155- 200530399 In other aspects, the invention provides a recombinant Listeria bacterium comprising a performance cassette, wherein the performance cassette includes the following: ( a) a polynucleotide encoding a polypeptide that is foreign to Listeria bacteria, wherein the polynucleotide is codon-optimized for expression in Listeria spp .; and (b) in an operable manner A promoter gene linked to a polynucleotide encoding the foreign polypeptide. Alternatively, in some specific embodiments, the Listeria is Listeria monocytogenes In other specific embodiments, the Listeria bacterium belongs to Listeria escherii, Listeria sclerotii, or Listeria harmless. In some embodiments, the Listeria bacterium is like The above-mentioned attenuated drug-reducing lines of Listeria bacteria. ^ + In another aspect, the present invention provides a recombinant Listeria including a recombinant nucleic acid molecule = a genus of bacteria (such as Listeria monocytogenes, wherein the recombinant nucleic acid molecule includes ( a) the first polynucleoside transcript encoding a non-Listeria spp. signal peptide, and (b) the same translational editing architecture as the first polynucleotide, encoding the second polybasic acid 1 of the polypeptide The recombinant nucleic acid molecule encodes a fusion protein that includes both a non-Listeria spp. Peptide and the polypeptide. In some embodiments, the Listeria spp. Is attenuated. For example, Cells spread, enter or proliferate into non-ϋ cells, and this Listeria is reduced. In some embodiments, the recombinant Listeria bacterial surface protein B, or both ActA and surface protein B is defective (e.g., a double deletion mutant). In some embodiments, the nucleic acid of the recombinant bacterium has been modified by reacting with a nucleic acid-targeted compound, such as a psoralen. Aspects The invention provides for the expression of K. spp., Listeria monocytogenes 98561.doc- 156- 200530399: Bacteria (such as Listeria monocytogenes encoding non-Listeria monocytogenes signal peptides of the first polymorphism card (such as non-Liszt Zhiyao, $ 个 夕 核 钱, encoding polypeptide words The second polynucleotide of the genus Polypeptide) is the same as the second polynucleotide of the m polynucleotide, and / or 、 /, the m-th transformation δ &quot; The promoter gene. The expression # :; and = fusion protein of non-Listeria spp. Peptide and the polypeptide. In one or two specific embodiments, 7 &lt; 4 &gt; cells spread 'into non-phagocytosis or ~ Colony 'attenuates the Listeria bacterium. In this example, the Listeria bacterium is defective on both a surface protein B: ^ surface protein B. In some embodiments, Has been used with nucleic acid-targeted compounds (such as psoralen Compound) to modify the nucleic acid of the recombinant bacterium. In some embodiments, the first polynucleotide, the second polynucleotide, or the first and second Codons for both polynucleotides-optimization. In some embodiments, 'for the performance in Listeria monocytogenes, the first polynucleotide and / or the second polynucleotide codon _Optimization. In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen, in some cases it may be a non-bacterial antigen. For example, the polypeptide, in some embodiments, is related to tumors Related antigens, or derived from such tumor-related antigens. For example, in some embodiments, the polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA , NY-ESO-1, WT-1, survival protein, gplOO ′ PAp, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, Mesothelin, PSCA, NY-ESO-1, 98561.doc 157- 200530399 WTM, Survivin Cytoplasm, gpl00, PAp, protease 3, spAs i, sp PAGE-4, TARP or CEA. For example, in some embodiments, the polypeptide is mesothelin, or a fragment or variant of mesothelin. In some other In specific embodiments, the polypeptide is NY-ESCM, or NY_ES〇 ~ + or a variant. In some specific embodiments, the antigen is an infectious disease antigen, or derived from an infectious disease antigen. In a preferred specific In embodiments, the signal peptide is bacterial. In some embodiments, the signal peptide is obtained from a bacterium belonging to the genus Bacillus, Staphylococcus or Lactococcus. For example, in some embodiments, the signal peptide is obtained from Bacillus anthracis, Bacillus subtilis, Staphylococcus aureus, or Lactococcus lactis. In some embodiments, the signal peptide is a secAl signal peptide, such as the Usp4Mf peptide from Lactococcus lactis or a protective antigen signal peptide from Bacillus anthracis. In some embodiments, the signal peptide is a secA2 signal peptide. In a further specific embodiment, the signal peptide is a Tat signal peptide, such as a Bacillus subtilis Tat signal peptide (e.g., pOD). The present invention further provides a recombinant bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nuclear gwen knife comprises: (a) a first polynucleoside encoding a bacterial autolysin, or a fragment or a catalytically active variant thereof; Acid; and a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule editor comprises the second polynucleotide Polynucleotide-encoded polypeptides and autolysins, or catalytically active fragments or protein variants of catalytically active variants' of which, in a protein chimera, the polypeptide is an autolysin, or a The fragments are fused with catalytically active variants, or are located in> gluten 'or its catalytically active fragments or catalytically active variants 98561.doc -158- 200530399. In some specific embodiments, the recombinant bacteria are intracellular bacteria, Pichia bacteria (eg, Listeria monocytogenes). In some cases, the "polypeptide" encoded by the second polynucleotide is foreign to the recombinant cell. Aspects of the present invention include Listeria monocytogenes performance cards Genus Bacteria, Hot in Pots 丨 ^^ 丨 丨 Atlas two performance cassettes encoding at least two separate

Of non-Listeria spp. For example, in some embodiments, the performance card includes a first polynuclear acid encoding a first non-Listeria polypeptide, and a second polynuclear bud encoding a second non-Listeria polypeptide. Acid, two promoter genes operably linked to the first and second multinucleate pupae. In some examples, the recombinant Listeria bacterium belongs to the species prokaryotic cell proliferation first and / or second non-Liszt; in some specific embodiments, the Streptozoa polypeptides include antigens (or fragments thereof). In some specific examples Φ, ssd. 0L bacteria, the performance card II = provides recombination including performance

Polynuclearization_: the first poly (::: number, the second polynucleic acid is the first polynucleotide, in which the structure is the same; the translation sequence is the same as the gw acid; ⑷ The third multi-core multi-core multi-core encoding of the second multi-H signal peptide is the same as the third: multi-core ㈣, where the fourth ⑺ has the same translation editing structure as the operable party; and the third Polynuclear m, fourth / nucleus, second polynucleic acid, animal gene, which makes this expression ::::: the first fusion protein of the peptide linked to the intergenic sequence, including: the first Signal peptide and the first protein and a fusion protein including the second signal peptide and the second polypeptide 98561.doc -159- 200530399. In some embodiments, the signal will be encoded for performance in bacteria One or more of the polynucleotide codons are optimized. In some embodiments, the third and / or fourth polynucleotide codons are optimized for performance in bacteria. In some embodiments In this case, the first and / or second polypeptide is heterologous to the recombinant bacteria (for example, the antigen in the middle). In some embodiments, the first and / or second signal state is a non-bacterial signal state. The first and / or second signal state may be natural or foreign to the recombinant bacteria. In some specific implementations In the example, the present recombinant bacterium is a Listeria bacterium, and the first and / or the second signal peptide is a non-Listeria g. In some specific embodiments, the intergenic sequence is monocytosis. Listeria aetA_pleB intergenic sequence. In some embodiments, the bacterium is Listeria monocytogenes. In another aspect, the present invention provides a bacterium comprising a recombinant nucleic acid molecule, including [multi-nucleus] encoding a signal peptide. ㈣, ⑻ encodes a second polynucleotide encoding a secreted protein or fragment thereof, wherein the second polynucleotide is the same translation editing framework as the first polynucleic acid, and ⑷ encodes the secreted protein or fragment thereof A third polynucleotide that is a heterologous polypeptide, wherein the third polynucleotide is the same translation editing framework as the first and second polynucleotides, and wherein the recombinant nucleic acid molecule encodes a signal A protein chimera of the polypeptide encoded by the second polynucleotide and the secreted protein or fragment thereof in the protein complex, or a polymorphism of the polymorphism with the secreted protein or fragment thereof, or located in the secreted protein Or a fragment thereof. In some embodiments, the bacterium is a Listeria bacterium. In some embodiments, where the bacterium is a Listeria bacterium, the third 98561.doc- 160-200530399 The polypeptide encoded by the nucleotide is foreign to Listeria. In some cases of carcass, the bacterium is Listeria monocytogenes. In some specific embodiments (such as In some of the above-mentioned viewpoints]) 'Integrate the expression cassette contained in the bacteria into the bacteria's genome. In other embodiments, the expression cassette contained in the bacteria is on the plastid inside the bacteria. Generally, the promoter gene used in the performance cassette will be a performance cassette suitable for performing a heterogeneous performance by a selected particular bacterial wounder. Those skilled in the art can easily identify which promoter gene is suitable for use in which bacteria. In some cases, the promoter gene is a bacterial promoter gene. In other specific embodiments, the promoter gene on the expression card E in bacteria is obtained from a bacterium belonging to the same genus as the bacteria containing the expression cassette. In other specific embodiments, the promoter gene on the expression cassette in bacteria is known from the promoter gene of a bacterium belonging to the same species as the bacteria containing the expression cassette. For example, if the bacterial mouse containing the expression cassette is in a Listeria monocytogenes species, the start-up gene used in the performance cassette at this time may be derived from a gene of Listeria, such as hly, if necessary. In other specific embodiments, the promoter is a constitutive promoter (for example, a P60 promoter) or a PrfA-dependent promoter (for example, an actA promoter). Furthermore, as mentioned above, the promoter genes of the expression cassette are, in some specific embodiments, constitutive promoter genes. In other embodiments, the promoter of the performance card is an inducible promoter, as described above. In some specific embodiments (for example, in some of the examples with the viewpoints of 98561.doc -161-200530399 mentioned above), the polypeptide encoded by the expression cassette in bacteria or the adherin comprising the polypeptide is Antigens or other therapeutically valuable protein stomachs, as described, for example, in Chapter W above. In some embodiments, a polypeptide or a fusion protein comprising a polypeptide is secreted from a bacterium. In some embodiments, the polypeptide expressed and / or secreted by the bacteria is heterologous to the bacteria. Therefore, in some specific embodiments, the present invention provides a signal that includes the expression of Kakun, and Listeria spp., Wherein the expression cassette includes a ⑷-encoded bacterial (Listeria or non-Listeria) signal The first polynucleotide of the peptide, where the first polynucleotide is codon-optimized for performance in Listeria; (b) a second polynucleotide encoding a non-Listeria antigen, Wherein the first polynucleotide is the same translation editing structure as the first polynucleotide; and (c) a promoter gene operably linked to the first and second polynucleotides, wherein the expression The cassette encodes a fusion protein including the signal peptide and the antigen. In a further specific embodiment, the Listeria is a strain of Listeria monocytogenes, such as the actA-inlB- strain. In some embodiments, the expression cassette has been integrated into the genome of a recombinant Listeria spp. In some embodiments, the second polynucleotide codon is optimized for performance in Listeria. The invention also provides a Listeria spp. Comprising a performance cassette, wherein the performance card E includes (a) the first polynucleic acid encoding a secA2 or Tat bacterial signal peptide, and (b) the same as the first polynucleotide Translational editing framework for a second polynucleotide encoding an antigen; and (c) a promoter gene operably linked to the first and second polynucleotides, wherein the expression cassette encoding includes the 泫 k number Fusion protein of peptide and the antigen. In some specific embodiments, the 98561.doc -162- 200530399 ', P. spp. L peptide 疋 Listeria. In other specific embodiments, the bacterial signal peptide is non-Listeria. In a further specific embodiment, the Listeria is a strain of Listeria monocytogenes, such as the actA inlB strain. In some embodiments, the expression card has been transcribed into the genome of a recombinant Listeria spp. In some specific embodiments, in order to be expressed in Listeria, a polynucleotide encoding a signal peptide (even if the signal peptide is a Listeria peptide) and / or a polynucleotide codon encoding an antigen optimize.

In a further specific embodiment, the present invention provides a recombinant Listeria comprising a performance card, wherein the performance card includes the following: a multinucleate encoding a non-Liszt g antigen, wherein As shown in Mycobacterium, the polynucleotide codon is optimized; and (b) a promoter gene operably linked to a polynucleotide encoding a foreign polypeptide. In some embodiments, the performance card further includes encoding a bacterial signal

In order to express in the Listeria genus, the codons of the nucleoside S are also maximized. In a specific embodiment, the bacterial signal peptide is of the genus Listeria. In another specific embodiment, the bacterial signal peptide is non-Listeria spp. In some embodiments, the bacterial signal peptide is a secAl signal peptide, a secA2 signal peptide, or a Tat signal peptide. In a further specific example, the Listeria is a strain of Listeria monocytogenes, such as actA inlB. In this specific example, the expression cassette has been integrated into the genome of the recombinant Listeria spp. In another embodiment, the present invention provides the following S. spp. Bacteria: The first polynucleotide encoding a signal peptide of a bacterium (Listeria or non-Listeria set 98561.doc -163-200530399), all eyes on ^ ^-', horses in Listeria In the performance, the most codon of one polynucleotide of this brother was studied. (B) The second polynucleotide of Shimafei-Liss transgenic antigen, 苴, 寺 n ,, ^ ^ and 弟A polynucleotide with the same trans #editing framework, in order to perform in the Listeria genus, this codon is optimized for nucleotide codons; and soil ten y and (C) in an operable manner with the first One and two polynucleotide-linked promoter genes, 1 ^ ^ ^ T 5 Hai performance cassette encoding 0 including the fusion protein of the #bleptide and the antigen ^ # Shi poor white In some specific embodiments, e Hai Listeria bacteria belong to the substance Listeria. In some specific embodiments, the Libanx k * idiopathic bacterium is an actA] nlB · mutant strain strain of Listeria monocytogenes. The present invention further provides a performance card comprising more than one described herein, such as Listeria. In special combinations, molecules of specific proteins can inhibit their appearance from recombinant bacteria, such as recombinant Listeria. One way to solve this problem is to separate the genes encoding the protein of interest at the level of the early-knife knife and fuse each part functionally with the sequence, which will write a plan for its secretion from bacteria (for example secAi, Kaba 2 or additional elements).-One method is to individually drive the recombinant Listeria spp. to express each part of the heterogeneous gene. Alternatively, the gene can be separated at the molecular level via bacterial chromosomes Individual components (including appropriate elements for secretion) are incorporated into the intergenic region using methods that are well established in the art, such as by dual gene exchange. Other examples are based on single polycistronic messages. Way, the genes that are now separated at the molecular level. According to this combination, scattered between the sequences encoding the protein, the genes that are separated at the molecular level will be the Shine-Dalgarno ribosome binding sequence in order to be more than 98561.doc -164-200530399 Resonant information again-to initiate protein synthesis. In other aspects, the present invention provides improved performance in recombinant bacteria such as Listeria and / Or methods of secreting heterologous polypeptides. Any of the polynucleotides, performance cassettes, and / or performance vectors described herein can be used in these methods. For example, the invention provides improved performance and / or secretion in Listeria A method for fusion of a heterologous polypeptide of a signal peptide includes compiling a sequence of a marsh peptide on the expression cassette, a sequence of a signal peptide encoding the expression cassette, or codon-optimization of both. The present invention also provides In Listeria, methods for improving the expression and / or secretion of heterologous polypeptides that combine with the signal peptide w include the use of signal peptides derived from sources other than Listeria and / or secretory pathways other than seca 1 The present invention also provides a method for producing a recombinant bacterium, such as a recombinant Listeria bacterium, comprising introducing a recombinant nucleic acid molecule, expression cassette, and / or expression vector described herein into a bacterium to produce a recombinant bacterium. For example, In some embodiments, the recombinant nucleic acid molecule includes (a) a first polynucleotide encoding a signal peptide that is natural to the bacterium, wherein , Optimize the first polynucleotide codon, and (b) the second polynucleotide encoding the polypeptide, wherein the second polynucleotide is the same translation as the first polynucleotide A framework in which a recombinant nucleic acid molecule encoding a fusion protein comprising the signal and the polypeptide is introduced into a bacterium to produce a recombinant bacterium. In some specific embodiments, the recombinant nucleic acid molecule includes (a) a non-secAl bacterial signal peptide encoding The first polynucleotide, and the second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, and the encoding package = the 98561 .doc -165- 200530399

The recombinant nucleic acid molecule of the signal peptide and the fusion protein of the polypeptide is introduced into bacteria to produce a recombinant bacteria. In some specific embodiments, the recombinant nucleic acid molecule introduced into the bacteria to produce a recombinant bacterium is a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes (a) a first polynucleotide encoding a non-Listeria signal peptide, And (b) a second polynucleotide encoding a polypeptide with the same translation editing architecture as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a non-Listeria signal peptide and the polypeptide . In some specific examples, the recombinant nucleic acid molecule used to generate the recombinant bacteria is a recombinant nucleic acid molecule that includes: (a) a bacterial autolysin, or a catalytically active fragment or a catalytically active variant thereof; A polynucleotide, and (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a protein Chimera, wherein the non-Listerial polypeptide is fused with autolysin or a fragment having catalytic activity or a variant having catalytic activity, or inserting autolysin or a fragment having catalytic activity or a variant having catalytic activity Inside. In some other specific embodiments, there is provided a method for generating a recombinant Listeria® fine g 'which includes cutting the expression of a polycistronic within a Listeria bacterium', wherein the performance cassette encodes at least two isolated Non-1 streptozoa polypeptides produce recombinant Listeria bacteria. IX. Pharmaceutical, Immunogen and / or Vaccine Compositions The present invention also provides a variety of different compositions, such as amidine compositions, immunogens, and: and vaccines. These compositions include any of the heavy and microbes described herein (see 'e.g., the Summary of the Invention, Implementation Methods I and chapters, and elsewhere in the fact sheet, including examples below). In some of these embodiments, the composition is isolated. For example, the present invention provides a pharmaceutical composition comprising: (i) a pharmaceutically acceptable carrier; and (Π) a recombinant bacterium described herein. For example, the present invention provides a pharmaceutical composition comprising: (i) a pharmaceutically acceptable carrier; and (ii) a recombinant bacterium including a performance cassette comprising a first multi-encoding signal peptide In order to: in the expression of bacteria *, optimize the first polynucleotide codon _, encoding a second polynucleotide of the polypeptide, wherein the second polynucleotide is the same as the first multinucleus The same translation and editing architecture of the uridine and the promoter gene operably linked to the first and second polynucleotides enable the expression cassette to encode a fusion protein including the signal peptide and the polypeptide. The present invention also provides a pharmaceutical composition, including: ⑴ a pharmaceutically acceptable carrier; and 表现 including a manifestation card of a recombinant bacterium, wherein the expression ^ includes a first polynucleotide encoding a non-secAl bacterial signal peptide, A second polynucleus encoding a polypeptide encoding a polypeptide with the same translation and editing architecture as the first polynucleotide, and a promoter gene operably linked to the first and second polynucleus is making the performance card It encodes a fusion protein including the signal peptide and the polymorphism. The present invention further provides a pharmaceutical composition 'comprising:' a pharmaceutically acceptable carrier; and (ii) including a recombinant Listeria bacterium which expresses a cut, and the performance card E includes the following: Is a polynucleotide of a foreign polypeptide, in which the polynucleotide is codon-optimized for expression in the genus Lisztium; and (b) the foreign polypeptide is encoded in a way that can be manipulated Polynucleotide linked promoter. 98561.doc -167- 200530399 The present invention also provides a pharmaceutical composition comprising: a pharmaceutically acceptable carrier; and (π) a recombinant Listeria bacterium including a performance cassette, the performance cassette comprising ... (A) the first polynucleotide encoding a non-Listeria spp. Peptide; (ii) a second polynucleotide encoding a polypeptide with the same translation editing architecture as the first polynucleic acid; and (c) A promoter gene operably linked to the first and second polynuclear #acids, wherein the performance card encodes a fusion protein including the non-Listeria signal peptide and the polypeptide. As used herein, "vehicle" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antifungals, isotonic and absorption delaying agents, buffer solutions, carrier solutions, suspensions Liquids, colloids and the like. Pharmaceutically acceptable carriers are well known to those skilled in the art and include any substance that, when mixed with an active ingredient, allows the ingredient to retain biological activity and does not react with the immune system of the individual. For example, pharmaceutically acceptable carriers include, but are not limited to, water, buffered physiological saline (e.g. 0.9 = physiological saline), emulsions' such as oil / water emulsions, and various types of wetting agents. Possible carriers also include, but are not limited to, oils (such as mineral oils), dextrose solutions, glycerol solutions, chalks, starches, salts, glycerol, and gelatin. Although any suitable carrier known to those skilled in the art can be used in the pharmaceutical composition, the type of carrier will vary depending on the mode of administration. The composition of the present invention can be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. In some embodiments &apos; for parenteral administration, such as subcutaneous injection, the carrier includes water, saline, alcohol, lipid, tincture, or a buffer solution. In two specific examples, for oral administration, any of the aforementioned carriers or 98561.doc 200530399 solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, Sucrose and magnesium carbonate.

Compositions comprising such carriers are formulated by conventional methods well known (see 'e.g. Remington's Pharmaceutical Sciences, 18th Edition A

Edited by Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and practice of Pharmacy, 20th Edition

Mack Publishing, 2000). In addition to pharmaceutical compositions, immunogenic compositions are also provided. For example, the present invention provides an immunogen composition comprising a recombinant bacterium described herein (see, for example, the recombinant bacterium described above, in the summary above, chapters 1 and M of the method of implementation, and elsewhere in the specification, Including examples below). In some specific embodiments, the immunogen composition includes a recombinant bacterium, wherein the polypeptide sequence that is part of the polypeptide expressed by the recombinant bacterium is in the form of an isolated protein, is a part of a fusion protein, or is embedded in a protein complex ( Depending on the recombinant nucleic acid molecule or performance cassette used), it is or includes an antigen. In other words, in some embodiments, the immunogen composition includes a recombinant bacterium that includes a recombinant nucleic acid molecule or expression cassette encoding a polypeptide that includes an antigen. Suitable antigens include, but are not limited to, any of those described herein (e.g., in Chapter ^ above). In some embodiments, the recombinant bacteria in the immunogen composition, when administered to the host (for example, a mammal, such as a human), to a degree sufficient to induce an immune response against the antigen, the performance includes the antigen Of peptides. In some embodiments, the immune response stimulated by the immunogen composition is a cell-mediated immune response. In some embodiments, the immune response stimulated by the immunogenic composition 98561.doc -169- 200530399 is a humoral immune response. In some embodiments, the immune response from the immunogen composition includes humoral and cellular-mediated immune responses. For example, 'an aspect of the present invention provides an immunogen composition comprising a recombinant bacterium' which: the bacterium includes a manifestation of what 'it includes the following: ⑷ a polynucleic acid encoding a signal peptide, wherein for the purpose of expression in bacteria, the first : A polynucleic acid codon-optimization; (b) a second polynucleic acid encoding an antigen, wherein the second polynucleic acid is the same translation editing structure as the first polynucleic acid; and An operably linked promoter gene linked to the first and second poly (pi) nucleotides enables the expression card E to encode a fusion protein comprising the signal peptide and the antigen. The present invention &amp; provides an immunogen composition comprising a recombinant bacterium, = the bacterium includes a performance card E, which includes the following: (i) a non-secAi, the first multinucleus 田 of the field bacteria L peptide; (b ) A second polynucleic acid encoding an antigen with the same translation editing structure as the first polynucleic acid; and ⑷ a promoter base linked to the first and second polynucleotides in an operable manner The 4 performance card was allowed to encode a fusion protein including the signal peptide and the antigen. The present invention includes an immunogen and a CT substance including a recombinant Listeria bacterium, wherein the recombinant Listeria bacterium includes an expression cassette, wherein: the expression cassette includes the following: The polynucleoside self-sense JE of the Mycobacterium antigen was optimized for its codon-expression in Listeria; and (b) a promoter gene operably linked to the polynucleic acid encoding the antigen. 98561.doc -170- 200530399 The present invention also provides an immunogen composition comprising a recombinant bacterium, the recombinant bacterium comprising a performance cassette comprising ... (a) the first polynucleoside encoding a non-Listeria signal peptide Acid; (b) a second polynucleotide encoding an antigen, which is the same translation editing framework as the first polynucleotide; and (c) operable with the first and second polynucleotides An acid-linked promoter, wherein the expression cassette encodes a fusion protein comprising a non-Listeria spp. Signal peptide and the antigen. In another aspect, the present invention provides an immunogen composition (or epidemic field) comprising a recombinant bacterium. The recombinant bacterium includes a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes (a) a signal peptide encoding a natural signal peptide to the bacterium. A first polynucleotide whose alpha is codon-optimized for performance in bacteria, and (b) a second polynucleotide encoding a polypeptide comprising an antigen, wherein the second Each polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein including the signal peptide and the polypeptide. In another aspect, the invention provides an immunogen composition (or vaccine) comprising a recombinant Listeria bacterium, wherein the recombinant bacterium includes a recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a signal peptide, wherein The first polynucleotide codon is optimized for performance in bacteria, and (b) the second polynucleotide encodes a polypeptide including the antigen, wherein the second polynucleotide is related to the first polynucleotide The same translation and editing architecture of uridine, wherein the recombinant nucleic acid molecule encodes a fusion protein including the signal peptide and the polypeptide. In another aspect, the present invention provides an immunogen composition (or epidemic field) including a recombinant bacterium, wherein the recombinant bacterium includes a recombinant nucleic acid molecule, wherein the recombinant nucleus 98561.doc -171-200530399 acid molecule includes a non-secAl bacterial signal peptide A first polynucleotide, and a second polynucleotide encoding a polypeptide including an antigen, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, and wherein the recombinant nucleic acid The molecule encodes a fusion egg comprising the signal peptide and the polypeptide. In another aspect, the invention provides an immunogen composition (or vaccine) comprising a recombinant Listeria bacterium, wherein the recombinant bacterium includes a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule Includes (a) the first polynucleic acid encoding a non-secAi bacterial signal peptide, and (b) the second polynucleotide encoding a polypeptide that is heterologous to the signal peptide or foreign to the bacterium Wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, and wherein the recombinant nucleic acid molecule encodes a fusion including the signal peptide and the polypeptide.合 protein. In some embodiments, the polypeptide encoded by the first polynucleotide includes an antigen. In another aspect, the invention provides an immunogen composition (or vaccine) comprising a recombinant Listeria bacterium, wherein the recombinant Listeria bacterium includes a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule includes It is said that the foreign polypeptide is a multinucleated polypeptide. In order to express it in the Listeria genus, the polypeptide: g: acid codon · optimization. In some embodiments, the foreign polypeptide includes an antigen. In another aspect, the invention provides an immunogen composition (or vaccine) comprising a recombinant Listeria bacterium, wherein the 4 groups of bacteria include a recombinant nucleic acid molecule, which includes a first encoding a non-Listeria signal peptide Polynuclear acid, and ⑻ A second polynucleotide encoding a polypeptide including an antigen, the second one is 98561.doc -172- 200530399 Nucleic acid is the same translation editing structure as the first polynucleotide And wherein the recombinant nucleic acid molecule encodes a fusion protein comprising both a non-Listeria spp. Signal peptide and the polypeptide. The present invention also provides an immunogen composition (or vaccine) including a recombinant bacterium, wherein the recombinant bacterium includes a nucleic acid molecule including (a) encoding a bacterial autolysin 'or a catalytically active fragment thereof or a catalytically active variant The first polynucleotide, and (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid molecule Encoding a protein chimera comprising a polypeptide encoded by the second polynucleotide and an autolysin, or a fragment or catalytically active variant thereof, wherein, in the protein chimera, the polypeptide is Or a catalytically active fragment or a catalytically active variant thereof fused or located therein. In some embodiments, the polypeptide encoded by the second polynucleotide includes an antibody. In another aspect, the invention provides an immunogen composition (or vaccine) comprising a recombinant Listeria bacterium, wherein the recombinant Listeria bacterium includes a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule encodes at least two isolated non- Listeria polypeptides, at least one of which includes an antigen. In another aspect, the present invention provides an immunogen composition (or vaccine) including a recombinant bacterium, wherein the recombinant protein includes a recombinant tritium molecule, which includes a first polynucleotide encoding a signal peptide and (b) a secreted protein. Or a fragment of a second polynucleotide thereof, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, and (c) encodes a heterologous fragment of the secreted protein Geqi fragment The third polynucleotide of the polypeptide, of which the second 98561.doc -173- 200530399 nucleosides § 相同 the same translation and editing framework as the first and second polynucleotides' where the recombinant nuclear sn The sub-encoding includes a signal peptide, a protein encoded by the third polynucleic acid, and a protein chimera of the secreted protein or a fragment thereof, and in the protein chimera, the third polynucleic acid encodes a polypeptide and The secreted protein or a fragment thereof is fused or located within the secreted protein or a fragment thereof. In some embodiments, the heterologous polypeptide encoded by the third polynuclear acid includes an antigen. σ By testing the ability of recombinant bacteria to stimulate the immune response in vitro or in a model system, determine the special form (and / or special performance cassette) of recombinant bacteria that is useful in immunogen compositions (or vaccines) . These immune cell responses can be measured in vitro and in vivo to determine whether the immune response of a particular recombinant bacterium (and / or a special performance card) is effective. One possibility is by using a population of This combined antigen-providing cell measures the presence of the protein or antigen of interest. Recombinant bacteria can be mixed with appropriate antigen-providing cells or cell lines, such as dendritic cells, and the protein or antigen recognized by dendritic cell pairs can be measured. The τ cell raises the antigen of the father. If the recombinant bacteria are expressing the protein or antigen in a sufficient amount, it will be processed into peptide fragments by dendritic cells and submitted to T cells as mhc type I or type II. For detection The protein or antigen to be submitted can be a cytoplasmic germline or a τ cell line that responds to a specific protein or antigen. The T cell can also be a τ cell fusion tumor, where the τ cell becomes permanent by fusion with a cancer cell line Immortal. Such τ-cell fusion tumors, T-cell pure germlines or τ-cell lines may include CD8 + or CD4 + T cells. This dendritic cell can be submitted to CD8 + or 98561.doc -174- 200530399 CD4 + T cells. CD8 + T cells are recognized as MHC class I antigens, and CD4 + are recognized as MHC class II antigens. T Cells will be stimulated by the submitted antigen, through the specific recognition of their T cell receptors, resulting in certain quantifiable proteins (such as using an ELISA assay, ELISPOT assay, or intracellular cytotoxic staining (ICS)), such as IL -2, tumor necrosis factor_a (TNF-a) or interferon-γ (IPN-γ)). These are techniques well known in the art and are also exemplified in the examples below (see, e.g., Example 21 below).

Alternatively, a fusion tumor that includes the reporter gene, such as β-galactosidase, may be activated when the T-cell fusion tumor is stimulated by the submitted antigen. The increase in the production of β-galactosidase can be quickly measured by its activity on substrates, such as aerosol red-B-galactosidase, resulting in a change in color. Color change can be measured directly as an indicator of the presence of a specific antigen.

Additional in vitro and in vivo methods for assessing the antigenic performance of the recombinant bacterial vaccine of the present invention are known to those skilled in the art. It is also possible to directly measure specific xenoantigens expressed by recombinant bacteria. For example, a radiolabeled amino acid can be added to a cell population, and the radioactive content incorporated into a particular protein can be determined. Proteins synthesized by cell populations can be separated, for example, by gel electrophoresis or capillary electrophoresis, and the radioactive content can be quantitatively measured to evaluate the degree of expression of a particular quality. Alternatively, a non-radioactive protein can be expressed, and various methods can be used to develop its color, such as eusa measurement or gel electrophoresis and western blotting method, using enzyme-linked antibodies or fluorescent labeling as provided in Example 21 below. Using some specific examples known to those skilled in the art, illustrate how techniques can be used to assess immunogenicity. For example, 98561.doc -175- 200530399

Elispot assay, intracellular cytotoxic staining assay (ICS), measurement of cytokine expression of stimulated spleen cells, and assessment of cytotoxic T cell activity in vitro and in vivo are all known in the art to Techniques for assessing immunogenicity. Representative illustrations of these techniques using model antigens are provided in Example 21. Representative measurements are also described in Examples 31A and 31E below. In addition, the therapeutic efficacy of a vaccine composition can also be assessed more directly by administering an immunogen composition or vaccine to an animal model, such as a mouse model, and then assessing survival or tumor growth. For example, survival can be measured after administration and challenge (for example, with tumors or pathogens). See, for example, the assay described in Example 20 * 3ib_d below. The tumor cells can be modified so that they can express the antigen or model antigen of interest, and then the tumor cells that express the antigen of interest can be implanted in mice to produce immunogenic compositions or vaccines for testing specific antigens. Immunogenic Mouse Pattern. Candidate immunity including recombinant bacteria expressing polypeptides can be used before tumor cell implantation (testing for the preventive efficacy of candidate compositions) or after tumor cell implantation in mice (testing for the therapeutic efficacy of 迖 and σ) The original composition or vaccine, the polypeptide comprising the antigen or model antigen of interest ', is used to vaccinate mice. For example, 'the standard techniques in this technique # may be used, including the appropriate vector encoding the expression antigens of the desired antigen or model antigen to metastasize π% mouse colon cancer cells. Pure germlines that express the antigen or model antigen can then be identified using standard techniques, such as flow cell impulses and Western blotting, at levels sufficient to be used in immunogenicity and / or potency determinations. 98561.doc 200530399 Alternatively, candidate compositions can be tested that include recombinant bacteria expressing an antigen that meets or is derived from an antigen that is endogenous to a tumor cell line (eg, reverse transcription gP70 tumor antigen AHjiCT26. Source, or irregular epitopes AH1_A5). In this type of assay, tumor cells can be implanted in animal models without further modification in order to express additional antigens. Candidate vaccines including the antigen can then be tested. As shown, a vaccine composition including the bacteria described herein is also provided. For example, the present invention provides an epidemic field that includes the recombinant bacteria described herein (see, for example, the Summary of the Invention, Methods of Implementation, Chapters and M, and _ elsewhere in the specification, including those described in the examples below) Recombinant bacteria), in which the polypeptide sequence is part of the polypeptide expressed by the recombinant bacteria, in the form of an isolated protein, a part of a fusion protein, or embedded in a protein shell (according to the recombinant nucleic acid molecule or performance cassette used) Is the antigen. Suitable antigens include any of those described herein (eg, in Chapter IV above). One aspect of the present invention provides a vaccine including a bacterium, wherein the bacterium includes a phenotype card E ′ which includes the following: (a) a first polynucleotide encoding a signal peptide, wherein the first Codons-optimized "(b) a second polynucleotide encoding an antigen, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide; and (c) A starter gene linked to the first and second polynucleotides in a cautious manner allows &quot; Hyperperformance Card E to encode a fusion protein comprising the signal peptide and the polypeptide. In another aspect, the present invention provides a vaccine including bacteria, wherein the bacteria packs a performance card! 'It includes the following: (a) encoding the non-secA 1 bacterial signal peptide 98561.doc -177- 200530399, the first nucleotide, (b) the same translation editing structure as the first polynucleotide, A second polynucleotide encoding an antigen; and (c) a promoter gene operably linked to the first and second multinuclear tadpoles, enabling the expression cassette to encode a fusion protein comprising the signal peptide and the polypeptide . In another aspect, the present invention provides an epidemic bacteria comprising a recombinant Listeria bacterium including a nucleic acid molecule, wherein the nucleic acid molecule includes the following: (1) a first multinucleate encoding a non-Listeria antigen, and In the genus Listeria, its codons are optimized, and (b) a promoter gene operably linked to a polynucleotide encoding an antigen. In another aspect, the present invention provides an epidemic field including a recombinant Listeria bacterium including a performance cassette comprising: (a) a first multi-core encoding a non-Listeria signal peptide (b) The first polynucleus: the same translation and editing framework, encoding a second polynuclear picric acid; and the "promoter gene linked to both the first and second polynucleotides in an operable manner", where the expression Card g encodes a fusion protein that includes both non-Listeria sig. And the antigen. In some embodiments, the vaccine composition includes an antigen-providing cell (APC), which has been infected with any of the recombinant bacteria described herein. f In some embodiments, the vaccine (or immunogen or pharmaceutical composition) does not include 2 donor cells (that is, the vaccine or composition is a bacterial-based vaccine or composition, not an APC-based vaccine Or composition). The method of administration of palatability and epidemic compositions (as well as pharmaceutical and immunogenic compositions) is known in the art and includes oral, intravenous, intradermal, intraperitoneal, intramuscular, intralymphatic, intranasal and Subcutaneous administration path. 98561.doc -178- 200530399 Vaccine formulations are known in the art, and in some embodiments, can include various additives such as preservatives (eg, sodium ethylmercaptothiosalicylate, 2-sodium Phenoxyethanol), stabilizers, adjuvants (such as aluminum hydroxide, aluminum phosphate, cytokines), antibiotics (such as neomycin, streptomycin), and other substances. In some embodiments &apos; the addition of a stabilizer, such as lactose or monosodium glutamate (MSG), stabilizes the vaccine formulation against various conditions, such as temperature changes or freeze-drying processes. In some embodiments, the vaccine formulation may also include a suspension fluid or diluent, such as sterile water, physiological saline, or isotonic buffered saline (eg, phosphate buffered to physiological pH). Vaccines can also contain small amounts of residual material from the manufacturing process. For example, in some embodiments, the vaccine composition is freeze-dried (i.e., freeze-dried). The freeze-dried preparation can be mixed with a sterile solution (such as a citric acid-bicarbonate buffer solution, buffered water, 0.4% physiological saline or the like) before administration. In some embodiments, the vaccine composition may further include additional components known in the art to improve the immune response to the vaccine, such as an adjuvant or a co-stimulatory molecule. In addition to those listed above, possible adjuvants also include chemokine and bacterial nucleic acid sequences, like CpG. In some specific embodiments, the vaccine includes antibodies that improve the immune response to the vaccine, such as CTLA4. In some embodiments, the co-stimulatory molecules include one or more selected from the group consisting of GM-CSF, IL-2, IL-12, IL-14, IL-15, α · 18, β7, β7.2 and B7 -A factor of a group of DCs, and may be incorporated into the vaccine composition of the present invention as needed. Other co-stimulatory molecules are known to those skilled in the art. 98561.doc • 179-200530399 The present invention provides a method for improving a pest or an immunogenic composition comprising a Listeria spp. That expresses an antigen. Any of the multicore, performance cards, and / or performance vectors described herein can be used in these methods. For example, the present invention provides a method for improving a vaccine or an immunogen including Listeria bacteria, and a mouthpiece, wherein the Listeria bacteria express a heterologous prion fusion with a signal peptide, including The sequence encoding the polypeptide, the sequence encoding the signal peptide representing the cassette, or both are codon-optimized. The present invention provides a method for improving a vaccine or immunogenic composition comprising a Listeria bacterium, wherein the Listeria bacterium exhibits a heterologous antigen fused to a signal peptide, including the use of a non-Listeria source and / or Signal peptides from secretory pathways other than seeA1. Methods of producing vaccines of the invention are also provided. For example, in a specific embodiment, a method of generating a vaccine comprising a recombinant bacterium (eg, a recombinant Listeria bacterium) includes introducing a recombinant nucleic acid molecule into a bacterium, introducing a performance cassette or a performance vector as described herein In bacteria, wherein the recombinant nucleic acid molecule, expression cassette or expression vector encodes an antigen. For example, in some embodiments, the 'recombinant nucleic acid molecule includes (a) a first polynucleotide encoding a signal peptide that is natural to the bacterium, wherein the first polynucleotide is expressed for performance in bacteria. Codon-optimization, and (b) a second polynucleotide encoding an antigen, wherein the second polynucleotide is the same translation editing framework as the first polypicrin, where the encoding includes the signal peptide The recombinant nucleic acid molecule of the fusion protein with the antigen is introduced into bacteria to produce a vaccine. In some embodiments, the recombinant nucleic acid molecule includes (a) a first polynucleotide encoding a non-secAl bacterial signal peptide, and (b) an 98561.doc -180-200530399 second polynucleotide encoding an antigen. 'Where the second polynucleic acid is the same translation editing structure as the first polynucleic acid, and wherein a recombinant nucleic acid molecule encoding a fusion protein including the signal and the polypeptide is incorporated into a bacterium to generate a vaccine. In some specific embodiments, the recombinant nucleic acid molecule introduced into the bacterium to produce a vaccine is a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises a first polynucleotide encoding a non-Listeria signal peptide, and The same translation and editing architecture as the first polynucleic acid, the second polynucleic acid encoding an antigen, wherein the recombinant nucleic acid molecule encodes a fusion protein including a non-Listeria signal peptide and the antigen. In some specific embodiments, the recombinant nucleic acid molecule used to generate the vaccine is a recombinant tritium molecule, including the first polynucleotide of the trichomonad bacterial autolysin or a catalytic fragment or a variant having a catalytic function And a second polynucleotide encoding a polypeptide, wherein the second multinucleus is the same translation and editing framework as the first multinucleus; the recombinant nucleic acid molecule encodes a protein chimera in the first multinucleus; wherein the non-Liszt The Mycobacterium polypeptide is fused with autolysin or a fragment having a catalytic function or a variant having a catalytic function, or is inserted into autolysin or a fragment having a catalytic function or a variant having a catalytic function. In some other specific embodiments, the method of producing a vaccine includes providing a Listeria bacterium, which includes encoding a polycistronic expression card and encoding the polycistronic expression card to encode at least two separate non- Liszt is transferred to Li Xi ^ Qiaofei Ziyan Fufu genus polypeptide, introduced into Listeria bacteria, to produce vaccines. Also provided is a kit comprising any of the recombinant nucleic acid molecules, expression cassettes, vectors, killers and / or compositions of the invention. X. Method of Use 98561.doc -181-200530399 Provides a variety of methods for using the recombinant bacteria or pharmaceutical, immunogen, or vaccine composition 'immunological response and / or preventing or treating conditions in a host. In some embodiments, the condition being treated or prevented is a disease. In some specific implementations, the disease is cancer. In some embodiments, the disease is H. In addition, recombinant bacteria can also be used: production and isolation of heterologous proteins, such as mammalian proteins. As used herein, "treatment" or "treatment" (for a condition or disease) is a method of obtaining favorable or desired results, including and preferably clinical results. For the purposes of this invention, Or desired results include, but are not limited to, the following-or more: improving conditions associated with the disease, curing the disease, reducing the severity of the disease, delaying the progression of the disease, reducing one or more disease-related symptoms, increasing the risk of disease The patient's quality of life, and / or prolonged survival. Also for the present invention, favorable or desired outcomes related to the condition include, but are not limited to, one or more of the following: improving the condition, curing the condition, reducing the severity of the condition , Delaying the progress of a condition, reducing one or more condition-related symptoms, increasing the quality of life of a person suffering from the condition, and / or prolonging survival. For example, specific uses in which the compositions described herein are used to treat cancer In the actual cases, the 'favorable or desired results include, but are not limited to, one or more of the following: reduction (or destruction) of the proliferation or reduction of neoplasms or cancer cells Metastasis of the cells found in cancer, shrinking the size of tumors, reducing symptoms caused by cancer, increasing the quality of life of those with cancer, reducing the dose of other medicines used to treat the disease, delaying the progression of cancer, and And / or prolong the survival of cancer patients. When used herein, &quot; prevent, disease, or &quot; protect the host &quot; from disease 98561.doc -182- 200530399 (in this context, the terms "including But is not limited to one or more of the following: discontinuing, delaying, hindering, slowing down, obstructing and / or delaying the onset or progression of the disease, stabilizing the progress of the disease, and / or delaying the development of the disease. "Prevention" Conditions or &quot; Protect the host from conditions (interchangeable in this context), including 4 is not limited to one or more of the following: suspension, delay, obstruction, slowing, obstruction, and / or delaying the onset or progression of conditions , The progress of a stable condition, and / or the state of delay; the development of a brother. The length of the prevention period can be changed depending on the disease or condition of the treatment and the history of the domain individual. For example, in the design When a vaccine is used to prevent or protect against an infectious disease caused by a pathogen, &quot; prevent &quot; disease: or "protect the host" from disease 'includes but is not limited to one or the following items. Suspension, Delay, Obstruction, Slow down, hinder and / or delay the onset of a pathogen-infected host, or the onset or progression of a disease associated with a pathogen-infected host, and / or stabilize the progress of a disease associated with a pathogen-infected host. Furthermore, For example, where the vaccine is an anti-cancer vaccine, "prevent disease &lt; &gt; 呆 Don't hurt the master &quot; Free from disease, including but not limited to one or more of the following: suspension, delay, obstruction, slowing, hindering and / or delaying the development of cancer or metastasis, cancer Progression or recurrence of cancer. The invention-aspect provides a method of inducing an immune response to an antigen in a host, comprising administering to the host an effective amount of a recombinant bacterium described herein, or an effective amount of a composition comprising a recombinant bacterium described herein ( (E.g., a pharmaceutical composition, an immunogen composition, or a vaccine) (see, for example, Summary of the Invention 'Implementing Methods, Article IX, and examples below). In some specific embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression card and / or expression vector in a recombinant bacterium includes an antigen, or an anti-98561.doc -183-200530399 original fusion protein or protein insert. Fit. For example, in one aspect, the present invention provides a method of inducing an immune response to an antigen in a host 'comprising administering to the host an effective amount of a composition including a recombinant bacterium, wherein the recombinant bacterium includes a performance cassette, which includes the following ... (a) the first polynucleotide encoding a signal peptide, wherein the first polynucleotide is codon-optimized for performance in bacteria; (b) the first polynucleotide encoding an antigen, wherein the The second polynucleotide is the same translation editing framework as the first polynucleotide; and (c) a promoter gene operably linked to the first and second polynucleotides, making the expression cassette It is possible to encode a fusion protein comprising the signal peptide and the antigen. In another aspect, the invention provides a method for inducing an immune response to an antigen in a host, comprising administering to the host an effective amount of a composition including a recombinant bacterium, the recombinant bacterium comprising a performance cassette, wherein the performance cassette includes the following : (A) the first polynucleotide encoding a non-secAl bacterial signal peptide; (b) the same translation and editing architecture as the first polynucleotide, the second multicore encoding the antigen, and In the M mode, Xiao Shoukou's second multinucleated promoter gene is connected to both, so that the performance card can encode a fusion protein including the h-shaped and the antigen. In another aspect, the present invention provides a method for inducing an immune response to a non-Listeria antigen in a host, comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium, said recombinant Listeria Bacterium bacteria include a nucleic acid molecule 'wherein the nucleic acid molecule includes the following: coding the polynuclear picric acid of the Matteria antigen and optimizing its codons in Listeria; and (b) being operable The method is linked to a promoter gene that encodes as many as 98561.doc -184- 200530399 nucleotides. In another aspect, the present invention provides a method for inducing an immune response to an antigen in a host, which comprises administering to the host an effective 3 mile recombinant Listeria monocytogenes #peptide and the polypeptide Fusion protein of both. In some embodiments of the method of inducing an immune response described herein, &apos; is administered to bacteria in the form of a pharmaceutical combination, an immunogen composition, and / or a vaccine composition. 'The recombinant Listeria bacterium includes a performance card, which includes the following: ⑷ encoding the non-. Listeria signal peptide of the _th multi-core m⑻ with the same translational editing structure as the first multi-core money , A second polynuclear m encoding an antigen, and a promoter gene operably linked to both the first and the second polynucleic acid, wherein the performance card encoding includes non-Liss in some embodiments, The immune response is an MHC class I immune response. In other specific embodiments, the immune response is a MHC class I immune response. In other embodiments, the immune response induced by administering the bacteria or composition is both an MHC class I and an MHC class II response. Thus, in some embodiments, the immune response includes a CD4 + T-cell response. In some embodiments, the immune response includes a CD8 + T-cell response. In some embodiments, the immune response includes both a CD4 + T-cell response and a CD8 + T-cell response. In some embodiments, the immune response includes a cellular response and / or a T-cell response. The B-cell response can be measured by determining the potency of the antibody against the antigen using methods known to those skilled in the art. In some embodiments, the immune response induced by the composition described herein is a humoral response. In other specific embodiments, the induced immune response is 98561.doc -185- 200530399. The epidemic response is a cellular immune response. In some embodiments, the immune response includes both cellular and humoral immune responses. In some embodiments, the immune response is antigen-specific. In some embodiments, the immune response is an antigen-specific τ-cell response. In addition to providing a method for inducing an immune response, the invention also provides a method for preventing or treating a condition in a host (e.g., an individual, such as a human patient). In some embodiments, the condition is a disease. The method comprises administering to the host a recombinant bacterium effective herein as described herein, or a composition comprising a recombinant bacterium described herein (see, e.g., the Summary of the Invention, Implementing Methods I, VIII, and IX) Chapter, or examples below). In some embodiments &apos; the disease is cancer. In some embodiments, the disease is an infectious disease. For example, one aspect of the present invention provides a method for preventing or treating a disease (or condition) in a host, comprising administering to the host an effective amount of a composition including a bacterium, wherein the bacterium includes a performance cassette comprising the following: ( a) the first polynucleotide encoding a signal peptide, wherein the first polynucleotide is codon-optimized for performance in bacteria; (b) encoding a polypeptide (eg, an antigen and / or therapeutic mammalian protein) A second polynucleotide, wherein the second nucleotide is the same translation editing framework as the first polynucleotide; and (C) interoperably interacts with the first and second multicores The nucleotide-linked promoter gene 'enables the performance card to encode a fusion protein comprising the signal peptide and the antigen. In another aspect, the invention provides the prevention or treatment of a disease (or condition) in a host, including administering to the host an effective amount of a group comprising recombinant bacteria 98561.doc 200530399, wherein the bacteria includes a performance cassette, and wherein The performance cassette includes the following: (a) the first polynucleotide encoding a non-secAl bacterial signal peptide; (b) the same translational editing architecture as the first polynucleotide, encoding a polypeptide (such as an antigen and (Or mammalian protein) second polynucleotide; and (c) a promoter gene operably linked to the first and second polynucleotides, enabling the performance card E to include the signal peptide and A fusion protein of this antigen. In another aspect, the present invention provides a method for preventing or treating a disease (or condition) in a host, comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium, wherein the bacterium includes a nucleic acid molecule, wherein the nucleic acid Molecules include the following: (a) Polynucleotides encoding non-Listeria spp. Polypeptides (such as antigens and / or therapeutic mammalian proteins), and their codons are optimized for performance in Listeria spp. And) a promoter gene operably linked to a polynucleotide encoding the antigen. In another aspect, the present invention provides a method for preventing or treating a disease (or condition) in a host, comprising administering to the host an effective amount of a substance including a recombinant Listeria spp., A field strain, and a substance, the bacteria Includes a performance cassette that includes: (a) a first polynucleotide encoding a non-Listeria spp. Signal peptide; encoding a polypeptide (such as an antigen and / Or treating a second polynucleotide of 1 * born milk animal protein); and (c) a promoter gene operably linked to both the first and second polynucleotides, where the expression cassette is 5 Encodes a fusion protein that includes both a non-Listeria signal peptide and the polypeptide. In some embodiments, the disease is cancer. In some specific implementations of 98561.doc -187- 200530399 cases, breast cancer, visceral cancer, and liver cancer are either to be treated or not. The disease is melanoma, pill cancer, Indian nest cancer, squamous :: Cancer, lung cancer, brain cancer, gall cancer, or prostate cancer. In two; and gastrointestinal cancer, cervical cancer, kidney cancer, color. In the embodiment, the cancer is a black monk. In some alternatives, the cancer is also pancreatic cancer. In some ^ r. _ / Is for ... bowel cancer. In some specific embodiments, the cancer is stigma adenocarcinoma. In these two-in-one embodiments, the cancer is metastatic. In other specific embodiments, the ^ M ^ μ disease is an autoimmune disease. In other specific embodiments, &amp;μ;C; is an infectious disease, or other diseases caused by a pathogen, such as a virus, bacteria, jellyfish pain or protozoa. In some embodiments &apos; the disease is an infectious disease. In two specific embodiments, the use of recombinant bacteria in preventing or treating cancer includes delivering the recombinant bacteria to cells of an individual's immune system, preventing or treating existing cancer, or individuals with increased risk factors, such as environmental storm roads and / Or family tendencies. In other specific embodiments, the use of recombinant bacteria for the prevention or treatment of cancer includes the delivery of recombinant bacteria to individuals who have had a tumor removed or who have had cancer in the past but are currently temporarily relieved. In some embodiments, a composition comprising a recombinant bacterium described herein is administered to a host to induce a CD4 + T-cell response in the host. In some other specific embodiments, a composition comprising a recombinant bacterium described herein is administered to a host to induce a CD8 + T-cell response in the host. In some embodiments, a composition comprising a recombinant bacterium described herein is administered to a host to induce both a CD4 + T-cell response and a CD8 + T-cell 98561.doc -188- 200530399 response in the host. The efficacy of humans or other therapeutic conditions can be assessed in individuals, such as mice. Recognize the mouse model as potency in humans ^ Assess and define the vaccine of the invention. The possibility of using the money model to demonstrate the efficacy of the vaccine in vivo. The ability of a vaccine to provide preventive or therapeutic efficacy against a particular disease can be assessed. For example, in the case of an infectious disease, a mouse population can be vaccinated with a suitable vaccine of the present invention in a desired amount, and the recombinant bacteria express an antigen related to the infectious disease. Mice were then infected with infectious agents associated with the vaccine antigens and evaluated for protection against infection. The progression of infectious diseases can be observed relative to the control group (not vaccinated, or only vaccinated with bacteria or bacteria without appropriate antigens). In the case of a cancer vaccine, a tumor cell pattern is available, in which the desired expression can be expressed either before (treatment mode) or after (prevention mode) inoculation with a composition comprising the bacteria containing the desired tumor antigen of the present invention. Tumor cell lines of tumor antigens were injected into the mouse population. Vaccination with recombinant bacteria containing tumor antigens can be compared with control group populations that have not been vaccinated, inoculated with a vehicle, or inoculated with recombinant bacteria that exhibit irrelevant antigens. Vaccine efficacy in such models is evaluated from the perspective of tumor volume as a function of time after tumor injection, or from the perspective of survival populations as a function of time after tumor injection (e.g., Example 31 ID). In a specific embodiment, the tumor volume in a mouse vaccinated with a composition comprising a recombinant bacterium is about 5% lower than the tumor volume in a mouse that has not been vaccinated or vaccinated, or a bacterium that exhibits an unrelated antigen, About 10%, about 25%, about 50%, about 75%, about 90%, or about 100%. In other specific examples, 98561.doc -189- 200530399 at least about 10, about i7, or about a day after tumor implantation in a mouse, differences in tumor volume are observed. In a specific embodiment, the median survival time in mice vaccinated with a composition that includes recombinant bacteria is at least about 2, longer than in mice that have not been vaccinated or vaccinated with a vehicle or bacteria that exhibit unrelated antigens. About 5, about 7 or at least about 10 days. The host (i.e., individual) in the methods described herein is any spinal animal, preferably mammals, including domestic animals, race animals, and primates, including humans. In some embodiments, the host is a mammal. In some embodiments, the host is human. Recombinant bacteria or compositions comprising the strain can be delivered by any suitable method, such as intradermal, -p, &amp; subcutaneous intraperitoneal, intravenous, intramuscular, intralymphatic, oral or intranasal, Hereby, any path related to a particular malignant or infectious disease or other condition. The composition including the recombinant bacteria and the immunostimulant can be simultaneously, continuously, or slashed to the master. Examples of immunostimulants include, but are not limited to, IE. , IL-12, GMCSF, m 1 ^ iL B'BT.I'bu, and B7_dc and il] 4. Additional examples of stimulants are provided in Chapter K above. When using an effective amount of bacteria, composition, or immunogen composition in this document, Α, 上, 上-> ^ ° f is enough to make a favorable or desired result. With regard to preventive uses, the right outcomes of right-hand brake phase disease include the elimination or reduction of the severity of the disease or the delay of the onset of the disease, including the biochemical, electrophysiological, and therapeutic effects of the disease and / or仃 is the symptom, its complications, and the intermediate pathological phenotype of the middle brother who appears during the development of the disease. As for therapeutic use, 98561.doc 200530399 favorable or desired results include clinical results, such as inhibition or suppression of the disease, reduction of one or more symptoms (biochemical, histological, and / or behavioral), including the disease Complications, and intermediate pathological phenotypes that occur during the development of the disease, increase the quality of life of those suffering from the disease, reduce the dose of other medicines needed to treat the disease, increase the effectiveness of other medicines, delay the progress of the disease, and / or Extend patient survival. The effective dose can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly accomplish prevention or treatment. As is known clinically, along with other drugs, compounds, or pharmaceutical compositions, the effective content of the drug, compound, or pharmaceutical composition may or may not be reached. Therefore, for the administration of one or more therapeutic agents, an effective dose can be considered, and if the same or more other preparations are possible or achieve the desired result, a single preparation can be considered to be administered in an effective amount. In some embodiments, for the treatment of cancer, the effective content includes a content that results in a desired immune response, wherein the immune response slows the growth of the target tumor, reduces the size of the tumor, or preferably eliminates the tumor completely. Administration of the vaccine can be repeated at appropriate intervals and can be administered simultaneously at different locations in the individual being vaccinated. In some embodiments, to prevent cancer, the effective content includes agents s that will produce a protective immune response as a result, which significantly reduces the likelihood that an individual will develop cancer. Vaccination The ingestion method may consist of a single dose, or it may be repeated at appropriate intervals until a protective immune response is established. In some embodiments, an individual's cancer treatment may be initiated when the individual has been diagnosed with cancer, or may be combined with other treatments. For example, 98561.doc -191-200530399 may be used to treat an individual whose tumor has been surgically removed or to be treated with radiation therapy or by chemotherapy in order to reduce or exclude any residual tumors in the individual, or to reduce the recurrence of cancer. risk. In some embodiments, an individual's cancer prevention treatment will begin in an individual with an increased risk of developing certain cancers due to environmental conditions or genetic advantages. The dosage of the pharmaceutical composition or vaccine administered to the host will vary depending on the host's species, the size of the host, and the condition or disease of the host. The dosage of the composition will also depend on the frequency and route of administration of the composition. The precise dose is chosen by the individual physician from the perspective of the patient to be treated. In some embodiments, the pharmaceutical composition, immunogen composition, or vaccine used in the method includes a recombinant bacterium that includes a recombinant nucleic acid molecule, a performance cassette, and / or a performance vector as described herein. In some embodiments, 'the recombinant bacterium is a modified and / or mutated bacterium, such as applied on June 30, 2004, US Patent Application No. 10 / 883,599 by Thomas W. Dubensky, Jr. et al. , Entitled, "Modified Free-Living Microbes, Vaccine Compositions and Methods of Use Thereof", US Patent Publication No. 2004 / 〇228877 No. and those described in U.S. Patent Publication No. 2004/0197343, each of which is incorporated herein by reference in its entirety. In some embodiments, a single dose includes such modified and / or mutant species of bacteria, Or any other pharmaceutical composition or vaccine of recombinant bacteria described herein, including from about 102 to 200 g, bacillus organisms. In other specific embodiments, a single dose includes about 10 To about 100 bacterial organisms. In another specific embodiment 98561.doc 200530399 'early-dose * includes from about 106 to about ι &quot; bacterial organisms. In another In the detailed examples, a 'single dose of a pharmaceutical composition or vaccine includes from about to about bacterial organisms. In another specific embodiment, an earlier dose! A pharmaceutical composition or vaccine includes from about 107 to about 109 Bacterial organisms. ★ In some embodiments, a single-dose includes at least about bacterial organisms. In some embodiments, a single-dose composition includes at least about 1 × 10 5 organisms. In other specific embodiments, a single The dosage, and &amp; substance or disease field includes at least about χχ 〇 06 bacterial organisms. In another embodiment, a 'single-dose composition or vaccine includes at least about χχ 〇 07 bacterial organisms. In some specific In embodiments, a single dose of a pharmaceutical composition, immunogen, or cloud or disease field includes a recombinant, modified, or canine variant bacterium described herein, which includes from about i CFU / kg to about 1x10 CFU / kg (CFU = colony forming unit). In some embodiments, a single dose of the composition includes from about 100 CFU / kg to about 0 CFU / A kg. In other specific embodiments, a single dose of the composition or vaccine includes from about 1 x 102 CFU / kg to about 1 x 108 cfu / kg. In another specific embodiment, a single dose of the composition or vaccine includes from about 1 x 10 3 CFU / kg to about 1 × 10 8 CFU / kg. In other specific embodiments, a single dose of the composition or vaccine includes from about ixio4 cfu / kg to about 1 × 107 cfu / kg. In some embodiments, a &apos; single-dose composition includes at least about 1 CFU / kg. In some octahedral embodiments, a single dose of the composition includes at least about 10 Cfu / 98561.doc -193- 200530399 kg. In other specific embodiments, a single dose of the composition or vaccine includes at least about 1 x 102 cfu / kg. In another specific embodiment, a single dose of a composition or vaccine includes at least about 1xlo3cfu / kg. In another specific embodiment, a single dose of a composition or vaccine includes at least about 1 x 104 CFU / kg. In some embodiments, methods known in the art can be used to infer the appropriate (and effective) dose content of one host, such as a mouse, from the ld50 data of another host, such as a human. In some embodiments, the pharmaceutical composition, immunogenic composition, or vaccine includes an antigen-providing cell, such as a dendritic cell, which has been included in the recombinant nucleic acid molecule, expression cassette, and / or expression vector described herein Recombinant bacterial infection. In some embodiments, the bacterium has been modified and / or mutated, such as US Patent Application No. 10 / 883,599 filed on June 30, 2004, and US Patent Publication No. 2004/0228877 and Those described in US 2004/0197343 are each fully incorporated herein by reference. For example, the following: International Application No. PCT / US2004 / 23881, filed July 23, 2004, by Thomas W. Dubensky, Jr. et al., Entitled "Providing-Antigen Cell Vaccines, and Methods of Use (Eight 1 ^ 1§611-Presenting Cell Vaccines and Methods of Use Thereof) ,; US Patent Application No. 10 / 883,599, filed on June 30, 2004; US Patent Publication No. 2004/0228877; and US Patent Publication Case No. US 2004/0197343 describes vaccines based on such donor-antigen-based cells, each of which is incorporated herein by reference in its entirety. In some embodiments, based on donor-antigen-based cells Individual doses of the vaccine 9856I.doc -194- 200530399 include bacteria 'such as those described herein, including cells that provide -antigen between about 乂 χ 乂 03 and about 1 抗原 1010. In some specific embodiments Individual doses of the vaccine include between about 1 x 105 and about 1 x 10 9 cells that provide antigens. In some embodiments, individual doses of the vaccine include between about 1 x 10 7 and about 1 x 10 cells. In some embodiments, the dosage unit is preferably administered multiple times in a day, or over a period of one week, one month, or one or more years. In some embodiments, the dosage is administered daily. The unit lasts for several days, or once a week for several weeks. The present invention further provides any of the recombinant bacteria described herein (ie, any bacteria including recombinant nucleic acid molecules, expression cassettes or vectors described herein) for use in manufacturing Use of a pharmaceutical to induce an immune response to an antigen in a host ', wherein the polypeptide encoded by the recombinant nucleic acid molecule, expression kakun and / or vector in the bacterium includes an antigen. In some specific embodiments, the antigen Is a heterologous antigen. The present invention also provides the use of a recombinant bacterium described herein for the manufacture of a medicament for the prevention or treatment of a condition (eg, a disease, such as cancer or an infectious disease) in a host. The present invention also provides The use of a recombinant bacterium described herein to induce an immune response to an antigen in a host, wherein the recombinant nucleic acid molecule, The polypeptides encoded by the cassettes and / or vectors include antigens. The invention further provides the use of recombinant bacteria described herein to prevent or treat conditions (such as diseases) in a host. The invention also provides the induction of MHC class I antigens or MHC A method of presenting a class II antigen on a donor-antigen cell, comprising contacting a cell described herein with a donor-antigen cell. 98561.doc -195- 200530399 The present invention further provides a method of A method of immune response comprising the steps of: (i) contacting a recombinant bacterium described herein with a host-provided antigen-providing cell under appropriate conditions for a period of time sufficient to load the antigen-providing cell; and The donor-antigen-producing cells are administered to a host. Recombinant nucleic acid molecules, performance cards and other possible uses for bacteria will be recognized by those skilled in the art. For example, the recombinant nucleic acid molecules, expression cards E, vectors, and recombinant bacteria (and other host cells) described herein can be used to generate and isolate heterologous polym. In another aspect, the invention provides methods for expressing polypeptides in bacteria. Including the introduction of a performance card or vector described herein into a bacterium (eg, via metastatic infection, transformation, or conjugation); and ⑻ growth of the bacterium in culture under conditions suitable for protein expression. In other aspects, the invention provides a method of producing an isolated polypeptide 'comprising the following: (i) introducing a performance cassette or vector described herein into a bacterium (eg, via metastatic infection, transformation, or conjugation); Conditions τ suitable for the performance of egg clams, growing the clams in bacterial culture, and (C) isolating proteins from the cell culture of bacteria. Appropriate methods of transformation, metastatic infection, and co-consumption are well known to those skilled in the art, such as cultivating and growing bacteria, and isolating secretory or non-secretory proteins from cell culture. Examples The following examples are provided to explain, but not limit, the invention. Example I Preparation of a representative mutant Listeria strain The Listeria strain was derived from 1 () 4 () 3s (Bish () p et al., L Immunol. 9856l.doc -196- 200530399 139: 2005 (1987 )). By means of SOE-PCR and dual gene exchange using established methods (Gamilli et al., Mol. Microbiol. 8: 143 (1993)), the Listeria spp. Lines deleted from the framework with the specified genes were produced. The mutant strain line LLO L461T (DP-L4017) is described in Glomski et al., J. Cell. Biol. 156: 1029 (2002), which is incorporated herein by reference. The actA-mutant is the DP-L3078 strain described in Skoble et al., J. of Cell Biology, 150: 527-5 37 (2000), which is hereby incorporated by reference in its entirety, which has eliminated its original Phage. (The elimination of prophages is described in (Lauer et al., J. Bacteriol. 184: 4177 (2002); US Patent Publication No. 2003/0203472)). U.S. Provisional Application No. 60 / 446,051 filed February 6, 2003 under L4029 / uvrAB (see, for example, Example 7 of the application), and actAivrAB is described in U.S. Patent Publication No. 2004/0197343 · Construction of strains. Under the provisions of the Budapest Treaty on the Deposit of Internationally Recognized Microorganisms for the Purposes of Patent Procedures, DP-L4029uvrAB (Listeria monocytogenes actA7uvrAB_double-deletion mutant) was deposited in American typical cultures on October 3, 2003 Collection Center (ATCC), 10801 University Blvd, Manassas, Virginia 20110-2209, United States of American, and named PTA-5 563. Additional descriptions of the mutant Listeria spp. Are provided in the following applications or publications, all of which are incorporated herein by reference: U.S. Patent Publication No. 2004/0228877; U.S. Patent Publication No. 2004 / 0 1973 43; PCT International Application No. PCT / US2004 / 23 881 filed on July 23, 2004; and US Patent Application No. 10 / 883,599 filed on June 30, 2004. In addition, the representative treaty Listeria monocytogenes AactAAinlB has been doubled on October 3, 2003 under the provisions of the Budapest Treaty on the Deposit of Microorganisms Recognized by the Patent Procedure Country 98561.doc -197- 200530399. Deletion mutants are deposited at the American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, Virginia 20110, 2209,

United States of America and named PTA-5562. A non-limiting example of a method for deleting genes in Listeria monocytogenes produces attenuated mutants provided in Example 24 below. Example 2 Constructing a Listeria strain expressing AH1 / OVA or AH1-A5 / OVA. Preparation of a truncated form of model antigen ovalbumin (OVA), immunodominant epitope derived from mouse colorectal cancer (CT26 ), Called AH1 (SPSYVYHQF (sequence number 72)), and the altered epitope AH1-A5 (SPSYAYHQF (sequence number 73); Slansky et al., Immunity, 13: 529-538 (2000)) Mutant Listeria strains. Use a pPL2 integration vector (Lauer et al., J. Bacteriol. 184: 4177 (2002); U.S. Patent Publication No. 2003/0203472) to navigate OVA and a single copy containing a single copy integrated into a harmless location in the Listeria genome AH1-A5 / OVA recombinant Listeria strain. A. Constructing Listeria spp. (DP-L4056) expressing OVA First, a hemolysin-deleted LLO including a fusion with truncated OVA was prepared, and an antigen expression card g (pPL2 / LLO-OVA) was incorporated into the pPL2 integration vector. By introducing PPL2 / LLO-OVA into the phage-depleted Listeria monocytogenes strain DP-L4056 at the PSA (Phage from ScottA) attachment site, the Listeria spp. Were derived -OVA vaccine strain. Hemolysin-deleted LLO was amplified using PCR using the following template and primers: 98561.doc -198- 200530399 Source: DP-L4056 genomic DNA primers: forward (KpnI-LLO nucleotides 1257-1276): 5, -CTCTGGTACCTCCTTTGATTAGTATATTC ( Preface, J. No. 74) (Tm: LLO · Specific: 52 ° C. Overall: 80 ° C.) Reverse (BamHI-XhoI_LLO Nucleotide 281 1-2792): S ^ CAATGGATCCCTCGAGATCATAATTTACTTCATC CC (Sequence No. 75 ) (Tm: LLO-specific: 52 ° C. Overall: 102 ° C.) PCR was also used to extend the truncated OVA using the following templates and primers: Source: pDP3616 from DP-E3616 E. coli Somatic DNA (Higgins et al., Mol. Molbiol. 31: 1631-1641 (1999)). Primers: Forward (XhoI_NcoI OVA cDNA nucleotides 174-186): 5, -ATTTCTCGAGTCCATGGGGGGTTCTCATCATC (sequence number 76) (Tm: OVA-specific: 60 ° C. Overall: 88 ° C.) Reverse (Xhol-Notl- Hindlll): 5, -GGTGCTCGAGTGCGGCCGCAAGCT! Y queue No. 77) (Tm: overall: 82t.) A draft to complete the construction process involves cutting the LLO expansion region using κρηΙ and BamHI, and inserting the KpnI / BamHI vector into the pPL2 vector ( pPL2-LLO). The OVA extension was then cut with Xhol and Notl and inserted into pPL2-LLO, which had been cut with Xhol / Notl, 98561.doc -199- 200530399. (Note: The pPL2 vector does not contain any Xhol positions; pDP-3616 contains an Xhol position, which is used in the design of the OVA reverse primer). The construct pPL2 / LLO-OVA (KpnI-LLO-XhoI-OVA-NotI) was confirmed by restriction analysis and sequenced. The plastid PPL2 / LLO-OVA was introduced into E. coli by transformation, and then introduced via conjugation and integrated into Listeria (DP-L4056), exactly as described by Lauer et al. (Or into other In the desired Listeria strain, such as inlB_ mutant or inlB_actA · double mutant). B. Constructing a Listeria strain expressing AH1 / OVA or AH1-A5 / OVA To prepare a Listeria expressing AH1 / OVA or AH1-A5 / OVA antigen sequences, first prepare an antigen-carrying insert from an oligonucleotide And then ligated into the vector pPL2 / LLO-OVA (prepared as described above). The following oligonucleotides were used to make AH1 or AH1-A5 inserts: AH1 epitope insert (Clal-PstI compatible ends): Upper oligo (above AH1): 5, -CGATTCCCCTAGTTATGTTTACCACCAATTTGCTG CA (sequence number 78) Lower oligo (below AH1): 5, -GCAAATTGGTGGTAAACATAACTAGGGGAAT (sequence number 79) AH1-A5 epitope insert (Clal-Avall compatible ends): The sequence of AH1-A5 epitope is SPSYAYHQF (Serial No. 73) (5, -AGT CCA AGT TAT GCA TAT CAT CAA TTT-3 '(Serial No. 80)). 98561.doc -200- 200530399 Above: 5'-CGATAGTCCAAGTTATGCATATCATCAATTTGC (Sequence No. 81) Below: 5, _GTCGCAAATTGATGATATGCATAACTTGGACTAT (Sequence No. 82) Mix the specific epitope oligonucleotide pairs together at equal mole ratios , Heat to 95 ° C for 5 minutes. The oligonucleotide mixture was then allowed to cool slowly. Then, at a molar ratio of 200 to 1, the ligated oligonucleotide pairs were linked to pPL2-LLO / OVA plastids prepared by digestion with the relevant restriction enzymes. The identity of the new construct can be confirmed by restriction analysis and / or sequencing. This plastid can then be introduced into E. coli by transformation, and then introduced and integrated into the Listeria spp. (DP-L4056) via conjugation, exactly as described by Lauer et al., Or other thoughts. Within the Listeria spp. Line, such as the actA_ mutant line (DP_L0429), the LLO L461T line (DP-L4017), or the actA- / uvrAB_ line (DP-L4029uvrAB). Example 3. Construction of Listeria spp. Polynucleotides and expression cassettes A. Selection vectors Insert selected heterologous antigen expression cassette molecular constructs into pPL2 (Lauer et al., J. Bacteriol. 2002) or pAM401 (Wirth et al.) , J. Bacteriol. 1M: 83 1-836), modified to contain a multiple selection sequence of pPL2 (Aatll small fragment, 171 base pairs), inserted into the tetracycline resistance gene, dull Xbal and Nrul cognitive locations (pAM401-MCS, Figure 32). Typically, the hly promoter gene and (selected) signal peptide sequences are inserted between the pK2 and pam401-MCS plastid vectors, which are unique to the κρηΙ and BamHI positions. 98561.doc -201-200530399 The selected EphA2 gene (sometimes modified to include terminal and C-terminal epitope tags; see description below) is then selected for the BamHI and SacI positions unique to these constructs between. The molecular structure based on the PAM401-MCS plastid carrier was introduced into the selected Listeria monocytogenes strain by electropenetration, and it has also used the method commonly used by this artist to utilize lysobacteria Enzyme treated. The expected plastid structure in Listeria-transferred organisms was confirmed by isolating DNA by restriction enzyme analysis from colonies formed on BHI agar plates (10 μg / ml) containing aerobicin. The expression and secretion of heterologous proteins were measured using a recombinant Listeria spp. Transformed with a cassette construct based on various pAM401-MCS-based heterologous proteins. According to the method previously described [Lauer et al., J. Bacteriol. 184, 4177-4186 (2002)], a PPL2-based heterologous protein expression cassette construct was inserted into the genome of the selected Listeria strain Within the tRNAArg gene. In short, the pPL2 heterologous protein expression card was first introduced into the E. coli host strain SM10 by electro-penetration or by chemical tools [Simon et al., Bi0 / Techn〇1〇gy 1: 784 · 791 (1983)]. Subsequently, plastid-based plastids were transferred from the transformed SM10 into the selected Listeria strains by conjugation. Drugs containing 7.5 micrograms of aeromycin per milliliter and 200 micrograms of streptomycin per milliliter

Use forward primer NC16 (5f- select individual colonies and select by PCR, gtcaaaacatacgctcttatg-3 '(sequence number 94 98561.doc • 202-200530399)) and reverse primer PL95 (5f-acataatcagtccaaagtagatgc_3' (sequence number 95) )) The primer pair. The selected colonies had a PPL2-based plastid, which was inserted into the tRNAArg gene in the genome of the selected Listeria strain to generate a diagnostic DNA amplification region with 499 base pairs. B. Promoter Gene The heterologous protein expression cassette contains a prfA-dependent hly promoter gene, which controls the transcription of the gene encoding Listerialysin O (LLO) and is activated in the microenvironment of infected cells. Using the primer pair shown below, the nucleotides 205 5 86-206000 (414 base pairs) were amplified from Listeria monocytogenes, strain DP-L4056 by PCR. The expanded region includes the hly promoter and the first 28 amino acids of LLO, including the secAl signal peptide (see above) and the PEST functional site. The expected sequence for this region of Listeria monocytogenes strain EGD can be found in GenBank (accession number: gi 丨 16802048 | ref | NC—003210.1 | [16802048]). The primers used in the PCR reaction are as follows: Primer pair: forward (Kpnl-LLO nucleotide 1257-1276): 5, -CTCTGGTACCTCCTTTGATTAGTATATTC (sequence number 74) reverse (BamHI-LLO nucleotide): 5, -CTCTGGATCCATCCGCGTGTTTCTTTTCG ( SEQ ID No. 84) (Subscripts are added to the recognition position of the endonuclease restriction enzyme) According to the manufacturer's instructions, the 422 base pair PCR amplification region was cloned 98561.doc -203-200530399 into the plastid vector pCR- XL-TOPO (Invitrogen, Carlsbad, CA). The nucleotide sequence of Listeria-specific test bases in the pCR-XL-TOPO-hly promoter plastid pure line was determined. Compared with the EGD line, the Listeria monocytogenes line DP-L4056 contains 8 nucleotide base changes and is flanked by the prfA box in the hly promoter gene. As shown in Figure 1 below, the hly promoter genes of the Listeria monocytogenes DP-L405 6 and EGD lines are lined up. By digestion with κρηΙ and BamHI, from the pCR-XL-TOPO-hly promoter plastid germline, 422 base pairs of DNA equivalent to the hly promoter and the secAl LLO signal peptide were released, and according to the skilled artist The well-known traditional method is to clone into pPL2 plastid vector (Lauer et al. 2002 J. Bact.). This plastid is called pPL2-hlyP (natural). C. Shine-Dalgarno sequence A poly-purine Shine-Dalgarno sequence is contained at the 3 'end of the promoter gene. It is necessary for the 30S ribosomal subunit (via 16S rRNA) to coincide with the heterologous gene RNA transcript and to initiate translation. The Shine-Dalgarno sequence usually has the following consensus sequence: 5'-NAGGAGGU-N5_1 ()-AUG (start codon) -3 '(sequence number 85; sequence numbers 125-129). There are variant poly-purine Shine-Dalgarno sequences. It is worth noting that the Listeria hly gene encoding Listeria lysin O (LLO) has the following Shine-Dalgarno sequence: AAGGGAGAGTGAAACCCATG (sequence number 70) (the Shine-Dalgarno sequence is underlined and Bold indicates the translation start codon). 98561.doc -204- 200530399 Example 4. Polynucleotide encoding a fusion protein including secAl signal peptide (LLO) and human EphA2. The encoding of the fusion with the secAl signal peptide (110 signal peptide) plus LLO PEST sequence is shown in Figure 2. A full-length human EphA2 antigen expressing the Kaka sequence. The amino acid sequence of the fusion protein encoded by the expression card g is shown in FIG. 3. Example 5. Codon-Optimization of Extracellular Functional Sites of Human EphA2 (EX2) In order to be expressed in Listeria monocytogenes, the extracellular functional site of human EphA2 (amino acid 25-526 ) Sequence codons-optimization. The natural nucleotide sequence encoding the extracellular functional site of human EphA2 is shown in Fig. 4. The nucleotide sequence for optimal codon usage in Listeria is shown in FIG. 5. The amino acid sequence of the extracellular functional site of human EphA2 is shown in FIG. Example 6. Polynucleotide A encoding a fusion protein including extracellular functional sites of the secAl signal peptide (LLO) and huEphA2 (EX2). &Gt; Polynucleotide which has a synergistic, code, and optimized function. The sequence of the polynucleotide encoding the extracellular functional site of the human EphA2 antigen fused to the secA1 signal peptide (LL0 signal peptide) plus the LLOPE ST sequence is presented. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. 8. B_ Codon-optimized expression of extracellular functional sites of human EphA2. The extracellular site of human EphA2 antigen encoded with the secAi signal peptide (LLO signal peptide) plus LLO PEST sequence is shown in Figure 9 The performance of 98561.doc -205-200530399 Ka Kuang of the sequence 'In order to express in Listeria monocytogenes, the sequence codons encoding the extracellular functional site of EphA2-optimized. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. C. Codon-optimized seca 1 signal peptide and codon _ optimized performance of extracellular functional parts of human EphA2. The cassette is shown in Figure 11 with the secA1 signal peptide (LL0 signal peptide) plus LL. 〇PEST sequence fusion human EphA2 antigen extracellular function site performance card 1 £ sequence 'wherein for expression in Listeria monocytogenes, the extracellular function site, signal peptide and pEST sequence encoding EphA2 Sequence all codons-optimized. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. Example 7. Codons encoding fusion proteins including extracellular functional sites of the Tat signal peptide (Bacillus subtilis phod) and huEphA2 (EX2). Optimized performance cassettes. The encoding and Tat signal peptide (Subtilis The sequence of the expression cassette of the extracellular functional site of the EphA2 antigen fused with B. bacilli (PhD). In order to express in Listeria monocytogenes, all the sequences encoding the extracellular functional site and signal peptide of EphA2 are coded. Sub_optimization. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in Figure j4. Example 8 · Codons for intracellular functional sites of human EphA2 (CO) _ The optimization effect has been designed to encode human intracellular functional sites (amino acids 558_975) of human EphA2 for Listeria monocytogenes Sequence Codons. 98561.doc -206-200530399 Optimization. The natural nucleotide sequence encoding a functional site in human EphA2 is shown in Fig. 15. The nucleotide sequence for optimal codon usage in Listeria is shown in FIG. 16. The amino acid sequence of the intracellular functional site of human EphA2 is shown in FIG. Example 9. Polynucleotide A encoding a fusion protein comprising a functional site in the cell of the secAl signal peptide (LLO) and huEphA2 (CO). A polynucleotide without a codon-optimization effect. The encoding and secAl signals are shown in FIG. 18 Peptide (LLO) plus LLO PEST sequence fused human EphA2 antigen sequence of the polynucleotide in the functional site of the polynucleotide. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. B. Codon-optimized expression of intracellular functional sites in human EphA2. Figure 20 shows the intracellular functional sites encoding the huEphA2 antigen fused to the secAl signal peptide (LLO signal peptide) plus LLO PEST sequence. The expression cassette sequence, in which the sequence codons encoding the intracellular functional site of EphA2 are optimized for expression in Listeria monocytogenes. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. 21. C. The performance cassette with codon-optimized secA1 signal peptide and codon-optimized human EphA2 intracellular functional sites. The encoding and secAl signal peptide (LLO signal peptide) plus l0PEST are shown in Figure 22. The expression of the intracellular functional site of the sequence-fused EphA2 antigen. The Kaka sequence, in order to express in Listeria monocytogenes, the sequence encoding the intracellular functional site, signal peptide and PEST sequence of EphA2 98561.doc- 207- 200530399 All codons-optimization. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. Example 10. Codons encoding optimized fusion proteins including intracellular functional sites including the phoD signal peptide of bacillus subtilis and huEphA2 (CO) -optimized performance cassette. The encoding and Tat signal peptide (bacillus subtilis phOD) is shown in FIG. 24. The sequence of the expression cassette of intracellular functional sites of the fused EphA2 antigen, in order to express in Listeria monocytogenes, the sequences of intracellular functional sites and signal peptides encoding EphA2 were all codon-optimized. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. 25. Example 11. Codon-optimized performance cassette encoding a fusion protein including LLO signal peptide and] VY-ESO-1 To express the human testicular cancer antigen NY-ESO-1 in Listeria monocytogenes ( GenBank accession number NM_001327), design performance cassette. The sequence of a performance cassette encoding NY-ESO-1 fused to the secAl signal peptide (LLO) plus LLOPEST sequence is shown in FIG. For expression in Listeria monocytogenes &apos; the sequence codon-encoding of the antigen and signal peptide will be optimized in this expression cassette. The amino acid sequence of the fusion protein encoded by the expression cassette is shown in FIG. 27. Example 12. Codon-optimized expression cassette encoding an antigen fused to a non-Listeria secAl signal peptide (Lactococcus lactis usp45) To use non-Listeria secA 1 signal peptide, the A heterogeneous antigen was expressed in Listeria monocytogenes, and a cassette was designed for expression. The amino acid sequence of the USp45 signal peptide from Lactococcus lactis is shown below (Steidler et al., Nature Biotechnology, 21: 785-9 (2003)), its natural code 98561.doc -208-200530399 sequence, and Optimized codon sequence in Listeria monocytogenes. Amino acid sequence: MKKKIISAILMSTVILSAAAPLSGVYATIX sequence No. 46) Signal peptidase recognition position ... VYA_DT (sequence No. 55) Natural nucleotide sequence: 5, ATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTAC AGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTA CGCTGACACA3 'in order to increase the number of cells Codons with optimized expression in Listeria monocytogenes: 5, ATGAAAAAAAAAATTATTAGTGCAATTTTAATGAGTA CAGTTATTTTAAGTGCAGCAGCACCATTAAGTGGTGTTT ATGCAGATACA3, (Sequence No. 87) Figure 28 shows a sequence of a partially expressed cassette, including an operable manner and encoding the Usp45 signal peptide The codon-optimized sequence ligated hly promoter from Listeria monocytogenes. To represent a fusion protein comprising a Usp45 signal peptide and the desired antigen, this sequence can be mixed with a codon-optimized or un-codon-optimized antigen sequence. Example I3. Codons encoding the antigen fused to the secA2 signal peptide (P60)-optimized performance card E and vector A. Designed codons-optimized performance cassette. In order to use the secA2 secretion pathway, the number of monocytes is increased. A heterologous antigen was expressed in Listeria and a cassette was designed for expression. The amino acid sequence of the p60 signal peptide of Listeria monocytogenes 98561.doc -209- 200530399 from Listeria monocytogenes, its natural code sequence, and its expression in Listeria monocytogenes are shown below Optimized password sequence. Amino acid sequence: MNMKKATIAATAGIAVTAFAAPTIASA'ST (sequence number 48) Signal peptidase recognition position: ASA-ST (sequence number 57) Natural nucleotide sequence: 5, ATGAATATGAAAAAAGCAACTATCGCGGCTACAGCTG GGATTGCGGTAACAGCATTTGCTGCGCCAACAATCGCAT CCGCAAGCACT3f (sequence number 90) Codons with optimized expression in Listeria monocytogenes: 5, ATGAATATGAAAAAAGCAACAATTGCAGCAACAGCAG GTATTGCAGTTACAGCATTTGCAGCACCAACAATTGCAA GTGCAAGTACA3, (Sequence 4 No. 91) The sequence of a partial expression cassette is shown in FIG. 29, which includes an operable method and The natural sequence encoding the p60 signal peptide is linked to the hly promoter gene from Listeria monocytogenes. The sequence of a partially expressed cassette is shown in FIG. 30, which includes an hly promoter gene derived from Listeria monocytogenes that is operatively linked to a codon encoding the p60 signal peptide. B. Construction of pPL2-hlypro_p60 A performance cassette is also constructed in which the sequence encoding the antigen is structurally inserted into one or more positions in the code sequence of the p60 gene. The following describes construction instructions for a part of the performance cassette that 98561.doc -210- 200530399 can be used to architecturally insert the antigen sequence into the p60 sequence. The performance cassette for this section contains the hly promoter gene. Using the following primers and pPL2-hlyP-OVA as the first template (same as pPL2 / LLO-OVA in Example 2A above), individual preliminary PCR reactions using Pfx or Vent polymerase were performed: pPL2-5F: 5, -GACGTCAATACGACTCACTATAG (sequence number 92) p60-WyP-23m: 5, -CTTTTTTCATCATCATGGGTTTCACTCTCCTTCTAC (sequence number 93) The size of the replication region obtained was 285 base pairs. Individual preliminary PCR reactions using Pfx or Vent polymerase were also performed using pCR-TOPO-p60, which was co-operated as a second template using the following primers. (The vector pCR-TOPO-p60 is made from the pCR-TOPO vector obtained from Invitrogen, Carlsbad, California, in which the genomic p60 sequence from Listeria monocytogenes has been inserted. Any other available Of many alternative sources of p60 password sequences as insert templates). The primers used in this PCR reaction are as follows: hlyP-p60-lF: 5f-AAGGAGAGTGAAACCCATGAATATGAAAAAAGCAAC (sequence number 88) pCR-TOPO-2283R: 5'-GTGTGATGGATATCTGCAGAATTC (sequence number 89) The size of the replication region obtained is 15 10 Base pairs. The PCR reaction was then purged with an S6 column (Bio-Rad Laboratories, Hercules, California). A secondary PCR reaction was then performed using approximately 5 microliters each of the preliminary PCR reactions as 98561.doc -211-200530399 as the template. The following primers were used for the secondary PCR reaction: KpnI-LLO 1257F (previously used bow): 5fCTCTGGTACCTCCTTTGATTAG TATATTC (sequence number 74) and pCR-TOPO-2258R: 5, -CCCTTGGG GATCCTTAATTATACG (sequence number U) 83) . The size of the obtained replication region was 171 5 base pairs. Analysis of the size of the expected replication region in all PCR reactions was confirmed by agarose gel analysis. Digest with BamHI, clear the secondary PCR reaction, and clear again with κρηΙ digest. Then connect the hlyP-p60 gene fragment (1 ^ 111-63111111) (Fig. 3 0) to 卩? 1 ^ 2 and modified 28] \ 1401 (pAM401-MCS; Figure 32) between the BamHI and KρηΙ positions of both plastids. Then digested with BamHI / KpnI (1697, 6024 base pairs) and Hindlll (210, 424, 3460, 3634 base pairs) to confirm the construction of pPL2-p60 plastids. It was also confirmed that the PstI position in pPL2-p60 plastids is unique. (KpnI / PstI digestion will also produce fragments of 736 and 6985 base pairs). Construction of pAM401-p60 plastids (KρηΙ / PstI and KρηΙ / BamHI fragments from p60 are the same as the pPL2 construct). A large number of preparations for each plastid are then separated using methods known to those skilled in the art. The desired coding antigen sequence can then be inserted into the p 6 0 sequence, and using the same translation architecture as the p 60 sequence, using techniques well known to those skilled in the art. Generally, insertions or populations should leave the complete N-terminal signal peptide sequence of p60. The C-terminal autolysin sequence of p60 should also be left intact. Example 14. Codon-optimizing sequence encoding human mesothelin for expression in Listeria monocytogenes. Codons encoding human mesothelin, cancer antigen-optimal 98561 are shown in Figure 33 .doc -212- 200530399 Polynucleotide sequence. "One", shown in Listeria monocytogenes, -The sequence codons shown in Fig. 32 are optimized. The polypeptide sequence not encoded by the sequence in Fig. 32 is shown in Fig. Μ Example 15. Codon-encoding sequence for mouse mesothelin-optimization for expression in Listeria monocytogenes 9-14, Listed in Figure 35, the codons encoding the old gas mesothelin, the cancer antigen, are shown. Optimized polynucleotide sequence. In order for ~ j to appear in Listeria polycytogenes, the sequence codons-ΛA + alpha-shown in Figure 35 have been optimized. The polypeptide sequence encoded by the sequence in Figure 35. Example 16. Through dual gene exchange, the seedling integrates the performance cassette into the Listeria chromosome. An integration vector such as pPL2 can be used to follow the heterogeneous performance card into Listeria In the chromosome of the genus, it becomes another possible choice. In short, by spreading the bacteria on a BHI slow-lipid medium containing chloramphenicol (10 μg / ml) and culturing at a temperature allowed by the pit, Choose to pKSV7-xenogeneic Self-permeability bacteria that penetrate into the body. By substituting several individual colonies for multiple generations at a non-permissible temperature of 41 t in a medium containing aeromycin, a single cross is selected into the bacterial chromosome. Integrative effect. Finally, plastid excision and excision (dual crossover) were completed by subculturing several individual colonies for multiple generations at a permissible temperature of 30.0% in aerobicin-free 6111 medium. The PCR confirmed that the heterologous protein expression cassette was integrated into the bacterial chromosome, using primer pairs that expanded to limit the region from the heterologous protein expression cassette to the bacterial chromosomal target sequence, but not included in the pKSV7 plastid vector construct. 98561.doc- 213 · 200530399 Example 17 · In order to express in the selected recombinant mononuclear cell 増 Listeria polyclonal strains, colony and insert EphA2 into the pPL2 carrier To insert various pPL2-signal peptide expression cassettes, select colonies EphA2 penetrates the outer (EX2) and cytoplasmic (CO) functional sites of EphA2 on the side of the membrane helix. Use genes equivalent to natural mammalian sequences, or Codon-optimized genes for EphA2 EX2 and CO functional sites expressed in Listeria monocytogenes. In order to optimize EphA2 EX2 and EphA2 CO codons, use 20 amine groups in Listeria spp. Optimal codons for acids (see Table 3 above). By extending overlapping oligonucleotides, the codon-optimized EphA2 EX2 and CO functional sites are synthesized using techniques commonly used by those skilled in the art. By Nucleotide sequencing confirmed the extended sequences of all synthetic EphA2 constructs. In Figure 4-6 (EX2 sequence) and Figure 15-17 (CO functional site sequence), the original amines of EphA2 EX2 and CO functional site are shown Acid sequence, along with its natural and codon-optimized nucleotide sequence. In addition, in order to detect the expression and secretion of EphA2 using antibodies specific to FLAG or protein by Western blot analysis, insert FLAG into the amine and end terminals of synthetic EphA2 EX2 and CO genes in the architecture, respectively. (Stratagene, La Jolla, CA) and myc epitope tags. Therefore, the expressed protein has the following sequence of elements: NH2-signal peptide-FLAG-EphA2-myc-C02. The following shows the FLAG and myc epitope tag amino acid and codon-optimized nucleotide sequences: FLAG: 98561.doc -214- 200530399 5'-GATTATAAAGATGATGATGATAAA (Sequence No. 96) NH2-DYKDDDDK-C02 (Sequence No. 97)

Myc: 5f-GAACAAAAATTAATTAGTGAAGAAGATTTA (sequence number 98) NH2-EQKLISEEDL-C02 (sequence number 99) Example 18 · Detection of heterologous proteins synthesized and secreted by western blot analysis | Bacteria precipitated by triacetic acid (TCA) Western blot analysis of culture fluids determined EphA2 protein synthesized and secreted from various selected recombinant Listeria-EphA2 strains. Briefly, a Listeria mid-log phase culture grown in BHI medium was collected in a 50 ml conical centrifuge tube, the cells were pelleted, and ice-cold TCA was added to the bacterial culture [6%] of the supernatant was finished and incubated on ice for a minimum of 90 minutes or overnight. The TCA-precipitated protein was collected by centrifugation at 2400X for 20 minutes at 4 ° C. The pellet was then resuspended in a 300-600 microliter volume containing 15 micrograms / φml of phenol red TE, ρΗ8.0. The sample is dissociated by rotation. If necessary, adjust the pH of the sample by adding ΝΗΟΗ until the color turns pink. For electrophoresis, add 100 μl of 4X SDS loading buffer solution at 90 ° C. (: Incubate for 10 minutes, prepare all samples. Then centrifuge the samples at 14,000 rpm for 5 minutes in a microcentrifuge, and collect the supernatant and store at -20 ° C. As for Western blot analysis, A method commonly used by this artist 'is to load 20 microliters of the prepared lysate (equivalent to the culture fluid obtained from i-4x109 bacteria) into a 4-12% SDS-PAGE gel, electrophoresis, and protein 98561.doc -215-200530399 Transfer to PDDF membrane. In order to culture with the antibody, prepare the transferred membrane by incubating with 5% dehydrated milk powder in PBS for 2 hours at room temperature and stir. Use according to The following antibodies were diluted with PBST buffer solution (0.1% Tween 20 in PBS): (1) rabbit anti-Myc polyclonal antibody (ICL laboratories, Newberg, Oregon) diluted at 1: 00: 00; (2) mouse anti-FLAG monoclonal antibody (Stratagene, La Jolla, CA) diluted 1: 2,000; and (3) rabbit anti-EphA2 (carboxyl terminal-specific) multiclonal antibody (sc-924, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). By anti-goat conjugated with horseradish peroxidase Or secondary culture of anti-mouse antibodies, and the detection of the ECL chemiluminescence assay kit (Amersham), exposure of the film to assess the specific binding of the antibody to the protein target. Example 19. Recombinant Liszt encoding EphA2 in various forms Mycobacterium

EphA2 protein A. Listeria: [line DP-L4029 (actA) or DP_L4017 (LLO L461T)] Performance cassette construct: LLOss-PEST-CO-EphA2 Genetically combines the natural sequence of EphA2 CO functional site with the natural The secAl LLO sequence was fused and, as described above, a heterologous antigen expression cassette under the control of the Listeria hly promoter gene was inserted into the pPL2 plastid between the Kpη1 and SacI positions. As described above, pPL2-EphA2 plastid construct was introduced by conjugating into the Listeria strains DP-L4029 (actA-) and DP-L4017 (L461T LLO). Figure 37 shows the results of Western blot analysis of TC A-precipitated bacterial culture fluids of 4029-EphA2 CO and 4017-EphA2 CO. This analysis confirmed that the recombinant Listeria spp. Was designed to contain a heterologous protein table 98561.doc -216- 200530399, which contains a natural sequence equivalent to secAl and an EphA2 CO fusion protein, secreting multiple EphA2-specific fragments. , Which is lower than the expected molecular weight of 52 kDa, confirming the need to modify the performance cassette. B. Listeria: [DP-L4029 (actA-)] Performance cassette construct: 1. Natural LLOss-PEST-FLAG_EX2-EphA2-myc-codon-optimized 2. (codon-optimized LLOss-PEST- (codon-optimized) FLAG-EX2_EphA2-myc genetically makes the natural secAl LLO signal peptide sequence or codon-optimized secAl LLO signal for expression in Listeria The peptide sequence was fused with the codon-optimized EphA2 EX2 functional site sequence for expression in Listeria, and in accordance with the above, the heterologous antigen expression cassette under the control of the Listeria hly promoter gene was cloned at κρηΙ Inserted into the pPL2 plastid from the SacI site. As described above, pPL2-EphA2 plastid construct was introduced by conjugating into the Listeria strain DP-L4029 (actA). FIG. 38 shows the TCA-Shendian bacteria culture fluid of Listeria actA encoding a natural or codon-optimized secAl LLO signal peptide fused to a codon-optimized EphA2 EX2 functional site. result. This analysis confirmed the sequence of both the signal peptide and the heterologous protein optimized for optimal codon utilization in Listeria monocytogenes, and as a result, the expected full-length EphA2 EX2 functional site protein. When only the codon-optimization of the EphA2 codon sequence was performed, the full-length EphA2 EX2 functional site protein did not perform well. Heterologous protein expression (fragment) when using a codon-optimized Listeria monocytogenes LLO secAl signal peptide for expression in Listeria monocytogenes 98561.doc -217- 200530399 Or full length). C. Listeria: [DP_L4029 (actA)] Performance cassette construct: 3. Natural LLOss-PEST- (Codon-optimized # 1 ^ 0-EphA2_CO-myc 4. Codon-optimized LLOss -PEST- (codon-optimized) FLAG-EphA2_CO-myc 5. Codon-optimized PhoD- (codon-optimized) FLAG-EphA2_CO-myc genetically converts the natural secAl LLO signal peptide sequence , Or codon-optimized for expression in Listeria secAl LLO signal peptide sequence, or codon-optimized for expression in Listeria from the phoD gene of Bacillus subtilis The Tat signal peptide is fused to the codon-optimized EphA2 CO functional site sequence for expression in Listeria, and according to the above, a heterologous antigen expression card under the control of the Listeria hly promoter Cassette, inserted into the PAM401-MCS between the positions of κρηΙ and SacI. As described above, the pAM401-EphA2 plastid construct was introduced into the Listeria strain DP-L4029 (actA) by electrical penetration. Figure 39 shows the coding Natural or fused to codon-optimized EphA2 CO functional sites Codon-optimized secAl LLO signal peptide, or codon-optimized subtilis phoD Tat signal peptide Listeria TC A-precipitated bacterial culture fluid Western blotting 98561.doc -218- 200530399 This analysis confirmed that once the signal peptide and the sequence of the heterologous protein optimized for optimal codon utilization in Listeria monocytogenes were used again, the results showed the expected full-length EphA2 CO Functional site protein. In addition, the expected expression and secretion of the full-length EphA2 CO functional site protein results from the encoding of a codon-optimized EphA2 CO functional site fusion codon-optimized recombinant Bacillus subtilis phoD Tat signal peptide This result confirms a novel and unexpected discovery that signal peptides from different bacterial species can be used to write a program for secreting heterologous proteins from recombinant Listeria φ genera. Only codon-optimization of the EphA2 sequence The expression of full-length EphA2 CO functional site proteins is poor. When used in order to express in Listeria monocytogenes, Codon-optimized signal peptide, the highest degree of heterologous protein expression. D. pCDNA4 encoding pCDNA4 encoding full-length EphA2 infection of 293 cell performance cassette construct: 6. pCDNA4-EphA2 The EphA2 gene was cloned into the pCDNA4 (Invitrogen, Carlsbad, CA), a performance plastid based on the eukaryotic CMV promoter gene. Figure 40 shows the results of Western blot analysis of lysates prepared from 293 cells infected with pCDNA4-EphA2 plastid transfer, and confirmed the abundant expression of full-length EphA2 protein in mammalian cells. Example 20 Therapeutic efficacy of recombinant Listeria spp. Using codon-optimized EphA2 in Balb / C mice carrying CT26 tumors encoding human EphA2 98561.doc -219- 200530399 are provided in Figures 41-44 The following data confirm the following: Coded as 0VA.AH1 (MMTV gp70 immunodominant epitope) or OVA.AHl-A5 (MMTV gp70 immunodominant epitope, with irregular changes that increase T-cell receptor binding) Recombinant Listeria spp. Immunized Balb / C mice bearing CT26.24 (huEphA2 +) lung tumors for long-term survival.

The EphA2 CO functional site is highly immunogenic, and when immunized with a recombinant Listeria spp. That encodes a codon-optimized or natural EphA2 CO functional site sequence, it carries CT26.24 (huEphA2 +) in lung tumors. In terms of survival of Balb / C mice, a significant long-term increase was observed (Figure 43).

The immunogenicity of EphA2 EX2 functional sites is poor, and only when carrying a codon-optimized EphA2 EX2 functional site sequence codon-optimized secAl signal peptide for recombinant Listeria immunization, carry was observed CT26.24 (huEphA2 +) lung tumor increased survival in Balb / C mice. When immunized with a recombinant Listeria sec. Natural secAl signal peptide fused to a sequence codon-optimized EphA2 EX2 functional site sequence, no therapeutic efficacy was observed in mice (Figure 42). The benefits of using both the codon-optimized secAl signal peptide and the EphA2 EX2 functional site sequence are supported by statistically significant therapeutic anti-tumor efficacy, as shown in Table 4 below. 98561.doc 220- 200530399 Table 4. Logarithmic-permutation test comparing survival curves shown in Figure 42. Intermediate survival (days) in experimental group. Significance (p value) for HBSS group. For actA-natural secAl / EphA2EX2 group. Significance (P value) HBSS 19--actA 20 NS ♦ NS actA-natural secAl-EphA2 EX2 (natural) 19 NS actA-natural secAl-EphA2 EX2 (codon-optimized) 24 0.0035 NS actA -Codon-optimized secAl-EphA2 EX2 (codon-optimized) 37 0.0035 0.0162 actA-natural secAl-EphA2 CO (codon-optimized) &gt; 99 0.0035 0.0015 Obviously, even with pCDNA4-EphA2 Infected 293 cells in vivo and produced extremely high levels of protein expression. The pCDNA4-EphA2 plastids were used to immunize Balb / C mice carrying CT26.24 (huEphA2 +) lung tumors. Figure 44). For the study of treatment of tumors in vivo, female Balb / C mice IV were implanted into 5xl05 CT26 cells stably expressing EphA2. After 3 days, the old mice were randomly divided into groups and inoculated with various recombinant Listeria strains encoding EphA2 at IV. In some cases (referred to in the figure), mice were inoculated with 100 .. # micrograms of PCDNA4 plastids or pCDNA4-EphA2 plastids in the anterior tibialis muscle. As a positive control group, mice were inoculated with a recombinant Listeria strain strain IV encoding the OVA.AHI or OVA.AH1-A5 protein chimera. Mice were vaccinated 3 and 14 days after tumor cell implantation. Hanks balanced salt solution (HBSS) buffer solution or unmodified Listeria spp. Were injected into mice as a negative control group. All experimental groups contained 5 · mice. As for survival studies, mice were sacrificed when they started showing any symptoms of urgency or hard breathing. 98561.doc -221-200530399 Example 21: Evaluation of antigen-specific immune response after vaccination Various methods in vitro and in vivo can be used to evaluate the vaccine of the present invention. -Some assays involve the analysis of antigens derived from the spleen of vaccinated mice. Specific T cell analysis. Non-limiting examples of methods for estimating immune responses in vitro and in vivo are provided in this example. The antigens mentioned in the representative descriptions of these assays are model antigens and are not necessarily those generated using recombinant nucleic acid molecules, expression cassettes and / or expression vectors described herein. Those skilled in the art will readily recognize the assays described in this example and can quickly use them to evaluate bacteria including recombinant nucleic acid molecules, expression cassettes and / or expression vectors described herein in vitro or in vivo Within the immune response. For example, C57B1 / 6 or Balb / C is inoculated by intravenous injection of a Listeria strain expressing OVA (or other appropriate antigen) of 0.1 LD50. Seven days after the vaccination, the spleen cells of the mice (usually 3 mice per group) were harvested by flattening the spleen on ice-cold rpmi 丨 media, or a single cell suspension was prepared therefrom. Alternatively, similar Harvest mouse lymph nodes, prepare single cell suspensions, and replace spleen cells in the following assays. Generally, for vaccines administered intravenously or intraperitoneally, spleen cells are evaluated, and intramuscularly, subcutaneously or intradermally. The administered vaccine evaluates spleen cells and cells derived from lymph nodes. Unless otherwise mentioned, all antibodies used in these examples can be obtained from Pharmingen, San Diego, CA. EUSPOT assay: Use of OVA antigen The Listeria strain was used as an example, and the ELISPOT assay was used to evaluate the quantitative frequency of antigen-specific τ cells produced when immunized in mouse mode 98561.doc -222- 200530399. Assessing the antigen-specific τ cells is OVA Specific CD8 + or LLO specific CD8 + or CD4 + T cells. This OVA antigen model assesses the immune response to heterologous tumor antigens inserted into the vaccine 'And can be replaced by any antigen of interest. The LLO antigen is specific to Listeria. By detecting the release of cytokines (such as INF-γ) when a specific antigen is recognized, specific τ cells can be evaluated. PVDF-based 96-well culture plates (BD Bio sciences, San Jose, CA) were coated with anti-mouse IFN-γ monoclonal antibody (mAb R4; 5 μg / ml) at 4 ° C overnight. At room temperature Rinse the plate with 200 microliters of complete RPMI and block for 2 hours. Add 2x105 cells per well to spleen cells from vaccinated mice (or uninoculated control mice) and incubate at 37 ° C at 37 ° C. Culture in the presence of peptides at various concentrations ranging from 0.01 to 10 μM for 20-22 hours. Peptides for OVA and LLO are SL8, MHC class I epitope of OVA, LLO190 (NEKYAQAYPNVS (sequence number 100 No.) Invitrogen), MHC class II epitope of Listerialysin 0 (Listeria antigen), LL0296 (VAYGRQVYL (SEQ ID NO. 101), MHC class I antigen of Listeria lysin 0 Position, or LL091 (GYKDGNEYI (SEQ ID NO. 102)), M of Listerialysin 0 HC class I epitope. Use LLO190 * LLO296 in C57B1 / 6 mode and LL091 in Balb / C mode. After washing, dilute the culture plate with PBS to 0.5 μg / ml. IFN-γ specific secondary biotinylated antibody (XMG1.2) — cultured. After 2 hours of incubation at room temperature, 'wash the plate' and dilute with PBS at 37 C, containing 1% BSA's 1-nanometer golden goat anti-biotin conjugate (GAB-1; 1: 200 dilution; Ted Pella, Redding, CA) —Kei 98561.doc -223-200530399 was raised for 1 hour. After thorough rinsing, in order to spread the dots, the plate was incubated with the substrate (silver raising kit; 30 ml / well; Ted Pella) at room temperature for 2 to 10 minutes. The culture plate was then rinsed with distilled water to stop the mass reaction. After the plate had been air-dried, the points in each well were calculated using an automatic ELISPOT plate reader (CTL, Cleveland, OH). The cytokine response was expressed as the number of IFN-γ dot-forming cells (SFCs) to OVA-specific T cells or Listeria-specific T cells per 2xl05 spleen cells. Intracellular cytokine staining assay (ICS): To further assess antigen-specific CD8 + or CD4 + T cells and correlate results with those obtained from ELISPOT assays, perform ICS and evaluate cells by flow cell count analysis . In the presence of Brefeldin A (Pharmingen), spleen cells from vaccinated and control mice were combined with SL8 (stimulating OVA-specific CD8 + cells) or LL019〇 (stimulating LLO-specific CD4 + cells). ) Cultivate for 5 minutes. Brefeldin A inhibits the secretion of cytokines produced when T cells are stimulated. Spleen cells cultured with unrelated MHC class I peptides were used as a control group. Spleen cells stimulated with PMA (phorbol-12-myristic acid-π-acetate, Sigma) 20 ng / ml and ionomycin (Sigma) 2 ug / ml were used as IFN-γ and TNF-α Positive control group for intracellular cytokine staining. In order to detect the cytokine expression of the cytoplasm, the cells were stained with FITC-anti-CD4 mAb (RM4-5) and PerCP-anti-CD8 mAb (53-6.7), fixed and permeated with Cytofix / CytoPerm solution (Pharmingen). Anti-TNF-amAb (MP6-XT22) and APC-co-anti-IFN-γ mAb (XM &lt;} 1.2) were stained for 30 minutes. Flow cell count analysis (FACScalibur, Becton Dickinson, Mountain View, CA) was used to determine 98561.doc -224-200530399 the percentage of cells expressing intracellular IFN-γ and / or TNF-α, and CELLQuest software (Becton Dickinson Immunocytometry System) analyzes the data. Because the fluorescent labels on various antibodies can be identified by FACScalibur, appropriate cells can be confirmed by opening the door for CD8 + and CD4 + stained with anti-IFN-γ or anti-TNF-α. Stimulated spleen cells exhibit cytokines: The control group and vaccinated C57B1 / 6 mice can also be evaluated for cytokine secretion from mouse spleen cells. Spleen cells were stimulated with SL8 or LL019 () for 24 hours. A control group was stimulated with an unrelated peptide HSV_gB2 (Invitrogen, SSIEFARL, Sequence No. 124). Supernatants of the stimulated cells were collected, and the T helper-1 and T helper 2 cytokines were determined using an ELISA assay (eBiosciences, CO) or a cell counting bead array kit (Pharmingen). Evaluation of cytotoxic T cell activity: OVA-specific CD8 + T cells were further evaluated by evaluating their cytotoxic activity in vitro or directly in the living body of C57B 1/6 mice. CD8 + T cells recognize and lyse their individual target cells in an antigen-specific manner. The chromium release assay was used to determine cytotoxicity in vitro. 10: 1 ratio, irradiated EG7.0VA cells (EL-4 tumor cell line metastasis infection to show 0VA, ATCC, Manassas, VA) or 100 nM SL8 stimulation inexperienced and inoculated with Listeria-OVA Spleen cells from (internal) mice in order to expand OVA-specific T cells in the spleen cell population. After 7 days in culture, in standard 4-hour 51Cr-release assays, EL-4 cells (ATCC, Manassas, VA) pulsed with EG7.0VA or SL8 were used as standard stem cells, while only EL-4 cells were negative In the control group, the cytotoxic activity of effector cells was determined. 98561.doc -225-200530399 YAC-1 cell line (ATCC, Manassas, VA) was used as a target to determine the activity of NK cells in order to distinguish the activity attributed to T cells from the activity attributed to NK cells. Calculate the percentage of specific cytotoxicity at 100x (experimental release-spontaneous release) / (maximum release-spontaneous release). Spontaneous release was determined by culturing target cells in the absence of effector cells. The maximum release was determined by lysing the cells with a 0.1% tee X-100. If the spontaneous release &lt; 20% maximum release, the experiment is considered effective for analysis. To evaluate the in vitro cytotoxic activity of OVA-specific CD8 + T cells, spleen cells obtained from inexperienced C57B 1/6 mice were divided into two equal portions. Each group was pulsed with 0.5 μg / ml specific peptide, target (SL8) or control group (HSV_gB2) for 90 minutes at 37 ° C. The cells were then washed 3 times with the medium 'and twice with PBS + 0.1% BSA. Cells were resuspended at ΐχΐ07 cells per ml in warm PBS + 0.10 / 〇BSA (10 ml or less) to utilize carboxyfluorescein diacetate succinimide (CFSE, Molecular Probes, Eugene, OR). To the target cell suspension, add 125 μl of 5 mM CFSE stock solution and mix the sample by rotation. In the control cell suspension, a 10-fold dilution of the CFSE stock solution was added and the remote samples were mixed by rotation. The cells were incubated at 37 ° C for 10 minutes. Staining was stopped by adding a large volume (40 ml) of ice-cold PBS. Cells were washed twice with pbs at room temperature and then resuspended and counted. Each cell suspension was diluted to 50 x 106 cells per milliliter, mixed with ΙΟΟμΙ per ethnic group, and injected via the tail vein of unexperienced or vaccinated mice. After 2-24 hours, the spleen was harvested and analyzed by flow cell counting for a total of 5x106 cells. List the high (standard stem) and low (control) fluorescence peaks and use the ratio of the two to establish 98561.doc -226- 200530399 Percentage of standard stem cell lysis. Cytotoxicity assays in vivo allow the assessment of antigen-specific τ cell lytic activity without the need for retesting in vitro. In addition, this assay evaluates T cell function in its natural environment. Example 22 · Human EphA2-specific immunity induced by inoculating Balb / C mice with a Listeria strain expressing EphA2 at two-week intervals to represent intracellular functional sites of hEphA2 (Lister in Figure 45 HEphA2-ICD) Listeria L461T, or extracellular functional site of hEphA2 expressed from a codon-optimized sequence for expression in Listeria monocytogenes (in Figure 45 The Listeria △ actA (actA) strain of Listeria spp. HEphA2-ECD) was immunized to Balb / C mice (n = 3). (In this article, the intracellular functional site of hEphA2 is otherwise referred to as hEphA2-ICD, hEphA2 ICD, EphA2 CO, or CO. At the end of this article, the extracellular functional site of hEphA2 is otherwise referred to as hEphA2-ECD, hEphA2 ECD, EphA2 EX2, or EX2. ). Mice were euthanized 6 days after the last immunization, and the spleens were harvested and pooled. Regarding the ELISPOT assay, P815 cells expressing full length hEphA2 or cell lysates prepared from these cells were re-stimulated cells in vitro. Parental P8 15 cells or lysates of the cells were used as a negative control group. Cells were also stimulated with recombinant hEphA2 Fc fusion protein. Colonies (SFCs) forming IFN-γ positive spots were measured using a 96-well point reader. As shown in Figure 45, increased IFN-ySFCs were observed using spleen cells derived from mice inoculated with Listeria-hEphA2. Cells expressing hEphA2 or lysate of cells stimulated both, with the result that IFN-ySFC was increased, suggesting that EphA2-specific CD8 + and CD4 + T cell responses. Spleen cells obtained from mice inoculated with the parental Listeria control group 98561.doc -227- 200530399 did not demonstrate an increase in IFN-ySFC. Example 23: EphA2-specific anti-tumor requires CD4 + and CD8 + T cell responses. Balb / C mice were intravenously inoculated with 2xl05 CT26-hEphA2 on day 0 (n = 10). On days 1 and 3, CD4 + cells or CD8 + T-cells were depleted by injection of 200 micrograms of anti-CD4 (ATCC fusion tumor GK1.5) or anti-CD8 (ATCC fusion tumor 2.4-3), which Confirmed by FACS analysis (data not shown). On day 4, mice were immunized intravenously with Listeria L461T expressing hEphA2 ICD with 0.1 LDm and monitored for survival. As shown in Figure 46, the CD4 + and CD8 + depleted groups were unable to confirm the anti-tumor response seen in non-τ cell depleted animals. The data are summarized in Table 5 below: Table 5 Intermediate survival in the vaccinated group P vs. number of HBSS survivors (day 67) HBSS 17-0 Listeria-hEphA2-ICD &gt; 67 &lt; 0.0001 7 Listeria -hEphA2-ICD + anti-CD4 19 0.03 2 Listeria-hEphA2-ICD + anti-CD8 24 0.0002 0 The foregoing data indicate that both CD4 + and CD8 + T cells are required for optimal inhibition of tumor growth.

Example 24. Deletion of iniB from the Listeria spp. By dual gene exchange. In some embodiments, the bacteria including the recombinant nucleic acid molecules and cassettes described herein are mutant Listeria spp. For example, in some embodiments, the bacteria including the recombinant nucleic acid molecule and the expression cassette is a Listeria monocytogenes strain in which the actA gene, the inlB gene, or both have been deleted. A representative method for producing deletion mutants in Listeria is described below. 98561.doc -228 · 200530399 can be achieved by dual gene exchange, as described by Cami lli et al., Mol. Microbiol. 8: 143-147 (1993), from Listeria DP-L4029 (or from Other selected mutant strains or deleted surface protein B gene (inlB) from Listeria. Conjugation overlap extension (SOE) PCR can be used to prepare constructs for use in dual gene exchange procedures. The source of the surface protein B gene is the sequence listed in GenBank accession number AL591975 (Listeria monocytogenes strain EGD, complete genome, fragment 3/12; inlB gene region: nucleotides 97008-98963), all in The references are incorporated herein, and / or the sequences listed in GenBank Accession No. NC_003210 (Listeria monocytogenes strain EGD, complete genome, inlB gene region: nucleotides 457008-458963) are all cited by reference This method is incorporated herein. In the preliminary PCR reaction, the following templates and primers were used to expand approximately 1,000 base pairs upstream and downstream of the sequences from the 5 'and 3' ends of the Listeria inlB gene, respectively: Template : DL-L4056 or DP-L4029 genomic DNA primer pair 1 (used to expand the upstream region from the 5 'end of inlB):

Lm-96031F: 5f-GTTAAGTTTCATGTGGACGGCAA AG (Serial No. 103) (Tm: 72 ° C)

Lm- (3 丨 inlB-R +) 97020R: 5 丨-AGGTCTTTTTCAGTTAACTATCCTCTCCTTGA TTCTAGTTAT (Sequence No. 104) (Tm: 114 ° C) (The sequence with the subscript line is complementary to the downstream region of the InlB carboxyl terminal.) (Base pair): 1007 98561.doc -229-200530399 Primer pair 2 (used to expand the downstream region from the 3 'end of inlB): Lm- (5finlB-F +) 9891 1F: 5,-CAAGGAGAGGATAGTTAACTGAAAAAGACCTAA AAAAGAAGGC (sequence No. 105) (Tm: 118 ° C) (Subscripted sequence is complementary to the upstream region of the InlB amino terminal)

Lm-99970R: 5, -TCCCCTGTTCCTATAATTGTTAGCTC (SEQ ID NO: 106) (Tm: 74 ° C) Enlarged region size (base pair): 1074 In a secondary PCR reaction, a preliminary PCR amplification region is fused via SOEPCR fusion. Complementarity between the reverse primer of pair 1 and the forward primer of pair 2. As a result, the inlB code sequence was precisely deleted: nucleotides 9702 1-989 10 = 1889 base pairs. The following templates and primers were used in the secondary PCR reaction: Template: Cleaned preliminary PCR reaction Primer pair:

Lm-96043F: 5, -GTGGACGGCAAAGAAACAACCAAAG (Serial No. 107) (Tm: 74 ° C)

Lm-99964R: 5, -GTTCCTATAATTGTTAGCTCATTTTTTTC (Sequence No. 108) (Tm: 74 ° C) (Enlarged region size (base pairs): 2033) The draft to complete this construction process is as follows: Performed using Vent DNA Polymerase (NEB) A preliminary PCR reaction (3 temperature cycles) was performed, and 10 microliters of washed Listeria DP-L405 6 or DP-L4029 was cultured overnight at 30 ° C. The expected size of the enlarged region of Streptozoa (1007 base pairs and 1074 base pairs) was confirmed by a 1% agarose gel with Li 98561.doc • 230- 200530399. The preliminary PCR reaction was gel purified, and DNA was eluted with GeneClean (BIO 101). A secondary PCR reaction was performed, using approximately equal amounts of each preliminary reaction as a template (approximately 5 μl). The expected size of the Listeria extended region obtained from the secondary PCR reaction (2033 base pairs) was confirmed on a 1% agarose gel. An adenosine residue was added to the 3 'end of the Listeria dl inlB extension region with Taq polymerase. The Listeria dl inlB extension was then inserted into the PCR2.1-TOPO vector. PCR2.1-TOPO-dlinlB plastid DNA was digested with Xhol and KpηI, and a 2123 base pair fragment was gel purified. A fragment of 1 ^ 111 / y} 1012123 base pairs was inserted into a pKSV7 vector that had been digested with κρηΙ and Xhol and treated with CIAP (pKSV7_dl inlB). The accuracy of the dl inlB sequence in pKSV7-dl inlB was then confirmed. The inlB gene was deleted from the desired Listeria strain by using the pKSV7-dl inlB plastid by dual gene exchange. Example 25. To construct recombinant Listeria, the signal peptide codon-optimization is provided in Table 6 below. Some representative codons in the performance cassettes available in recombinant Listeria-optimized signals are provided. Peptide. 98561.doc -231-200530399 Table 6. Amino acid sequences of signal peptides used to construct a representative signal peptide secretion pathway of recombinant Listeria spp. Signal peptidase position (,) The natural sequence is expressed for Lm the codon-optimized sequence of the gene [genus / species] secAl MKKIMLV FITLILVSL PIAQQTEA KDASAFN KENSISSM APPASPPA SPKTPIEK KHAD (serial No. 109) 1 TEA'K D (serial No. 54) ATGAAAAAAATAATGCT AGTTTTTATTACACTTAT ATTAGTTAGTCTACCAA TTGCGCAACAAACTGA AGCAAAGGATGCATCT GCATTCAATAAAGAAAA TTCAATTTCATCCATGG CACCACCAGCATCTCCG CCTGCAAGTCCTAAGA CGCCAATCGAAAAGAA ACACGCGGAT (Serial No. 110) ATGAAAAAAATTATGTT AGTTTTTATTACATTAAT TTTAGTTAGTTTACCAA TTGCACAACAAACAGA AGCAAAAGATGCAAGT GCATTTAATAAAGAAAA TAGTATTAGTAGTATGG CACCACCAGCAAGILCAO ACCAGCAAGPL TSG Vaccine (Sequence No. 113) MSA (Sequence No. 113) Sequence No. 55) ATGAAAAAAAAGATTAT CTCAGCTATTTTAATGTC TACAGTGATACTTTCTG CTGCAGCCCCGTTGTCA GGTGTTTACG CTGACAC A (sequence number 86) ATGAAAAAAAAAATTAT TAGTGCAATTTTAATGA GTACAGTTATTTTAAGT GCAGCAGCACCATTAA GTGGTGTTTATGCAGAT ACA (sequence number 87) Usp45 [Lactococcus lactis] MKKRKVL IPLMALSTI LTGTGCA TCGTACGATGACGAAEVACGAAG CAGAAGTT (sequence number 111) ATGAAAAAACGTAAAG TTTTAATTCCATTAATGG CATTAAGTACAATTTTA GTTAGTAGTACAGGTAA TTTAGAAGTTATTCAAG CAGAAGTT (sequence, J number 114) pag (protective antigen) [anthrax] secA2 MNMKKAT IAATAGIA VTAFAA48 sequence IASTAG 48 ATGAATATGAAAAAAGC AACTATCGCGGCTACAG CTGGGATTGCGGTAACA GCATTTGCTGCGCCAAC AATCGCATCCGCAAGCA CT (sequence No. 90) ATGAATATGAAAAAAGC AACAATTGCAGCAACA GCAGGTATTGCAGTTAC AGCATTTGCAGCACCA ACAATTGCAAGTGCAA GTACA (serial No. 91) iap and invasion-related protein P60 [monocytogenes Listeria] Tat MAYDSRF DEWVQKL KEESFQNN TFDRRKFI QGAGKIA GLSLGLTI AQSVGAF ( sequence Column No. 53) VGA'F (Serial No. 62) ATGGCATACGACAGTCG TTTTGATGAATGGGTAC AGAAACTGAAAGAGGA AAGCTTTCAAAACAATA CGTTTGACCGCCGCAAA TTTATTCAAGGAGCGGG GAAGATTGCAGGACTTT CTCTTGGATTAACGATT GCCCAGTCGGTTGGGG CCTTT (Serial No. 112) ATGGCATATGATAGTCG TTTTGATGAATGGGTTC AAAAATTAAAAGAAGA AAGTTTTCAAAATAATA CATTTGATCGTCGTAAA TTTATTCAAGGTGCAGG TAAAATTGCAGGTTTAA GTTTAGGTTTAACAATT GCACAAAGTGTTGGTG CATTT (Serial No. 115) PhoD alkaline phosphatase The sequence shown by the enzyme [Bacillus subtilis] 1 includes a PEST sequence obtained from LLO. Example 26. Codon-Optimized Performance Cassette Including Protective Antigen (PA) Signal Peptide for Anthracis Bacteria To Use Non-Listeria spp.-232- 98561.doc 200530399 in Listeria monocytogenes The secAl signal peptide represents a heterologous antigen, and a cassette is designed for expression. The amino acid sequence (GenBank Accession No. NC_007322) derived from the protective antigen (PA) of Bacillus anthracis (Ba) is shown below, its natural code sequence, and its best performance for expression in Listeria monocytogenes Optimized password sequence. Amino acid sequence: MKKRKVLIPLMALSTILVSSTGNLEVIQAEV (sequence number 47) Signal peptidase recognition position: IQA'EV (sequence number 56) Natural nucleotide sequence: φ ATGAAAAAACGAAAAGTGTTAATACCATTAATGGCATT GTCTACGATATTAGTTTCAAGCACAGGTAATTTAGAGGT GATTC sequence of AGGCA Codons optimized for Listeria monocytogenes: ATGAAAAAACGTAAAGTTTTAATTCCATTAATGGCATTA ATGGCATTAAGTACAATTTTAGTTAGTAGTACAGGTAAT TTAGAAGTTATTCAAGCAGAAGTT (Sequence ^ No. 114) · Figure 47 shows the sequence of a partially expressed cassette, which includes a sequence derived from monocytogenes The hly promoter gene of the bacterium is operably linked to the codon-optimized sequence encoding the Ba PA signal peptide. This sequence can be mixed with codon-optimized or un-codon-optimized antigen sequences to represent a fusion protein that includes the PA signal peptide of Anthrax anthracis and the desired antigen. Example 27. Codon-optimized expression and secretion of both the antigen signal peptide and tumor antigen from a recombinant Listeria spp. That includes a codon-optimized performance cassette, providing weights from 98561.doc-233 · 200530399 Effective expression and secretion in Listeria: codon-optimization of both signal peptides and genetic elements encoding heterologous proteins, providing a recombinant listeria from human tumor antigens including water-repellent functional sites. Optimal secretion of the basic vaccine. Efficient secretion of antigens from cytosolic bacteria requires efficient submission via the MHC Class I pathway, and CD8 + 孓 cell induction 'is therefore directly related to the efficacy of Listeria-based vaccines. Secretion of two human tumor antigens that bind to malignant cell membranes, Mesothelin and NY_ESCM from recombinant Listeria, which are associated with pancreatic and ovarian cancer (mesothelin) and melanoma (NY-ES〇-1) Related immune targets have been optimized in other solid tumors via codon-optimization of both the antigen and signal peptide codon sequences. Construction of various expression cassettes including hly promoter genes, which are linked to natural or codon-optimized sequences encoding signal peptides related to secAl or other secretory pathways including secA2 and Twin-Arg translocation (Tat), It is fused in architecture to selected human tumor antigens-human NY-ESO-1 or human mesothelin. (See Examples 11-14 and 25 above for antigen sequences and / or signal sequences). The Western blot analysis of the tCA · precipitated culture fluid of Listeria spp. Grown in BHI broth was used to evaluate the synthesis and secretion of heterologous proteins from recombinant Listeria spp. (For Western blot analysis, use methods similar to those described in Example 18 above). The results of these experiments are shown in Figures 48A-C. When the signal peptide code sequence is used for codon utilization in Listeria monocytogenes, including those derived from Listeria monocytogenes, and the operably linked foreign antigen code sequence is most When optimized, it was observed that recombinant full-length tumor antigens were efficiently expressed and secreted from the sterilized Li 98561.doc -234-200530399. Figure 48A shows the expression and / or secretion of ΔactA monocytogenes Listeria monocytogenes from a construct having a construct including an LLO signal peptide fused to human mesothelin, using both lLO and mesothelin Human mesothelin. By Western blot analysis of TCA-precipitated bacterial culture fluid, no secretion of the expected full-length mesothelin (62 kDa) was observed for these constructs, but only a few small fragments were observed (Figure 48A) . FIG. 48B shows the expression and / or secretion of human mesothelin from Listeria monocytogenes Δact A by a plastid (pAM401) containing a construct containing various signal peptides fused to human mesothelin. Dot analysis. In each construct ', the mesothelin codon sequence was codon-optimized for expression in Listeria monocytogenes. Where specified, a signal peptide codon sequence containing a natural sequence ") or codon-optimized Γ codon-optimized, for expression in Listeria monocytogenes is used. Affinity is used. _ Purified multiple anti-human / mouse antibodies were prepared by injecting rabbits with the selected peptide and IFA to detect the secreted mesothelin. As shown in runway 3-5 and 8-9 of Figure 48B Obviously, the secretion of full-length mesothelin (62 kDa) was observed only when the signal peptide and the mesothelin codon sequence were codon-optimized for expression in Listeria. This observation also obviously includes Listeria-derived ^ -like 's derived from the bacterial LLO and p60 proteins relative to the secAl and secA2 secretion pathways, respectively, both of which contain uncommon codons. (The LLO PEST sequence is also included in the LLO signal peptide, and Also codon-optimized its codon). When the codon-optimized Listeria LL0 signal peptide and the codon-optimal link were connected, see 98561.doc 200530399 'T, to Wang Effective secretion of long mesothelin (62 kDa) (run 8. Figure, but not when using the natural code sequence of the Listeria LL0 signal peptide (Figure 7 of the runway-48B). In addition, the # codon · optimized Listeria layer p⑽ signal skin and codons are the best When optimizing the mesothelin connection, secretion of full-length mesothelin (62 gal) was observed (Runway 3, Tucher), but not when using the natural code sequence of the Listeria signal peptide (Runway 6, Figure (Through). Finally, when codon-optimization is obtained from the best signal peptide from a bacterial species different from Listeria monocytogenes in an operable manner to codon-optimize the epithelin connection, Secretion of full-length mesothelin (62 kDa) was observed (Figure 48β). Signal peptides supplied from Bacillus anthracis protective antigen (BaPA), or signal peptides derived from Lactobacillus lactis Usp45 protein (li Usp45), prepared from A program to efficiently secrete full-length mesothelin (62 kDa) in the recombinant Listeria σσ line (Figure 48B, runways 4 and 5). The Bacillus subtilis PhoD signal peptide (Bs phoD) was also prepared to efficiently secrete full-length from Listeria Mesothelin pattern (Figure 48B, runway 9). Has approximately 62 The band of molecular weight of 0, 00 is consistent with mesothelin, and the paired double band may be consistent with the non-cut and cut mesothelin peptide (ie, partially cut). Figure 48C shows that the band includes The expression / secretion of NY_ESCM in Listeria monocytogenes AactAAinlB of the construct encoding the sequence encoding the lL0 signal peptide 'The LL0 signal peptide is fused to the sequence encoding human NY-ESO-1' Both are in Listeria Codon-optimized for performance in genera. Detection of secreted NY-ESCM using a NY-ESO-1 monoclonal antibody. In this example, 'the best codon for each amino acid is used, as if by The meaning is from the frequency of every 1000 passwords in the code sequence of the Listeria genus 98561.doc -236-200530399 (http: // www &gt; kazusa.or.jp/c〇don/cgibin/ showcodon.cgi? species = Listeria + monocytogenes + [gbbct]), to synthesize signal peptides and tumor antigen functional sites. Signal peptides related to secAl, secA2, or twin-Arg translocation (Tat) secretion pathways from Listeria and other Gram-positive bacteria, to write programs for efficient secretion of human tumor antigens from recombinant Listeria . Surprisingly, the signal peptides obtained from the Listeria proteins LLO and p60 each contain rare codons (frequency per 1,000 codons &lt; 10), in order to be efficiently secreted from the recombinant Listeria Mesothelin and NY-ESO-1 require optimization of these sequences (Figure 48B). When linked to the sec A1 peptide No. 4 from the anaerobic protective antigen (pagA) and Lactococcus lactis Usp45, and the Tat signal peptide from the phosphodiesterase / alkaline phosphatase D gene (phoD) of Bacillus subtilis At the same time, mesothelin secretion was also observed. Using signal peptides from different secretory pathways, determine whether a particular pathway will: facilitate optimal secretion of heterologous proteins. For example, the Tat pathway is used to secrete cheap proteins in bacteria, and the Bacillus subtilis phoD protein is secreted via this mechanism. It was originally hypothesized that by folding before delivery, tumor antigens that contain significant water-repellent functional sites, such as secondary secretions, may be helpful. However, these results indicate that the codons for both the signal peptide and the tumor antigen encode a sequence rather than a secretory pathway Optimal effect is the initial need for effective secretion of mammalian proteins. Importantly, compared to the parental Listeria Δ_Α / ΔBin strain Che: The phenotype of the recombinant vaccine using any route for tumor antigen secretion was not significantly affected. The intermediate lethality of Listeria spp. 98561.doc -237- 200530399 (LD50) is lxl08cfu in C57BL / 6 mice. The incorporation of the tumor antigen expression card IE into the stable single copy position within the harmless position on the chromosome of Listeria △ act A / Δ inlB using the pPL2 integration vector &apos; The LD50 of the tumor antigen encoding Listeria ΔactA / ΔinlB was within 5 times of Listeria ΔactA / ΔinlB. Example 28. Construction of a bicistronic hEphA2 expression vector as a non-limiting example, providing the construction of an antigen expression cassette in which the external (EX2) and internal (CO; kinase death) functions of hEphA2 occur from bicistronic information Part of the performance. By functionally linking Ba PA and Bs PhoD signal peptides to EX2 and CO functional sites, respectively, secretion of EX2 and CO functional sites is completed. In the construction of the bicistronic hEphA2 expression cassette, codon-optimized human EphA2 kinase death plastids are used, which is called phEphA2KD. (EphA2 is a tyrosine kinase receptor, but deprived of kinase activity by mutations from K to M at the active site of the enzyme). The password sequence of phEphA2KD is shown in FIG. The phEphA2KD sequence in Figure 49 includes a codon-optimized codon sequence that deletes hEphA2 that penetrates the functional site of the membrane, and contains unique 5 * and 3f BamHI and SacI restriction positions, helping to construct a functional antigen performance card cassette. In the sequence shown in Fig. 49, the Mlul cognitive sequence is shown in bold. A sub-fragment of human EphA2 synthesized between two Mlul restriction enzyme recognition sites (deletion of the membrane-penetrating functional site, kinase-death) (by means known in the art, such as by the recruitment of nuclear picric acid Synthesis, PCR and / or Klenow fill up or similar). During synthesis, precisely at the junction between the extracellular and intracellular functional sites of EphA2 Fine 98561.doc -238-200530399, which is separated in the natural protein by penetrating the functional site of the membrane through water repellency, and insert the actA-plcB gene Between areas. The sequence of the Mlul sub-fragment of codon-optimized human EphA2, which contains the intergenic region of actA-plcB (the intergenic region is shown in bold) is shown in FIG. In addition, the codon-optimized Bs phoD signal peptide is located at the 31 end of the actA-plcB intergenic sequence and is fused to the codon of the downstream EphA2 CO functional site in the framework. By replacing the corresponding region in the human EphA2 sequence of the dead human EphA2 sequence by the transmembrane deletion kinase shown in FIG. 49, a functional human EphA2 bicistronic was assembled in place of the Mini fragment containing the actA-plcB intergenic region and the Bs phoD signal peptide. Cassette. The resulting sequence contains unique BamHI and SacI restriction enzyme recognition positions at its 5 'and 31 ends, respectively, which helps to insert and functionally link the hly promoter gene with the original signal peptide, such as Ba PA. Therefore, the seven sequentially functional elements of the bicistronic human EphA2 antigen expression cassette are as follows: hly promoter gene-Ba PA signal peptide-EX functional site EphA2-stop codon-actA-plcB intergenic region (with Shine-Dalgarno sequence) -Bs PhoD signal peptide-CO functional site EphA2-stop codon. Preferably, all EphA2 and signal peptide codon sequences are codon-optimized. Recombinant Listeria strains that express and secrete EphA2 EX and CO functional sites can be derived by the methods explained in this application using pAM401, pKSV7, or pPL1 and pPL2 integration vectors. Detect the expression and secretion of EphA2 protein by western analysis of bacterial lysates, using methods described and / or familiar with this art. 98561.doc -239-200530399 Example 29. Expression and secretion of an antigen derived from recombinant Listeria spp. Which includes the antigen_bacterial protein and Zhao Zhao In some embodiments of the present invention, a signal peptide is fused to its heterologous protein Sequence codon optimization for both partners. In some specific embodiments, it is desirable to place the codon-optimized heterologous protein sequence within a defined region of the protein to secrete its natural form from the Listeria spp. Functionally, the heterologous protein sequence is placed within the selected sequence of the secreted Listeria protein sequence, and a protein chimera having the same molecular weight as the secreted protein can be synthesized and secreted. The secretion of heterologous proteins can be promoted by utilizing the mechanism of the host Listeria bacterium required for optimal secretion of autologous bacterial proteins. The molecular chaperone helps to secrete selected bacterial proteins. As a non-limiting example, a protein chimera produced between Listeria monocytogenes protein P60 and human tumor antibodies, mesothelin. A protein chimera is produced by placing the human tumor antigen mesothelin precisely within the amino acid position 70 within the Listeria monocytogenes protein ρ60 (although it should be understood that any desired Heterologous protein coding sequences to produce protein complexes). The protein chimera contains the best codon for the Listeria genus at p60 amino acid and the entire mesothelin codon sequence. In addition, the P60-human mesothelin protein chimera is functionally linked to the Listeria monocytogenes hly promoter gene and incorporated into the ppL2 vector, which is then used to produce expression and secrete humans as described herein Mesothelin Recombinant Listeria Monocytogenes Strain Described below to construct optimal expression and secrete p60_human mesothelin protein chimera 98561.doc -240- 200530399

See Lenz et al. (2003 PNAS, 2003). A reselected heterologous protein sequence of a selected protein chimera between selected monocytogeneic Listeria white matter sequences is placed in the Listeria gene, and the monocytogenes and bacteria are Optimal secretion of the interaction of the chaperone and the secretory device, and the functional activity of the protein chimeric Listeria polystera protein. It is desirable to functionally place heterologous proteins within the single 3secA2-dependent proteins NamA and p60 'for retention Peptidoglycan cell wall hydrolase activity of these proteins (see 10 0 ·· 12 4 3 2 -12 4 3 7), for example regarding

Description of SecA2-dependent NamA and P60 proteins). In some specific embodiments, a heterologous protein code sequence between a chromosome sequence (ss) and a cell wall binding functional site (LySM) and a catalytic functional site Lyz-2 (NamA) and a p60-functional site (p60) is desired. Functional placement (Lenz et al. (2003)). In some embodiments, the expression of the antigen or heterologous protein is functionally linked to a prfA-dependent promoter gene. The expression of heterologous proteins themselves is induced in recombinant Listeria-infected cells. The first step in the construction of the p60_mesothelin protein chimera involves the DNA synthesis of the prfA-dependent hly promoter gene, which is functionally linked to a DNA sequence encoding 70 amino acids before p60, with The best codon for secretion in Agrobacterium. (In some embodiments, codon usage can be further modified to avoid excessive regions of RNA secondary structure, which may inhibit protein translation efficiency 98561.doc -241-200530399). A DNA sub-fragment corresponding to the -70N-terminal p60 amino acid of the hly promoter gene was synthesized. (This can usually be done by gene synthesis methods known in the art, such as by oligonucleotide synthesis, PCR and / or Klenow filling or the like). The sequence of the first 70 amino acids from p60 of Listeria monocytogenes strain 10403S is shown below: MNMKKATIAATAGIAVTAFAAPTIASASTVVVEAGDTLW GIAQSKGTTVDAIKKANNLTTDKIVPGQKLQ (SEQ ID NO: 116) The person skilled in the art can know that there are multiple monocyte augmentations Laboratory and field isolates of Listeria-encoding genes, including p60, can contain variability at both the nucleotide and amino acid sequence levels, but are essentially the same genes and proteins. In addition, those skilled in the art will know that genes from any laboratory or field isolate (including food-borne or clinical strains) of Listeria monocytogenes can be used to construct protein chimeras. The synthetic DNA sequence corresponding to the hly promoter gene -70N-terminal p60 amino acid is shown in FIG. In addition, the codons encoding p60 amino acid residues 69 (L) and 70 (Q) were modified to contain a unique PstI enzyme recognition sequence to facilitate functional insertion of heterologous sequences. In addition, the 5 'end of the synthetic sub-fragment contains a unique κρηΙ enzyme recognition sequence. The 447 base pair digested sub-fragments with κρηΙ and PstI were ligated into the corresponding κρηΙ and PstI positions of the pPL2 vector, and digested with κρηΙ and PstI enzymes, and bovine intestinal alkaline phosphatase (CIAP ) Digestive treatment. This plastid is called pPL2_hlyP_Np60 CodOp. Subsequently, the remainder of the native p60 gene 98561.doc-242-200530399 was selected between the unique PstI and BamHI positions and cloned into the pPL2-hlyP-Np60 CodOp plastid. The remaining p60 gene was cloned by PCR, using a proofreading that contains a thermostable polymerase, and the following primer pairs: Forward primer: 5f-CGC CTGCAGGTAAATAATGAGGTTGCTG (Order) J No. 117) Reverse primer: 5'-CGCGGATCCTTAATTATACGCGACCGAAG ( SEQ ID NO: 118) Digestion of the 1241 base pair expansion region with PstI and BamHI, and ligation of the purified 1235 base pairs into pPL2-hlyP-Np60 CodOp plastid digested with PstI and BamHI and treated with CIAP . The plastid contains the complete Listeria monocytogenes p60 gene, has the best codon for 1-77 amino acids, and the natural codon for 78-478 amino acids. Functionally linked to the Listeria monocytogenes hly promoter gene. This plastid is called pPL2-hlyP-Np60 CodOp (1-77), and the sequence of the KpnI-BamHI sub-fragment is shown in Fig. 52, which contains hlyP functionally linked to the p60 coding sequence. The expected sequence of pPL2-hlyP-Np60 CodOp (l-77) plastid was confirmed by sequencing. The next step in the construction is to functionally insert a heterologous protein coding sequence at a unique PstI position like pPL2-hlyP-Np60 CodOp (1-77) plastid, which is located between the N-terminal signal peptide sequence and P60. The first LysM cell wall binds between functional sites, thereby preserving the normal biological function of the Listeria monocytogenes protein. 98561.doc -243-200530399 As a non-limiting example, for optimal performance in Listeria monocytogenes, the human mesothelin codon was optimized and this protein was inserted like pPL2_hlyP-Np60 CodOp (l-77) is within the unique PstI position of the plastid. Specifically, a thermostable polymerase having proofreading activity was used, and the following primer pairs were used to colonize the full-length human mesothelin-containing plastid described in Example 27 to delete signal peptide and GPI cross-linker functional sites. Full-length mesothelin or mesothelin (mesothelin ASP / AGPI), containing the best codons for performance in Listeria monocytogenes: 1. Full-length forward primer (huMeso 3F): 5, -AAACTGCAGGCATTGCCAACTGCACGTCC (Sequence number 119) reverse primer (hMeso 1935R): 5f-AAACTGCAGAGCTAATGTACTGGCTAATAATAATGCT AAC (sequence number 120) 2. △ signal peptide, AGPI anchor forward primer (huMeso 133F): 5f-CGCCTGCAGCGTACATTAGCAGGTGAAACAGG (reverse primer 121) (HuMeso 1770R): 5, -CGCCTGCAGGCCTTGTAAACCTAAACCTAATGTATC (SEQ ID NO: 122) Purify 1932 base pairs (full-length mesothelin) and 1637 base pairs (mesothelin △ SPMGPI), digest with PstI, purify, and connect to Plastid 98561.doc -244- 200530399 pPL2-hlyP-Np60 CodOp (1-77) in the unique PstI position, digested with PstI, and treated with CIAP. The invariant N-CO orientation of p60 and mesothelin functional sites was confirmed by endonuclease mapping. These plastids are referred to as pPL2-hlyP-Np60 CodOp (l-77) -mesothelin and pPL2-hlyP-Np60 CodOp (l-77) -mesothelin ASPMGPI, and as described in the examples incorporated herein, Introduce it into selected Listeria monocytogenes strains (such as AactAAinlB double deletion mutants). Fig. 53 shows the Kpnl-BamHI sub-fragment of plastid pPL2-hlyP_Np60 CodOp (l-77) -mesothelin, which contains the hly promoter gene functionally linked to the gene encoding the p60-human mesothelin protein chimera. The sequence of the KpnI-BamHI sub-fragment of the plastid pPL2-hlyP-Np60 CodOp (l-77) -mesothelin Δ SP / AGPI is shown in FIG. 54, which contains functionally similar to p60-human mesothelin ASP / The AGPI protein chimera encodes a gene-linked hly promoter. Western analysis of expression and secretion of p60-mesothelin protein chimeras As discussed in the examples, the expression and secretion results of selected xenogeneic antigens effectively induce MHC class I · restricted CD8 + T cell responses. By western analysis and by the method described in the examples incorporated herein, tests were performed using mesothelin-specific multiclonal antibodies against pPL2-hlyP-Np60 CodOp (1-77) -mesothelin or pPL2-hlyP -Np60 CodOp (1-77) -Mesothelin ASPMGPI plastids of tRNA-Arg chromosomes inserted into the recombinant Listeria monocytogenes AactAAinlB double deletion mutants expressed and secreted into the protein protein zygotes. For proteins presented in some runways, the designation of human mesothelin is designated 98561.doc -245-200530399 (△ SP △ GPI, also referred to herein as △ SS △ gpi, △ SP / ZXGPI, △ ssmgh Etc.) are as follows: The deletion signal sequence (Δδρ) corresponds to 34 amino acids at the N-terminal of human mesothelin (for the sequence of human mesothelin, see, for example, FIG. 34 or GenBank accession number BC009272). The deleted GPI (AGPI) functional site is equivalent to 42 amino acids at the c-terminal, starting with the amino acid residue Gly_Ile_pro and ending with the amino acid residue Thr_Leu-Ala (see, for example, FIG. 34). The results of this analysis confirm that from recombinant Listeria monocytogenes _ efficiently expressed and secreted a protein chimera including p60 and accurately inserted into human mesothelin or human mesothelin ASP / ZiGPI (in the architecture, Insert the amino acid 70 of p60 between the N-terminal signal peptide and the first of two LysM cell wall binding functionalities). See Figure 55. (The axis of FIG. 55 shows the molecular weight of the protein in the trapezoidal run-out trapezoid on the far left. ^ gpi's expected expression and secretion of protein chimeras. In runway 2 and 4, the performance of human mesothelin △ φ △ GPI is significantly increased compared to full-length mesothelin. On runways 3 and 4 In the protein chimera shown in Figure 5, the reliable N-terminal p60 amino acid was used. In the conjugation that ran in runway 丨 and 2 of Figure 55, the amino acids butyl and v were deleted at positions 29 and 64, respectively. The runway 5 shows the expression and secretion of the PA signal peptide of P. anthracis fused to human △ SPAGpi_mesothelin (in which the signal peptide and mesothelium were expressed for the expression in Listeria monocytogenes). Codon-optimization), while runway 6 shows the expression and secretion of full-length human mesothelin fusion 2LL0 (in order to list in Listeria monocytogenes 98985.doc -246-200530399 Signal peptide and mesothelin code sequence Codon_optimization). Runway 8 shows the protein expressed by J293, a human cell line, while Runway 7 shows the protein expressed and secreted by J293 containing plastids encoding full-length human mesothelin (J293 / full-length). Runway 10 shows proteins expressed and secreted from Listeria spp. Where endogenous endogenous 60 has been deleted. 较低 The lower box in Figure 55 shows the use of multiple strains of α-ρ60 antibodies for Listeria monocytogenes Western analysis of p6O secretion. The results confirm that an equal amount of [.secreted protein is loaded on the gel. The results confirm that p60 can be used as a molecular chaperone for secreting heterologous proteins and facilitates submission of MHC class I pathways. Example 30 · Additional examples of expression and secretion of antigen by recombinant Listeria monocytogenes A. Expression of intracellular functional sites (ICD) of EphA2 from bicistronic constructs using non-Listeria signal peptides Figure 56 Western blot analysis of intracellular functional sites (ICD) that express and secrete EphA2 from bicistronic information using non-Listeria, non-secA1 signal sequences.

EphA2 is a protein shell that includes extracellular functional sites (ECD) and intracellular functional sites (ZCD). Excluding Listeria △ actA △ inlB, it shows bicistronic mRNAs. Among them, bicistronic mRNAs are separated polypeptides that encode extracellular and intracellular functional sites of Epha2. In order to be expressed in Listeria monocytogenes, all the coding sequences (the Bacillus subtilis phod signal peptide, the anthracis protective antigen signal peptide, and the Lactococcus lactis Usp45 signal peptide) used in the constructs were encoded. Sequence codon optimization. It is also 98561.doc -247- 200530399. It is expressed in Listeria monocytogenes, and the sequence codons encoding ECD and ICD functional sites are optimized. The Listeria promoter hly obtained from the LLO gene was used as the promoter in these constructs. The integration vector pPL2 was used to integrate a performance card encoding bicistronic mRNA into the Listeria genome. Western blot analysis using a variety of bacteria / valley fractions using standard techniques is used to detect and measure the EphA2 function site in the accumulated cells. The results confirmed that the intracellular functional site of Epha2 was expressed and secreted from the bicistronic construct using a non-Listeria signal peptide 'encoded by the codon-optimized sequence. The performance constructs include: (1) a codon-optimized sequence encoding a secretory sequence of Lactococcus lactis Usp45 linked in an operable manner (functionally) with the codon sequence of the extracellular functional site (the first polypeptide) of EphA2 , And a codon-optimized sequence encoding the secretory signal of Bacillus subtilis phod (operator 1) linked to the codon sequence of the extracellular functional site (second polypeptide) of EphA2 in an operable manner; and (2) encoding A codon-optimized sequence of the anthracis anthracis compliance-secreting sequence operably linked to the codon sequence of the extracellular functional site (first polypeptide) of EphA2, and a cell encoding operatively linked to EphA2 Codon_optimized sequence of phoD secretory sequence linked to the codon sequence of the internal functional site (second polypeptide) (Runway 2-3 (two different pure germline); see construction in Example 28 above Description of the performance cassette). The control group study (runway 4) used the attenuated parental Listeria strain ΔαοΐΑΔίπΙB strain to confirm that there were detectable cross-reactivity of various contents in the ink dots of some control groups. Runways 1-3 show slow-moving bands and fast-moving bands, where the fast-moving band is equivalent to the intracellular functional unit 98561.doc -248-200530399 (ICD). Intracellular functional sites of EphA2 expressed from all constructs (Runway 1-3) were observed in all three bacterial lysates. Runway 4 (control group) shows only slowly moving bands. Since there are no antibodies available for extracellular functional sites, the expression / secretion of extracellular functional sites was not determined. B · Plastid-based expression and secretion of rat mesothelin, which becomes a function of N-terminal fusion with various codon-optimized signal peptides Figure 57 shows the use of various signal peptides, including non-Listeria signals Peptide and non-secAl signal peptide, plastid-based expression and secretion of mouse mesothelin expressed from the codon-optimized mesothelin codon sequence. It shows that the expression and secretion of plastid-based mouse mesothelin become the function of N-terminal fusion with various signal peptides encoded by codon-optimized sequences. In all cases, the sequence codon _ of the signal peptide encoding the mesothelin fusion protein was optimized, and the mouse mesothelin codon sequence was codon-optimized for expression in Listeria monocytogenes . The expression and secretion of mouse mesothelin from Listeria monocytogenes were measured, wherein the Listeria genus contained PAM401 plastids, and wherein the plastids encode mesothelin. Various plastid-based constructs were tested in which the signal peptide was altered. Western blotting was performed on proteins recovered from various fractions of secreted proteins (A), cell walls (B), and cell lysates (c). For each lysate, runway 丨 _2 shows mouse mesothelin expressed as a fusion with the signal sequence of the protective antigen of Bacillus anthracis, and runway 3-4 shows a fusion with the Lsp lactis Usp45 signal sequence Mouse mesothelin, runway% 6 shows mouse mesothelin expressed in the form of fusion with the Bacillus subtilis phoD signal sequence, and runway shows mouse mesothelin expressed in the form of fusion with p60 signal sequence, running 98561.doc -249- 200530399 Lanes 9-10 show mouse mesothelin expressed as a fusion with the LL0 signal sequence, and runway 11 shows the protein expressed by the control group Listeria. The results confirmed that the highest expression and secretion were found at the signal sequences including the protective antigen of the bacillus carboniferae L (sequence 1_2) and the Bacillus subtilis pHOD signal sequence (sequence 5_6). C. Human mesothelin expression and secretion based on the chromosome of Listeria monocytogenes. Figure 58 shows human mesothelin in various bacterial cell lysates (ie, secreted proteins, cell niches; glutenin products). Western blot analysis of expression and secretion based on the chromosome of Listeria monocytogenes. When fused to non-Listeria secAl and non-secAl signal peptides, it is necessary to test the expression and knife of human mesothelin. Listeria bacteria in the text test are △ button 丨 A / △ Listeria spp., And are as follows: Listeria s.acta / AinlB (control group Listeria spp., Not designed to express mesothelium ) (Runway 1); Listeria spp. (Runway 2-3) encoding the Anthrax anthracis protective antigen signal sequence fused to ass / a GPI human mesothelin; Encoding to △ SS / AGpi human mesothelin fusion Listeria spp. PhoD signal sequence (Runway 4_5); Listeria spp. (Runway 6_7) coding for anthrax protective antigen signal sequence fused to full-length human mesothelin; coding for fusion to full-length human mesothelin Listeria spp. For the PhOD signal sequence of Bacillus subtilis (runway 8-9). In order to be expressed in Listeria monocytogenes, the sequence encoding the signal sequence fused to mesothelin in all Listeria species described above will be densely-optimized. In addition, in each construct, in order to be expressed in Listeria monocytogenes, the mesothelin codon sequence (Δ SS / Δ GPI and full 98561.doc -250- 200530399 long) codon-most optimization. In each of the above-mentioned Listeria spp. Expressing mesothelin, the mesothelin expression cassette was inserted into the Listeria chromosome via the integration of pPL2. Where human mesothelin was designed to delete its signal sequence and delete the water-repellent region (gpi region), the southernmost performance using the phoD secretion sequence of Bacillus subtilis appeared (runway 4-5). Example 31. Additional examples of immunogenicity and anti-tumor efficacy of recombinant Listeria monocytogenes vaccines The following examples disclose the results of vaccination with Listeria spp. According to the invention, such as vaccine-dependent cytokine expression Stimulation, vaccine-dependent survival of animals with tumors, reduction of vaccine-dependence on tumor metastasis, and reduction of vaccine-dependence on tumor volume. A. Immunogenicity of Listeria vaccine including P-60 model antigen chimera Figures 59A and B show that by expressing Listeria spp. Which is a p60-antigen chimera or LLO signal peptide-antigen fusion protein, xenogeneic Antigen is delivered to the MHC class I pathway. The heterologous antigen used in this experiment was AH1-A5. It was inoculated with Listeria spp. Designed to include a p60 protein chimera expression cassette encoding ΑΗ1 · Α5 (fused to the OVA SL8 peptide) inserted into the p60 polypeptide sequence including the N-terminal p60 signal peptide sequence ("A p60-based construct, or a Listeria spp. Designed to encode an LLO signal peptide, linked to a nucleic acid encoding the same antigen, AH1-A5 embedded in OVA (" LLO-based construct '·). Both constructs use the Listeria starter gene hly ° p60 疋 Listeria peptidoglycan autolysin, which is secreted by the secA2 pathway, and LLO is Listeria lysin. 98561 .doc -251-200530399 To produce a p60-based construct, the nucleic acid encoding p60 is designed to contain a PstI colony position, where the pstl colony position represents a silent mutation, that is, the result is in the encoded amino acid sequence No change. The pstI position is located between the N-terminal signal sequence in the p60 sequence and the first of two LysM cell wall binding functional sites. In the architecture, the encoding will include the AH1-A5 epitope (SPSYAYHQF (SEQ ID 73) No.)) and SL8 The polynucleotide of the original epitope (SIINFEKL (sequence number 123)) is inserted into the psti colony position. These epitope coding sequences are separated by a unique Xhol position, and in order to increase the number of monocytes The codon-optimization is performed for the specific bacteria. The effect of insertion into the PstI position occurs at the equivalent of nucleotide base number 199 of p60. For the performance of Listeria monocytogenes, the p60 code is used. The first amino acid codons of the sequence are optimized. Therefore, the codons that are best for performing in Listeria monocytogenes represent the first 27 amino acids corresponding to the signal peptide. The antigen expression cassette further contains unique 5, and 3 'KpnI and SacI positions. For the purpose of integration with the tRNA-gene adjacent position-specific integration of the Listeria monocytogenes genome, the PPL2 plastids were inserted separately. Within the MCS. The LL0-based construct includes a sequence encoding an LLO signal peptide and is operably linked to a nucleic acid encoding AH1_A5 within the OVA (without using any codon_optimization effect). Therefore, in this study, the signal peptide was obtained from Listeria ll10 or Listeria p60. By placing the construct in PPL2, it mediates the position-specific recombination of the Listeria genome Functioning vector and inserted into the Listeria genome 0 98561.doc -252- 200530399 Figures 59A and B show the performance of the inoculation (caudal vein) with the p60 signal sequence / autolysin as the AH1-A5 antigen of the p60 chimera The immune response to Listeria and the immune response to Listeria vaccinated with the ah 1-A5 antigen linked to the LLO signal sequence. In the X-axis of the figure, "Un st im," means that no peptide is added to the well (that is, the well is unstimulated), and "AH 1" means that AH1 nonapeptide is added to the well, and & quot AH1-A5, which means adding AH1-A5 nonapeptide to the well. Design all bacterial vaccines to contain integrated nucleic acids encoding AH 1-A5 (bacterial vaccines do not encode AH1) (see, for example, siansky et al .; Human (2000) Immunity 13: 529-538). Where the listeria is inoculated with a construct based on p60, the puppet is indicated by "p6〇" on the X-axis of the figure. For inoculation with Listeria spp. Including LLO-based constructs, the lines are represented by &quot; LLO "on the x-axis of the figure. The overall draft of Listeria vaccinations using p60-based constructs is as follows: (1) Mice were vaccinated with Listeria spp. (Tail vein (intravenous)) containing integrated nucleic acids, of which the integrated nucleic acids Encoding p6〇, containing a nucleic acid encoding AH1-A5 inserted at nucleotide 199 of p60. In other words, the nucleic acid encoding the AH 1 · A5 antigen is operably linked to the ρ60 signal sequence and ρ60 autolysin in the architecture. In order to express in monocytosis Listeria, 'optimize the nucleic acid codons encoding AH 1-Α5; (2) remove the spleen seven days after the sensation, (3) isolate the spleen cells and place them in the well In the 'and follow the instructions on the X-axis', the spleen cells were added with or without the shape (Figures 59 and 59), with the added person 111 (Figures 59 and 8) or with the added eight rules-8 5 ( Fig. 59B)-culture; (4) After adding the peptide, the cells were cultured for five hours, and then the presence of CD8 + T cells expressing IFNY was evaluated by FACS analysis. As for Lee 98561.doc -253-200530399, inoculation with Listeria spp. Which expresses LLO-based constructs, a similar draft is used. The results confirmed that where the added peptide was AH1 (Figure 59A) or where the added peptide was AH1-A5 (Figure 59B), the Listeria vaccine stimulated CD8 + T cells to express IFNY. Where the integrated AH1-A5 is operatively connected to the LLO signal peptide, the stimulus is higher, and when the integrated AH1-A5 is operatively connected to the p60 signal sequence, how much is the stimulus? Lower (Figures 59A and B). Figures 60A and B show experiments performed using the same Listeria spp. As described above, i.e. as in Figures 59A and B. Figure 60A shows the results of inoculating mice with Listeria ("p60") designed to contain a construct based on p60 or &quot; LLO "designed to contain a construct based on LLO. As indicated on the X-axis of FIG. 60A, peptide-free (unstimulated ;, imstimn) or LL091-99 peptide (, LL091n; Badovinac and Harty (2000) J. Immunol. 164 ·· 6444 -6452) supplemented with cell-based assays. The results confirm a similar immune response (IFNY manifestation), in which the Listeria vaccine contains p60-based constructs or LLO-based constructs. In Figure 60 A The stimulated immune response reflects the results derived from the cell-based strategy, which is due to the endogenous manifestation of natural LLO of Listeria. Figure 60B shows the design of Lees designed to contain a p60-based construct The result of inoculating mice with H. pylori, in which the hly promoter gene and signal peptide sequence are operably linked to a nucleic acid encoding AH1-A5, or in a L. Listeria inoculated mouse designed to contain an LLO-based construct Result, where hly Animal gene and signal peptide sequences are operably linked to the nucleic acid encoding AH1-A5 98561.doc -254-200530399. According to the instructions on the axis, the added peptide is peptide-free (unstimulated; |, 11 1111 &quot;) or 1) 60217.225 peptide ("1) 60-217"; 8 Xiao 8 et al. (1997) J. Biol. Chem. 272: 19261-19268). The stimulated immune response is reflected in Figure 60B Results obtained from cell-based assays are attributed to p10, which is endogenous to Listeria spp. For constructs based on 110, and to p60-based constructs, Combination of endogenous p60 and chimeric sequences expressing p60 protein. B. Therapeutic efficacy of Listeria spp. Expressing human mesothelin The results described in Figure 61 show the use of expressing human mesothelin (hu mesothelin) Listeria vaccinations prolonged survival in tumor-bearing mice, in which tumor cells in mice have been designed to express human mesothelin. The tumor cells are CT26 cells expressing human mesothelin, and the mouse For Balb / c mice. (All CT26 described in this article Cancer research involves Balb / c mice). In a performance cassette, the sequence encoding the non-Listeria signal sequence is operatively optimized with the codons encoding human mesothelin-optimized Sequence (deleted its signal sequence and GPI anchoring). In the Listeria vaccine, the expression cassette encoding the signal peptide fused to human mesothelin (AGpjass) was administered to tumor-bearing mice and the fusion protein pair was studied. Impact on tumor immune response. A performance cassette encoding a mesothelin fusion protein has been integrated into the Listeria chromosome. On day 0, 2xl05 CT26 cells (CT 26huMes0 +) expressing human mesothelin were injected intravenously into Balb / c mice. Mice were inoculated in the tail vein (intravenously). Inoculation with ie7 colony forming unit (CFU) Listeria (intravenous) occurred on day 3. 98561.doc -255-200530399

Figure 61 shows the percentage survival of mice to CT26 tumors expressing human mesothelin (shown on the y-axis) 'where the vaccine includes Hank's Balanced Salt Solution (HBSS) (pretend vaccine; &quot; HBSS "); Listeria △ actAMMBM has integrated performance card II showing SF-AH1A5 (positive control vaccine; nSF-AHl A5lf); or Listeria △ actAA inlB, which includes the encoding and human mesothelin (by codon -Optimized sequence coding) Performance cassette of the fused anthracnose protective antigen signal sequence (encoded by uncodon-optimized sequence), wherein the human mesothelin has the deleted signal sequence and the deleted code is water-resistant gpi-tin-like region (&quot; BaPA-huMeso △ gpi △ ss ,,). Listeria spp. SF-AH 1A5 construct and BaPA-huMeso △ gpiz \ ss construct to integrate on the chromosome These constructs are contained in the form of constructs. The nucleic acid molecule encoding SF-AH1A5 and the nucleic acid molecule encoding the BaPA-huMeso △ gpi △ ss construct have been integrated into the Listeria genome using pPL2. SF is derived from egg white protein The eight amino acid peptides are also known as SL8 (see, e.g., Shastri and Ganzalez (1993) J

Immunol. 150: 2724-2736). The abbreviations "SF-AH1A5,", "SF-AH1-A5", and "OVA / AH1-A5" mean AH1-A5 connected to the ovalbumin scaffold. "SF AH1-A5" means AH1-A5 (SPSYAYHQF (SEQ ID NO: 73)) and an SF peptide fused to the N-terminal of amino acids 138 to 386 of GenBank Accession No. 2 (ovalbumin). In this example, it encodes "SF-AH1 -A5 "polynuclear bite 'includes codon-optimized nucleic acid encoding AH 1-A5, and uncodon-optimized nucleic acid encoding ovalbumin-derived sequence. The results confirm the use of human mesothelin The single immunization of Listeria spp. Prolongs the survival of mice containing tumors expressing human mesothelin. Lee 98561.doc -256- 200530399 has been integrated on the chromosome and has been integrated with the △ signal sequence / △ gpi in humans. Corticoid-fused anthracnose protective antigen signal sequence (BaPA_huMeS0 gpi △ ss; filled square) has the highest percentage of survival. Survival is lowest in the "inoculation place" with the saline solution of the control group. C. Because of mesothelin -Dedicated anti-tumor efficacy in the expression of human mesothelin In tumor-bearing mice vaccinated by Mycobacterium spp., The degree of lung tumor nodules was reduced. The data in Figure 62 confirms that Listeria spp. Act A △ inlB, which expresses human mesothelin by vaccination, reduces the degree of lung tumor nodules, of which The tumor cells were designed to express human mesothelin. The mouse strain was Balb / c, and the lung tumor cells were CT26 cells harboring a vector expressing human mesothelin. On day 0, 2xl05 cells expressing human mesothelin were expressed. Dip 26 cells were administered intravenously to Balb / c mice. In the performance cassette, the sequences encoding various signal sequences are operatively linked to the codons encoding human mesothelin in the architecture_optimized Sequence ligation. The expression cassettes encoding various signal peptides fused to human mesothelin were administered to tumor-bearing mice via a Listeria vaccine including the expression cassettes. On day 3, ix107CFU / 10.00 micro L. Listeria vaccine was administered intravenously to mice bearing tumors. The negative control group was vaccinated with HBSS or Listeria. The positive control group was vaccinated with manifestations including AH1A5 (in the framework with the 0VA sequence Column fusion) of the Ova fusion protein of Listeria g. (By uncodon. Optimized performance card E encodes OVA fusion protein including AH1A5). On day 19, mice were sacrificed, their lungs were harvested, and calculated The number of lung tumor nodules. Listeria vaccine reduces the number of metastases in the lung. The control vaccine 98561.doc -257-200530399 only involves HBSS or Listeria △ actAA iniB. As a result, each lung 250 metastases, and an average of 135 metastases per lung. Listeria ("pAM-LLO-huMeson), which carries a plastid (pAM401) encoding an LLO signal peptide fused to human mesothelin, shows approximately 25 metastases per lung. For Listeria monocytogenes "Performance" optimizes the polynucleotide sequence codons of the pAM-LLO-huMeso plastid encoding the LLO signal peptide and the human mesothelin sequence. Fusion of the T code with human mesothelin (△ gpi / △ signal sequence) The integrated sequence of Listeria spp. ("BaPA-HuMesoZ \ gpiA ss") of the Bacillus anthracis protective antigen signal sequence (BaPA) also showed an average of about 25 metastases per lung. At BaPA-HuMes〇AgpiZ \ The polynucleotide encoding the protective antigen signal sequence of Bacillus anthracis in ss was not optimized by codons, and in order to be expressed in Listeria monocytogenes, humans encoding the deleted mesothelin signal peptide and GPI anchored Polynucleotide codon optimization of the mesothelin sequence. Figure 63 shows the results of a control study using mice including lung tumor nodules produced using CT26 parent target cells. Balb / c mice were used, but modified And note The field-type CT26 (on day 0, 2 x 105 cells (intravenously)). Studies have demonstrated that the anti-tumor efficacy of a Listeria vaccine expressing a mesothelin fusion protein is mesothelin-specific. In the performance cassette, the sequences encoding various signal peptides are operatively linked to the codons and optimized sequences encoding human mesothelin in the architecture. (The construct used in this example is the same as in the previous example. Those used in the experiments were the same, producing the data shown in Figure 62.) Via a Listeria vaccine expressing card E, the various peptides encoding human mesothelium 98561.doc -258-200530399 primes will be encoded. Performance card g is administered to the old-fashioned tumor. The inoculation is in the tail vein (lxl () ^: Fu / ⑽ microliter, intravenous, on day 3). In this particular study, tumor cells did not show human mesothelium The number of lung metastases was also measured where data were available. There were a total of five mice in each inoculation group. The negative control group was inoculated with hbss or Listeria △ actA △ ΙηΙ. Positive control Group vaccination involves performance Includes Listeria spp. OVA fused Listeria spp. (Without codon_optimization). The results are shown in Figure 63. The fork again indicates that it cannot survive, and each inoculation group contains 5 mice. As for the positive control group inoculation The mice survived, and the number of metastases detected in the lungs was about 25 per lung on average. Because tumor cells were not designed to express human mesothelin, the inoculation contained an LLO signal that expressed fusion with human mesothelin. Peptide plastids of Listeria (&quot; pAM_LLO-HuMes 0 ") mice did not survive. The protective antigen signal sequence of Bacillus anthracis which has been integrated on the chromosome and fused with human mesothelin (Agpi / △ signal sequence) (BaPA; encoded by the uncodon_optimized nuclear phonetic sequence) Listeria (&quot; BapA_HuMeso △ gpi △ ss ,,) Some mice survived but others failed to survive. D. Listeria inoculation with codon-optimized human mesothelin, reduced tumor volume Figure 64 shows humans obtained from a performance cassette that includes codon-optimized mesothelin codon sequence Mesotheliin inoculation with Listeria (△ aCtA △ iniB) reduced tumor volume. In the performance cassette, sequences encoding various signal sequences are operatively linked to the codon-optimized sequence 98561.doc -259- 200530399 encoding human mesothelin. The expression cards encoding various signal peptides of the human mesothelin fusion of the discoid human were administered to the carrying mice via a Listeria vaccine including the expression card E. In this study, mice bearing tumors were vaccinated with a Listeria vaccine expressing human mesothelin, including the following: Listeria monocytogenes Listeria monocytogenes carrying pAM40) plastids, expressed and secreted LLO signal peptide fused to human mesothelin (encoded by a sequence that is coded to make it visible in the Listeria monocytogenes g) ("paM opt · LLO-opt.huMeso"), carrying Listeria pAM401 plastid, a protective antigen signal sequence of Anthrax anthracis expressing fusion with human mesothelin (encoded by uncodon-optimized expression cassette) ("ρΑΜ n〇n_ opt. BaPA-opt. huMeso "); and a Listeria spp. that includes a performance cassette that integrates and encodes the anthracnose protective antigen signal sequence (encoded by uncodon _ optimized sequence) fused to human mesothelin, wherein the human Cortex has a deleted signal sequence and a deleted region that encodes a water-repellent, anchored peptide ("Non-opt. BaPA-opt.huMeso delgpi-ss"). In the study, Balb / c mice were implanted subcutaneously with 2xl05 c26 tumor colon tumor cells &apos; and designed to express human mesothelin (day 0). Five mice were included in each seed group. On day 3 after CT26 cell injection, mice were vaccinated intravenously with a non-Listeria control group or a 1x107 colony forming unit (CFU) listeria vaccine. The negative control group was immunized with HBSS. The positive control group was inoculated with Listeria spp. Which expressed SF-AH1 A5 (codon optimization). (SF is an eight amino acid peptide derived from ovalbumin, also known as SL8 (see, for example, Shastri and Ganzalez (1993) J_Immunol. 150: 2724-2736)). At various time points, the average tumor volume was determined. 98561.doc -260- 200530399 The results of this study are shown in Figure 64. The results confirmed that inoculation with Listeria spp., Which expresses human mesothelin fused to various signal peptides, reduces tumor volume, and inoculation with Listeria spp., Which expresses anthrax anthracis protective antigen signal peptide fused with human mesothelin Sexual (hollow circles and dotted lines). Listeria inoculated with Listeria spp., Which expresses human mesothelin fused to the LLO signal peptide, is protective (open triangle). Includes a performance cassette that has been integrated on the chromosome and encodes a Listerial signal peptide of the anthracnose protective antigen (non-codon_optimized nucleic acid) fused to human mesothelin (Δ§ρί / △ signal sequence). Bacterial inoculation (hollow oval and solid line) is also protective. Regarding the positive control group, Listeria (open squares) showing SF-AH1A5, which has been integrated on the chromosome, is also protective. The highest tumor volume and the earliest time for tumor growth occurred in mice receiving fake vaccines (11; 888). E. Immunogenicity of a Listeria vaccine expressing human mesothelin fused to a non-Listeria signal sequence Figure 65 depicts the immunogenicity of the Listeria △ actA / A inlB_ human mesothelin line Among them, the Listeria genus contains a nucleic acid that has been integrated on the chromosome, which encodes human mesothelin fused to the signal peptide of Bacillus anthracis (the most optimized 6 Jiang? 8111 ^ 8080-8181 88). The immune response was assessed using the ugly 11.8 d assay, which was sensitive to the performance of interferon-γ. This study includes the following steps: (1) Vaccinate (intravenous) mice (Balb / c mice or C57BL / 6 mice) with Listeria, which includes an integrated performance cassette that encodes human mesothelium (Coded by the codon-optimized sequence 'where the mesothelin signal peptide and water-repellent gpi-anchor sequence have been deleted) 98561.doc -261-200530399 fused anthracnose protective antigen signal peptide (by uncodon _ The most realistic sequence code), (2) 'remove the spleen after 7 days; (3) disperse the cells removed from the spleen in the wells. Each well receives approximately 20,000 spleen cells; (4) Add one of three media to the wells as directed. Spleen cells obtained from studies using mice received only culture media ("unstimulated ,,"), mesothelin peptide collection ("MesO collection"), or ρ60217 · 225 (, 'ρ6〇2ΐ7 ,,). Available from The spleen cells studied using C57BL / 6 that received media only ("unstimulated"), mesothelin peptide collection (,, Mes〇 collection ,,), or LLO296,4 ^! ^ 296,4 "). An ELISPOT assay was performed to determine the number of immune cells that responded to the added peptide (s). The mesothelin peptide collection includes 153 different peptides, which span the entire human mesothelin sequence. Each peptide is 15 amino acids long and overlaps with 11 adjacent amino acids. The results of EUSPOT measurement are shown in FIG. 65. The results indicate that a Listeria vaccine expressing human mesothelin fused to anthrax signal peptide can induce an immune response to mesothelin in Balb / c mice. In the mouse immune system, a higher IFN_y response to the epidermis of human Mesothelin than in the C57BL / 6 immune system was observed. The ELispoT signal to p6o or LL0 is in response to the naturally occurring p6o and LL0 proteins of Listeria. All publications, patents, patent applications, Internet addresses, and registration numbers / database sequences (including both polynucleotide and polypeptide sequences) described herein are incorporated herein by reference in their entirety. The extent is as if individual publications, patents, patent applications, Internet addresses or registration numbers / database sequences have been incorporated by reference individually and specifically. [Schematic description] 98561.doc -262-200530399 Figure 1 shows the hly promoter gene, which combines Listeria monocytogenes DP-L4056 (sequence number 1) (sequence below) and EGD strain (sequence number 2). ) (Sequence below) in line. Figure 2 shows the sequence of a polynucleotide encoding a fusion protein including the LLO signal peptide, the LLO PEST sequence, and the full-length human EphA2 antigen (Sequence No. 3). Fig. 3 shows the sequence (sequence number 4) of the fusion protein encoded by the polynucleotide shown in Fig. 2. Figure 4 shows the natural nucleotide sequence (sequence number 5) encoding the extracellular functional site (EX2) of human EphA2. Figure 5 shows the codon-optimized nucleotide sequence encoding human EphA2 extracellular functional site that has been codon-optimized for expression in Listeria monocytogenes (Sequence No. 6). Figure 6 shows the amino acid sequence of human EphA2 extracellular functional site (EX2) (sequence number 7). Figure 7 shows an uncodon-optimized polynucleic acid sequence (sequence number 8) encoding a fusion protein including an LLO signal peptide, an LLO PEST sequence, and a human extracellular functional site of EphA2. Fig. 8 shows the sequence of the fusion protein encoded by the code sequence shown in Fig. 7 (sequence number 9). Figure 9 shows the performance cassette (sequence number 10) including the hly promoter gene 'and encoding a fusion protein including the 110 signal peptide, the LLO PEST sequence, and the extracellular functional site of human EphA2. In this sequence, the sequence encoding the extracellular functional site of human EphA2 is codon-optimized for expression in Listeria monocytogenes. 98561.doc -263-200530399 Figure 10 shows the amino acid sequence (sequence number 11) encoded by the performance cassette of Figure 9. Figure 11 shows the performance cassette (sequence number 12) including the hly promoter gene and encoding a fusion protein including the 110 signal peptide, the 110 PE ST sequence, and the extracellular functional site of human EphA2. In this sequence, all of the sequences encoding the LLO signal peptide, LLO PEST, and human EphA2 extracellular functional site are codon-optimized for expression in Listeria monocytogenes. Fig. 12 shows the amino acid sequence (sequence number 13) encoded by the expression card shown in Fig. 11. Figure 13 shows a performance cassette that does not include the hly promoter gene &apos; and encodes a fusion protein including a pHOD Tat signal peptide and a human extracellular functional site of EphA2 (sequence number 14). In this sequence, the sequence codons encoding the phoD Tat signal peptide and the extracellular functional site of human EphA2 were optimized for expression in Listeria monocytogenes. FIG. 14 shows the amino acid sequence (sequence number 15) encoded by the expression cassette of FIG. 13. Figure 15 shows the natural nucleotide sequence (sequence number 16) encoding a functional site (CO) in human EphA2 cells. Figure 16 shows the codon-optimized nucleotide sequence (sequence number 17) encoding a functional site in human EphA2 cells that has been codon-optimized for expression in Listeria monocytogenes. Figure 17 shows the amino acid sequence (sequence number 18) of the functional site (EX2) in human EphA2 cells. Figure 18 shows the coding including LLO signal peptide, LLO PEST sequence and human 98561.doc -264- 200530399

The uncodon-optimized polynucleotide sequence of the fusion protein of the functional site in EphA2 cells (sequence number 19). Fig. 19 shows the sequence of the fusion protein encoded by the divergent code sequence shown in Fig. 18 (sequence number 20). Figure 20 shows the performance card S (sequence number 21) including a hly promoter gene and encoding a fusion protein including an LLO signal peptide, an LLO PEST sequence, and a functional site in human EphA2 cells. In this sequence, in order to be expressed in Listeria monocytogenes, the sequence encoding the functional site in human EphA2 cells was codon-optimized. Figure 21 shows the amino acid sequence (sequence number 22) encoded by the expression cassette of Figure 20. Fig. 22 shows the performance of card E (sequence number 23) including the hly promoter gene and encoding a fusion protein including the 110 signal peptide, the LLO PEST sequence, and a functional site in human EphA2 cells. In this sequence, in order to be expressed in Listeria monocytogenes, the sequences encoding the LLO signal peptide, LLO PEST, and the functional sites in human EphA2 cells were all codon-optimized. Figure 23 shows the amino acid sequence (sequence number 24) encoded by the expression cassette of Figure 22. Figure 24 shows a performance cassette (sequence number 25) that includes the hly promoter gene and encodes a fusion protein including a pHOD signal peptide and a functional site in human EphA2 cells. In this sequence, in order to be expressed in Listeria monocytogenes, the sequence codons encoding both the phoD Tat signal peptide and the human EphAhs intracellular functional site were optimized. FIG. 25 shows the amino acid sequence encoded by the expression cassette of FIG. 24 (Sequence No. 98561.doc -265-200530399 No. 26). Figure 26 shows a codon-optimized performance cassette (SEQ ID NO. 27) including the hly promoter gene and encoding a fusion protein including the 110 signal peptide and the NY-ESO-1 antigen. For expression in Listeria monocytogenes, the sequence codon-optimization of both the signal and the antigen will be coded. FIG. 27 shows the amino acid sequence (sequence number 28) encoded by the expression cassette of FIG. 26. Figure 28 shows a polynucleotide comprising an hly promoter gene operably linked to a codon-optimized sequence encoding a Usp45 signal peptide (sequence number 29). Figure 29 shows a polynucleotide comprising a hly promoter gene operably linked to a natural sequence encoding a p60 signal peptide (sequence number 30). Figure 30 shows a polynucleotide comprising a hly promoter gene operably linked to a codon-optimized sequence encoding a p60 signal peptide (sequence number 31). Figure 31 shows the sequence of hlyP-p60 gene fragment (sequence number 32). Fig. 32 (including Figs. 32A, 32B, and 32C) shows the sequence of pAM401-MCS (sequence number 33), and the pAM401 plastid contains a multiple selection site (MCS) derived from the PPL2 vector. Figure 33 shows the codon-optimized human mesothelin codon sequence that has been codon-optimized for expression in Listeria monocytogenes (Sequence No. 34). Figure 34 shows the amino acid sequence of human mesothelin (sequence number 35). Figure 35 shows the codon-optimized mouse mesothelin codon sequence that has been codon-optimized for expression in Listeria monocytogenes (sequence number 36). 98561.doc -266-200530399 Figure 36 shows the amino acid sequence of mouse mesothelin (sequence number 37). Figure 37 shows a Western blot analysis of the secreted protein in the recombinant Listeria spp. Obtained from a sequence encoding the functional site of native EphA2 CO. Figure 38 shows a Western blot analysis of secreted proteins in recombinant Listeria from natural or codon-optimized LLO secAl signal peptides encoding a codon-optimized EphA2 EX2 functional sequence. Figure 39 shows secretion in recombinant Listeria from encoding natural or codon-optimized LLO secAl signal peptides or codon-optimized Tat signal peptides fused to codon-optimized EphA2 CO functional sites Western blot analysis of proteins. Figure 40 shows western blot analysis of lysates from 293 cells 48 hours after infection with pCDNA4 plastid DNA encoding the full-length native EphA2 sequence. Figure 41 is a graph showing the long-term survival of Balb / C mice bearing CT26.24 (huEphA2 +) lung tumor immunized with recombinant Listeria spp. Encoding OVA.AH1 or OVA.AH1-A5. Figure 42 shows the increase in CT26.24 (huEphA2 +) lung tumors when immunized with recombinant Listeria spp. That encodes a codon-optimized secAl signal peptide fused to a codon-optimized EphA2 EX2 functional site sequence. Balb / C old-fashioned survival chart. Fig. 43 is a graph showing that the recombinant Listeria spp. Encoding the functional site of EphA2 CO immunized Balb / C mice bearing CT26.24 (huEphA2 +) lung tumors and given long-term survival. Fig. 44 shows the functional site encoding EphA2 CO, but no full length 98561.doc -267- 200530399

Recombinant Listeria spp. Of plastid DNA of EphA2 immunized Balb / C mice carrying CT26.24 (huEphA2 +) lung tumors, giving a graph of long-term survival. Figure 45 is a graph showing that Listeria spp. Expressing hEphA2 induces EphA2-specific CD8 + T cell responses. Figure 46 is a graph showing both CD4 + and CD8 + T cell responses that promote the anti-tumor efficacy of hEphA2 • of the Listeria spp. That express hEphA2. Figure 47 shows the sequence of the Listeria monocytogenes strain i04403hly promoter (sequence number 38), which is operably linked to a protective antigen signal peptide obtained from Bacillus anthracis and Divided in Listeria monocytogenes; must be codon-optimized. The inclusion of six additional nucleotides (5, -GGATCC-3,) corresponding to the recognition position of the B am hi restriction enzyme into the carboxy terminus of the signal peptide sequence helps to operatively interact with any selected Password sequence connection. The 5 end of the hly promoter gene contains a unique κρη I restriction enzyme recognition position. Figure 48 shows the full-length human tumor antigen efficiently expressed and secreted from the recombinant Listeria spp. Figure 48A shows the expression / distribution of mesothelin using a construct consisting of peptide LLO # fused to human mesothelin using natural codons. Figure 48B shows the expression / secretion of mesothelin using a construct including various signal peptides fused to codon-optimized human mesothelin for expression in Listeria. Figure 48C shows the expression / secretion of NY-ESO-1 using a construct that includes a codon-optimized iLL04 L-peptide fused to a human mesothelin fork codon-optimized NY-ESO-1. Figure 49 shows the cipher sequence of phEphA2KD (sequence number 39). 98561.doc -268-200530399 Figure 50 shows the codon-optimized Mlu I subfragment of human EphA2 (sequence number 40) containing the intergenic region of actA-plcB. Figure 51 shows the sequence of the hly promoter-70 N-terminal p60 amino acid (sequence number 41). Figure 52 shows the κρΙΙ-BamHI sub-fragment of the plastid pPL2-hlyP-Np60 CodOp (1-77) (sequence number 42). Figure 53 shows the KpnI-BamHI sub-fragment of plastid pPL2-hlyP-Np60 CodOp (l-77) -mesothelin (sequence number 43). Figure 54 shows the KpnI-BamHI sub-fragment of plastid pPL2-hlyP_Np60 CodOp (l-77) · mesothelin △ SPMGPI (sequence number 44). Figure 5-5 shows Western blot analysis of the expression and secretion of antigens from recombinant Listeria spp. Including antigen-bacterial protein chimeras. Figure 56 shows a Western blot analysis of intracellular functional sites (ICD) representing EphA2 from bicistronic information. Figure 57 shows the performance of plastid-based mouse mesothelin and secretion of western blots, which has been demonstrated to function in various bacterial isolates with various codons _ optimized signal peptide fusion N-terminal fusion function : Secreted protein (Figure 57A); cell wall (Figure 57B); and cell lysate (Figure 57C). Figure 58 shows a Western blot analysis of the performance and secretion of human mesothelin based on chromosomes in Listeria monocytogenes. Western blot analysis showing the expression of interstitial hormone in the lysates of various bacterial cells, resulting from the performance of the control group Listeria (not encoding mesothelin) and Listeria encoding mesothelin Fungus. Figures 59A and 59B are graphs showing delivery of a heterologous antigen 98561.doc -269- 200530399 (AH1-A5) to the MHC class i pathway by a Listeria vaccine. Listeria vaccines include Listeria (Figure 59A) expressing a P60-AH1-A5 protein chimera (AH1-A5 embedded in p60), or Listeria expressing a fusion protein that includes the LLO signal peptide and AH1-A5 Mycobacterium (Figure 59B). Figures 60A and 60B are diagrams showing the Listeria vaccine modulating the delivery of bacterial-specific antigens to the MHC Class I pathway, where the vaccine includes a Listeria spp. Expressing a ρ60_ΑΗ1_Α5 protein chimera (AH1_A5 embedded in p60) ( Figure 60A), or Listeria spp. (Figure 60B) showing a fusion protein including the LLO signal peptide and AH1-A5, and the test peptide added to the cell-based assay is a test-free peptide (untested Stimulated) (Fig. 60A), LL091-99 (Fig. 60A), no test case (Fig. 60B) or p602i7-225 (Fig. 60B). Figure 61 is a graph showing the therapeutic efficacy of Listeria in human mesothelin in vaccinated tumor-bearing mice in which tumor cells were designed to express human mesothelin. Fig. 62 is a graph showing the reduction of the degree of lung tumor nodules in tumor-bearing mice inoculated with Listeria spp. Expressing human mesothelin, in which tumor cells are designed to express human mesothelin. Figure 63 is a chart showing a control group study using CT 26 parental standard stem cells, that is, cells were not designed to express human mesothelin, confirming that the anti-tumor efficacy of the Lm-MeS0 vaccination is mesothelin-specific of. Fig. 64 is a graph showing the reduction of tumor volume by vaccination with Listeria spp. Which expresses codon-optimized human mesothelin. Figure 65 shows the results of the ELISPOT experiment, which shows the immunogenicity of the Listeria ΔactA / AinlB-human mesothelin strain, in which a nucleic acid encoding human mesothelin has been integrated into the Listeria genome. 98561.doc -270- 200530399 Sequence Listing &lt; 110 &gt; Cyrus Co., Ltd. &lt; 120 &gt; Recombinant Nucleic Acid Molecules, Expression Cassettes, Bacteria and Their Uses &lt; 130> 282172003941 &lt; 140> 093140281 &lt; 141 &gt; 2004-12-23 &lt; 150 &gt; US 60 / 616,750 &lt; 151 &gt; 2004-10-06 &lt; 150 &gt; US 60 / 615,287 &lt; 151 &gt; 2004-10-01 &lt; 150> US 60 / 599,377 &lt; 151 &gt; 2004-08-05 &lt; 150 &gt; PCT / US2004 / 23881 &lt; 151 &gt; 2004-07-23 &lt; 150 &gt; US 10 / 883,599 &lt; 151 &gt; 2004-06-30 &lt; 150 &gt; US 60 / 556,744 &lt; 151 &gt; 2004-03-26 &lt; 160 &gt; 129 &lt; 170 &gt; FastSEQ for Windows Version 4.0

&lt; 210 &gt; 1 &lt; 211〉 240 &lt; 212〉 DNA &lt; 213 &gt; Listeria monocytogenes ttttatgtgg aggcattaac 60 agctataaag caagcatata 120 ataattatca aaagagaggg 180 gaaggagagt gaaacccatg 240 &lt; 400> 1 ggtacctcccat attgtatggtatggta gtatttggca tattcctatc ggatagcaag aagcgaattt ttattaggtt ttaaagttac actagaataa cgccaatatt aaaaaatgta 98561.doc 200530399

&lt; 210〉 2 &lt; 211> 240 &lt; 212 &gt; DNA &lt; 213 &gt; Listeria monocytogenes &lt; 400 &gt; 2 ggtacctcct ttgattagta tattcctatc ttaaagtgac ttttatgttg aggcattatatcat acat agt agt agt agt gac agt agt agt aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240

&lt; 210> 3 &lt; 211 &gt; 3105 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223> Fusion Protein Coding Sequence &lt; 400 &gt; 3 atgaaaaaaa taatgctagt ttttattaca cttatattag ttagtctacc aattgcgcaatt 60 acattaga acat acat acat atccatggca 120 ccaccagcat ctccgcctgc aagtcctaag acgccaatcg aaaagaaaca cgcggatctc 180 gagctccagg cagcccgcgc ctgcttcgcc ctgctgtggg gctgtgcgct ggccgcggcc 240 gcggcggcgc agggcaagga agtggtactg ctggactttg ctgcagctgg aggggagctc 300 ggctggctca cacacccgta tggcaaaggg tgggacctga tgcagaacat catgaatgac 360 atgccgatct acatgtactc cgtgtgcaac gtgatgtctg gcgaccagga caactggctc 420

cgcaccaact gggtgtaccg aggagaggct gagcgtatct tcattgagct caagtttact 480 gtacgtgact gcaacagctt ccctggtggc gccagctcct gcaaggagac tttcaacctc 540 tactatgccg agtcggacct ggactacggc accaacttcc agaagcgcct gttcaccaag 600 attgacacca ttgcgcccga tgagatcacc gtcagcagcg acttcgaggc acgccacgtg 660 aagctgaacg tggaggagcg ctccgtgggg ccgctcaccc gcaaaggctt ctacctggcc 720 ttccaggata tcggtgcctg tgtggcgctg ctctccgtcc gtgtctacta caagaagtgc 780 cccgagctgc tgcagggcct ggcccacttc cctgagacca tcgccggctc tgatgcacct 840 tccctggcca ctgtggccgg cacctgtgtg gaccatgccg tggtgccacc ggggggtgaa 900 gagccccgta tgcactgtgc agtggatggc gagtggctgg tgcccattgg gcagtgcctg 960 tgccaggcag gctacgagaa ggtggaggat gcctgccagg cctgctcgcc tggatttttt 1020 aagtttgagg catctgagag cccctgcttg gagtgccctg agcacacgct gccatcccct 1080 gagggtgcca cctcctgcga gtgtgaggaa ggcttcttcc gggcacctca ggacccagcg 1140 tcgatgcctt gcacacgacc cccctccgcc ccacactacc tcacagccgt gggcatgggt 1200 gccaaggtgg agctgcgctg gacgccccct caggacagcg ggggccgcga ggacattgtc 1260 tacagcgtca cctg cgaaca gtgctggccc gagtctgggg aatgcgggcc gtgtgaggcc 1320 agtgtgcgct actcggagcc tcctcacgga ctgacccgca ccagtgtgac agtgagcgac 1380 98561.doc 200530399 ctggagcccc acatgaacta caccttcacc gtggaggccc gcaatggcgt ctcaggcctg 1440 gtaaccagcc gcagcttccg tactgccagt gtcagcatca accagacaga gccccccaag 1500 gtgaggctgg agggccgcag caccacctcg cttagcgtct cctggagcat ccccccgccg 1560 cagcagagcc gagtgtggaa gtacgaggtc acttaccgca agaagggaga ctccaacagc 1620 tacaatgtgc gccgcaccga gggtttctcc gtgaccctgg acgacctggc cccagacacc 1680 acctacctgg tccaggtgca ggcactgacg caggagggcc agggggccgg cagcagggtg 1740 cacgaattcc agacgctgtc cccggaggga tctggcaact tggcggtgat tggcggcgtg 1800 gctgtcggtg tggtcctgct tctggtgctg gcaggagttg gcttctttat ccaccgcagg 1860 aggaagaacc agcgtgcccg ccagtccccg gaggacgttt acttctccaa gtcagaacaa 1920 ctgaagcccc tgaagacata cgtggacccc cacacatatg aggaccccaa ccaggctgtg 1980 ttgaagttca ctaccgagat ccatccatcc tgtgtcactc ggcagaaggt gatcggagca 2040 ggagagtttg gggaggtgta caagggcatg ctgaagacat cctcggggaa gaaggaggtg 2100 c cggtggcca tcaagacgct gaaagccggc tacacagaga agcagcgagt ggacttcctc 2160 ggcgaggccg gcatcatggg ccagttcagc caccacaaca tcatccgcct agagggcgtc 2220 atctccaaat acaagcccat gatgatcatc actgagtaca tggagaatgg ggccctggac 2280 aagttccttc gggagaagga tggcgagttc agcgtgctgc agctggtggg catgctgcgg 2340 ggcatcgcag ctggcatgaa gtacctggcc aacatgaact atgtgcaccg tgacctggct 2400 gcccgcaaca tcctcgtcaa cagcaacctg gtctgcaagg tgtctgactt tggcctgtcc 2460 cgcgtgctgg aggacgaccc cgaggccacc tacaccacca gtggcggcaa gatccccatc 2520 cgctggaccg ccccggaggc catttcctac cggaagttca cctctgccag cgacgtgtgg 2580 agctttggca ttgtcatgtg ggaggtgatg acctatggcg agcggcccta ctgggagttg 2640 tccaaccacg aggtgatgaa agccatcaat gatggcttcc ggctccccac acccatggac 2700 tgcccctccg ccatctacca gctcatgatg cagtgctggc agcaggagcg tgcccgccgc 2760 cccaagttcg ctgacatcgt cagcatcctg gacaagctca ttcgtgcccc tgactccctc 2820 aagaccctgg ctgactttga cccccgcgtg tctatccggc tccccagcac gagcggctcg 2880 gagggggtgc ccttccgcac ggtgtccgag tggctggagt ccatcaagat gcagcagtat 2940 acggagc act tcatggcggc cggctacact gccatcgaga aggtggtgca gatgaccaac 3000 gacgacatca agaggattgg ggtgcggctg cccggccacc agaagcgcat cgcctacagc 3060 ctgctgggac &&;; &gt; 10 &gt; &lt; 210 Protein &lt; 400〉 4

Met Lys Lys He Met Leu Val Phe He Thr Leu lie Leu Val Ser Leu 15 10 15

Pro lie Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30

Glu Asn Ser lie Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45 98561.doc 200530399

Pro Lys Thr Pro lie Glu Lys Lys His Ala Asp Leu Glu Leu Gin Ala 50 55 60

Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys Ala Leu Ala Ala Ala 65 70 75 80

Ala Ala Ala Gin Gly Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala 85 90 95

Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp 100 105 110

Leu Met Gin Asn lie Met Asn Asp Met Pro lie Tyr Met Tyr Ser Val 115 120 125

Cys Asn Val Met Ser Gly Asp Gin Asp Asn Trp Leu Arg Thr Asn Trp 130 135 140

Val Tyr Arg Gly Glu Ala Glu Arg He Phe He Glu Leu Lys Phe Thr 145 150 155 160

Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu 165 170 175

Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn 180 185 190

Phe Gin Lys Arg Leu Phe Thr Lys lie Asp Thr He Ala Pro Asp Glu 195 200 205 lie Thr Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu Asn Val 210 215 220

Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala 225 230 235 240

Phe Gin Asp He Gly Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr 245 250 255

Tyr Lys Lys Cys Pro Glu Leu Leu Gin Gly Leu Ala His Phe Pro Glu 260 265 270

Thr He Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr 275 280 285

Cys Val Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met 290 295 300

His Cys Ala Val Asp Gly Glu Trp Leu Val Pro He Gly Gin Cys Leu 305 310 315 320

Cys Gin Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gin Ala Cys Ser 325 330 335

Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys 340 345 350

Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys 355 360 365

Glu Glu Gly Phe Phe Arg Ala Pro Gin Asp Pro Ala Ser Met Pro Cys 370 375 380

Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly 385 390 395 400

Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gin Asp Ser Gly Gly Arg 98561.doc -4- 200530399 405 410 415

Glu Asp lie Val Tyr Ser Val Thr Cys Glu Gin Cys Trp Pro Glu Ser 420 425 430

Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro 435 440 445

His Gly Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His 450 455 460

Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu 465 470 475 480

Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val Ser He Asn Gin Thr 485 490 495

Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser 500 505 510

Val Ser Trp Ser He Pro Pro Gin Gin Ser Arg Val Trp Lys Tyr 515 520 525

Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg 530 535 540

Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr 545 550 555 560

Thr Tyr Leu Val Gin Val Gin Ala Leu Thr Gin Glu Gly Gin Gly Ala 565 570 575

Gly Ser Arg Val His Glu Phe Gin Thr Leu Ser Pro Glu Gly Ser Gly 580 585 590

Asn Leu Ala Val He Gly Gly Val Ala Val Gly Val Val Leu Leu Leu 595 600 605

Val Leu Ala Gly Val Gly Phe Phe He His Arg Arg Arg Lys Asn Gin 610 615 620

Arg Ala Arg Gin Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gin 625 630 635 640

Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro 645 650 655

Asn Gin Ala Val Leu Lys Phe Thr Thr Glu He His Pro Ser Cys Val 660 665 670

Thr Arg Gin Lys Val He Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys 675 680 685

Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala He 690 695 700

Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gin Arg Val Asp Phe Leu 705 710 715 720

Gly Glu Ala Gly lie Met Gly Gin Phe Ser His His Asn lie lie Arg 725 730 735

Leu Glu Gly Val He Ser Lys Tyr Lys Pro Met Met He lie Thr Glu 740 745 750

Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly 755 760 765 98561.doc 200530399

Glu Phe Ser Val Leu Gin Leu Val Gly Met Leu Arg Gly lie Ala Ala 770 775 780

Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala 785 790 795 800

Ala Arg Asn He Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp 805 810 815

Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr 820 825 830

Thr Ser Gly Gly Lys lie Pro lie Arg Trp Thr Ala Pro Glu Ala lie 835 840 845

Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly lie 850 855 860

Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu 865 870 875 880

Ser Asn His Glu Val Met Lys Ala He Asn Asp Gly Phe Arg Leu Pro 885 890 895

Thr Pro Met Asp Cys Pro Ser Ala lie Tyr Gin Leu Met Met Gin Cys 900 905 910

Trp Gin Gin Glu Arg Ala Arg Arg Pro Lys Phe Ala Asp He Val Ser 915 920 925

He Leu Asp Lys Leu lie Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala 930 935 940

Asp Phe Asp Pro Arg Val Ser He Arg Leu Pro Ser Thr Ser Gly Ser 945 950 955 960

Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp Leu Glu Ser He Lys 965 970 975

Met Gin Gin Tyr Thr Glu His Phe Met Ala Ala Gly Tyr Thr Ala He 980 985 990

Glu Lys Val Val Gin Met Thr Asn Asp Asp lie Lys Arg He Gly Val 995 1000 1005

Arg Leu Pro Gly His Gin Lys Arg He Ala Tyr Ser Leu Leu Gly Leu 1010 1015 1020

Lys Asp Gin Val Asn Thr Val Gly He Pro lie 1025 1030 1035 &lt; 210〉 5 &lt; 211 &gt; 1506 &lt; 212 &gt; DNA &lt; 213〉 Hyundai Homo sapiens &lt; 400〉 5 cagggcaagg aagtggtact gctggacttt gctgcagctg gaggggagct cggacggctggcggggggggcgt gtgggacctg atgcagaaca tcatgaatga catgccgatc 120 tacatgtact ccgtgtgcaa cgtgatgtct ggcgaccagg acaactggct ccgcaccaac 180 98561.doc 200530399 tgggtgtacc gaggagaggc tgagcgtatc ttcattgagc tcaagtttac tgtacgtgac 240 tgcaacagct tccctggtgg cgccagctcc tgcaaggaga ctttcaacct ctactatgcc 300 gagtcggacc tggactacgg caccaacttc cagaagcgcc tgttcaccaa gattgacacc 360 attgcgcccg atgagatcac cgtcagcagc gacttcgagg cacgccacgt gaagctgaac 420 gtggaggagc gctccgtggg gccgctcacc cgcaaaggct tctacctggc cttccaggat 480 atcggtgcct gtgtggcgct gctctccgtc cgtgtctact acaagaagtg ccccgagctg 540 ctgcagggcc tggcccactt ccctgagacc atcgccggct ctgatgcacc ttccctggcc 600 actgtggccg gcacctgtgt ggaccatgcc gtggtgccac cggggggtga agagccccgt 660 atgcactgtg cagtggatgg cgagtggctg gtgcccattg ggcagtgcct gtgccaggca 720 g gctacgaga aggtggagga tgcctgccag gcctgctcgc ctggattttt taagtttgag 780 gcatctgaga gcccctgctt ggagtgccct gagcacacgc tgccatcccc tgagggtgcc 840 acctcctgcg agtgtgagga aggcttcttc cgggcacctc aggacccagc gtcgatgcct 900 tgcacacgac ccccctccgc cccacactac ctcacagccg tgggcatggg tgccaaggtg 960 gagctgcgct ggacgccccc tcaggacagc gggggccgcg aggacattgt ctacagcgtc 1020 acctgcgaac agtgctggcc cgagtctggg gaatgcgggc cgtgtgaggc cagtgtgcgc 1080 tactcggagc ctcctcacgg actgacccgc accagtgtga cagtgagcga cctggagccc 1140 cacatgaact acaccttcac cgtggaggcc cgcaatggcg tctcaggcct ggtaaccagc 1200 cgcagcttcc gtactgccag tgtcagcatc aaccagacag agccccccaa ggtgaggctg 1260 gagggccgca gcaccacctc gcttagcgtc tcctggagca tccccccgcc gcagcagagc 1320 cgagtgtgga agtacgaggt cacttaccgc aagaagggag actccaacag ctacaatgtg 1380 cgccgcaccg agggtttctc cgtgaccctg gacgacctgg ccccagacac cacctacctg 1440 gtccaggtgc aggcactgac gcaggagggc cagggggccg gcagcagggt gcacgaattc 1500 cagacg 1506 &lt; 210> 6 &lt; 211 &gt; 1506 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence ttccaggtgg tgcaagtagt 400> 6 caaggtaaag aagttgtttt attagatttt gcagcagcag gtggtgaatt aggttggtta 60 acacatccat atggtaaagg ttgggattta atgcaaaata ttatgaatga tatgccaatt 120 tatatgtata gtgtttgtaa tgttatgagt ggtgatcaag ataattggtt acgtacaaat 180 tgggtttatc gtggtgaagc agaacgtatt tttattgaat taaaatttac agttcgtgat 240 tgtaatagtt; - &lt; 220 &gt; &lt;; 223 & gt optimized sequence & lt codon tgtaaagaaa catttaattt atattatgca 300 gaaagtgatt tagattatgg tacaaatttt caaaaacgtt tatttacaaa aattgataca 360 attgcaccag atgaaattac agttagtagt gattttgaag cacgtcatgt taaattaaat 420 gttgaagaac gtagtgttgg tccattaaca cgtaaaggtt tttatttagc atttcaagat 480 attggtgcat gtgttgcatt attaagtgtt cgtgtttatt ataaaaaatg tccagaatta 540 ttacaaggtt tagcacattt tccagaaaca attgcaggta gtgatgcacc aagtttagca 600 acagttgcag gtacatgtgt tgatcatgca gttgttccac caggtggtga agaaccacgt 660 atgcattgtg cagttgatgg tgaatggtta gttccaattg gtcaatgttt atgtcaagca 720 98561.doc 200530399 ggttatgaaa aagttgaaga tgcatgtcaa gcatgtagtc caggtttttt taaatttgaa 780 gcaagtgaa a gtccatgttt agaatgtcca gaacatacat taccaagtcc agaaggtgca 840 acaagttgtg aatgtgaaga aggttttttt cgtgcaccac aagatccagc aagtatgcca 900 tgtacacgtc caccaagtgc accacattat ttaacagcag ttggtatggg tgcaaaagtt 960 gaattacgtt ggacaccacc acaagatagt ggtggtcgtg aagatattgt ttatagtgtt 1020 acatgtgaac aatgttggcc agaaagtggt gaatgtggtc catgtgaagc aagtgttcgt 1080 tatagtgaac caccacatgg tttaacacgt acaagtgtta cagttagtga tttagaacca 1140 catatgaatt atacatttac agttgaagca cgtaatggtg ttagtggttt agttacaagt 1200 cgtagttttc gtacagcaag tgttagtatt aatcaaacag aaccaccaaa agttcgttta 1260 gaaggtcgta gtacaacaag tttaagtgtt agttggagta ttccaccacc acaacaaagt 1320 cgtgtttgga aatatgaagt tacatatcgt aaaaaaggtg atagtaatag ttataatgtt 1380 cgtcgtacag aaggttttag tgttacatta gatgatttag caccagatac aacatattta 1440 gttcaagttc aagcattaac acaagaaggt caaggtgcag gtagtcgtgt tcatgaattt 1500 caaaca 1506 &lt; 210> 7 &lt; 211 &gt; 502 &lt; 212> PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400 &gt; 7

Gin Gly Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu 15 10 15

Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gin 20 25 30

Asn lie Met Asn Asp Met Pro lie Tyr Met Tyr Ser Val Cys Asn Val 35 40 45

Met Ser Gly Asp Gin Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg 50 55 60

Gly Glu Ala Glu Arg lie Phe He Glu Leu Lys Phe Thr Val Arg Asp 65 70 75 80

Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn 85 90 95

Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gin Lys 100 105 110

Arg Leu Phe Thr Lys lie Asp Thr lie Ala Pro Asp Glu He Thr Val 115 120 125

Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg 130 135 140

Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gin Asp 145 150 155 160

He Gly Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys 165 170 175

Cys Pro Glu Leu Leu Gin Gly Leu Ala His Phe Pro Glu Thr He Ala 180 185 190 98561.doc 200530399

Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp 195 200 205

His Ala Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala 210 215 220

Val Asp Gly Glu Trp Leu Val Pro lie Gly Gin Cys Leu Cys Gin Ala 225 230 235 240

Gly Tyr Glu Lys Val Glu Asp Ala Cys Gin Ala Cys Ser Pro Gly Phe 245 250 255

Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His 260 265 270

Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly 275 280 285

Phe Phe Arg Ala Pro Gin Asp Pro Ala Ser Met Pro Cys Thr Arg Pro 290 295 300

Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val 305 310 315 320

Glu Leu Arg Trp Thr Pro Pro Gin Asp Ser Gly Gly Arg Glu Asp He 325 330 335

Val Tyr Ser Val Thr Cys Glu Gin Cys Trp Pro Glu Ser Gly Glu Cys 340 345 350

Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu 355 360 365

Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr 370 375 380

Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser 385 390 395 400

Arg Ser Phe Arg Thr Ala Ser Val Ser lie Asn Gin Thr Glu Pro Pro 405 410 415

Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp 420 425 430

Ser lie Pro Pro Gin Gin Ser Arg Val Trp Lys Tyr Glu Val Thr 435 440 445

Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu 450 455 460

Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu 465 470 475 480

Val Gin Val Gin Ala Leu Thr Gin Glu Gly Gin Gly Ala Gly Ser Arg 485 490 495

Val His Glu Phe Gin Thr 500

&lt; 210 &gt; 8 &lt; 211 &gt; 1689 &lt; 212 &gt; DNA 98561.doc 200530399 &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223> fusion protein coding sequence &lt; 400> 8 atgaaaaaaa taatgctagt ttttattaca cttatattag ttagtctacc aattgcagca 60 caaaggatgc atctgcattc aataaagaaa attcaatttc atccatggca 120 ccaccagcat ctccgcctgc aagtcctaag acgccaatcg aaaagaaaca cgcggatctc 180 gagcagggca aggaagtggt actgctggac tttgctgcag ctggagggga gctcggctgg 240 ctcacacacc cgtatggcaa agggtgggac ctgatgcaga acatcatgaa tgacatgccg 300 atctacatgt actccgtgtg caacgtgatg tctggcgacc aggacaactg gctccgcacc 360 aactgggtgt accgaggaga ggctgagcgt atcttcattg agctcaagtt tactgtacgt 420 gactgcaaca gettccctgg tggcgccagc tcctgcaagg agactttcaa cctctactat 480 geegagtegg acctggacta cggcaccaac ttccagaagc gcctgttcac caagattgac 540 accattgcgc ccgatgagat caccgtcagc agegaetteg aggcacgcca cgtgaagctg 600 aacgtggagagg agcgctccgt ggggcctctggt ctggt ctgcc ctggcc ctggcc agg gcctggccca cttccctgag accatcgccg gctctgatgc accttccctg 780 gccactgtgg ccggcacctg tgtggaccat gccgtggtgc caccgggggg tgaagagccc 840 cgtatgcact gtgcagtgga tggcgagtgg ctggtgccca ttgggcagtg cctgtgccag 900 gcaggctacg agaaggtgga ggatgcctgc caggcctgct cgcctggatt ttttaagttt 960 gaggeatetg agagcccctg cttggagtgc cctgagcaca cgctgccatc ccctgagggt 1020 gccacctcct gcgagtgtga ggaaggette ttccgggcac ctcaggaccc agegtegatg 1080 ccttgcacac gacccccctc cgccccacac tacctcacag ccgtgggcat gggtgccaag 1140 gtggagctgc gctggacgcc ccctcaggac agcgggggcc gcgaggacat tgtctacagc 1200 gtcacctgcg aacagtgctg gcccgagtct ggggaatgcg ggccgtgtga ggccagtgtg 1260 cgctactcgg agcctcctca cggactgacc cgcaccagtg tgacagtgag cgacctggag 1320 ccccacatga actacacctt caccgtggag gcccgcaatg gcgtctcagg cctggtaacc 1380 agccgcagct tccgtactgc cagtgtcagc atcaaccaga cagagccccc caaggtgagg 1440 ctggagggcc gcagcaccac ctcgcttagc gtctcctgga gcatcccccc gccgcagcag 1500 agccgagtgt ggaagtaega ggtcacttac cgcaagaagg gagactccaa cagctacaat 1560 gtgcgccgca ccgagg gttt ctccgtgacc ctggacgacc tggccccaga caccacctac 1620 ctggtccagg tgcaggcact gacgcaggag ggccaggggg ccggcagcag ggtgcacgaa 1680 ttccagacg 1689 &lt; 210 &gt; 9 &lt; 211 &gt; PRT &lt; 212> ltlt &lt; 212> 9 98561.doc -10- 200530399

Met Lys Lys He Met Leu Val Phe lie Thr Leu He Leu Val Ser Leu 15 10 15

Pro He Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30

Glu Asn Ser lie Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45

Pro Lys Thr Pro He Glu Lys Lys His Ala Asp Leu Glu Gin Gly Lys 50 55 60

Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp 65 70 75 80

Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gin Asn lie Met 85 90 95

Asn Asp Met Pro He Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly 100 105 110

Asp Gin Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala 115 120 125

Glu Arg He Phe He Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser 130 135 140

Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr 145 150 155 160

Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gin Lys Arg Leu Phe 165 170 175

Thr Lys lie Asp Thr lie Ala Pro Asp Glu lie Thr Val Ser Ser Asp 180 185 190

Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly 195 200 205

Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gin Asp lie Gly Ala 210 215 220

Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu 225 230 235 240

Leu Leu Gin Gly Leu Ala His Phe Pro Glu Thr lie Ala Gly Ser Asp 245 250 255

Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val 260 265 270

Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala Val Asp Gly 275 280 285

Glu Trp Leu Val Pro lie Gly Gin Cys Leu Cys Gin Ala Gly Tyr Glu 290 295 300

Lys Val Glu Asp Ala Cys Gin Ala Cys Ser Pro Gly Phe Phe Lys Phe 305 310 315 320

Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro 325 330 335

Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg 340 345 350

Ala Pro Gin Asp Pro Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala 98561.doc • 11-200530399 355 360 365

Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg 370 375 380

Trp Thr Pro Pro Gin Asp Ser Gly Gly Arg Glu Asp He Val Tyr Ser 385 390 395 400

Val Thr Cys Glu Gin Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys 405 410 415

Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr 420 425 430

Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr 435 440 445

Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe 450 455 460

Arg Thr Ala Ser Val Ser He Asn Gin Thr Glu Pro Pro Lys Val Arg 465 470 475 480

Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp Ser lie Pro 485 490 495

Pro Pro Gin Gin Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys 500 505 510

Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser 515 520 525

Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gin Val 530 535 540

Gin Ala Leu Thr Gin Glu Gly Gin Gly Ala Gly Ser Arg Val His Glu 545 550 555 560

Phe Gin Thr &lt; 210〉 10 &lt; 211 &gt; 1989 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequences &lt; 220〉 &lt; 223〉 Performance cassette, encoding fusion protein &lt; 400〉 10 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aaaaaaataa tgctagtttt tattacactt atattagtta gtctaccaat tgcgcaacaa 300 actgaagcaa aggatgcatc tgcattcaat aaagaaaatt caatttcatc catggcacca 360 ccagcatctc cgcctgcaag tcctaagacg ccaatcgaaa agaaacacgc ggatggatcc 420 98561.doc -12- 200530399 gattataaag atgatgatga taaacaaggt aaagaagttg ttttattaga ttttgcagca 480 gcaggtggtg aattaggttg gttaacacat ccatatggta aaggttggga tttaatgcaa 540 aatattatga atgatatgcc aatttatatg tatagtgttt gtaatgttat gagtggtgat 600 caagataatt ggttacgtac aaattgggtt tatcgtggtg aagcagaacg tatttttatt 660 gaattaaaat ttacagttcg tgattgtaat agttttccag gtggtgc aag tagttgtaaa 720 gaaacattta atttatatta tgcagaaagt gatttagatt atggtacaaa ttttcaaaaa 780 cgtttattta caaaaattga tacaattgca ccagatgaaa ttacagttag tagtgatttt 840 gaagcacgtc atgttaaatt aaatgttgaa gaacgtagtg ttggtccatt aacacgtaaa 900 ggtttttatt tagcatttca agatattggt gcatgtgttg cattattaag tgttcgtgtt 960 tattataaaa aatgtccaga attattacaa ggtttagcac attttccaga aacaattgca 1020 ggtagtgatg caccaagttt agcaacagtt gcaggtacat gtgttgatca tgcagttgtt 1080 ccaccaggtg gtgaagaacc acgtatgcat tgtgcagttg atggtgaatg gttagttcca 1140 attggtcaat gtttatgtca agcaggttat gaaaaagttg aagatgcatg tcaagcatgt 1200 agtccaggtt tttttaaatt tgaagcaagt gaaagtccat gtttagaatg tccagaacat 1260 acattaccaa gtccagaagg tgcaacaagt tgtgaatgtg aagaaggttt ttttcgtgca 1320 ccacaagatc cagcaagtat gccatgtaca cgtccaccaa gtgcaccaca ttatttaaca 1380 gcagttggta tgggtgcaaa agttgaatta cgttggacac caccacaaga tagtggtggt 1440 cgtgaagata ttgtttatag tgttacatgt gaacaatgtt ggccagaaag tggtgaatgt 1500 ggtccatgtg aagcaagtgt tcgttatagt gaaccaccac atggtttaac acgtaca agt 1560 gttacagtta gtgatttaga accacatatg aattatacat ttacagttga agcacgtaat 1620 ggtgttagtg gtttagttac aagtcgtagt tttcgtacag caagtgttag tattaatcaa 1680 acagaaccac caaaagttcg tttagaaggt cgtagtacaa caagtttaag tgttagttgg 1740 agtattccac caccacaaca aagtcgtgtt tggaaatatg aagttacata tcgtaaaaaa 1800 ggtgatagta atagttataa tgttcgtcgt acagaaggtt ttagtgttac attagatgat 1860 ttagcaccag atacaacata tttagttcaa gttcaagcat taacacaaga aggtcaaggt 1920 gcaggtagtc gtgttcatga atttcaaaca gaacaaaaat taattagtga agaagattta 1980 tgagagctc 1989 &lt; 210> 11 &lt; 211 &gt; 581 &lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223> fusion protein &lt; 400 &gt; 11

Met Lys Lys lie Met Leu Val Phe lie Thr Leu lie Leu Val Ser Leu 1 5 10 15

Pro lie Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30

Glu Asn Ser lie Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45

Pro Lys Thr Pro He Glu Lys Lys His Ala Asp Gly Ser Asp Tyr Lys 5〇 55 60 98561.doc • 13- 200530399

Asp Asp Asp Asp Lys Gin Gly Lys Glu Val Val Leu Leu Asp Phe Ala 65 70 75 80

Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly 85 90 95

Trp Asp Leu Met Gin Asn lie Met Asn Asp Met Pro He Tyr Met Tyr 100 105 110

Ser Val Cys Asn Val Met Ser Gly Asp Gin Asp Asn Trp Leu Arg Thr 115 120 125

Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg lie Phe He Glu Leu Lys 130 135 140

Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys 145 150 155 160

Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly 165 170 175

Thr Asn Phe Gin Lys Arg Leu Phe Thr Lys lie Asp Thr lie Ala Pro 180 185 190

Asp Glu He Thr Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu 195 200 205

Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr 210 215 220

Leu Ala Phe Gin Asp He Gly Ala Cys Val Ala Leu Leu Ser Val Arg 225 230 235 240

Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gin Gly Leu Ala His Phe 245 250 255

Pro Glu Thr lie Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala 260 265 270

Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro 275 280 285

Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro He Gly Gin 290 295 300

Cys Leu Cys Gin Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gin Ala 305 310 315 320

Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu 325 330 335

Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys 340 345 350

Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gin Asp Pro Ala Ser Met 355 360 365

Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly 370 375 380

Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gin Asp Ser Gly 385 390 395 400

Gly Arg Glu Asp He Val Tyr Ser Val Thr Cys Glu Gin Cys Trp Pro 405 410 415

Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu 98561.doc -14- 200530399 420 425 430

Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu 435 440 445

Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser 450 455 460

Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val Ser He Asn 465 470 475 480

Gin Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser 485 490 495

Leu Ser Val Ser Trp Ser lie Pro Pro Gin Gin Ser Arg Val Trp 500 505 510

Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn 515 520 525

Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro 530 535 540

Asp Thr Thr Tyr Leu Val Gin Val Gin Ala Leu Thr Gin Glu Gly Gin 545 550 555 560

Gly Ala Gly Ser Arg Val His Glu Phe Gin Thr Glu Gin Lys Leu lie 565 570 575

Ser Glu Glu Asp Leu 580 &lt; 210〉 12 &lt; 211 &gt; 1989 &lt; 212〉 DNA &lt; 213> Artificial sequence &lt; 220> &lt; 223〉 Performance cassette, encoding fusion protein &lt; 400 &gt; 12 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aaaaaaatta tgttagtttt tattacatta attttagtta gtttaccaat tgcacaacaa 300 acagaagcaa aagatgcaag tgcatttaat aaagaaaata gtattagtag tatggcacca 360 ccagcaagtc caccagcaag tccaaaaaca ccaattgaaa aaaaacatgc agatggatcc 420 gattataaag atgatgatga taaacaaggt aaagaagttg ttttattaga ttttgcagca 480 gcaggtggtg aattaggttg gttaacacat ccatatggta aaggttggga tttaatgcaa 540 aatattatga atgatatgcc aatttatatg tatagtgttt gtaatgttat gagtggtgat 600 caagataatt ggttacgtac aaattgggtt tatcgtggtg aagcagaacg tatttttatt 660 gaattaaaat ttacagttcg tgattgtaat agttttccag gtggtgcaag tagttgtaaa 720 gaaacattta atttatatta tgcagaaagt gatttagatt atggtacaaa ttttcaaaaa 780 98561.doc -15- 200530399 cgtttattta caaaaattga tacaattgca ccagatgaaa ttacagttag tagtgatttt 840 gaagcacgtc atgttaaatt aaatgttgaa gaacgtagtg ttggtccatt aacacgtaaa 900 ggtttttatt tagcatttea agatattggt gcatgtgttg cattattaag tgttcgtgtt 960 tattataaaa aatgtccaga attattacaa ggtttagcac attttccaga aacaattgca 1020 ggtagtgatg caccaagttt agcaacagtt gcaggtacat gtgttgatca tgcagttgtt 1080 ccaccaggtg gtgaagaacc aegtatgeat tgtgcagttg atggtgaatg gttagttcca 1140 attggtcaat gtttatgtca ageaggttat gaaaaagttg aagatgcatg tcaagcatgt 1200 agtccaggtt tttttaaatt tgaagcaagt gaaagtccat gtttagaatg tccagaacat 1260 acattaccaa gtccagaagg tgcaacaagt tgtgaatgtg aagaaggttt ttttcgtgca 1320 ccacaagatc cagcaagtat gccatgtaca cgtccaccaa gtgcaccaca ttatttaaca 1380 gcagttggta tgggtgcaaa agttgaatta cgttggacac caccacaaga tagtggtggt 1440 cgtgaagata ttgtttatag tgttacatgt gaacaatgtt ggccagaaag tggtgaatgt 1500 ggtccatgtg aagcaagtgt tcgttatagt gaaccaccac atggttt aac acgtacaagt 1560 gttacagtta gtgatttaga accacatatg aattatacat ttacagttga ageaegtaat 1620 ggtgttagtg gtttagttac aagtcgtagt tttcgtacag caagtgttag tattaatcaa 1680 acagaaccac caaaagttcg tttagaaggt cgtagtacaa caagtttaag tgttagttgg 1740 agtattccac caccacaaca aagtcgtgtt tggaaatatg aagttacata tcgtaaaaaa 1800 ggtgatagta atagttataa tgttcgtcgt acagaaggtt ttagtgttac attagatgat 1860 ttagcaccag atacaacata tttagttcaa gttcaagcat taacacaaga aggtcaaggt 1920 gcaggtagtc gtgttcatga atttcaaaca gaacaaaaat taattagtga agaagattta 1980 tgagagctc 1989

&lt; 210 &gt; 13 &lt; 211 &gt; 581 &lt; 212> PRT &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; fusion protein &lt; 400> 13

Met Lys Lys lie Met Leu Val Phe lie Thr Leu lie Leu Val Ser Leu 1 5 10 15

Pro lie Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30

Glu Asn Ser lie Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45

Pro Lys Thr Pro lie Glu Lys Lys His Ala Asp Gly Ser Asp Tyr Lys 5〇 55 60

Asp Asp Asp Asp Lys Gin Gly Lys Glu Val Val Leu Leu Asp Phe Ala 65 70 75 80

Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly 85 90 95

Trp Asp Leu Met Gin Asn He Met Asn Asp Met Pro lie Tyr Met Tyr 100 105 110 98561.doc -16- 200530399

Ser Val Cys Asn Val Met Ser Gly Asp Gin Asp Asn Trp Leu Arg Thr 115 120 125

Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg lie Phe lie Glu Leu Lys 130 135 140

Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys 145 150 155 160

Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly 165 170 175

Thr Asn Phe Gin Lys Arg Leu Phe Thr Lys lie Asp Thr lie Ala Pro 180 185 190

Asp Glu He Thr Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu 195 200 205

Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr 210 215 220

Leu Ala Phe Gin Asp lie Gly Ala Cys Val Ala Leu Leu Ser Val Arg 225 230 235 240

Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gin Gly Leu Ala His Phe 245 250 255

Pro Glu Thr He Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala 260 265 270

Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro 275 280 285

Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro He Gly Gin 290 295 300

Cys Leu Cys Gin Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gin Ala 305 310 315 320

Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu 325 330 335

Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys 340 345 350

Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gin Asp Pro Ala Ser Met 355 360 365

Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly 370 375 380

Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gin Asp Ser Gly 385 390 395 400

Gly Arg Glu Asp He Val Tyr Ser Val Thr Cys Glu Gin Cys Trp Pro 405 410 415

Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu 420 425 430

Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu 435 440 445

Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser 450 455 460

Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val Ser He Asn 98561.doc -17- 200530399 465 470 475 480

Gin Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser 485 490 495

Leu Ser Val Ser Trp Ser lie Pro Pro Gin Gin Ser Arg Val Trp 500 505 510

Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn 515 520 525

Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro 530 535 540

Asp Thr Thr Tyr Leu Val Gin Val Gin Ala Leu Thr Gin Glu Gly Gin 545 550 555 560

Gly Ala Gly Ser Arg Val His Glu Phe Gin Thr Glu Gin Lys Leu He 565 570 575

Ser Glu Glu Asp Leu 580 &lt; 210 &gt; 14 &lt; 211 &gt; 1968 &lt; 212> DNA &lt; 213> Artificial sequence &lt; 220> &lt; 223> Performance cassette, encoding fusion protein &lt; 400 &gt; 14 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 gcatacgaca gtcgttttga tgaatgggta cagaaactga aagaggaaag ctttcaaaac 300 aatacgtttg accgccgcaa atttattcaa ggagcgggga agattgcagg actttctctt 360 ggattaacga ttgcccagtc ggttggggcc tttggatccg attataaaga tgatgatgat 420 aaacaaggta aagaagttgt tttattagat tttgcagcag caggtggtga attaggttgg 480 ttaacacatc catatggtaa aggttgggat ttaatgcaaa atattatgaa tgatatgcca 540 atttatatgt atagtgtttg taatgttatg agtggtgatc aagataattg gttacgtaca 600 aattgggttt atcgtggtga agcagaacgt atttttattg aattaaaatt tacagttcgt 660 gattgtaata gttttccagg tggtgcaagt agttgtaaag aaacatttaa tttatatta t 720 gcagaaagtg atttagatta tggtacaaat tttcaaaaac gtttatttac aaaaattgat 780 acaattgcac cagatgaaat tacagttagt agtgattttg aagcacgtca tgttaaatta 840 aatgttgaag aacgtagtgt tggtccatta acacgtaaag gtttttattt agcatttcaa 900 gatattggtg catgtgttgc attattaagt gttcgtgttt attataaaaa atgtccagaa 960 ttattacaag gtttagcaca ttttccagaa acaattgcag gtagtgatgc accaagttta 1020 gcaacagttg caggtacatg tgttgatcat gcagttgttc caccaggtgg tgaagaacca 1080 cgtatgcatt gtgcagttga tggtgaatgg ttagttccaa ttggtcaatg tttatgtcaa 1140 98561.doc -18 - 200530399 gcaggttatg aaaaagttga agatgcatgt caagcatgta gtccaggttt ttttaaattt 1200 gaagcaagtg aaagtccatg tttagaatgt ccagaacata cattaccaag tccagaaggt 1260 gcaacaagtt gtgaatgtga agaaggtttt tttcgtgcac cacaagatcc agcaagtatg 1320 ccatgtacac gtccaccaag tgcaccacat tatttaacag cagttggtat gggtgcaaaa 1380 gttgaattac gttggacacc accacaagat agtggtggtc gtgaagatat tgtttatagt · 1440 gttacatgtg aacaatgttg gccagaaagt ggtgaatgtg gtccatgtga agcaagtgtt 1500 cgttatagtg aaccaccaca tggtttaaca cgtacaagtg tta cagttag tgatttagaa 1560 ccacatatga attatacatt tacagttgaa gcacgtaatg gtgttagtgg tttagttaca 1620 agtcgtagtt ttcgtacagc aagtgttagt attaatcaaa cagaaccacc aaaagttcgt 1680 ttagaaggtc gtagtacaac aagtttaagt gttagttgga gtattccacc accacaacaa 1740 agtcgtgttt ggaaatatga agttacatat cgtaaaaaag gtgatagtaa tagttataat 1800 gttcgtcgta cagaaggttt tagtgttaca ttagatgatt tagcaccaga tacaacatat 1860 ttagttcaag ttcaagcatt aacacaagaa ggtcaaggtg caggtagtcg tgttcatgaa 1920 tttcaaacag aacaaaaatt aattagtgaa gaagatttat gagagctc 1968

&lt; 210〉 15 &lt; 211〉 574 &lt; 212〉 PRT &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; fusion protein &lt; 400 &gt; 15

Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gin Lys Leu Lys Glu 15 10 15

Glu Ser Phe Gin Asn Asn Thr Phe Asp Arg Arg Lys Phe He Gin Gly 20 25 30

Ala Gly Lys lie Ala Gly Leu Ser Leu Gly Leu Thr lie Ala Gin Ser 35 40 45

Val Gly Ala Phe Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gin Gly 50 55 60

Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly 65 70 75 80

Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gin Asn He 85 90 95

Met Asn Asp Met Pro He Tyr Met Tyr Ser Val Cys Asn Val Met Ser 100; 105 110

Gly Asp Gin Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu 115 120 125

Ala Glu Arg lie Phe He Glu Leu Lys Phe Thr Val Arg Asp Cys Asn 130 135 140

Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr 145 150 155 160

Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gin Lys Arg Leu 98561.doc -19- 200530399 165 170 175

Phe Thr Lys lie Asp Thr lie Ala Pro Asp Glu lie Thr Val Ser Ser 180 185 190

Asp Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg Ser Val 195 200 205

Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gin Asp He Gly 210 215 220

Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro 225 230 235 240

Glu Leu Leu Gin Gly Leu Ala His Phe Pro Glu Thr He Ala Gly Ser 245 250 255

Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala 260 265 270

Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala Val Asp 275 280 285

Gly Glu Trp Leu Val Pro lie Gly Gin Cys Leu Cys Gin Ala Gly Tyr 290 295 300

Glu Lys Val Glu Asp Ala Cys Gin Ala Cys Ser Pro Gly Phe Phe Lys 305 ^ 310 315 320

Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu 325 330 335

Pro Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe 340 345 350

Arg Ala Pro Gin Asp Pro Ala Ser Met Pro Cys Thr Arg Pro Pro Ser 355 360 365

Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val Glu Leu 370 375 380

Arg Trp Thr Pro Pro Gin Asp Ser Gly Gly Arg Glu Asp He Val Tyr 385 390 395 400

Ser Val Thr Cys Glu Gin Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro 405 410 415

Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu Thr Arg 420 425 430

Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe 435 440 445

Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser 450 455 460

Phe Arg Thr Ala Ser Val Ser He Asn Gin Thr Glu Pro Pro Lys Val 465 470 475 480

Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp Ser lie 485 490 495

Pro Pro Pro Gin Gin Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg 500 505 510

Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe 515 520 525 98561.doc -20- 200530399

Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gin 530 535 540

Val Gin Ala Leu Thr Gin Glu Gly Gin Gly Ala Gly Ser Arg Val His 545 550 555 560

Glu Phe Gin Thr Glu Gin Lys Leu lie Ser Glu Glu Asp Leu 565 570 &lt; 210 &gt; 16 &lt; 211 &gt; 1254 &lt; 212 &gt; DNA &lt; 213〉 Modern Homo sapiens &lt; 400 &gt; 16

caccgcagga ggaagaacca gcgtgcccgc cagtccccgg aggacgttta cttctccaag 60 tcagaacaac tgaagcccct gaagacatac gtggaccccc acacatatga ggaccccaac 120 caggctgtgt tgaagttcac taccgagatc catccatcct gtgtcactcg gcagaaggtg 180 atcggagcag gagagtttgg ggaggtgtac aagggcatgc tgaagacatc ctcggggaag 240 aaggaggtgc cggtggccat caagacgctg aaagccggct acacagagaa gcagcgagtg 300 gacttcctcg gcgaggccgg catcatgggc cagttcagcc accacaacat catccgccta 360 gagggcgtca tctccaaata caagcccatg atgatcatca ctgagtacat ggagaatggg 420 gccctggaca agttccttcg ggagaaggat ggcgagttca gcgtgctgca gctggtgggc 480 atgctgcggg gcatcgcagc tggcatgaag tacctggcca acatgaacta tgtgcaccgt 540 gacctggctg cccgcaacat cctcgtcaac agcaacctgg tctgcaaggt gtctgacttt 600 ggcctgtccc gcgtgctgga ggacgacccc gaggccacct acaccaccag tggcggcaag 660 atccccatcc gctggaccgc cccggaggcc atttcctacc ggaagttcac ctctgccagc 720 gacgtgtgga gctttggcat tgtcatgtgg gaggtgatga cctatggcga gcggccctac 780 tgggagttgt ccaaccacga ggtgatgaaa gccatcaatg atggcttccg gctccccaca 840 cccatggact gcccctccgc catctaccag ctcatgatgc agtgctggca gcaggagcgt 900

gcccgccgcc ccaagttcgc tgacatcgtc agcatcctgg acaagctcat tcgtgcccct 960 gactccctca agaccctggc tgactttgac ccccgcgtgt ctatccggct ccccagcacg 1020 agcggctcgg agggggtgcc cttccgcacg gtgtccgagt ggctggagtc catcaagatg 1080 cagcagtata cggagcactt catggcggcc ggctacactg ccatcgagaa ggtggtgcag 1140 atgaccaacg acgacatcaa gaggattggg gtgcggctgc ccggccacca gaagcgcatc 1200 gcctacagcc tgctgggact caaggaccag gtgaacactg tggggatccc catc 1254 &lt; 210> 17 &lt; 211 &gt; 1254 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequences &lt; 220 &gt; &lt; 223 &gt; Codon-Optimized Sequences &lt; 400 &gt; 17 98561.doc -21-200530399 cacagacgta gaaaaaatca acgtgctcga caatccccag aagatgtgta tttttctcgaa acc atacgtacga agacccaaat 120 caagcagtat taaaatttac aacagaaata cacccaagtt gtgttacaag acaaaaagtt 180 attggagcag gtgaattcgg agaggtatat aaaggtatgt taaaaacatc atcaggtaaa 240 aaagaagttc cggttgcaat taaaacctta aaggcaggat atacagaaaa acagcgagtt 300 gattttttag gtgaagcagg aattatgggt caatttagcc atcataatat tattcgtttg 360 g aaggagtaa taagtaaata taaaccaatg atgattatta cagaatacat ggaaaacggt 420 gctttagata aatttttacg tgaaaaggat ggtgaattta gtgttttaca attggttggt 480 atgttaagag gaattgctgc aggtatgaaa tatttagcta atatgaatta tgttcaccgt 540 gatttggcag caagaaatat cctagtcaat tccaatttag tatgtaaagt tagtgatttt 600 ggtttaagca gagtattaga agacgatcca gaggcaacct atacaacatc gggaggtaaa 660 attcctattc gttggacagc accagaagct atcagttacc gtaaatttac aagtgcatca 720 gacgtgtgga gttttgggat tgtaatgtgg gaagttatga catatggaga aagaccatat 780 tgggaattaa gtaatcatga agttatgaaa gcaattaacg atggatttag attaccaact 840 ccgatggatt gtccatctgc catttatcaa ctaatgatgc aatgttggca acaagaaaga 900 gcacgacgtc caaaatttgc agatattgtt agtattttag acaaattaat tcgtgcacca 960 gatagtttaa aaactttagc agactttgat cctcgtgtta gtattcgatt accaagtacg 1020 tcaggttccg aaggagttcc atttcgcaca gtctccgaat ggttggaatc aattaaaatg 1080 caacaataca ccgaacactt tatggcagca ggttacacag caatcgaaaa agttgttcaa 1140 atgacaaatg atgatattaa acgtattgga gttagattac caggccacca gaaacgtatt 1200 gcatattctt tattag gttt aaaagatcaa gttaataccg tgggaattcc aatt 1254 &lt; 210 &gt; 18 &lt; 211 &gt; 456 &lt; 212〉 PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400〉 18

Val His Glu Phe Gin Thr Leu Ser Pro Glu Gly Ser Gly Asn Leu Ala 15 10 15

Val He Gly Gly Val Ala Val Gly Val Val Leu Leu Leu Val Leu Ala 20 25 30

Gly Val Gly Phe Phe He His Arg Arg Arg Lys Asn Gin Arg Ala Arg 35 40 45

Gin Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gin Leu Lys Pro 50 55 60

Leu Lys Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro Asn Gin Ala 65 70 75 80

Val Leu Lys Phe Thr Thr Glu He His Pro Ser Cys Val Thr Arg Gin 85 90 95

Lys Val lie Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys Gly Met Leu 100 105 110

Lys Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala lie Lys Thr Leu 115 120 125

Lys Ala Gly Tyr Thr Glu Lys Gin Arg Val Asp Phe Leu Gly Glu Ala 98561.doc • 22- 200530399 130 135 140

Gly lie Met Gly Gin Phe Ser His His Asn lie He Arg Leu Glu Gly 145 150 155 160

Val lie Ser Lys Tyr Lys Pro Met Met lie lie Thr Glu Tyr Met Glu 165 170 175

Asn Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly Glu Phe Ser 180 185 190

Val Leu Gin Leu Val Gly Met Leu Arg Gly He Ala Ala Gly Met Lys 195 200 205

Tyr Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn 210 215 220 lie Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu 225 230 235 240

Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser Gly

245 250 255

Gly Lys lie Pro He Arg Trp Thr Ala Pro Glu Ala He Ser Tyr Arg 260 265 270

Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly He Val Met Trp 275 280 285

Glu Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn His 290 295 300

Glu Val Met Lys Ala He Asn Asp Gly Phe Arg Leu Pro Thr Pro Met 305 310 315 320

Asp Cys Pro Ser Ala lie Tyr Gin Leu Met Met Get Cys Trp Gin Gin 325 330 335

Glu Arg Ala Arg Arg Pro Lys Phe Ala Asp lie Val Ser He Leu Asp 340 345 350

Lys Leu lie Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe Asp 355 360 365

Pro Arg Val Ser lie Arg Leu Pro Ser Thr Ser Gly Ser Glu Gly Val 370 375 380

Pro Phe Arg Thr Val Ser Glu Trp Leu Glu Ser lie Lys Met Gin Gin 385 390 395 400

Tyr Thr Glu His Phe Met Ala Ala Gly Tyr Thr Ala lie Glu Lys Val 405 410 415

Val Gin Met Thr Asn Asp Asp lie Lys Arg He Gly Val Arg Leu Pro 420 425 430

Gly His Gin Lys Arg He Ala Tyr Ser Leu Leu Gly Leu Lys Asp Gin 435 440 445

Val Asn Thr Val Gly He Pro He 450 455 &lt; 210 &gt; 19 &lt; 211 &gt; 1437 98561.doc -23-200530399 &lt; 212〉 DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; fusion protein coding sequence &lt; 400> 19 atgaaaaaaa taatgctagt caaactgaag caaaggatgc ccaccagcat ctccgcctgc gagcaccgca ggaggaagaa aagtcagaac aactgaagcc aaccaggctg tgttgaagtt gtgatcggag caggagagtt aagaaggagg tgccggtggc gtggacttcc tcggcgaggc ctagagggcg tcatctccaa ggggccctgg acaagttcct ggcatgctgc ggggcatcgc cgtgacctgg ctgcccgcaa tttggcctgt cccgcgtgct aagatcccca tccgctggac agcgacgtgt ggagctttgg tactgggagt tgtccaacca acacccatgg actgcccctc cgtgcccgcc gccccaagtt cctgactccc tcaagaccct acgagcggct cggagggggt atgcagcagt atacggagca cagatgacca acgacgacat atcgcctaca gcctgctggg &lt; 210 &gt; 20 &lt; 211〉 479 &lt; 212〉 PRT &lt; 213 &gt; Artificial Sequences &lt; 220〉 &lt; 223> Fusion Protein ttttattaca cttatattag ttagtctacc aggacg agcaga agagat agagat cgccagtccc cg gaggacgt ttacttctcc 240 cctgaagaca tacgtggacc cccacacata tgaggacccc 300 cactaccgag atccatccat cctgtgtcac tcggcagaag 360 tggggaggtg tacaagggca tgctgaagac atcctcgggg 420 catcaagacg ctgaaagccg gctacacaga gaagcagcga 480 cggcatcatg ggccagttca gccaccacaa catcatccgc 540 atacaagccc atgatgatca tcactgagta catggagaat 600 tcgggagaag gatggcgagt tcagcgtgct gcagctggtg 660 agctggcatg aagtacctgg ccaacatgaa ctatgtgcac 720 catcctcgtc aacagcaacc tggtctgcaa ggtgtctgac 780 ggaggacgac cccgaggcca cctacaccac cagtggcggc 840 cgccccggag gccatttcct accggaagtt cacctctgcc 900 cattgtcatg tgggaggtga tgacctatgg cgagcggccc 960 cgaggtgatg aaagccatca atgatggctt ccggctcccc 1020 cgccatctac cagctcatga tgcagtgctg gcagcaggag 1080 cgctgacatc gtcagcatcc tggacaagct cattcgtgcc 1140 ggctgacttt gacccccgcg tgtctatccg gctccccagc 1200 gcccttccgc acggtgtccg agtggctgga gtccatcaag 1260 cttcatggcg gccggctaca ctgccatcga gaaggtggtg 1320 caagaggatt ggggtgcggc tgcccggcca ccagaagcgc 1380 actcaaggac caggtgaaca ctgtggggat ccccatc 1437 &lt; 400 &gt; 20

Met Lys Lys lie Met Leu Val Phe He Thr Leu lie Leu Val Ser Leu 15 10 15

Pro He Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30 98561.doc -24- 200530399

Glu Asn Ser lie Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45

Pro Lys Thr Pro He Glu Lys Lys His Ala Asp Leu Glu His Arg Arg 50 55 60

Arg Lys Asn Gin Arg Ala Arg Gin Ser Pro Glu Asp Val Tyr Phe Ser 65 70 75 80

Lys Ser Glu Gin Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His Thr 85 90 95

Tyr Glu Asp Pro Asn Gin Ala Val Leu Lys Phe Thr Thr Glu lie His 100 105 110

Pro Ser Cys Val Thr Arg Gin Lys Val lie Gly Ala Gly Glu Phe Gly 115 120 125

Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu Val 130 135 140

Pro Val Ala He Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gin Arg 145 150 155 160

Val Asp Phe Leu Gly Glu Ala Gly He Met Gly Gin Phe Ser His His 165 170 175

Asn He lie Arg Leu Glu Gly Val lie Ser Lys Tyr Lys Pro Met Met 180 185 190 lie lie Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu Arg 195 200 205

Glu Lys Asp Gly Glu Phe Ser Val Leu Gin Leu Val Gly Met Leu Arg 210 215 220

Gly He Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val His 225 230 235 240

Arg Asp Leu Ala Ala Arg Asn lie Leu Val Asn Ser Asn Leu Val Cys 245 250 255

Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro Glu 260 265 270

Ala Thr Tyr Thr Thr Ser Gly Gly Lys He Pro He Arg Trp Thr Ala 275 280 285

Pro Glu Ala He Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val Trp 290 295 300

Ser Phe Gly lie Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg Pro 305 310 315 320

Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala He Asn Asp Gly 325 330 335

Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala lie Tyr Gin Leu 340 345 350

Met Met Gin Cys Trp Gin Gin Glu Arg Ala Arg Arg Pro Lys Phe Ala 355 360 365

Asp He Val Ser He Leu Asp Lys Leu He Arg Ala Pro Asp Ser Leu 370 375 380

Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser He Arg Leu Pro Ser 98561.doc -25- 200530399 385 390 395 400

Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp Leu 405 410 415

Glu Ser lie Lys Met Gin Gin Tyr Thr Glu His Phe Met Ala Ala Gly 420 425 430

Tyr Thr Ala lie Glu Lys Val Val Gin Met Thr Asn Asp Asp lie Lys 435 440 445

Arg lie Gly Val Arg Leu Pro Gly His Gin Lys Arg lie Ala Tyr Ser 450 455 460

Leu Leu Gly Leu Lys Asp Gin Val Asn Thr Val Gly lie Pro lie 465 470 475

&lt; 210 &gt; 21 &lt; 211 &gt; 1737 &lt; 212> DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; performance cassette, encoding fusion protein &lt; 400 &gt; 21 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aaaaaaataa tgctagtttt tattacactt atattagtta gtctaccaat tgcgcaacaa 300 actgaagcaa aggatgcatc tgcattcaat aaagaaaatt caatttcatc catggcacca 360 ccagcatctc cgcctgcaag tcctaagacg ccaatcgaaa agaaacacgc ggatggatcc 420

gattataaag atgatgatga taaacacaga cgtagaaaaa atcaacgtgc tcgacaatcc 480 ccagaagatg tgtatttttc gaaaagtgaa caattaaaac cattaaaaac ttatgttgat 540 ccgcatacgt acgaagaccc aaatcaagca gtattaaaat ttacaacaga aatacaccca 600 agttgtgtta caagacaaaa agttattgga gcaggtgaat tcggagaggt atataaaggt 660 atgttaaaaa catcatcagg taaaaaagaa gttccggttg caattaaaac cttaaaggca 720 ggatatacag aaaaacagcg agttgatttt ttaggtgaag caggaattat gggtcaattt 780 agccatcata atattat teg tttggaagga gtaataagta aatataaacc aatgatgatt 840 attacagaat acatggaaaa cggtgcttta gataaatttt tacgtgaaaa ggatggtgaa 900 tttagtgttt tacaattggt tggtatgtta agaggaattg ctgcaggtat gaaatattta 960 getaatatga attatgttea ccgtgatttg gcagcaagaa atatcctagt caattccaat 1020 ttagtatgta aagttagtga ttttggttta ageagagtat tagaagaega tccagaggca 1080 acctatacaa catcgggagg taaaattcct attegttgga cagcaccaga agctatcagt 1140 taccgtaaat ttacaagtgc atcagacgtg tggagttttg ggattgtaat gtgggaagtt 1200 atgacatatg gagaaagacc atattgggaa ttaagtaatc atgaagttat gaaagcaatt 1260 aacgatggat tta gattacc aactccgatg gattgtccat ctgccattta tcaactaatg 1320 atgcaatgtt ggcaacaaga aagageaega cgtccaaaat ttgcagatat 1380 98561.doc • 26 200530399 ttagacaaat taattcgtgc accagatagt ttaaaaactt tagcagactt tgatcctcgt 1440 gttagtattc gattaccaag tacgtcaggt tccgaaggag ttccatttcg cacagtctcc 1500 gaatggttgg aatcaattaa aatgcaacaa tacaccgaac actttatggc agcaggttac 1560 acagcaatcg aaaaagttgt tcaaatgaca aatgatgata ttaaacgtat tggagttaga 1620 ttaccaggcc accagaaacg tattgcatat tgttagtatt tctttattag gtttaaaaga tcaagttaat 1680 accgtgggaa ttccaattga acaaaaatta atttccgaag aagacttata agagctc 1737

&lt; 210〉 22 &lt; 211〉 497 &lt; 212〉 PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt;

&lt; 223 &gt; Fusion protein &lt; 400〉 22

Leu He Leu Val Ser Leu 15 Ala Ser Ala Phe Asn Lys 30 Ala Ser Pro Pro Ala Ser 45 Asp Gly Ser Asp Tyr Lys 60 Asn Gin Arg Ala Arg Gin 75 80 Glu Gin Leu Lys Pro Leu 95 Asp Pro Asn Gin Ala Val

Cys Val Thr Arg Gin Lys 125 Tyr Lys Gly Met Leu Lys 140 Ala He Lys Thr Leu Lys 155 160 Phe Leu Gly Glu Ala Gly 175 lie Arg Leu Glu Gly Val 190 Thr Glu Tyr Met Glu Asn 205 Asp Gly Glu Phe Ser Val 220 Ala Ala Gly Met Lys Tyr

Met Lys Lys lie Met Leu Val Phe lie Thr 1 5 10

Pro He Ala Gin Gin Thr Glu Ala Lys Asp 20 25

Glu Asn Ser lie Ser Ser Met Ala Pro Pro 35 40

Pro Lys Thr Pro He Glu Lys Lys His Ala 50 55

Asp Asp Asp Asp Lys His Arg Arg Arg Lys 65 70

Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser 85 90

Lys Thr Tyr Val Asp Pro His Thr Tyr Glu 100 105

Leu Lys Phe Thr Thr Glu lie His Pro Ser 115 120

Val lie Gly Ala Gly Glu Phe Gly Glu Val 130 135

Thr Ser Ser Gly Lys Lys Glu Val Pro Val 145 150

Ala Gly Tyr Thr Glu Lys Gin Arg Val Asp 165 170

He Met Gly Gin Phe Ser His His Asn lie 180 185 lie Ser Lys Tyr Lys Pro Met Met He lie 195 200

Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys 210 215

Leu Gin Leu Val Gly Met Leu Arg Gly lie 98561.doc -27- 200530399 225 230 235 240

Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn lie 245 250 255

Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser 260 265 270

Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser Gly Gly 275 280 285

Lys lie Pro lie Arg Trp Thr Ala Pro Glu Ala He Ser Tyr Arg Lys 290 295 300

Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly He Val Met Trp Glu 305 310 315 320

Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn His Glu 325 330 335

Val Met Lys Ala He Asn Asp Gly Phe Arg Leu Pro Thr Pro Met Asp 340 345 350

Cys Pro Ser Ala He Tyr Gin Leu Met Met Gin Cys Trp Gin Gin Glu 355 360 365

Arg Ala Arg Arg Pro Lys Phe Ala Asp He Val Ser lie Leu Asp Lys 370 375 380

Leu He Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe Asp Pro 385 390 395 400

Arg Val Ser lie Arg Leu Pro Ser Thr Ser Gly Ser Glu Gly Val Pro 405 410 415

Phe Arg Thr Val Ser Glu Trp Leu Glu Ser He Lys Met Gin Gin Tyr 420 425 430

Thr Glu His Phe Met Ala Ala Gly Tyr Thr Ala He Glu Lys Val Val 435 440 445

Gin Met Thr Asn Asp Asp lie Lys Arg He Gly Val Arg Leu Pro Gly 450 455 460

His Gin Lys Arg He Ala Tyr Ser Leu Leu Gly Leu Lys Asp Gin Val 465 470 475 480

Asn Thr Val Gly He Pro lie Glu Gin Lys Leu lie Ser Glu Glu Asp 485 490 495

Leu &lt; 210 &gt; 23 &lt; 211 &gt; 1737 &lt; 212 &gt; DNA &lt; 213〉 artificial sequence &lt; 220 &gt; &lt; 223〉 performance cassette, encoding fusion protein 98561.doc -28- 200530399 &lt; 400> 23 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aaaaaaatta tgttagtttt tattacatta attttagtta gtttaccaat tgcacaacaa 300 acagaagcaa aagatgcaag tgcatttaat aaagaaaata gtattagtag tatggcacca 360 ccagcaagtc caccagcaag tccaaaaaca ccaattgaaa aaaaacatgc agatggatcc 420 gattataaag acgatgatga taaacacaga cgtagaaaaa atcaacgtgc tcgacaatcc 480 ccagaagatg tgtatttttc gaaaagtgaa caattaaaac cattaaaaac ttatgttgat 540 ccgcatacgt acgaagaccc aaatcaagca gtattaaaat ttacaacaga aatacaccca 600 agttgtgtta caagacaaaa agttattgga gcaggtgaat tcggagaggt atataaaggt 660 atgttaaaaa catcatcagg taaaaaagaa gttccggttg caattaaaac ctt aaaggca 720 ggatatacag aaaaacagcg agttgatttt ttaggtgaag caggaattat gggtcaattt 780 agccatcata atattattcg tttggaagga gtaataagta aatataaacc aatgatgatt 840 attacagaat acatggaaaa cggtgcttta gataaatttt tacgtgaaaa ggatggtgaa 900 tttagtgttt tacaattggt tggtatgtta agaggaattg ctgcaggtat gaaatattta 960 gctaatatga attatgttca ccgtgatttg gcagcaagaa atatcctagt caattccaat 1020 ttagtatgta aagttagtga ttttggttta agcagagtat tagaagacga tccagaggca 1080 acctatacaa catcgggagg taaaattcct attcgttgga cagcaccaga agctatcagt 1140 taccgtaaat ttacaagtgc atcagacgtg tggagttttg ggattgtaat gtgggaagtt 1200 atgacatatg gagaaagacc atattgggaa ttaagtaatc atgaagttat gaaagcaatt 1260 aacgatggat ttagattacc aactccgatg gattgtccat ctgccattta tcaactaatg 1320 atgcaatgtt ggcaacaaga aagagcacga cgtccaaaat ttgcagatat tgttagtatt 1380 ttagacaaat taattcgtgc accagatagt ttaaaaactt tagcagactt tgatcctcgt 1440 gttagtattc gattaccaag tacgtcaggt tccgaaggag ttccatttcg cacagtctcc 1500 gaatggttgg aatcaattaa aatgcaacaa tacaccgaac actttatggc agcaggttac 156 0 acagcaatcg aaaaagttgt tcaaatgaca aatgatgata ttaaacgtat tggagttaga 1620 ttaccaggcc accagaaacg tattgcatat tctttattag gtttaaaaga tcaagttaat 1680 accgtggga &lt; 212 &lt; 210 &210; 210 &gt; 210 & 210 〉 Fusion protein &lt; 400〉 24

Met Lys Lys He Met Leu Val Phe He Thr Leu lie Leu Val Ser Leu 15 10 15

Pro lie Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30

Glu Asn Ser He Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 98561.doc -29- 200530399 35 40 45

Pro Lys Thr Pro lie Glu Lys Lys His Ala Asp Gly Ser Asp Tyr Lys 50 55 60

Asp Asp Asp Asp Lys His Arg Arg Arg Lys Asn Gin Arg Ala Arg Gin 65 70 75 80

Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gin Leu Lys Pro Leu 85 90 95

Lys Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro Asn Gin Ala Val 100 105 110

Leu Lys Phe Thr Thr Glu He His Pro Ser Cys Val Thr Arg Gin Lys 115 120 125

Val He Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys Gly Met Leu Lys 130 135 140

Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala He Lys Thr Leu Lys

145 150 155 160

Ala Gly Tyr Thr Glu Lys Gin Arg Val Asp Phe Leu Gly Glu Ala Gly 165 170 175

He Met Gly Gin Phe Ser His His Asn He lie Arg Leu Glu Gly Val 180 185 190

He Ser Lys Tyr Lys Pro Met Met lie lie Thr Glu Tyr Met Glu Asn 195 200 205

Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly Glu Phe Ser Val 210 215 220

Leu Gin Leu Val Gly Met Leu Arg Gly lie Ala Ala Gly Met Lys Tyr 225 230 235 240

Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn He 245 250 255

Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser 260 265 270

Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser Gly Gly 275 280 285

Lys He Pro lie Arg Trp Thr Ala Pro Glu Ala He Ser Tyr Arg Lys 290 295 300

Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly He Val Met Trp Glu 305 310 315 320

Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn His Glu 325 330 335

Val Met Lys Ala He Asn Asp Gly Phe Arg Leu Pro Thr Pro Met Asp 340 345 350

Cys Pro Ser Ala He Tyr Gin Leu Met Met Gin Cys Trp Gin Gin Glu 355 360 365

Arg Ala Arg Arg Pro Lys Phe Ala Asp lie Val Ser He Leu Asp Lys 370 375 380

Leu He Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe Asp Pro 385 390 395 400 98561.doc -30- 200530399

Arg Val Ser lie Arg Leu Pro Ser 405

Phe Arg Thr Val Ser Glu Trp Leu 420

Thr Glu His Phe Met Ala Ala Gly 435 440

Gin Met Thr Asn Asp Asp He Lys 450 455

His Gin Lys Arg lie Ala Tyr Ser 465 470

Asn Thr Val Gly lie Pro He Glu 485 Leu

Thr Ser Gly Ser Glu Gly Val Pro 410 415

Glu Ser lie Lys Met Gin Gin Tyr 425 430

Tyr Thr Ala lie Glu Lys Val Val 445

Arg He Gly Val Arg Leu Pro Gly 460

Leu Leu Gly Leu Lys Asp Gin Val 475 480

Gin Lys Leu He Ser Glu Glu Asp 490 495

&lt; 210 &gt; 25 &lt; 211> 1716 &lt; 212> DNA &lt; 213> artificial sequence &lt; 220> &lt; 223> performance cassette, encoding fusion protein &lt; 400> 25 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgta ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta 240

gcatacgaca gtcgttttga tgaatgggta cagaaactga aagaggaaag ctttcaaaac 300 aatacgtttg accgccgcaa atttattcaa ggagcgggga agattgcagg actttctctt 360 ggattaacga ttgcccagtc ggttggggcc tttggatccg attataaaga tgatgatgat 420 aaacacagac gtagaaaaaa tcaacgtgct cgacaatccc cagaagatgt gtatttttcg 480 aaaagtgaac aattaaaacc attaaaaact tatgttgatc cgcatacgta cgaagaccca 540 aatcaagcag tattaaaatt tacaacagaa atacacccaa gttgtgttac aagacaaaaa 600 gttattggag caggtgaatt cggagaggta tataaaggta tgttaaaaac atcatcaggt 660 aaaaaagaag ttccggttgc aattaaaacc ttaaaggcag gatatacaga aaaacagcga 720 gttgattttt taggtgaagc aggaattatg ggtcaattta gccatcataa tattattcgt 780 ttggaaggag taataagtaa atgatgatta atataaacca cctatacaac atcgggaggt 1080 ttacagaata catggaaaac 840 ggtgctttag ataaattttt acgtgaaaag gatggtgaat ttagtgtttt acaattggtt 900 ggtatgttaa gaggaattgc tgcaggtatg aaatatttag ctaatatgaa ttatgttcac 960 cgtgatttgg cagcaagaaa tatcctagtc aattccaatt tagtatgtaa agttagtgat 1020 tttggtttaa gcagagtatt agaagacgat ccagaggcaa aaaattecta ttegttg gac agcaccagaa gctatcagtt accgtaaatt tacaagtgca 1140 tcagacgtgt ggagttttgg gattgtaatg tgggaagtta tgacatatgg agaaagacca 1200 98561.doc -31 - 200530399 tattgggaat taagtaatca tgaagttatg aaagcaatta acgatggatt tagattacca 1260 actccgatgg attgtccatc tgccatttat caactaatga tgcaatgttg gcaacaagaa 1320 agagcacgac gtccaaaatt tgcagatatt gttagtattt tagacaaatt aattcgtgca 1380 ccagatagtt taaaaacttt agcagacttt gatcctcgtg ttagtattcg attaccaagt 1440 acgtcaggtt ccgaaggagt tccatttcgc acagtctccg aatggttgga atcaattaaa 1500 atgcaacaat acaccgaaca ctttatggca gcaggttaca cagcaatcga aaaagttgtt 1560 caaatgacaa atgatgatat taaacgtatt ggagttagat taccaggcca ccagaaacgt 1620 attgcatatt ctttattagg tttaaaagat caagttaata ccgtgggaat tccaattgaa 1680 caaaaattaa tttccgaaga agacttataa gagctc 1716 &lt; 210> 26 &lt; 211 &gt; 490 &lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220> &lt; 223 &gt; Fusion protein &lt; 400 &gt; 26

Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gin Lys Leu Lys Glu 15 10 15

Glu Ser Phe Gin Asn Asn Thr Phe Asp Arg Arg Lys Phe He Gin Gly 20 25 30

Ala Gly Lys He Ala Gly Leu Ser Leu Gly Leu Thr He Ala Gin Ser 35 40 45

Val Gly Ala Phe Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys His Arg 50 55 60

Arg Arg Lys Asn Gin Arg Ala Arg Gin Ser Pro Glu Asp Val Tyr Phe 65 70 75 80

Ser Lys Ser Glu Gin Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His 85 90 95

Thr Tyr Glu Asp Pro Asn Gin Ala Val Leu Lys Phe Thr Thr Glu lie 100 105 110

His Pro Ser Cys Val Thr Arg Gin Lys Val He Gly Ala Gly Glu Phe 115 120 125

Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu 130 135 140

Val Pro Val Ala lie Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gin 145 150 155 160

Arg Val Asp Phe Leu Gly Glu Ala Gly lie Met Gly Gin Phe Ser His 165 170 175

His Asn lie lie Arg Leu Glu Gly Val lie Ser Lys Tyr Lys Pro Met 180 185 190

Met He lie Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu 195 200 205 98561.doc -32- 200530399

Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gin Leu Val Gly Met Leu 210 215 220

Arg Gly lie Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val 225 230 235 240

His Arg Asp Leu Ala Ala Arg Asn lie Leu Val Asn Ser Asn Leu Val 245 250 255

Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro 260 265 270

Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys lie Pro lie Arg Trp Thr 275 280 285

Ala Pro Glu Ala lie Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val 290 295 300

Trp Ser Phe Gly He Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg 305 310 315 320

Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala lie Asn Asp 325 330 335

Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala He Tyr Gin 340 345 350

Leu Met Met Gin Cys Trp Gin Gin Glu Arg Ala Arg krg Pro Lys Phe 355 360 365

Ala Asp lie Val Ser He Leu Asp Lys Leu He Arg Ala Pro Asp Ser 370 375 380

Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser He Arg Leu Pro 385 390 395 400

Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp 405 410 415

Leu Glu Ser He Lys Met Gin Gin Tyr Thr Glu His Phe Met Ala Ala 420 425 430

Gly Tyr Thr Ala He Glu Lys Val Val Gin Met Thr Asn Asp Asp He

435 440 445

Lys Arg He Gly Val Arg Leu Pro Gly His Gin Lys Arg lie Ala Tyr 450 455 460

Ser Leu Leu Gly Leu Lys Asp Gin Val Asn Thr Val Gly He Pro He 465 470 475 480

Glu Gin Lys Leu He Ser Glu Glu Asp Leu 485 490 &lt; 210 &gt; 27 &lt; 211 &gt; 966 &lt; 212 &gt; DNA &lt; 213> Artificial sequence &lt; 220> &lt; 223> Performance cassette, encoding fusion protein 98561. doc -33- 200530399 &lt; 400 &gt; 27 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aaaaaaatta tgttagtttt tattacatta attttagtta gtttaccaat tgcacaacaa 300 acagaagcaa aagatgcaag tgcatttaat aaagaaaata gtattagtag tatggcacca 360 ccagcaagtc caccagcaag tccaaaaaca ccaattgaaa aaaaacatgc agatggatcc 420 caagcagaag gtcgcggaac aggaggaagt acaggagatg cagacggacc aggaggacca 480 ggaataccag acggaccagg aggaaatgca ggaggcccag gcgaagcagg cgcaacagga 540 ggaagaggac caagaggagc aggagcagca cgagcatcag gaccaggagg cggagcacca 600 agaggaccac atggcggagc ggcaagcgga ttaaatggat gttgtagatg tggagcacgc 660 ggaccagaat caaga ctttt agaattttat ttagccatgc catttgcaac cccaatggaa 720

gcagaattag cacgaagatc attagcacaa gatgccccac cattaccagt accaggagtt 780 ttattaaaag agtttacagt atcaggcaat attttaacaa tacgtttaac agcagcagac 840 catcgtcaat tacaactatc tatcagttca tgtttacaac aattatcctt attaatgtgg 900 attacacaat gttttttacc agttttttta gcacaaccac catcaggaca aagaagataa 960 gagctc 966 &lt; 210> 28 &lt; 211 &gt; 240 &lt; 212> PRT &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; fusion protein &lt; 400 &gt; 28

Met Lys Lys lie Met Leu Val Phe lie Thr Leu lie Leu Val Ser Leu 15 10 15

Pro lie Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30

Glu Asn Ser He Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45

Pro Lys Thr Pro lie Glu Lys Lys His Ala Asp Gly Ser Gin Ala Glu 50 55 60

Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly 65 70 75 80

Pro Gly lie Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu 85 90 95

Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala Arg 100 105 110

Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro His Gly Gly Ala 115 120 125 98561.doc • 34- 200530399

Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg Gly Pro Glu! 3〇 135 140

Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met 145 150 155 160

Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gin Asp Ala Pro Pro Leu 165 170 175

Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn lie 18〇 185 190

Leu Thr lie Arg Leu Thr Ala Ala Asp His Arg Gin Leu Gin Leu Ser 195 200 205 lie Ser Ser Cys Leu Gin Gin Leu Ser Leu Leu Met Trp lie Thr Gin 210 215 220

Cys Phe Leu Pro Val Phe Leu Ala Gin Pro Pro Ser Gly Gin Arg Arg 225 230 235 240

&lt; 210> 29 &lt; 211 &gt; 330 &lt; 212> DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; Partial performance cassette including codon-optimized sequence &lt; 400 &gt; 29 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtgtagatagatagat

aaaaaaaaaa ttattagtgc aattttaatg agtacagtta ttttaagtgc agcagcacca 300 ttaagtggtg tttatgcaga tacaggatcc 330 &lt; 210 &gt; 30 &lt; 211 &gt; 330 &lt; 212> DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; box ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 98561.doc -35- 200530399 aatatgaaaa aagcaactat cgcggctaca gctgggattg cggtaacagc atttgctgcg 300 ccaacaatcg catccgcaag cactggatcc 330 &lt; 210 &gt; 31 &lt; 211 &gt; 330 &lt; 212 &gt; DNA &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Partial performance cassette including codon-optimized sequence &lt; 400 &gt; 31 ggtacctcct ttgattagta atttgttaat gacgtcaaaa atattgcgtt tcatctttag gtggcaa gtatttggca aatatgaaaa aagcaacaat ccaacaattg caagtgcaag &lt; 210> 32 &lt; 211 &gt; 1702 &lt; 212 &gt; DNA &lt; 213 &gt; human Sequence tattcctatc ttaaagttac ggatagcaag actagaataa aagcgaattt cgccaatatt ttattaggtt aaaaaatgta tgcagcaaca gcaggtattg tacaggatcc ttttatgtgg aggcattaac 60 agctataaag caagcatata 120 ataattatca aaagagaggg 180 gaaggagagt gaaacccatg 240 cagttacagc atttgcagca 300 330

&lt; 220 &gt; &lt; 223 &gt; hlyP-p60 gene fragment &lt; 400〉 32 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aatatgaaaa aagcaactat cgcggctaca gctgggattg cggtaacagc atttgctgcg 300 ccaacaatcg catccgcaag cactgtagta gtcgaagctg gtgatactct ttggggtatc 360 gcacaaagta aagggactac tgttgacgca attaaaaaag caaacaattt aacaacagat 420 aaaatcgtac caggtcaaaa attacaagta aataatgagg ttgctgctgc tgaaaaaaca 480 gagaaatctg ttagcgcaac ttggttaaac gtccgtagtg gcgctggtgt tgataacagt 540 attattacgt ccatcaaagg tggaacaaaa gtaactgttg aaacaaccga atctaacggc 600 tggcacaaaa ttacttacaa actggtttcg cgatggaaaa tgacactatt tgggctttat ccgtaaaata ttaacggtaa atacttaact 660 gacaaagcag taagcactcc agttgcacca acacaagaag tgaaaaaaga aactactact 720 caacaagctg cacctgctgc agaaacaaaa actgaagtaa aacaaactac acaagcaact 780 acacctgcgc ctaaagtagc agaaacgaaa gaaactccag tagtagatca aaatgctact 840 acacacgctg ttaaaagcgg cggtgtttct 900 gttcaagaca ttatgtcat g gaataattta tcttcttctt ctatttatgt aggtcaaaag 960 cttgctatta aacaaactgc taacacagct actccaaaag cagaagtgaa aacggaagct 1020 98561.doc -36- 200530399 ccagcagctg aaaaacaagc agctccagta gttaaagaaa atactaacac aaatactgct 1080 actacagaga aaaaagaaac agcaacgcaa caacaaacag cacctaaagc accaacagaa 1140 gctgcaaaac cagctcctgc accatctaca aacacaaatg ctaataaaac aaatacaaat 1200 acaaatacaa atacaaatac aaacaatact aatacaaata caccatctaa aaatactaat 1260 acaaactcaa atactaatac gaatacaaac tcaaatacga atgctaatca aggttcttcc 1320 aacaataaca gcaattcaag tgcaagtgct attattgctg aagctcaaaa acaccttgga 1380 aaagcttatt catggggtgg taacggacca actacatttg attgctctgg ttacactaaa 1440 tatgtatttg ctaaagcggg aatctccctt ccacgtactt ctggcgcaca atacgctagc 1500 actacaagaa tctctgaatc tcaagcaaaa cctggtgatt tagtattctt tgactatggt 1560 agcggaattt ctcacgttgg tatctacgtt ggtaatggtc aaatgattaa cgcgcaagac 1620 aatggcgtta aatacgataa catccacggc tctggctggg gtaaatatct agttggcttc 1680 ggtcgcgtat aattaaggat cc 1702

&lt; 210〉 33 &lt; 211 &gt; 9808 &lt; 212〉 DNA &lt; 213〉 artificial sequence &lt; 220〉 &lt; 223 &gt; pAM401-MCS plastid &lt; 400> 33 ctttaaacgt ggatcatttt ctttaaattt atgctgacga cctttgaatt tgcctttttt cttgatt tatt ttttttcgtt ctgattctgc 120 ttgatacttg tacaattcaa tgacaaggct attaatcaaa cgccttaaat tttcatcttc 180 aataccattc attgagggta aatttaagac ttccagggtt gcccccttaa tttgaatttg 240 attcatcaat tctgttaatt ctttattatt tcgtcctaat cgatctaatt cagtaacaat 300 aacaatatcc ccttcacgaa tatagttaag catagcttgt aattgtgggc gttcgaccga 360 ttgaccgctt aatttgtctg aaaagacctt agaaacgccc tgtaacgctt gtaattgccg 420

atctaagttc tgttctttgc tactgacacg tgcataacca attttagcca ttttcaacca 480 acctctaaaa ttctctcggt tgcaataacc aatcagcaat atctactttt tcaatttcaa 540 attgcttatc agaaattgtc ttttcgtaag cgataaaatc ttgcgcatat tgttgctcat 600 taaaaatagc caccactteg tcattttcta aaactcgata aataaatttt ttcattttac 660 tcctcctatt atgcccaact taaatgacct attcaccaag tcaattatac tgctaaaatc 720 atattaggac aaataggtat actctattga cctataaatg atagcaactt aaaagatcaa 780 gtgttcgctt cgctctcact gcccctcgac gttttagtag cctttccctc aettegttea 840 gtccaagcca actaaaagtt ttcgggctac tctctccttc tccccctaat aattaattaa 900 aatcttactc tgtatatttc tgctaatcat tcactaaaca acaaacacgt gcaaagaaaa tcgccttcag taccttcagc 1260 960 atcatagata taaatgtaat ggcatagtgc gggttttatt ttcagcctgt ategtageta 1020 aacaaatcga gttgtgggtc cgttttgggg cgttctgcca atttgtttag agtttcttga 1080 ataaatgtac gttctaaatt aaaegaaget gtcagcgcct ttatataget ttctcgttct 1140 tettttttta atttaatgat cgatagcaac aatgatttaa cactagcaag ttgaatgcca 1200 ccatttcttc ctggtttaat cttaaagaaa atttcctgat aatttatcta atgt ccgttc aggaatgcct agcacttctc taatctcttt tttggtcgtc 1320 getaaataag gcttgtatac atcgcttttt tegetaatat aagccattaa atettettte 1380 98561.doc -37- 200530399 cattctgaca aatgaacacg ttgacgtteg cttctttttt tettgaattt aaaccaccct 1440 tgacggacaa ataaatcttt actggttaaa tcacttgata cccaagcttt gcaaagaatg 1500 gtaatgtatt ccctattage cccttgatag ttttctgaat aggcacttet aacaattttg 1560 attaettett tttcttctaa gggttgatct aategattat taaactcaaa catattatat 1620 tcgcacgttt egattgaata gcctgaacta aagtaggeta aagagagggt aaacataacg 1680 ctattgegee ctactaaacc cttttctcct gaaaattteg tttcgtgcaa taagagatta 1740 aaccagggtt catctacttg ttttttgcct tctgtaccgc ttaaaaccgt tagaettgaa 1800 egagtaaage ccttattatc tgtttgtttg aaagaccaat cttgccattc tttgaaagaa 1860 taacggtaat tgggatcaaa aaattctaca ttgtccgttc ttggtatacg agcaatccca 1920 aaatgattgc aegttagate aactggcaaa gactttccaa aatatteteg gatattttgc 1980 gagattattt tggctgcttt gacagattta aattctgatt ttgaagteae atagactggc 2040 gtttctaaaa caaaatatgc ttgataacct ttatcagatt tgataattaa egtaggeata 2 100 aaacctaaat caatagctgt tgttaaaata tegettgetg aaatagtttc tttttccgtg 2160 tgaatatcaa aatcaataaa gaaggtattg atttgtctta aattgttttc agaatgtcct 2220 ttagtgtatg aacggttttc gtctgcatac gtaccataac gataaacgtt tggtgtccaa 2280 tgcgtaaatg tatettgatt ttcgtgaatc gettettegg aagtcagaac aacgccacgt 2340 ccgccaatca tgcttttttt tgagcgatac gcaaaaatag cccctttact tttacctggc 2400 ttggtagtga ttgagcgaat tttactattt ttaaatttgt actttaacaa geegteatga 2460 agcacagttt ctacaacaaa agggatattc attcagctgt tctcctttct tacgaaaatt 2520 aattagttag aagetaegat caaagttgaa tcacaacaaa aaaggcaatc aactaagttt 2580 ttettaattg attgcctggt atettettaa agaettgaaa tcccctcaaa aacccgatat 2640 aatgggttta cagatattta agtatctgat taataaagta attaaatact ttaccaaatt 2700 ttgggtctcg acttctttaa ttgattggtg gtaatcaatt aaggetegea gttaaaattt 2760 ctcaggcttt aactggtcgt ggctcttttt ttgtattctt tattcagttc gttgtttcgt 2820 tatatetagt atategettt ttaaaaaaat aagcaatgat ttcgtgcatt attcacacga 2880 aatcattgct tttttcttct tccatttcta actccaatgt tacttgttct gtttctggtt 2940 ct ggttctgt tggctcattt gggattaaat ccactactag cgttgagtta gttccgtctc 3000 taatageegg ttaagtaata gccggttaag tggtcaaact ttgggaaaat ctcaacccgc 3060 attaagtttt gatgccatga caatcgttgg aaatttgaac aaaactaatg ctaaaaagct 3120 atctgacttt atgagtgtag agccacaaat acgactttgg gatataette aaacaaagtt 3180 taaagctaag gcacttcaag aaaaagttta tategaatat gacaaagtaa aagcagatac 3240 ttgggataga cgtaatatgc gtgttgaatt taatcccaat aaactcacac atgaagaaat 3300 gatttggtta aaacaaaata ttatcgacta catggaagat gacggtttta caagattaga 3360 cttagctttt gattttgaag atgatttgag cgattactat gcaatgactg ataaagcagt 3420 taagaaaact gttttttatg gtcgtaatgg caagccagaa acaaaatatt ttggtgtccg 3480 tgatagtgat agatttatta gaatttataa taaaaaacaa gaacgtaaag ataacgcaga 3540 tgttgaagtt gtgtttgaac atttatggcg tgtagaagtt gaattaaaaa gagatatggt 3600 tgattactgg aatgattgtt ttaatgattt acacatcttt gaaacctgcg tgggctactt 3660 tagaaaaaat taatgagcaa gctatggttt atactttgtt gcatgaagaa agtatgtggg 3720 gaaagctaag taagaatact aagactaaat ttaaaaaatt gattagagaa atatctccaa 3780 ttgattta ac ggaattaatg aaategaett taaaagcgaa cgaaaaacaa ttgcaaaagc 3840 agattgattt ttggcaacgt gaatttaggt tttggaagta aaataagttt tatttgataa 3900 aaattgctaa ttcagtataa ttaatattta cgaggtgaca taacgtatga aaaaatcaga 3960 ggattattcc tcctaaatat aaaaatttaa aatttaggag gaagttatat atgactttta 4020 atattattga attagaaaat tgggatagaa aagaatattt tgaacactat tttaatcagc 4080 98561.doc -38- 200530399 aaactactta tagcattact aaagaaattg atattacttt gtttaaagat atgataaaaa 4140 agaaaggata tgaaatttat ccctctttaa tttatgcaat tatggaagtt gtaaataaaa 4200 ataaagtgtt tagaacagga attaatagtg agaataaatt aggttattgg gataagttaa 4260 atcctttgta tacagttttt aataagcaaa ctgaaaaatt tactaacatt tggactgaat 4320 ctgataaaaa cttcatttct ttttataata attataaaaa tgacttgctt gaatataaag 4380 ataaagaaga aatgtttcct aaaaaaccga tacctgaaaa caccataccg atttcaatga 4440 ttccttggat tgattttagt tcatttaatt taaatattgg taacaatagc agctttttat 4500 tgcctattat tacgataggt aaattttata gtgagaataa taaaatttat ataccagttg 4560 ctctgcaact tcatcattct gtatgtgatg gttaccatgc ttcactattt atgaa tgaat 4620 ttcaagatat aattcatagg gtagatgatt ggatttagtt tttagatttt gaaagtgaat 4680 ttaattttat acacgtaagt gatcataaaa tttatgaacg tataacaacc acattttttg 4740 gttgcttgtg gttttgattt tgaatttggt tttgaactta tggactgatt tattcagtcc 4800 attttttgtg cttgcacaaa aactagcctc gcagagcaca cgcattaatg acttatgaaa 4860 cgtagtaaat aagtctagtg tgttatactt tacttggaag atgcaccgaa taaaaaatat 4920 tgaagaacaa ctagcaaaag attttaaaga gttattttat tttaagtctt tataacatga 4980 gtgaagcgaa tttttaaatt tcgatagaaa tttttacatc aaaaagcccc ctgtcaaaat 5040 tgacgaaggg ggttttttgg cgcacgcttt tcgttagaaa tatacaagat tgaaaatcgt 5100 gtataagtgc gccctttgtt ttgaacttag cacgttacat caatttttta aaatgatgta 5160 taagtgcgcc cttttaaatt ttgagtgatt atattttttg agttagaaaa agggattggg 5220 aaaatttccc aaaataattt aaaaaataag caaaaatttt cgatagagaa tgtgctattt 5280 tttgtcaaag gtgtatacct tgactgtgct tgctgttaca ttaagtttat ttttaagtta 5340 ttaaaaaaga aatagctttt aaagtttggc tcgctgtcgc tttataaagc tgattgactt 5400 ttgattgcaa actacttaaa gaaaacaaac tcggactatt cgttttcttc tctttggttt 5460 gaacatcagc aattatcccc tcttgattgc ctattttagc ttgtttagaa gaaacaaaag 5520 ctaaaagctc ctcttgggtt ttaaaacgct gtgtggggct tagaacgccc ttaaacgacc 5580 cttggtttac ttttatacta gcttccacct cgaaaaaagg ttctttttta aaattctcta 5640 tggcttcctg gcgctgaaaa aataaggtat aaggtgggcg tttgaacacg tcctagtgaa 5700 aatgtacctt gtacgcccct tctgttgtaa atttaacgta tacaaagggc ttgcgttcat 5760 gccgatcaac caatcggcaa tttggcgtgt ttgcgcttct tgataaaagg gatagtaatt 5820 cattccaggt tgcaaatttt gaaaaccgct tcggattaca tctttttcta agctattgat 5880 ccatagtctt ttaaatgttt tatcttttga aaaggcattt gctttatgga taatcgacca 5940 ggcgatattt tcaccttctc tgtcgctatc tgttgcaaca ataattgtat ttgccttttt 6000 gagaagttct gcaacaattt taaactgctt tcccttatct tttgcaactt caaaatcgta 6060 tcgatcagga aaaatcggca aagattcaag tttccaattt tgccactttt cgtcataatg 6120 acctggttct gctaattcca ctaaatgccc aaaaccaaag gtgataaacg tttcatctgt 6180 aaatagtggg tctttgatct caaaataacc gtcttttttg gtgctttgtt ttaaagcact 6240 tgcgtaggct aatgcctggc ttggtttttc agctaaaata accgtactca ttaactatcc 6300 c tcttttcat tgttttttct ttgatcgact gtcacgttat atcttgctcg ataccttcta 6360 aacgttcggc gattgattcc agtttgttct tcaacttctt tatcggataa accattcaaa 6420 aacaaatcga aagcatggat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat 6480 atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca 6540 attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg 6600 cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta 6660 caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca 6720 gcggtccagt gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct 6780 98561.doc -39- 200530399 gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc 6840 cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgca atacgactca 6900 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acggtatcga 6960 atcgaattcc tgcagcccgg gggatccact agttctagag cggccgccac cgcggtggag 7020 ctccagcttt tgttcccttt agtgagggtt aatgctagaa atattttatc tgattaataa 7080 taagcttgat gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc ttgctctga a aacgaaaaaa 7140 ccgccttgca gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta 7200 actggcttgg aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc 7260 atgacttcaa gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt 7320 gcatgtcttt ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac 7380 tgaacggggg gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt 7440 gtcaggcgtg gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa 7500 ggcaggaaca ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag 7560 tcctgtcggg tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg 7620 gcggagccta tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc 7680 ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga 7740 acgaccgagc gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc 7800 tgctgacgca ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct 7860 catcagtgcc aacatagtaa gccagtatac actccgctag cgctgatgtc cggcggtgct 7920 tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag ggacagctga taga aacaga 7980 agccactgga gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac 8040 ccgacgcact ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa 8100 atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg 8160 ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg 8220 tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca 8280 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc 8340 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa 8400 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc 8460 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg 8520 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct 8580 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt 8640 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct 8700 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact 8760 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc 8820 cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta 8880 atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt 8940 attggtgccc ttaaacgcct ggtgctacgc ctgaataagt gataataagc ggatgaatgg 9000 cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg ttaaatagcc 9060 gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt gaccgtgtgc 9120 ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat aattgacgat 9180 atgatcattt attctgcctc ccagagcctg ataaaaacgg ttagcgcttc gttaatacag 9240 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg 9300 tgcagggcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg 9360 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga 9420 cgatagtcat gccccgcgcc caccggaagg agctaccgga cagcggtgcg gactgttgta 9480 98561.doc -40- 200530399 actcagaata agaaatgagg ccgctcatgg cgttgactct cagtcatagt atcgtggtat 9540 caccggttgg ttccactctc tgttgcgggc aacttcagca gcacgtaggg gacttccgcg 9600 tttccagact ttacgaaaca cggaaaccga agaccattca tg ttgttgct caggtcgcag 9660 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattea ttctgctaac 9720 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 9780 cccgtggcca ggacccaacg ctgcccga 9808 &lt; 210> 34 &lt; 211 &gt; 1869 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; codon - optimized sequence &lt; 400 &gt; 34 atggcattgc caactgcacg tccattacta ggtagttgcg gtacaccagc actaggttct 60 ttattatttt tgttattttc tctaggttgg gttcaaccaa gtcgtacatt agcaggtgaa 120 acaggtcaag aagcagcacc aettgaeggt gtattaaega atccaccaaa tatatcaagt 180 ttaagtccac gtcaattatt aggttttcca tgtgcagaag tttcaggttt aagtacagaa 240 cgtgtccgtg agttageagt tgeattagea caaaaaaacg ttaaattatc tacagaacag 300 ttacgttgtt tagcccatag attaagegaa ccaccagaag aettagatge acttccttta 360 gaccttcttt tattettaaa tccagatgca ttttcaggac cacaagcatg tacacgtttt 420 tttagtcgaa ttacaaaagc caatgttgat ttattacctc gtggggctccgatgggtggt agggt agggt ggt ag atttacctg gtcgtttcgt tgeagaatea 600 gcagaagtgt tattaccgag attagtttea tgcccaggac etttagatea agatcaacaa 660 gaggeageta gageagetet tcaaggagga ggcccaccat atggcccacc aagtacatgg 720 agtgttteta caatggatgc gttaagaggt ttattaccgg ttttaggaca accaattatt 780 cgtagtattc cacaaggcat tggcgtcaac gtagtteteg tgatccgtct 840 tggcgacaac cagaacgtac 960 tgggaattag aagcatgtgt tgtageagea aattctacgt ccaagatttc gtagagaagt agaaaaaacg 900 gcgtgtccta gtggcaaaaa agcacgtgaa attgatgaaa gtttaatttt ttataaaaaa egatgeagea ttactagcta cacaaatgga tcgtgttaat 1020 gctattccat tcacatatga acaattagat gttttaaagc ataaattaga egaattatat 1080 ccacaaggtt atccagaatc agttattcaa catttaggtt aettattttt aaaaatgagt 1140 ccagaagaca tacgcaaatg gaatgttaca agtttagaaa cattaaaagc gcttttagaa 1200 gttaacaaag gtcatgaaat gagtccacaa gttgctacgt taattgatag attegttaaa 1260 ggccgtggtc aattagataa agataettta gatacattaa cagcatttta tcctggctac 1320 ttatgcagtt tatcaccaga agaattaagt tccgttccac egagtagtat ctgggcagtt 1380 cgtccgcaag atttagatac atgcgaccca cgtcaattag atg ttttata tccaaaagca 1440 agattagett tccaaaatat gaacggtagt gaatatttcg taaaaattca atccttttta 1500 ggtggtgcac caactgaaga tctaaaagca ttaagccaac aaaatgtaag tatggattta 1560 getaegttta tgaaattacg tacagatgca gttctaccat taacagttgc agaagttcaa 1620 98561.doc -41 - 200530399 aaattattag gtccacacgt agaaggatta aaagcagaag aacgtcaccg tccagttcgc 1680 gattggattt tacgtcaacg tcaagatgat ttagatacat taggtttagg tttacaaggc 1740 ggtattccga atggatattt agtgttagat ttatctgttc aagaagcatt aagtggtaca 1800 ccgtgtttat taggtccagg tccagtttta acagtgttag cattattatt agccagtaca I860 ttagcttaa 1869 &lt; 210〉 35 &lt; 211〉 622 &lt; 212 &gt; PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400〉 35

Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro 15 10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gin 20 25 30

Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gin Glu Ala Ala Pro Leu 35 40 45

Asp Gly Val Leu Thr Asn Pro Pro Asn He Ser Ser Leu Ser Pro Arg 50 55 60

Gin Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu 65 70 75 80

Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gin Lys Asn Val Lys Leu 85 90 95

Ser Thr Glu Gin Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro 100 105 110

Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro 115 120 125

Asp Ala Phe Ser Gly Pro Gin Ala Cys Thr Arg Phe Phe Ser Arg He 130 135 140

Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gin 145 150 155 160

Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu 165 170 175

Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu 180 185 190

Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu 195 200 205

Val Ser Cys Pro Gly Pro Leu Asp Gin Asp Gin Gin Glu Ala Ala Arg 210 215 220

Ala Ala Leu Gin Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp 225 230 235 240

Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly 245 250 255

Gin Pro He He Arg Ser He Pro Gin Gly He Val Ala Ala Trp Arg 98561.doc -42- 200530399 260 265 270

Gin Arg Ser Ser Arg Asp Pro Ser Trp Arg Gin Pro Glu Arg Thr He 275 280 285

Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser 290 295 300

Gly Lys Lys Ala Arg Glu He Asp Glu Ser Leu He Phe Tyr Lys Lys 305 310 315 320

Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gin Met 325 330 335

Asp Arg Val Asn Ala lie Pro Phe Thr Tyr Glu Gin Leu Asp Val Leu 340 345 350

Lys His Lys Leu Asp Glu Leu Tyr Pro Gin Gly Tyr Pro Glu Ser Val 355 360 365

He Gin His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp He 370 375 380

Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu 385 390 395 400

Val Asn Lys Gly His Glu Met Ser Pro Gin Val Ala Thr Leu lie Asp 405 410 415

Arg Phe Val Lys Gly Arg Gly Gin Leu Asp Lys Asp Thr Leu Asp Thr 420 425 430

Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu 435 440 445

Leu Ser Ser Val Pro Pro Ser Ser He Trp Ala Val Arg Pro Gin Asp 450 455 460

Leu Asp Thr Cys Asp Pro Arg Gin Leu Asp Val Leu Tyr Pro Lys Ala 465 470 475 480

Arg Leu Ala Phe Gin Asn Met Asn Gly Ser Glu Tyr Phe Val Lys lie 485 490 495

Gin Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser 500 505 510

Gin Gin Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr 515 520 525

Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gin Lys Leu Leu Gly 530 535 540

Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg 545 550 555 560

Asp Trp He Leu Arg Gin Arg Gin Asp Asp Leu Asp Thr Leu Gly Leu 565 570 575

Gly Leu Gin Gly Gly He Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser 580 585 590

Val Gin Glu Ala Leu Ser Gly Thr Pro Cys Leu Leu Gly Pro Gly Pro 595 600 605

Val Leu Thr Val Leu Ala Leu Leu Leu Ala Ser Thr Leu Ala 610 615 620 98561.doc -43- 200530399 &lt; 210〉 36 &lt; 211 &gt; 1878 &lt; 212〉 DNA &lt; 213 &gt; Artificial Sequences &lt; 220 &gt; &lt; 223 &gt; Codon-Optimized Sequences &lt; 400> 36 atggcattac caacggctcg cccattatta ggttcttgtg gttcaccaat ttgtagtcgc 60 agttttttat tattattact atctttaggt tggattccgc gtttacaaac acaaaccact 120 aaaacaagtctgggggaggagat

ttaccaacag gcttatttct tggtcttaca tgtgaagaag ttagtgattt aagtatggaa 240 caagcaaaag gtttagcgat ggcggttcgc caaaaaaata ttacattacg tggtcatcaa 300 ttacgttgtt tagcacgtcg tttaccacga catttaacag atgaagaatt aaatgctcta 360 ccattagact tattattatt tttaaatcca gcaatgtttc caggtcaaca agcatgtgcc 420 cattttttca gtttaatttc gaaagcaaat gtagatgttt taccgagacg tagcttagaa 480 cgtcaacgtc ttttaatgga agcattaaaa tgtcaaggtg tttatggttt ccaagttagt 540 gaagcagatg ttcgtgcact tggtggttta gcttgtgatt taccagggaa atttgtagca 600 cgttctagtg aagtattatt accatggtta gcaggttgtc aaggtccatt agatcaaagt 660 caagaaaaag cagttcgtga agtcttacgt agtggtcgta ctcaatatgg cccacctagc 720 aaatggagtg ttagtacgtt agatgcatta caaagtttag tagctgtttt agatgaaagt 780 attgttcaga gtattccaaa agatgtgaaa gcagagtggt tacaacatat ttcccgtgac 840 ccatctcgtt taggtagtaa attaacagtt attcatccac gttttcgccg cgacgcagaa 900 caaaaagcat gtccaccagg taaagaacca tataaagtag atgaagattt aattttttat 960 cagaattggg aattagaagc ctgtgttgat ggtacaatgt tagcacgtca aatggattta 1020 gttaatgaaa ttccattt ac atatgaacaa ttaagtatct ttaaacataa attagataaa 1080

acatatccac aaggttatcc agaatcgtta attcaacaat taggtcattt ttttcgttat 1140 gttagtccag aagacattea tcaatggaat gttacaagtc cagatacagt taaaacttta 1200 ttaaaagtta gtaaaggtca aaaaatgaat gctcaagcaa ttgcattagt egeatgttat 1260 ttacgtggag gtggtcaatt agatgaagat atggttaaag cattagggga tattccatta 1320 tcatatttat gtgatttctc cccacaagac ttacattcag ttccaagtag tgttatgtgg 1380 ttagttggtc cacaaggttt agataaatgt agtcaacgtc atttaggttt actttatcaa 1440 aaagcatgta gtgcgtttca aaatgttagt ggtttagaat attttgaaaa aatcaaaaca 1500 tttttaggag gtgcatctgt aaaagattta cgcgcattaa gtcaacataa tgtaagtatg 1560 gatatcgcaa catttaaacg tttacaagtc gatagtetag ttggtcttag tgtagcagaa 1620 gttcaaaaat tattagggee gaatattgta gatttaaaaa cagaagaaga taaaagtcca 1680 gttcgtgact ggttatttcg acaacatcag aaagaettag ategtettgg attaggttta 1740 caaggtggta ttccaaatgg ttatttagtt ttagatttta atgtacgtga agcatttagt 1800 tcaagagcga gtttattagg tccaggtttt gtgttaattt ggattccagc attactacca 1860 gcacttcgtt tatcataa 1878 &lt; 210> 37 98561 .doc -44- 200530399 &lt; 211 &gt; 6 25 &lt; 212 &gt; PRT &lt; 213〉 Mouse &lt; 400〉 37

Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Ser Pro 15 10 15

He Cys Ser Arg Ser Phe Leu Leu Leu Leu Leu Ser Leu Gly Trp lie 20 25 30

Pro Arg Leu Gin Thr Gin Thr Thr Lys Thr Ser Gin Glu Ala Thr Leu 35 40 45

Leu His Ala Val Asn Gly Ala Ala Asp Phe Ala Ser Leu Pro Thr Gly 50 55 60

Leu Phe Leu Gly Leu Thr Cys Glu Glu Val Ser Asp Leu Ser Met Glu

65 70 75 80

Gin Ala Lys Gly Leu Ala Met Ala Val Arg Gin Lys Asn lie Thr Leu 85 90 95

Arg Gly His Gin Leu Arg Cys Leu Ala Arg Arg Leu Pro Arg His Leu 100 105 110

Thr Asp Glu Glu Leu Asn Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu 115 120 125

Asn Pro Ala Met Phe Pro Gly Gin Gin Ala Cys Ala His Phe Phe Ser 130 135 140

Leu He Ser Lys Ala Asn Val Asp Val Leu Pro Arg Arg Ser Leu Glu 145 150 155 160

Arg Gin Arg Leu Leu Met Glu Ala Leu Lys Cys Gin Gly Val Tyr Gly 165 170 175

Phe Gin Val Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys 180 185 190

Asp Leu Pro Gly Lys Phe Val Ala Arg Ser Ser Glu Val Leu Leu Pro 195 200 205

Trp Leu Ala Gly Cys Gin Gly Pro Leu Asp Gin Ser Gin Glu Lys Ala 210 215 220

Val Arg Glu Val Leu Arg Ser Gly Arg Thr Gin Tyr Gly Pro Pro Ser 225 230 235 240

Lys Trp Ser Val Ser Thr Leu Asp Ala Leu Gin Ser Leu Val Ala Val 245 250 255

Leu Asp Glu Ser lie Val Gin Ser He Pro Lys Asp Val Lys Ala Glu 260 265 270

Trp Leu Gin His lie Ser Arg Asp Pro Ser Arg Leu Gly Ser Lys Leu 275 280 285

Thr Val He His Pro Arg Phe Arg Arg Asp Ala Glu Gin Lys Ala Cys 290 295 300

Pro Pro Gly Lys Glu Pro Tyr Lys Val Asp Glu Asp Leu He Phe Tyr 305 310 315 320 98561.doc -45- 200530399

Gin Asn Trp Glu Leu Glu Ala Cys Val Asp Gly Thr Met Leu Ala Arg 325 330 335

Gin Met Asp Leu Val Asn Glu lie Pro Phe Thr Tyr Glu Gin Leu Ser 340 345 350

He Phe Lys His Lys Leu Asp Lys Thr Tyr Pro Gin Gly Tyr Pro Glu 355 360 365

Ser Leu He Gin Gin Leu Gly His Phe Phe Arg Tyr Val Ser Pro Glu 370 375 380

Asp He His Gin Trp Asn Val Thr Ser Pro Asp Thr Val Lys Thr Leu 385 390 395 400

Leu Lys Val Ser Lys Gly Gin Lys Met Asn Ala Gin Ala lie Ala Leu 405 410 415

Val Ala Cys Tyr Leu Arg Gly Gly Gly Gin Leu Asp Glu Asp Met Val 420 425 430

Lys Ala Leu Gly Asp He Pro Leu Ser Tyr Leu Cys Asp Phe Ser Pro 435 440 445

Gin Asp Leu His Ser Val Pro Ser Ser Val Met Trp Leu Val Gly Pro 450 455 460

Gin Gly Leu Asp Lys Cys Ser Gin Arg His Leu Gly Leu Leu Tyr Gin 465 470 475 480

Lys Ala Cys Ser Ala Phe Gin Asn Val Ser Gly Leu Glu Tyr Phe Glu 485 490 495

Lys He Lys Thr Phe Leu Gly Gly Ala Ser Val Lys Asp Leu Arg Ala 500 505 510

Leu Ser Gin His Asn Val Ser Met Asp He Ala Thr Phe Lys Arg Leu 515 520 525

Gin Val Asp Ser Leu Val Gly Leu Ser Val Ala Glu Val Gin Lys Leu 530 535 540

Leu Gly Pro Asn He Val Asp Leu Lys Thr Glu Glu Asp Lys Ser Pro

545 550 555 560

Val Arg Asp Trp Leu Phe Arg Gin His Gin Lys Asp Leu Asp Arg Leu 565 570 575

Gly Leu Gly Leu Gin Gly Gly lie Pro Asn Gly Tyr Leu Val Leu Asp 580 585 590

Phe Asn Val Arg Glu Ala Phe Ser Ser Arg Ala Ser Leu Leu Gly Pro 595 600 605

Gly Phe Val Leu He Trp He Pro Ala Leu Leu Pro Ala Leu Arg Leu 610 615 620

Ser 625 &lt; 210> 38 &lt; 211> 335 &lt; 212 &gt; DNA 98561.doc -46- 200530399 &lt; 213> Artificial sequence &lt; 220> &lt; 223> Partial performance card including codon-optimized sequence cassette &lt; 400> 38 gtacctcctt tgattagtat attcctatct taaagttact tttatgtgga ggcattaaca 60 tttgttaatg acgtcaaaag gatagcaaga ctagaataaa gctataaagc aagcatataa 120 tattgcgttt catctttaga agcgaatttc gccaatatta taattatcaa aagagagggg 180 tggcaaacgg tatttggcat tattaggtta aaaaatgtag aaggagagtg aaacccatga 240 aaaaacgtaa agttttaatt ccattaatgg cattaagtac aattttagtt agtagtacag 300 gtaatttaga agttattcaa gcagaagttg gatcc 335

&lt; 210> 39 &lt; 211 &gt; 2778 &lt; 212> DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; Partial performance cassette including codon-optimized sequence &lt; 400> 39 ggatcccaag gaaaagaagt cgtactttta gatttcgcag cagcaggagg agaattagga 60 tggttaactc atccatatgg caaaggctgg gatttaatgc aaaacattat gaacgatatg 120 ccaatttata tgtactccgt atgtaatgta atgagcggtg atcaagataa ctggttacgt 180 actaattggg tttatcgagg tgaagcagaa agaattttta ttgaacttaa atttactgtt 240 cgtgactgta atagttttcc aggaggggcc tcatcatgta aagaaacatt caatctatat 300 tatgccgaaa gcgatcttga ttatggtaca aatttccaaa aacgtttatt tactaaaatt 360 gatacaatag ctccagatga aatcactgta agttccgatt ttgaagctcg tcatgtaaaa 420

ttaaatgtag aagaacgcag tgttggtcca ctaactagaa aaggatttta tcttgctttc 480 caagatatag gggcttgcgt agcattgtta tccgttcgtg tatactataa aaaatgtcca 540 gaactacttc aaggcttagc acattttcca gaaacaattg cgggctcaga tgcgccatca 600 cttgcaactg tggcgggtac atgtgttgat catgctgttg tgccaccagg aggagaggaa 660 cctcgcatgc actgtgcagt agatggtgaa tggttagttc ctattggtca atgtttatgt 720 caagccggtt atgaaaaagt tgaagatgct tgtcaagcat gctccccagg tttttttaaa 780 ttcgaagcta gtgaatctcc atgcttagaa tgtccagaac acacattacc aagtccagaa 840 ggtgcaacgt cctgtgaatg cgaagaaggt ttttttcgtg ccccacaaga tccagcctca 900 atgccttgta cacgaccgcc ttctgctcca cactatttaa cagccgtagg aatgggcgct 960 aaagtagagt tacgatggac accgcctcaa gatagtggag gccgtgaaga tattgtttat 1020 tccgttactt gtgaacaatg ctggccagaa agtggtgaat gcgggccttg cgaagcatca 1080 gttagatatt cggaaccacc acacgggtta actagaacta gtgtcacagt atcagactta 1140 gaaccacaca tgaattatac atttacagtt gaggcacgta atggagtatc tggtttagtt 1200 acatcacgct cttttcgcac agcatcggtc tctattaacc aaactgaacc gccaaaagta 1260 agattagaag ggcg ttcgac aacatcactt tccgtaagtt ggtcaattcc accaccacaa 1320 caatcacgcg tttggaaata tgaagttaca tacagaaaaa aaggagattc gaatagttat 1380 98561.doc -47- 200530399 aatgttagac gtacagaagg attcagcgta accctagatg atttagctcc agatacaaca 1440 tatttagtac aggtgcaagc attaacacaa gaaggacaag gggcgggctc acgagtteat 1500 gaatttcaaa cattacatag aagaagaaag aatcaaagag cacgtcaatc accagaagat 1560 gtttattttt caaagtctga acagttgaaa ccattgaaaa cctatgttga tccacacaca 1620 tacgaagacc caaaccaagc ggtccttaaa tttacaaccg aaattcatcc atcatgcgta 1680 actcgtcaaa aagtgatcgg agctggagaa ttcggggagg tatacaaagg catgttgaaa 1740 acctcaagtg gtaaaaaaga agttcctgta gcaattatga ctcttaaagc agggtataca 1800 gaaaaacaac gagttgattt tttaggcgaa gctggtatca tgggacaatt ttcgcatcat 1860 aatataatta gaettgaagg tgttatctct aaatataaac caatgatgat tattactgaa 1920 tatatggaaa acggtgcttt agataaattt etaegegaaa aagatggtga attttctgtc 1980 cttcaattag ttggtatgtt acgtggcatc gctgcaggta tgaaatatct tgccaacatg 2040 aattatgtac atagagattt ageggetega aatattcttg taaattccaa tttagtgtgc 2 100 aaagttagtg atttcggttt aagtcgagta ttagaagatg atccagaagc aacctatact 2160 acttcggggg gtaaaattcc gatccgttgg acagcaccgg aagcaatttc atategtaaa 2220 tttacatctg caagcgatgt ttggagtttc ggaattgtga tgtgggaagt aatgacatac 2280 ggcgaacgtc catattggga attgtcaaac catgaagtaa tgaaagcgat taacgatggt 2340 ttcagattac caaccccaat ggactgtcca tcagcaattt atcaactaat gatgcaatgc 2400 tggcaacaag aaagagetag aagacctaaa tttgcagaca ttgtttcaat tttagacaaa 2460 etaattegtg cgccagatag tcttaaaacc etagetgatt tcgatccacg cgtatcaatt 2520 cgtcttccat caacatcggg atctgaaggt gttcctttta gaacagtaag cgagtggtta 2580 gaategatta aaatgcaaca gtatacagaa cattttatgg cagccggata cacagcaatt 2640 gaaaaagttg tgcaaatgac aaatgatgat attaaacgta ttggagtgcg tctacctggc 2700 caccaaaaac gtattgetta ctccctttta ggtttaaaag accaagtaaa tacagtcgga 2760 attccaatat gagagctc 2778 &lt; 210 &gt; 40 &lt; 211 &gt; 1382 &lt; 212 &gt; DNA &lt; 213> Artificial sequence &lt; 220> &lt; 223 &gt; Codon-optimized sequence subfragment containing intergenic region &lt; 400> 40 acgcgtttgg aaatatgaag ttacatacag aaaaaaagga gattegaata gttataatgt 60 tagacgtaca gaaggattca gcgtaaccct agatgattta gctccagata caacatattt 120 agtacaggtg caagcattaa cacaagaagg acaaggggcg ggctcacgag ttcatgaatt 180 tcaaacatta taaaaacaca gaacgaaaga aaaagtgagg tgaatgatat ggcatatgat 240 agtcgttttg atgaatgggt tcaaaaatta aaagaagaaa gttttcaaaa taatacattt 300 gategtegta aatttattca aggtgcaggt aaaattgcag gtttaagttt aggtttaaca 360 attgcacaaa gtgttggtgc atttcataga agaagaaaga atcaaagagc acgtcaatca 420 ccagaagatg tttatttttc aaagtctgaa cagttgaaac cattgaaaac ctatgttgat 480 ccacacacat acgaagaccc aaaccaagcg gtccttaaat ttacaaccga aattcatcca 540 teatgegtaa ctcgtcaaaa agtgatcgga gctggagaat tcggggaggt atacaaaggc 600 atgttgaaaa cctcaagtgg taaaaaagaa gttcctgtag caattatgac tettaaagea 660 98561.doc -48- 200530399 gggtatacag aaaaacaacg agttgatttt ttaggcgaag ctggtatcat gggacaattt 720 tcgcatcata atataattag acttgaaggt gttatctcta aatataaacc aatgatgatt 780 attactgaat atatggaaaa cggtgcttta gataaatttc tacgcgaaaa agatggtgaa 840 ttttctgtcc ttcaat tagt tggtatgtta cgtggcatcg ctgcaggtat gaaatatctt 900 gccaacatga attatgtaca tagagattta gcggctcgaa atattcttgt aaattccaat 960 ttagtgtgca aagttagtga tttcggttta agtcgagtat tagaagatga tccagaagca 1020 acctatacta cttcgggggg taaaattccg atccgttgga cagcaccgga agcaatttca 1080 tatcgtaaat ttacatctgc aagcgatgtt tggagtttcg gaattgtgat gtgggaagta 1140 atgacatacg gcgaacgtcc atattgggaa ttgtcaaacc atgaagtaat gaaagcgatt 1200 aacgatggtt tcagattacc aaccccaatg gactgtccat cagcaattta tcaactaatg 1260 atgcaatgct ggcaacaaga aagagctaga agacctaaat ttgcagacat tgtttcaatt 1320 ttagacaaac taattcgtgc gccagatagt cttaaaaccc tagctgattt cgatccacgc 1380 gt 1382 &lt; 210〉 41 &lt; 211〉 447 &lt; 212 &gt; DNA &lt; 213 &gt; &gt; &k; &gt; artificial sequence &lt;220; 400〉 41 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt agggata agtatagatata agcgaattt cgccaatatt agtgata gaaggagagt gaaacccatg 240 aatatgaaaa aagctacgat tgcagctaca gccggcattg ccgtaacagc ttttgcagca 300 ccaactattg cctcagcctc tacagttgtt gtcgaagcag gagacacatt atggggaatc 360 gcacaatcaa aaggtacaac ggttgatgct attaaaaaag cgaataattt aacaacagat 420 aaaatcgtgc caggtcaaaa actgcag 447 &lt; 210> 42 &lt; 211 &gt; 1683 &lt; 212 &gt; DNA &lt; 213> Artificial Sequence &lt; 220> &lt; 223> plastid views fragment &lt; 400 &gt; 42 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 98561.doc -49- 200530399 aatatgaaaa aagctacgat tgcagctaca gccggcattg ccgtaacagc ttttgcagca 300 ccaactattg cctcagcctc tacagttgtt gtcgaagcag gagacacatt atggggaatc 360 gcacaatcaa aaggtacaac ggttgatgct attaaaaaag cgaataattt aacaacagat 420 aaaatcgtgc caggtcaaaa actgcaggta aataatgagg ttgctgctgc tgaaaaaaca 480 gagaaatctg ttagcgcaac tt ggttaaac gtccgtactg gcgctggtgt tgataacagt 540 attattacgt ccatcaaagg tggaacaaaa gtaactgttg aaacaaccga atctaacggc 600 tggcacaaaa ttacttacaa cgatggaaaa actggtttcg ttaacggtaa atacttaact 660 gacaaagcag taagcactcc agttgcacca acacaagaag tgaaaaaaga aactactact 720 caacaagctg cacctgttgc agaaacaaaa actgaagtaa aacaaactac acaagcaact 780 acacctgcgc ctaaagtagc agaaacgaaa gaaactccag taatagatca aaatgctact 840 acacacgctg tcaaaagcgg tgacactatt tgggctttat ccgtaaaata cggtgtttct 900 gttcaagaca ttatgtcatg gaataattta tcttcttctt ctatttatgt aggtcaaaag 960 cttgctatta aacaaactgc taacacagct actccaaaag cagaagtgaa aacggaagct 1020 ccagcagctg aaaaacaagc agctccagta gttaaagaaa atactaacac aaatactgct 1080 actacagaga aaaaagaaac agcaacgcaa caacaaacag cacctaaagc accaacagaa 1140 gctgcaaaac cagctcctgc accatctaca aacacaaatg ctaataaaac gaatacaaat 1200 acaaatacaa acaatactaa tacaccatct aaaaatacta atacaaactc aaatactaat 1260 acgaatacaa actcaaatac gaatgctaat caaggttctt ccaacaataa cagcaattca 1320 agtgcaagtg ctattattgc tgaagctcaa aaaca ccttg gaaaagctta ttcatggggt 1380 ggtaacggac caactacatt tgattgctct ggttacacta aatatgtatt tgctaaagcg 1440 ggtatctccc ttccacgtac atctggcgca caatatgcta gcactacaag aatttctgaa 1500 tctcaagcaa aacctggtga tttagtattc ttcgactatg gtagcggaat ttctcacatt 1560 ggtatttatg ttggtaatgg tcaaatgatt aacgcgcaag acaatggcgt taaatacgat 1620 aacatccacg gctctggctg gggtaaatat ctagttggct tcggtcgcgt ataataagga 1680 tcc 1683 &lt; 210> 43 &lt; 211> 3552 &lt; 212 &gt; DNA &lt; 213> artificial sequence &lt; 220> &lt; 223> plastid subfragment &lt; 400> 43 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaga 60 atttgttaat 180tgatatctatt agctata agctta agctata gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aatatgaaaa aagctacgat tgcagctaca gccggcattg ccgtaacagc ttttgcagcatgagcatca acca t 420 aaaatcgtgc caggtcaaaa actgcaggca ttgccaactg cacgtccatt actaggtagt 480 tgcggtacac cagcactagg ttctttatta tttttgttat tttctctagg ttgggttcaa 540 ccaagtcgta cattagcagg tgaaacaggt caagaagcag caccacttga cggtgtatta 600 98561.doc -50- 200530399 acgaatccac caaatatatc aagtttaagt ccacgtcaat tattaggttt tccatgtgca 660 gaagtttcag gtttaagtac agaacgtgtc cgtgagttag cagttgcatt agcacaaaaa 720 aacgttaaat tatctacaga acagttacgt tgtttagccc atagattaag cgaaccacca 780 gaagacttag atgcacttcc tttagacctt cttttattct taaatccaga tgcattttca 840 ggaccacaag catgtacacg tttttttagt cgaattacaa aagccaatgt tgatttatta 900 cctcgtgggg ctcctgaaag acaacgttta ttacctgctg cattagcatg ctggggtgtt 960 cgcggtagct tattaagtga agccgatgtt cgtgctttag ggggtttagc atgtgattta 1020 cctggtcgtt tcgttgcaga atcagcagaa gtgttattac cgagattagt ttcatgccca 1080 ggacctttag atcaagatca acaagaggca gctagagcag ctcttcaagg aggaggccca 1140 ccatatggcc caccaagtac atggagtgtt tctacaatgg atgcgttaag aggtttatta 1200 ccggttttag gacaaccaat tattcgtagt attccacaag gcattgtagc agcatggcgt 1260 caacgtagtt ctcgtgatcc gtcttggcga caaccagaac gtacaattct acgtccaaga 1320 tttcgtagag aagtagaaaa aacggcgtgt cctagtggca aaaaagcacg tgaaattgat 1380 gaaagtttaa ttttttataa aaaatgggaa ttagaagcat gtgtcgatgc agcattacta 1440 gctacacaaa tggatcgtgt taatgctatt ccattcacat atgaacaatt agatgtttta 1500 aagcataaat tagacgaatt atatccacaa ggttatccag aatcagttat tcaacattta 1560 ggttacttat ttttaaaaat gagtccagaa gacatacgca aatggaatgt tacaagttta 1620 gaaacattaa aagcgctttt agaagttaac aaaggtcatg aaatgagtcc acaagttgct 1680 acgttaattg atagattcgt taaaggccgt ggtcaattag ataaagatac tttagataca 1740 ttaacagcat tttatcctgg ctacttatgc agtttatcac cagaagaatt aagttccgtt 1800 ccaccgagta gtatctgggc agttcgtccg caagatttag atacatgcga cccacgtcaa 1860 ttagatgttt tatatccaaa agcaagatta gctttccaaa atatgaacgg tagtgaatat 1920 ttcgtaaaaa ttcaatcctt tttaggtggt gcaccaactg aagatctaaa agcattaagc 1980 caacaaaatg taagtatgga tttagctacg tttatgaaat tacgtacaga tgcagttcta 2040 ccattaacag ttgcagaagt tcaaaaatta ttaggtccac acgtagaagg attaaa agca 2100 gaagaacgtc accgtccagt tcgcgattgg attttacgtc aacgtcaaga tgatttagat 2160 acattaggtt taggtttaca aggcggtatt ccgaatggat atttagtgtt agatttatct 2220 gttcaagaag cattaagtgg tacaccgtgt ttattaggtc caggtccagt tttaacagtg 2280 ttagcattat tattagccag tacattagct ctgcaggtaa ataatgaggt tgctgctgct 2340 gaaaaaacag agaaatctgt tagcgcaact tggttaaacg tccgtactgg cgctggtgtt 2400 gataacagta ttattacgtc catcaaaggt ggaacaaaag taactgttga aacaaccgaa 2460 tctaacggct ggcacaaaat tacttacaac gatggaaaaa ctggtttcgt taacggtaaa 2520 tacttaactg acaaagcagt aagcactcca gttgcaccaa cacaagaagt gaaaaaagaa 2580 actactactc aacaagctgc acctgttgca gaaacaaaaa ctgaagtaaa acaaactaca 2640 caagcaacta cacctgcgcc taaagtagca gaaacgaaag aaactccagt aatagatcaa 2700 aatgctacta cacacgctgt caaaagcggt gacactattt gggctttatc cgtaaaatac 2760 ggtgtttctg ttcaagacat tatgtcatgg aataatttat cttcttcttc tatttatgta 2820 ggtcaaaagc ttgctattaa acaaactgct aacacagcta ctccaaaagc agaagtgaaa 2880 acggaagctc cagcagctga aaaacaagca gctccagtag ttaaagaaaa tactaacaca 2 940 aatactgcta ctacagagaa aaaagaaaca gcaacgcaac aacaaacagc acctaaagca 3000 ccaacagaag ctgcaaaacc agctcctgca ccatctacaa acacaaatgc taataaaacg 3060 aatacaaata caaatacaaa caatactaat acaccatcta aaaatactaa tacaaactca 3120 aatactaata cgaatacaaa ctcaaatacg aatgctaatc aaggttcttc caacaataac 3180 agcaattcaa gtgcaagtgc tattattgct gaagctcaaa aacaccttgg aaaagcttat 3240 tcatggggtg gtaacggacc aactacattt gattgctctg gttacactaa atatgtattt 3300 98561.doc -51 - 200530399 gctaaagcgg gtatctccct tccacgtaca tctggcgcac aatatgctag cactacaaga 3360 atttctgaat ctcaagcaaa acctggtgat ttagtattct tcgactatgg tagcggaatt 3420 tctcacattg gtatttatgt tggtaatggt caaatgatta acgcgcaaga caatggcgtt 3480 aaatacgata acatccacgg ctctggctgg ggtaaatatc tagttggctt cggtcgcgta 3540 taataaggat cc 3552 &lt; 210 &gt; 44 &lt; 211 &gt; 3327 &lt; 212> DNA &lt; 213 &gt; Artificial sequence &lt; 220>

&Lt; 223> plastid views fragment &lt; 400> 44 ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac 60 atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata 120 atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg 180 gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg 240 aatatgaaaa aagctacgat tgcagctaca gccggcattg ccgtaacagc ttttgcagca 300 ccaactattg cctcagcctc tacagttgtt gtcgaagcag gagacacatt atggggaatc 360 gcacaatcaa aaggtacaac ggttgatgct attaaaaaag cgaataattt aacaacagat 420 aaaatcgtgc caggtcaaaa actgcagcgt acattagcag gtgaaacagg tcaagaagca 480 gcaccacttg acggtgtatt aacgaatcca ccaaatatat caagtttaag tccacgtcaa 540 ttattaggtt ttccatgtgc agaagtttca ggtttaagta cagaacgtgt ccgtgagtta 600 gcagttgcat tagcacaaaa aaacgttaaa ttatctacag aacagttacg ttgtttagcc 660 catagattaa gcgaaccacc agaagactta gatgcacttc ctttagacct tcttttattc 720 ttaaatccag atgcattttc aggaccacaa gcatgtacac gtttttttag tcgaattaca 780 aaagccaatg ttgatttatt acctcgtggg gctcctgaaa gacaacg ttt attacctgct 840

gcattagcat gctggggtgt tcgcggtagc ttattaagtg aagccgatgt tcgtgcttta 900 gggggtttag catgtgattt acctggtcgt ttcgttgcag aatcagcaga agtgttatta 960 ccgagattag tttcatgccc aggaccttta gatcaagatc aacaagaggc agctagagca 1020 gctcttcaag gaggaggccc accatatggc ccaccaagta catggagtgt ttctacaatg 1080 gatgcgttaa gaggtttatt accggtttta ggacaaccaa ttattcgtag tattccacaa 1140 ggcattgtag cagcatggcg tcaacgtagt tctcgtgatc cgtcttggcg acaaccagaa 1200 cgtacaattc tacgtccaag atttcgtaga gaagtagaaa aaacggcgtg tcctagtggc 1260 aaaaaagcac gtgaaattga tgaaagttta attttttata aaaaatggga attagaagca 1320 tgtgtcgatg cagcattact agctacacaa atggatcgtg ttaatgctat tccattcaca 1380 tatgaacaat tagatgtttt aaagcataaa ttagacgaat tatatccaca aggttatcca 1440 gaatcagtta ttcaacattt aggttactta tttttaaaaa tgagtccaga agacatacgc 1500 aaatggaatg ttacaagttt agaaacatta aaagcgcttt tagaagttaa caaaggtcat 1560 gaaatgagtc cacaagttgc tacgttaatt gatagattcg ttaaaggccg tggtcaatta 1620 gataaagata ctttagatac attaacagca ttttatcctg gctacttatg cagtttatca 1680 ccagaaga at taagttccgt ttacgtacag atgcagttct accattaaca gttgcagaag ttcaaaaatt attaggtcca 1980 cacgtagaag gattaaaagc agaagaacgt caccgtccag ttcgcgattg gattttacgt 2040 caacgtcaag agtatctggg cagttcgtcc gcaagattta 1740 gatacatgcg acccacgtca attagatgtt ttatatccaa aagcaagatt agctttccaa 1800 98561.doc -52- 200530399 aatatgaacg gtagtgaata tttcgtaaaa attcaatcct ttttaggtgg tgcaccaact 1860 gaagatctaa aagcattaag ccaacaaaat gtaagtatgg atttagctac gtttatgaaa 1920 tccaccgagt atgatttaga tacattaggt ttaggtttac aaggcctgca ggtaaataat 2100 gaggttgctg ctgctgaaaa aacagagaaa tctgttagcg caacttggtt aaacgtccgt 2160 actggcgctg gtgttgataa cagtattatt acgtccatca aaggtggaac aaaagtaact 2220 gttgaaacaa ccgaatctaa cggctggcac aaaattactt acaacgatgg aaaaactggt 2280 ttcgttaacg gtaaatactt aactgacaaa gcagtaagca ctccagttgc accaacacaa 2340 gaagtgaaaa aagaaactac tactcaacaa gctgcacctg ttgcagaaac aaaaactgaa 2400 gtaaaacaaa ctacacaagc aactacacct gcgcctaaag tagcagaaac gaaagaaact 2460 ccagtaatag atcaaaatgc tactacacac gctgtcaaaa gcggtgacac tattt gggct 2520 ttatccgtaa aatacggtgt ttctgttcaa gacattatgt catggaataa tttatcttct 2580 tcttctattt atgtaggtca aaagcttgct attaaacaaa ctgctaacac agctactcca 2640 aaagcagaag tgaaaacgga agctccagca gctgaaaaac aagcagctcc agtagttaaa 2700 gaaaatacta acacaaatac tgctactaca gagaaaaaag aaacagcaac gcaacaacaa 2760 acagcaccta aagcaccaac agaagctgca aaaccagctc ctgcaccatc tacaaacaca 2820 aatgctaata aaacgaatac aaatacaaat acaaacaata ctaatacacc atctaaaaat 2880 actaatacaa actcaaatac taatacgaat acaaactcaa atacgaatgc taatcaaggt 2940 tcttccaaca ataacagcaa ttcaagtgca agtgctatta ttgctgaagc tcaaaaacac 3000 cttggaaaag cttattcatg gggtggtaac ggaccaacta catttgattg ctctggttac · 3060 actaaatatg tatttgctaa agcgggtatc tcccttccac gtacatctgg cgcacaatat 3120 gctagcacta caagaatttc tgaatctcaa gcaaaacctg gtgatttagt attcttcgac 3180 tatggtagcg gaatttctca cattggtatt tatgttggta atggtcaaat gattaacgcg 3240 caagacaatg gcgttaaata cgataacatc cacggctctg gctggggtaa atatctagtt 3300 ggcttcggtc gcgtataata aggatcc 3327

&lt; 210 &gt; 45 &lt; 211 &gt; 26 &lt; 212 &gt; PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400> 45

Met Lys Lys lie Met Leu Val Phe He Thr Leu He Leu Val Ser Leu 15 10 15

Pro He Ala Gin Gin Thr Glu Ala Lys Asp 20 25 &lt; 210 &gt; 46 &lt; 211〉 29 &lt; 212 &gt; PRT &lt; 213 &gt; Lactococcus lactis &lt; 4〇0 &gt; 46 98561.doc-53-200530399

Met Lys Lys Lys lie lie Ser Ala lie Leu Met Ser Thr Val lie Leu 15 10 15

Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Asp Thr 20 25 &lt; 210〉 47 &lt; 211 &gt; 31 &lt; 212 &gt; PRT &lt; 213 &gt; Bacillus anthracis &lt; 400〉 47

Met Lys Lys Arg Lys Val Leu He Pro Leu Met Ala Leu Ser Thr He 15 10 15

Leu Val Ser Ser Thr Gly Asn Leu Glu Val lie Gin Ala Glu Val 20 25 30

&lt; 210> 48 &lt; 211 &gt; 29 &lt; 212 &gt; PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400 &gt; 48

Met Asn Met Lys Lys Ala Thr He Ala Ala Thr Ala Gly He Ala Val 15 10 15

Thr Ala Phe Ala Ala Pro Thr lie Ala Ser Ala Ser Thr 20 25

&lt; 210〉 49 &lt; 211〉 54 &lt; 212〉 PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400〉 49

Met Gin Lys Thr Arg Lys Glu Arg He Leu Glu Ala Leu Gin Glu Glu 15 10 15

Lys Lys Asn Lys Lys Ser Lys Lys Phe Lys Thr Gly Ala Thr He Ala 20 25 30

Gly Val Thr Ala lie Ala Thr Ser lie Thr Val Pro Gly lie Glu Val 35 40 45

He Val Ser Ala Asp Glu 50 98561.doc -54- 200530399 &lt; 210 &gt; 50 &lt; 211 &gt; 28 &lt; 212 &gt; PRT &lt; 213 &gt; Bacillus anthracis &lt; 400 &gt; 50

Met Lys Lys Leu Lys Met Ala Ser Cys Ala Leu Val Ala Gly Leu Met 15 10 15

Phe Ser Gly Leu Thr Pro Asn Ala Phe Ala Glu Asp 20 25

&lt; 210 &gt; 51 &lt; 211 &gt; 31 &lt; 212〉 PRT &lt; 213 &gt; Staphylococcus aureus &lt; 400> 51

Met Ala Lys Lys Phe Asn Tyr Lys Leu Pro Ser Met Val Ala Leu Thr 1 5 10 15

Leu Val Gly Ser Ala Val Thr Ala His Gin Val Gin Ala Ala Glu 20 25 30

&lt; 210> 52 &lt; 211 &gt; 59 &lt; 212 &gt; PRT

&lt; 213 &gt; Listeria monocytogenes &lt; 400> 52

Met Thr Asp Lys Lys Ser Glu Asn Gin Thr Glu Lys Thr Glu Thr Lys 15 10 15

Glu Asn Lys Gly Met Thr Arg Arg Glu Met Leu Lys Leu Ser Ala Val 20 25 30

Ala Gly Thr Gly He Ala Val Gly Ala Thr Gly Leu Gly Thr lie Leu 35 40 45

Asn Val Val Asp Gin Val Asp Lys Ala Leu Thr 50 55 &lt; 210 &gt; 53 &lt; 211 &gt; 53 &lt; 212 &gt; PRT &lt; 213> Bacillus 98561.doc -55- 200530399 &lt; 400 &gt; 53

Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gin Lys Leu Lys Glu 15 10 15

Glu Ser Phe Gin Asn Asn Thr Phe Asp Arg Arg Lys Phe lie Gin Gly 20 25 30

Ala Gly Lys He Ala Gly Leu Ser Leu Gly Leu Thr He Ala Gin Ser 35 40 45

Val Gly Ala Phe Gly 50

&lt; 210〉 54 &lt; 211 &gt; 5 &lt; 212〉 PRT &lt; 213> Listeria monocytogenes &lt; 400> 54

Thr Glu Ala Lys Asp 1 5 &lt; 210〉 55 &lt; 211〉 5 &lt; 212〉 PRT &lt; 213 &gt; Lactococcus lactis

&lt; 400 &gt; 55

Val Tyr Ala Asp Thr 1 5 &lt; 210〉 56 &lt; 211〉 5 &lt; 212 &gt; PRT &lt; 213> Bacillus anthracis &lt; 400 &gt; 56

He Gin Ala Glu Val 1 5 98561.doc -56- 200530399

&lt; 210 &gt; 57 &lt; 211〉 5 &lt; 212 &gt; PRT &lt; 213> Listeria monocytogenes &lt; 400> 57

Ala Ser Ala Ser Thr 1 5

&lt; 210 &gt; 58 &lt; 211 &gt; 5 &lt; 212 &gt; PRT

&lt; 213〉 Listeria monocytogenes &lt; 400> 58

Val Ser Ala Asp Glu 1 5 &lt; 210 &gt; 59 &lt; 211> 5 &lt; 212 &gt; PRT &lt; 213 &gt; Bacillus anthracis &lt; 400 &gt; 59

Ala Phe Ala Glu Asp 1 5

&lt; 210 &gt; 60 &lt; 211〉 5 &lt; 212 &gt; PRT &lt; 213 &gt; Staphylococcus aureus &lt; 400> 60

Val Gin Ala Ala Glu 1 5 &lt; 210〉 61 98561.doc 57- 200530399

&lt; 211〉 5 &lt; 212 &gt; PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400> 61

Asp Lys Ala Leu Thr 1 5 &lt; 210 &gt; 62 &lt; 211 &gt; 5 &lt; 212 &gt; PRT &lt; 213 &gt; Bacillus subtilis

&lt; 400 &gt; 62

Val Gly Ala Phe Gly 1 5 &lt; 210 &gt; 63 &lt; 211 &gt; 9 &lt; 212〉 PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400〉 63

Val Leu Gin Glu Leu Asn Val Thr Val 1 5

&lt; 210 &gt; 64 &lt; 211 &gt; 9 &lt; 212 &gt; PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400〉 64

Ser Leu Leu Phe Leu Leu Phe Ser Leu 1 5 &lt; 210 &gt; 65 &lt; 211 &gt; 9 &lt; 212 &gt; PRT &lt; 213〉 Hyundai Homo sapiens 98561.doc -58- 200530399 &lt; 400 &gt; 65

Val Leu Pro Leu Thr Val Ala Glu Val 1 5 &lt; 210〉 66 &lt; 211〉 9 &lt; 212 &gt; PRT &lt; 213> Hyundai Homo sapiens &lt; 400 &gt; 66

Glu Leu Ala Val Ala Leu Ala Gin Lys 1 5

&lt; 210 &gt; 67 &lt; 211 &gt; 9 &lt; 212 &gt; PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400 &gt; 67

Ala Leu Gin Gly Gly Gly Pro Pro Tyr 1 5

&lt; 210 &gt; 68 &lt; 211> 9 &lt; 212 &gt; PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400 &gt; 68

Phe Tyr Pro Gly Tyr Leu Cys Ser Leu 1 5 &lt; 210〉 69 &lt; 211〉 9 &lt; 212 &gt; PRT &lt; 213〉 Modern Homo sapiens &lt; 400〉 69

Leu Tyr Pro Lys Ala Arg Leu Ala Phe 1 5 98561.doc -59- 19 200530399

&lt; 210> 70 &lt; 211 &gt; 19 &lt; 212 &gt; DNA &lt; 213 &gt; Listeria monocytogenes &lt; 400> 70 aaggagagtg aaacccatg

&lt; 210 &gt; 71 &lt; 211 &gt; 37 &lt; 212 &gt; DNA &lt; 213 &gt; Listeria monocytogenes &lt; 400 &gt; 71 37 taaaaacaca gaacgaaaga aaaagtgagg tgaatga &lt; 210 &gt; 72 &lt; 211> 9 &lt; 212 &gt; PRT &lt; 213 &gt; Mus musculus &lt; 400 &gt; 72

Ser Pro Ser Tyr Val Tyr His Gin Phe 1 5 &lt; 210〉 73 &lt; 211〉 9 &lt; 212 &gt; PRT &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Altered epitope &lt; 400> 73

Ser Pro Ser Tyr Ala Tyr His Gin Phe 1 5 &lt; 210〉 74 &lt; 211〉 29 &lt; 212 &gt; DNA &lt; 213> artificial sequence 98561.doc 60- 200530399 &lt; 220> &lt; 223 &gt; PCR primers &lt; 400〉 74 ctctggtacc tcctttgatt agtatattc &lt; 210〉 75 &lt; 211〉 36 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223> PCR Primer &lt; 400 &gt; 75 caatggatcc ctcgagatca taatttactt catccc &lt; 76 &lt; 211 &gt; 32 &lt; 212 &gt; DNA &lt; 213〉 Artificial sequence &lt; 220〉 &lt; 223〉 PCR primer &lt; 400> 76 atttctcgag tccatggggg gttctcatca tc &lt; 210 &gt; 77 &lt; 211 &gt; 25 &lt; 212 &gt; DNA &lt; 213> artificial sequence &lt; 220> &lt; 223> PCR primer &lt; 400 &gt; 77 ggtgctcgag tgcggccgca agctt &lt; 210 &gt; 78 &lt; 211> 37 &lt; 212> DNA &lt; 213> artificial sequence &lt; 220 &gt; -61-98561.doc 37200530399 &lt; 223 &gt; Synthetic Oligonucleotide &lt; 400 &gt; 78 cgattcccct agttatgttt accaccaatt tgctgca &lt; 210 &gt; 79 &lt; 211 &gt; 31 &lt; 212 &gt; DNA &lt; 213> Artificial sequence &lt; 220 〉 &Lt; 223 &gt; Synthetic ribotrophic acid &lt; 400 &gt; 79 gcaaattggt ggt aaacata actaggggaa t &lt; 210 &gt; 80 &lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt; 213> artificial sequence 31

&lt; 220> &lt; 223> altered epitope coding sequence &lt; 400 &gt; 80 agtccaagtt atgcatatca tcaattt &lt; 210> 81 &lt; 211> 33 &lt; 212> DNA &lt; 213 &gt; artificial sequence 27

&lt; 220> &lt; 223> Synthetic Oligonucleotide &lt; 400 &gt; 81 cgatagtcca agttatgcat atcatcaatt tgc &lt; 210 &gt; 82 &lt; 211 &gt; 34 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence 33 &lt; 220 &gt; &lt; 223 &gt; Synthetic Oligonucleotide 98561.doc 62- 34 200530399 &lt; 400〉 82 gtcgcaaatt gatgatatgc ataacttgga ctat &lt; 210〉 83 &lt; 211 &gt; 24 &lt; 212 &gt; DNA &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223〉 PCR primer &lt; 400> 83 cccttgggga tccttaatta tacg &lt; 210 &gt; 84 &lt; 211 &gt; 29 &lt; 212〉 DNA &lt; 213> Artificial sequence &lt; 220> &lt; 223> PCR primer &lt; 400 &gt; 84 ctctggatcc atccgcgtgt ttcttttcg &lt; 210〉 85 &lt; 211 &gt; 16 &lt; 212 &gt; RNA &lt; 213〉 artificial sequence &lt; 220 &gt; &lt; 223〉 consensus sequence &lt; 220〉 &lt; 221 &gt; misc-feature &lt; 222〉 1, 9, 10, 11, 12, 13 &lt; 223> n = A, U, C or G &lt; 400 &gt; 85 naggaggunn nnnaug

&lt; 210〉 86 &lt; 211〉 87 &lt; 212〉 DNA 24

29

98561.doc 63- 16 200530399 &lt; 213 &gt; Lactococcus lactis &lt; 400 &gt; 86 atgaaaaaaa agattatctc agctatttta atgtctacag tgatactttc tgctgcagcc 60 ccgttgtcag gtgtttacgc tgacaca 87 &lt; 210 &gt; 87 &lt; 211> DNA> 211> Sequence &lt; 220 &gt;

&lt; 223 &gt; Codon-Optimized Sequence &lt; 400> 87 atgaaaaaaa aaattattag tgcaatttta atgagtacag ttattttaag tgcagcagca 60 ccattaagtg gtgtttatgc agataca 87 &lt; 210> 88 &lt; 211> 36 &lt; 212 &gt; DNA &lt; 213 &gt; 〉 &Lt; 223〉 PCR primers &lt; 400 &gt; 88 aaggagagtg aaacccatga atatgaaaaa agcaac 36

&lt; 210 &gt; 89 &lt; 211 &gt; 24 &lt; 212> DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223> PCR primer &lt; 400 &gt; 89 gtgtgatgga tatctgcaga attc

&lt; 210〉 90 &lt; 211〉 87 &lt; 212 &gt; DNA &lt; 213> Listeria monocytogenes 98561.doc -64- 24 200530399 &lt; 400> 90 atgaatatga aaaaagcaac tatcgcggct acagctggga ttgcggtaac agcatttgct 60 gcgccaactc ag &lt; 210> 91 &lt; 211 &gt; 87 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; codon-optimized sequence &lt; 400> 91

atgaatatga aaaaagcaac aattgcagca acagcaggta ttgcagttac agcatttgca 60 gcaccaacaa ttgcaagtgc aagtaca 87 &lt; 210〉 92 &lt; 211〉 23 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220> cactactac; taggacac # 223 twenty three

&lt; 210〉 93 &lt; 211〉 36 &lt; 212〉 DNA &lt; 213〉 artificial sequence &lt; 220〉 &lt; 223〉 PCR primers &lt; 400 &gt; 93 cttttttcat attcatgggt ttcactctcc ttctac 36 &lt; 210〉 94 &lt; 211〉 21 &lt; 212〉 DNA &lt; 213〉 Artificial sequence &lt; 220〉 &lt; 223〉 PCR primer 98561.doc 65- 21 200530399 &lt; 400> 94 gtcaaaacat acgctcttat c &lt; 210 &gt; 95 &lt; 211 &gt; 24 &lt; 212 &gt; DNA &lt; 213> artificial sequence &lt; 220 &gt; &lt; 223> PCR primer &lt; 400> 95 acataatcag tccaaagtag atgc &lt; 210 &gt; 96 &lt; 211> 24 &lt; 212> DNA &lt; 213> artificial sequence &lt; 220 &gt; &lt; 223 &gt; codon-optimized sequence &lt; 400> 96 gattataaag atgatgatga taaa &lt; 210 &gt; 97 &lt; 211 &gt; 8 &lt; 212> PRT &lt; 213> artificial sequence &lt; 220> &lt; 223 &gt; synthetic Antigenic Mapping &lt; 400> 97

Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 &lt; 210 &gt; 98 &lt; 211〉 30 &lt; 212 &gt; DNA &lt; 213〉 Artificial sequence &lt; 220〉 24

twenty four

98561.doc 66- 200530399 &lt; 223 &gt; codon-optimized sequence &lt; 400 &gt; 98 gaacaaaaat taattagtga agaagattta &lt; 210 &gt; 99 &lt; 211〉 10 &lt; 212〉 PRT &lt; 213 &gt; Hyundai Homo sapiens &lt; 400〉 99

Glu Gin Lys Leu lie Ser Glu Glu Asp Leu 1 5 10

&lt; 210〉 100 &lt; 211〉 12 &lt; 212〉 PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400 &gt; 100

Asn Glu Lys Tyr Ala Gin Ala Tyr Pro Asn Val Ser 1 5 10

&lt; 210〉 101 &lt; 211〉 9 &lt; 212〉 PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400〉 101

Val Ala Tyr Gly Arg Gin Val Tyr Leu 1 5

&lt; 210 &gt; 102 &lt; 211> 9 &lt; 212 &gt; PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400 &gt; 102

Gly Tyr Lys Asp Gly Asn Glu Tyr lie 1 5 -67- 98561.doc 200530399 &lt; 210 &gt; 103 &lt; 211〉 25 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223〉 PCR Primer &lt; 400 &gt; 103 25

gttaagtttc atgtggacgg caaag &lt; 210> 104 &lt; 211 &gt; 42 &lt; 212 &gt; DNA &lt; 213> artificial sequence &lt; 220 &gt; &lt; 223> PCR primers &lt; 400> 104 aggtcttttt cagttaacta tcctctcctt gattctagtt at 42 &lt; 210> 105 &lt; 211〉 43 &lt; 212〉 DNA &lt; 213〉 artificial sequence &lt; 220>

&lt; 223〉 PCR primers &lt; 400> 105 43 caaggagagg atagttaact gaaaaagacc taaaaaagaa ggc &lt; 210 &gt; 106 &lt; 211> 26 &lt; 212> DNA &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223> PCR primer &lt; 400 &gt; 106 tcccctgttc ctataattgt tagctc 26 &lt; 210 &gt; 107 98561.doc -68 · 200530399 &lt; 211 &gt; 25 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223> PCR primers &lt; 400 &gt; 107 gtggacggca aagaaacaac caaag 25 &lt; 210 &gt; 108 &lt; 211> 29 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence

&lt; 220 &gt; &lt; 223 &gt; PCR primers &lt; 400> 108 gttcctataa ttgttagctc atttttttc 29

&lt; 210〉 109 &lt; 211〉 59 &lt; 212 &gt; PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400> 109

Met Lys Lys lie Met Leu Val Phe lie Thr Leu lie Leu Val Ser Leu 15 10 15

Pro He Ala Gin Gin Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30

Glu Asn Ser He Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45

Pro Lys Thr Pro He Glu Lys Lys His Ala Asp 50 55

&lt; 210〉 110 &lt; 211〉 177 &lt; 212 &gt; DNA &lt; 213 &gt; Listeria monocytogenes &lt; 400> 110 atgaaaaaaa taatgctagt ttttattaca cttatattag ttagtctacc aattgcgcaa 60 caaactgaag caaaggatgc atctgaa561tc atdocacatacdocacatacdocacatca.doc -200530399 ccaccagcat ctccgcctgc aagtcctaag acgccaatcg aaaagaaaca cgcggat 177 &lt; 210〉 111 &lt; 211 &gt; 93 &lt; 212〉 DNA &lt; 213 &gt; Bacillus anthracis &lt; 400〉 111 atgaaaaaac gaaaagtagtatcat agt gct agg gat agg gat

&lt; 210〉 112 &lt; 211〉 156 &lt; 212> DNA &lt; 213 &gt; Bacillus subtilis &lt; 400 &gt; 112 atggcatacg acagtcgttt tgatgaatgg gtacagaaac tgaaagagga aagctttcaa 60 aacaatacgt & ttggaccgccg caaatttatt caaggagcgggcggggggcgg &lt; 211 &gt; 177 &lt; 212〉 DNA &lt; 213〉 artificial sequence &lt; 220>

&lt; 223 &gt; codon-optimized sequence &lt; 400> 113 atgaaaaaaa ttatgttagt ttttattaca ttaattttag ttagtttacc aattgcacaa 60 caaacagaag caaaagatgc aagtgcattt aataaagaaa atagtattag tagtatggca 120 ccaccagcaa agt cctccaccag

&lt; 210 &gt; 114 &lt; 211> 93 &lt; 212> DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223〉 codon-optimized sequence &lt; 400> 114 atgaaaaaac gtaaagtttt aattccatta atggcattaa gtacaatttt agttagtagt 60 acaggtaatt tagaagagatta tca gtt 93 98561.doc -70- 200530399 &lt; 210 &gt; 115 &lt; 211〉 156 &lt; 212〉 DNA &lt; 213〉 artificial sequence &lt; 220〉 &lt; 223 &gt; codon-optimized sequence &lt; 400 &gt; 115 atggcatatg atagtcgttt tgatgaatgg gttcaaaaat taaaagaaga aagttttcaa 60 aataatacat ttgatcgtcg taaatttatt caaggtgcag gtaaaattgc aggtttaagt 120 ttaggtttaa caattgcaca aagtgttggt gcattt 156

&lt; 210 &gt; 116 &lt; 211 &gt; 70 &lt; 212 &gt; PRT &lt; 213 &gt; Listeria monocytogenes &lt; 400> 116

Met Asn Met Lys Lys Ala Thr lie Ala Ala Thr Ala Gly lie Ala Val! 5 10 15

Thr Ala Phe Ala Ala Pro Thr lie Ala Ser Ala Ser Thr Val Val Val 20 25 30

Glu Ala Gly Asp Thr Leu Trp Gly He Ala Gin Ser Lys Gly Thr Thr 35 40 45

Val Asp Ala lie Lys Lys Ala Asn Asn Leu Thr Thr Asp Lys He Val 50 55 60

Pro Gly Gin Lys Leu Gin, 65 70

&lt; 210〉 117 &lt; 211〉 28 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220> &lt; 223〉 PCR Primer &lt; 400〉 117 cgcctgcagg taaataatga ggttgctg 28 &lt; 210 &gt; 118 &lt; 211〉 29 9856l.doc -71-200530399 &lt; 212 &gt; DNA &lt; 213〉 artificial sequence &lt; 220〉 &lt; 223〉 PCR primers &lt; 400 &gt; 118 cgcggatcct taattatacg cgaccgaag &lt; 210> 119 &lt; 211 &gt; 29 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220> &lt; 223> PCR Primer &lt; 400> 119 aaactgcagg cattgccaac tgcacgtcc &lt; 210> 120 &lt; 211 &gt; 40 &lt; 212> DNA &lt; 213> Artificial Sequence &lt; 220> &lt; 223> PCR primers &lt; 400> 120 tggctaataa taatgctaac aaactgcaga gctaatgtac &lt; 210 &gt; 121 &lt; 211> 32 &lt; 212 &gt; DNA &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223> PCR primer &lt; 400> 121 cgcctgcagc gtacattagc aggtgaaaca gg

&lt; 210 &gt; 122 &lt; 211 &gt; 36 &lt; 212> DNA -72- 98561.doc 36 200530399 &lt; 213> Artificial sequence &lt; 220> &lt; 223> PCR primer &lt; 400 &gt; 122 cgcctgcagg ccttgtaaac ctaaacctaa tgtatc &lt; 210〉 123 &lt; 211 &gt; 8 &lt; 212〉 PRT &lt; 213〉 Red original chicken &lt; 400〉 123

Ser lie lie Asn Phe Glu Lys Leu 1 5 &lt; 210> 124 &lt; 211 &gt; 8 &lt; 212 &gt; PRT &lt; 213 &gt; Herpes simplex virus &lt; 400 &gt; 124

Ser Ser He Glu Phe Ala Arg Leu 1 5

&lt; 210 &gt; 125 &lt; 211 &gt; 17 &lt; 212> RNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223> consensus sequence &lt; 220> &lt; 221 &gt; other types of features &lt; 222 &gt; 1, 9, 10, 11, 12, 13, 14 &lt; 223 &gt; n = A, U, C or G &lt; 400 &gt; 125 naggaggunn nnnnaug 17 &lt; 210〉 126 98561.doc • 73- 200530399 &lt; 211〉 18 &lt; 212 &gt; RNA &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223〉 Consensus sequence &lt; 220> &lt; 221〉 Other types of features &lt; 222〉 1, 9, 10, 11, 12, 13, 14 , 15 &lt; 223 &gt; n = A, U, C or G &lt; 400 &gt; 126 18 naggaggunn nnnnnaug &lt; 210〉 127 &lt; 211〉 19 &lt; 212 &gt; RNA &lt; 213〉 artificial sequence &lt; 220〉 &lt; 223> Consistency sequence &lt; 220 &gt; &lt; 221> Other types of features &lt; 222 &gt; 1, 9, 10, 11, 12, 13, 14, 15, 16 &lt; 223 &gt; n = A, U, C or G &lt; 400〉 127 19 naggaggunn nnnnnnaug &lt; 210〉 128 &lt; 211 &gt; 20 &lt; 212 &gt; RNA &lt; 213〉 artificial sequence &lt; 220〉 &lt; 223> consensus sequence &lt; 220 &gt; &lt; 221 &gt; Other types of features &lt; 222 &gt; 1, 9, 10, 11, 12, 13, 14, 15, 16, 17 &lt; 223 &gt; n = A, U, C or G &lt; 400 &gt; 128 98561.doc -74 · 200530399 naggaggunn nnnnnnnaug 20 &lt; 210 &gt; 129 &lt; 211 &gt; 21 &lt; 212 &gt; RNA &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223 &gt;-Consistent sequence &lt; 220 &gt; * &lt; 221> Other types of features

&lt; 222〉 1, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 &lt; 223> η = A, U, C or G 21

&lt; 400〉 129 naggaggunn nnnnnnnnau g

98561.doc -75-

Claims (1)

200530399 10. Scope of patent application: 1. A recombinant nucleic acid molecule, including: (a) the first polynucleotide encoding a signal peptide that is natural to bacteria; Codons-optimization; and (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombination The nucleic acid molecule encodes a fusion protein including the signal peptide and the polypeptide. 2. The recombinant nucleic acid molecule according to claim 1, wherein the bacterium is a Listeria bacterium. 3. The recombinant nucleic acid molecule according to claim 1, wherein the polypeptide encoded by the second polynucleotide comprises a polypeptide selected from a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen, an infectious disease antigen, and an infectious disease antigen A group of antigens consisting of peptides. 4. The recombinant nucleic acid molecule according to claim 1, wherein the polypeptide encoded by the second polynucleic acid is heterologous to the signal peptide and foreign to bacteria or both. 5. The recombinant nucleic acid molecule according to claim 1, wherein the signal peptide is an LLO signal peptide obtained from Listeria monocytogenes or a P60 signal peptide obtained from Listeria monocytogenes. 6. A performance card comprising a recombinant nucleic acid molecule as claimed in claim 1, further comprising a promoter gene operatively linked to the first and second polynucleotides of the recombinant nucleic acid molecule. 98561.doc 200530399 7. 8. 9. 10. 11. 12. A recombinant bacterium comprising a recombinant nucleic acid molecule as claimed in claim 1, wherein in order to be expressed in the recombinant bacterium, the first polynucleotide codon- optimize. The use of a recombinant bacterium as described in item 7 in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide includes the antigen. A recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises: (a) a first polynucleotide encoding a signal peptide, wherein the first Polynucleotide codon optimization; and (b) a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is the same translation editing framework as the first polynucleotide, wherein the recombinant nucleic acid The molecule encodes a fusion protein comprising the signal peptide and the polypeptide. The recombinant Listeria bacterium of claim 9, comprising a performance cassette comprising a recombinant nucleic acid molecule, wherein the performance cassette further comprises first and second polynucleotides operatively associated with the recombinant nucleic acid molecule. A promoter gene linked to both. The recombinant Listeria bacterium of claim 9, wherein the Listeria bacterium belongs to the species Listeria monocytogenes. For example, the recombinant Listeria bacterium of claim 9, wherein the polypeptide encoded by the second polynucleotide includes a polypeptide selected from a tumor-related antigen, a polypeptide derived from a tumor-associated antigen, 98561.doc 200530399, and an infectious disease. An antigen and a group of antigens derived from an infectious disease polypeptide. 13. The recombinant Listeria bacterium of claim 12, wherein the polypeptide encoded by the second polynucleic acid comprises a member selected from the group consisting of K-Ras, H-Ras, N-Ras, 12-K-Ras, and mesothelium. Antigen, psCA, NY-ESO-1, WT-1, survival protein, gplOO, PAP, protease 3, SPAS-1, sp 17, pAGE-4, TARP, and CEA, or include antigens derived from K_Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WTM, survival protein, gpl〇〇, pAp, protease 3, SPAS-1, SP-17, Polypeptides of antigens consisting of PAGE-4, TARP and CEA. 14. The recombinant Listeria bacterium of claim 13, wherein the polypeptide encoded by the second polynucleotide includes mesothelin or an antigenic fragment or antigenic variant thereof 'or includes NY-ESO-1 or Its antigenic fragments or antigenic variants. 15. The recombinant Listeria bacterium of claim 14, wherein the polypeptide encoded by the second polynucleotide includes human mesothelin that deletes its signal peptide and the functional site of the Gpi cross-linking agent. 16. The recombinant Listeria bacterium of item 9 as described above, wherein the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. 1 7. The recombinant Listeria bacterium of claim 9, wherein the signal peptide is foreign to the Listeria bacterium. 18. The recombinant Listeria bacterium of claim 9, wherein the signal peptide is natural to the Listeria bacterium. 98561.doc 200530399 19. The recombinant Listeria monocytogenes according to claim 9, wherein the signal peptide is selected from the LLO signal peptide obtained specifically from the drop of monocytogenes Listeria, and is obtained from Usp45 signal peptide of Lactococcus lactis, protective antigen signal peptide obtained from S. anaerobes, P60 signal peptide obtained from monocytic cytotoxicity Li Zhe surname Stele and phoD signal obtained from Bacillus subtilis Signaling of a group of peptides. 20. If the recombinant Listeria bacterium of claim 9, is attenuated for cell propagation, entry into non-phagocytic cells, or proliferation. 21. The recombinant Listeria bacterium of claim 9, which is defective in light, surface protein B or both ActA and surface protein B. 22. In the case of the recombinant Listeria bacterium of claim 9, the nucleic acid of the recombinant bacterium has been modified by reacting with the compound targeted by the nucleic acid '. 23. An immunogenic composition or vaccine comprising a recombinant Listeria bacterium of claim 9. 24. Use of a recombinant Listeria bacterium, such as kiss item 9, in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide includes an antigen. 25. Use of a recombinant Listeria bacterium of the type 9 as claimed in the manufacture of a medicinal product for preventing or treating a disease in a host. 26. A recombinant nucleic acid molecule comprising: (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide; and () a second horse which is a heterologous polypeptide to the peptide # 5HAI # In the polynucleoside, the far second polynucleotide is the same translation editing structure as the first polynucleotide, and 98561.doc 200530399 wherein the recombinant nucleic acid molecule encodes a fusion protein including the signal peptide and the polypeptide. 27. The recombinant nucleic acid molecule of claim 26, wherein the first polynucleotide, the second polynucleotide, or both the first and the second polynucleotide are codon-optimized for expression in bacteria. 28. The recombinant nucleic acid molecule of claim 26, wherein the signal peptide is a Listeria signal peptide. 29. The recombinant nucleic acid molecule of claim 26, wherein the signal peptide is a secA2 signal peptide or a Tat signal peptide. 30. The recombinant nucleic acid molecule according to claim 26, wherein the signal peptide is a p60 signal peptide obtained from Listeria monocytogenes or a phoD signal peptide obtained from Bacillus subtilis. 31. The recombinant nucleic acid molecule of claim 26, wherein the polypeptide encoded by the second polynucleotide comprises a polypeptide selected from a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen, an infectious disease antigen, and an infectious disease antigen Antigens of a group of peptides. 32. A performance cassette comprising a recombinant nucleic acid molecule as claimed in claim 26, further comprising a promoter gene operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule. 33. A recombinant bacterium comprising a recombinant nucleic acid molecule as claimed in claim 26. 34. The recombinant bacterium of claim 33, which is a Listeria bacterium. 35. The use of a recombinant bacterium as described in claim 33 in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide includes an antigen. 98561.doc 200530399 36. Recombinant Listeria bacteria comprising a recombinant nucleic acid molecule, wherein the nucleic acid molecule comprises: (a) a first polynucleotide encoding a non-secAi bacterial signal peptide; and (b) an The second polynucleotide is heterologous to the signal peptide, or a foreign polypeptide to the bacterium or both, wherein the second polynucleotide is the same translation as the first polynucleotide Read the framework, wherein the recombinant nucleic acid molecule encodes a fusion protein including the signal peptide and the polypeptide. 37. The recombinant Listeria bacterium according to item 36, comprising a performance cassette comprising a recombinant nuclear rune knife, wherein the performance cassette further comprises first and first operatively associated with the recombinant nucleic acid molecule. A promoter gene to which two polynucleotides are linked. 38. The recombinant Listeria bacterium of claim 36, wherein the first polynucleotide, the second polynucleotide, or the first and second polynucleotides are expressed in order to be expressed in a Listeria bacterium. Both codon-optimized. 39. The recombinant Listeria bacterium of item # 36, wherein the bacterium belongs to the species Listeria monocytogenes. 40. The reconstituted Li Si surname Yisi A ^ f as in claim 36, wherein the non-secA 1 signal peptide is a non-Listeria signal peptide. 41. Recombinant Listeria + Terhalus as described in claim 36, in which the non-secAl signal peptide 疋 Listeria is #. 42. The recombined Listeria spp. Special halogen bacterium according to claim 36, wherein the signal peptide is a secA2 signal peptide. 98561.doc 200530399 43. The recombinant Listeria spp. According to claim 42, wherein the recombinant nucleic acid molecule further comprises: (c) the same translation and editing framework as the first and second polynucleotides, encoding secA2 autolysis A third polynucleotide of a protein or fragment thereof, wherein the second polynucleotide is located within the third polynucleotide or between the first and third polynucleotides. 44. The recombinant Listeria bacterium of claim 36, wherein the signal peptide is a Tat signal peptide. 45. The recombinant Listeria bacterium of claim 36, wherein the signal peptide is a phoD signal peptide obtained from Bacillus subtilis or a p60 signal peptide obtained from Listeria monocytogenes. 46. The recombinant Listeria bacterium of item 36, wherein the polypeptide encoded by the second polynucleotide includes a polypeptide selected from a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen, an infectious disease antigen, and An antigen derived from a group of polymorphisms of an infectious disease antigen. 47. The recombinant Listeria bacterium of item 46, wherein the polypeptide encoded by the second polynuclear picric acid comprises a member selected from the group consisting of K-Ras, H-Ras, N-Ras, 12-K-Ras, and mesothelium. Group of antigens, including PSA, PSCA, NY-ESO-1, WT-1, survival protein, gplOO, PAP, protease 3, SpAS-Bu SP-17, PAGE-4, TARP and CEA From K-Ras, H-Ras, N-Ras, 12-K-Ras, Mesothelin, PSCA, NY-ESCM, WT-B Survivin, gpl00, PAp, Protease 3, SPAS-1, SP-17, Polypeptides of antigens consisting of PAGE-4, TARP and CEA. 98561.doc 200530399 48. 49. 50. 51. 52. 53. 54. 55. 56. The recombinant Listeria bacterium of claim 47, wherein the polypeptide encoded by the second polynucleic acid is sauvignon R + The main L ^ is mesothelin, or an antigenic fragment or antigenic variant thereof. For example, the recombinant Listeria g. Bacterium of claim 48, wherein the polypeptide encoded by the second polynucleotide includes deletion of its signal peptide and target human mesothelin. For example, the recombinant Listeria bacterium belonging to item 36 of β month is attenuated for cell-to-cell propagation, entry into non-phagocytic cells, or proliferation. For example, the recombinant Listeria spp. G of claim 36 is defective in secret, surface protein B or both ActA and surface protein B. For example, the recombinant Listeria bacterium of claim 36 has been modified by reacting with a nucleic acid-targeted compound to modify the nucleic acid of the recombinant bacterium. -An immunogen composition or vaccine comprising a recombinant Listeria bacterium of claim 36. A use of a recombinant Listeria bacterium of claim 36 in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide includes an antigen. A use of a recombinant Listeria bacterium of claim 36 in the manufacture of a pharmaceutical product for preventing or treating a condition in a host. A recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises: (a) a first polynucleotide encoding a non-Listeria spp. Signal peptide; and (b) The same translation and editing architecture of two polynucleotides encodes the second polynucleic acid of the polypeptide, 98561.doc 200530399, wherein the recombinant nucleic acid molecule encodes a fusion protein including the non-Listeria spp. Signal peptide and the polypeptide. Recombinant Listeria bacterium of the month Seeker 5 6 includes a performance card including a recombinant nuclear knife. The performance card also includes the first-and the second in an operable manner with the recombinant nuclear gt molecule. Prokaryotic genes linked to both polynuclear picric acids. Recombinant Listeria bacterium of Seeker 56 in July, in order to express in Listeria bacterium, the first polynucleotide, the second polynucleotide I, or both the first and the second polynucleotide Codon-optimization. 59. U Item 56 of the recombinant Listeria bacterium, wherein the bacterium is Listeria monocytogenes. 60. The recombinant Listeria bacterium of claim 56, wherein the signal peptide is bacterial. • The recombinant Listeria bacterium of claim 60, wherein the signal line is derived from a Gram-positive bacterium. 62. The recombinant Listeria bacterium of claim 6i, wherein the signal peptide is derived from a bacterium belonging to the genus Bacillus, Staphylococcus or Lactococcus. 63. The recombinant Listeria bacterium of claim 62, wherein the signal peptide is selected from the group consisting of a Usp45 signal peptide obtained from Lactococcus lactis, a protective antigen signal peptide obtained from Bacillus anthracis and a pOD signal obtained from Bacillus subtilis. Group of signal peptides. 64. The recombinant Listeria bacterium of claim 56, wherein the polypeptide encoded by the second polynucleic acid includes a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen 98561.doc 200530399 tumor, and an infectious disease. The antigen and the polypeptide derived from an infectious disease antigen are selected from the group of antigens. 65. The recombinant Listeria bacterium of claim 64, wherein the polypeptide encoded by the second polynucleotide comprises a member selected from the group consisting of K-Ras, H-Ras, N-Ras, 12-K-Ras, and mesothelin , PSCA, NY-ESO-1, WT-1, survival protein, gplOO, PAP, protease 3, SpAS-1, SP-17, PAGE-4, TARP and CEA, or include antigens derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY_ESCM, WTM, survival proteins, gpl〇〇, pAp, protease 3, SPAS-1, SP-17, PAGE- 4. Polypeptides of antigens composed of TARP and CEA. 66. The recombinant Listeria bacterium of item 6 5 as described above, wherein the polypeptide encoded by the second polynucleotide includes mesothelin, or an antigenic fragment or an antigenic variant thereof. 67. The recombinant Listeria bacterium of claim 66, wherein the second Nucleotide-encoded polypeptides include deletion of their signal peptide and Gpi-anchored human mesothelin. 68. The recombinant Listeria bacterium of claim 56 'is attenuated for cell-to-cell propagation, entry into non-phagocytic cells, or proliferation. 69. The recombinant Listeria bacterium of claim 56, which is defective in ActA, surface protein B, or both ActA and surface protein B. 70. The recombinant Listeria bacterium of claim 56, wherein the nucleic acid of the recombinant bacterium has been modified by reacting with a nucleic acid-targeted compound. 98561.doc -10- 200530399 71.-An immunogenic composition or vaccine comprising a recombinant Listeria bacterium of claim 56. 72.-The use of a recombinant Listeria bacterium of claim 56 in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide in # includes an antigen. 73.-The use of a recombinant Listeria bacterium of claim 56 in the manufacture of a pharmaceutical product for preventing or treating a disease in a host. 74. A recombinant nucleic acid molecule comprising: (a) a first polynucleotide encoding a bacterial autolysin, or a catalytically active fragment or a catalytically active variant thereof; and (b) encoding a The bacterial autolysin is a second polynucleoside of a heterologous polypeptide, wherein the second polynucleotide is the same translation editing structure as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes The polypeptide encoded by the second polynucleotide and the autolysin, or a catalytic chimeric fragment or a protein chimera of the catalytically active variant, wherein in the protein chimera, the polypeptide and the autolysin Or the catalytically active fragment or catalytically active variant is fused, or is located in the autolysin, or the catalytically active fragment or catalytically active variant. 75. A recombinant bacterium comprising a recombinant nucleic acid molecule as claimed in item 74. 76. The use of a recombinant bacterium as claimed in claim 75 in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide includes an antigen. 98561.doc 11 200530399 77 · A recombinant Listeria bacterium comprising a polycistronic performance card Kuang-Zhongxixi cistron performance card g encodes at least two isolated non-_ Listeria polypeptides . 78.-Use of a recombinant Listeria bacterium of the type 77 as claimed in claim 77 for the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the non-_ Listeria monocytogenes encoded by the polycistronic expression card E Is a polypeptide-including antigens. 79. A recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises a polynuclear #acid encoding a polypeptide that is foreign to the Listeria bacterium, wherein This polynucleotide is codon-optimized. 80. A use of a recombinant Listeria bacterium of claim 79 in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the foreign polypeptide encoded by the second polynucleotide includes an antigen. 8 1 · A recombinant nucleic acid molecule comprising: (a) the first polynucleic acid encoding # ^ tiger-like; (b) a second polynucleotide encoding a secreted protein or a fragment thereof, wherein the second Each polynucleotide is the same translation editing framework as the first polynucleotide, and (c) a third polynucleotide encoding a polypeptide that is heterologous to the secreted protein or fragment thereof, wherein the third polynucleotide The polynucleotide is the same translation editing framework as the first and second polynucleotides, wherein the recombinant nucleic acid molecule encodes a protein insert including a signal peptide, a polypeptide encoded by a third polynucleotide, and a secreted protein or fragment thereof And in the protein chimera, a polypeptide encoded by the third polynucleic acid is fused to the secreted protein or a fragment thereof, or is located within the secreted protein or a fragment thereof. 82. A recombinant bacterium comprising a recombinant nucleic acid molecule as claimed in item 81. 83. The use of a recombinant bacterium as claimed in claim 82 in the manufacture of a pharmaceutical product that induces an immune response to an antigen in a host, wherein the polymorphism encoded by the third polynucleotide includes the antigen. 98561.doc 13-