CN1921884A - Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof - Google Patents
Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof Download PDFInfo
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- CN1921884A CN1921884A CNA200480042020XA CN200480042020A CN1921884A CN 1921884 A CN1921884 A CN 1921884A CN A200480042020X A CNA200480042020X A CN A200480042020XA CN 200480042020 A CN200480042020 A CN 200480042020A CN 1921884 A CN1921884 A CN 1921884A
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- listera
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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
The cross reference of related application
Require following each U.S. Provisional Application No. rights and interests according to 35 U.S. C. § 119 (e) the application, wherein the disclosure of each all is incorporated herein by reference at this: U.S. Provisional Application series No.60/616,705, denomination of invention is " a bacterial expression box; the method for bacterial vaccine compositions and use thereof ", Thomas W.Dubensky, Jr etc., application on October 6th, 2004 (Docket No.282173003923); U.S. Provisional Application series No.60/615,287, denomination of invention is " bacterial expression box, a bacterial vaccine compoistion and method of use ", Thomas W.Dubensky, Jr etc., application on October 1st, 2004 (Docket No.282173003922); U.S. Provisional Application series No.60/599, application on August 5th, 377,2004; U.S. Provisional Application series No.60/556, application on March 26th, 744,2004; U.S. Provisional Application series No.60/541, application on February 2nd, 515,2004; With U.S. Provisional Application series No.60/532, the application in 24, of 598,2003 on Decembers.In addition, this application is following each part continuation application in first to file, and wherein the disclosure of each all is incorporated herein by reference at this: international application No.PCT/US2004/23881, application on July 23rd, 2004; U.S. Patent application series No.10/883, application on June 30th, 599,2004; U.S. Patent application series No.10/773, application on February 6th, 618,2004; With U.S. Patent application series No.10/773, application on February 6th, 792,2004.
About the research of federal government's subsidy or the statement of exploitation
The present invention finishes under the government of the SBIR grant number No.1R43CA101421-01 that National Institute of Health gives supports.Government may enjoy certain right in the present invention.
Invention field
The present invention generally relates to new polynucleotide and the expression cassette that is used for comprising at the recombinant bacteria express polypeptide heterologous polypeptide.Especially, the present invention relates to be used for comprising of vaccine combination of new expression cassette and/or the recombinant bacteria of nucleic acid molecules.
Background of invention
Begun the microorganism exploitation as the vaccine of sending heterologous antigen.Containing coding and derive from the protein of different plant species or the microorganism of antigenic nucleotide sequence provides heterologous antigen to send by changing.Heterologous antigen for treatment or prevention especially by toxicity or fatal source such as cancer or pathogen (is for example sent, HIV or hepatitis B) disease or the disease that cause be particularly advantageous, the injection of wherein natural sensor or cancerous cell is potential deleterious to the receptor biological body, and proved attenuation or inactivating pathogens or cell to give in causing efficient immune be unsuccessful, or wherein can not guarantee the abundant attenuation of sensor or cancerous cell for certain.Recently, some bacterial isolates is developed as recombiant vaccine.For example; oral vaccine through changing the Salmonella (Salmonella) that can express the antigenic attenuation of Bai Shi plasmodium (Plasmodium berghei) ring spore has demonstrated can protect mice to malaria (Aggarwal etc.; 1990, J.Exp.Med.172:1083).
Listeria monocytogenes (Listeria monocytogenes) (Listera genus) is the facultative intracellular bacteria of Gram-positive, because it causes the ability of the cell-mediated response of effective CD4+/CD8+T-by I class and II class MHC antigen presentation approach, and its exploitation is used for the antigenic specificity vaccine.Referring to, for example, U.S. Patent No. 6,051,237,6,565,852 and 5,830,702.
Many years Listera have been studied as stimulation is congenital with model adaptability T cell dependency antibacterial immunity.The ability of Listera effective stimulus cellular immunity is based on its interior biocycle of born of the same parents.In case infection host, this antibacterial are comprised the phagocyte of macrophage and dendritic cell (DC) apace and are taken in the phagolysosome compartment.Most of subsequently antibacterial is degraded.The peptide that is formed by the proteolytic degradation of engulfing intravital pathogen that infects APC directly is loaded on the II class MHC molecule, the antigen of processing is expressed on the antigen-presenting cell surface by II class endosome approach, and the CD4+ that these MHC II-peptide complexes activation stimulate antibody to produce " assists " the T cell.Indoor at acidic region, activate some bacterial gene, comprise the cholesterol-dependent Lysin, LLO, its phagolysosome of can degrading is released into antibacterial in the kytoplasm compartment of host cell, and the Listera of survival is bred therein.Heterologous antigen need carry out from the beginning endogenous protein expression by Listera by effectively presenting of I class MHC approach.In the Cytoplasm of antigen-presenting cell (APC), take a sample and degraded by albuminous body with excretory protein by Listera is synthetic.Resulting peptide is shuttled back and forth enter endoplasmic reticulum and be loaded on the I class MHC molecule by TAP protein.MHC I-peptide complexes is delivered to cell surface, its with fully altogether stimulate (signal 2) combine to activate and stimulate cytotoxic T lymphocyte (CTL) to expand and discern the MHC I-peptide complexes of being presented on the tumor cell for example subsequently with connection TXi Baoshouti.In suitable microenvironment, the activated T cells targeting is also killed cancerous cell.
The mechanism that known Listera is presented heterologous antigen by MHC I classpath, expression of heterologous genes and new synthetic protein are directly related with initiation of CD8+T cell and/or activated usefulness to the excretory efficient that infects (antigen presentation) cell matter from antibacterial.Because the level that the Ag-specific T-cells causes is directly related with efficacy of vaccines, so heterologous protein is expressed and excretory efficient is directly related with efficacy of vaccines.
Therefore, this area need new method come the optimization heterologous protein express and excretory efficient to maximize based on the vaccine of Listera with based on the effect of the vaccines of other antibacterials.It also is useful that the optimization heterologous protein needs expression and excretory efficient in a large amount of heterologous proteins expression and the excretory bacterial host expression system therein.
Summary of the invention
The present invention always provides new polynucleotide, comprises the new recombinant nucleic acid molecules that is used at expression of antibacterial especially Listera and/or secrete polypeptide (for example, heterologous polypeptide), expression cassette, and carrier.In some embodiments, expression and/or secretion that these polynucleotide provide polypeptide to improve in antibacterial.The present invention always also provides and has comprised recombinant nucleic acid molecules, expression cassette, or the antibacterial of carrier, and the pharmaceutical composition that comprises this antibacterial, immunogenic composition and vaccine combination.These antibacterials and compositions can be used for induce immune response and treat and/or prevent multiple disease or other diseases, comprise cancer, infect and autoimmune.
On the one hand, the invention provides recombinant nucleic acid molecules, first polynucleotide that comprise the coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, and coded polypeptide (for example, antigen) second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein this recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, second polynucleotide also is that codon is optimized for the expression in antibacterial such as Listera.The present invention also provides the expression cassette that comprises this recombinant nucleic acid molecules and further comprise the promoter of this recombinant nucleic acid molecules that is operably connected.The carrier and the antibacterial that comprise this recombinant nucleic acid molecules and/or expression cassette also are provided, and the pharmaceutical composition that comprises this antibacterial, immunogenic composition and vaccine.Also provide the compositions of using this antibacterial or comprising this antibacterial to come the method for induce immune response and/or prevention or treatment host's disease such as disease.
On the other hand, the invention provides recombinant nucleic acid molecules, first polynucleotide that comprise the inherent signal peptide of (a) coding antibacterial, wherein first polynucleotide are that codon is optimized for the expression in this antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein this recombinant nucleic acid molecules coding comprises the fusion rotein of this signal peptide and this polypeptide.In some embodiments, be allogenic to signal peptide by the polypeptide of second polynucleotide encoding.In some embodiments, be external source to antibacterial by the polypeptide of second polynucleotide encoding.The present invention also provides the expression cassette that comprises this recombinant nucleic acid molecules and further comprise the promoter of this recombinant nucleic acid molecules that is operably connected.The carrier and the antibacterial that comprise this recombinant nucleic acid molecules and/or expression cassette also are provided, and the pharmaceutical composition that comprises antibacterial, immunogenic composition and vaccine.Also provide the compositions of using this antibacterial or comprising this antibacterial to come the method for induce immune response and/or prevention or treatment host's disease (for example, disease).
On the other hand, the invention provides the antibacterial of the reorganization Listera genus that comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotide of (a) coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression in Listera, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein this recombinant nucleic acid molecules coding comprises the fusion rotein of this signal peptide and this polypeptide.In some embodiments, be allogenic to signal peptide by the polypeptide of second polynucleotide encoding.In some embodiments, this polypeptide is an external source to the antibacterial that Listera belongs to.In some embodiments, this signal peptide is that Listera is inherent.Also provide the pharmaceutical composition that comprises Listera, immunogenic composition and vaccine.Also provide and used this Listera compositions of this Listera (or comprise) to come the method for induce immune response and/or prevention or treatment host disease (for example, disease).
On the other hand, the invention provides recombinant nucleic acid molecules, first polynucleotide that comprise the non-secA1 antibacterial signal peptide of encoding, and second polynucleotide of coded polypeptide (as antigen), wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein this recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, polypeptide and signal peptide are allogenic.In some embodiments, first and/or second polynucleotide are that codon is optimized for the expression in antibacterial such as Listera.The present invention also provides the expression cassette that comprises this recombinant nucleic acid molecules and further comprise the promoter of this recombinant nucleic acid molecules that is operably connected.The carrier and the antibacterial that comprise this recombinant nucleic acid molecules and/or expression cassette also are provided, and the pharmaceutical composition that comprises this antibacterial, immunogenic composition and vaccine.Also provide the compositions of using this antibacterial or comprising this antibacterial to come the method for induce immune response and/or treatment host's disease such as disease.
On the other hand, the invention provides the reorganization Listera that comprises recombinant nucleic acid molecules and belong to antibacterial, wherein this recombinant nucleic acid molecules comprises first polynucleotide of the non--secA1 antibacterial signal peptide of (a) coding, (b) coding is allogenic or to second polynucleotide of the polypeptide of antibacterial external source with signal peptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein this recombinant nucleic acid molecules coding comprises the fusion rotein of this signal peptide and this polypeptide.In some embodiments, be allogenic or be external source (that is, being allogenic) antibacterial to antibacterial by the polypeptide of second polynucleotide encoding and signal peptide, or both.Also provide the pharmaceutical composition that comprises this Listera, immunogenic composition and vaccine.Also provide and used this Listera compositions of this Listera (or comprise) to come the method for induce immune response and/or prevention or treatment host disease (for example, disease).
On the other hand, the invention provides recombinant nucleic acid molecules, wherein this recombinant nucleic acid molecules (for example comprises coding Listera allogenic polypeptide, cancer or non-Listera infectious disease antigen) polynucleotide, wherein the polynucleotide of encoding exogenous polypeptide are that codon is optimized for the expression in Listera.In some embodiments, this recombinant nucleic acid molecules further comprises the polynucleotide of the coded signal peptide that is arranged in the translation frame identical with the polynucleotide of coding Listera allogenic polypeptide.In some embodiments, this signal peptide is that the Listera antibacterial is inherent.In other embodiments, this signal peptide is an external source to the Listera antibacterial.In some embodiments, the polynucleotide of this coded signal peptide also are that codon is optimized for the expression in this Listera.The Listera that comprises this recombinant nucleic acid molecules also is provided.Also provide the pharmaceutical composition that comprises this Listera, immunogenic composition and vaccine.In addition, the invention provides use this reorganization Listera belong to antibacterial come induce immune response and/or prevention or treat host's disease (as, but be not limited to disease) method.
On the other hand, the invention provides the reorganization Listera that comprises expression cassette and belong to antibacterial, wherein this expression cassette (for example comprises coding Listera allogenic polypeptide, cancer or non-Listera infectious disease antigen) polynucleotide and the promoter of polynucleotide of the encoding exogenous polypeptide that is operably connected, wherein the polynucleotide of this encoding exogenous polypeptide are that codon is optimized for the expression in Listera.In some embodiments, this expression cassette further comprises the coded signal peptide polynucleotide (is natural or the signal peptide of external source to Listera) and that be operably connected promoter that are arranged in the translation frame identical with the polynucleotide of coding Listera allogenic polypeptide, makes this expression cassette express the fusion rotein that comprises this signal peptide and this allogenic polypeptide.In some embodiments, the polynucleotide of this coded signal peptide also are that codon is optimized for the expression in Listera.Also provide the pharmaceutical composition that comprises this Listera, immunogenic composition and vaccine.In addition, the invention provides this reorganization Listera of use and belong to the method that antibacterial comes induce immune response and/or prevention or treatment host's disease (for example, disease).
On the other hand, the invention provides recombinant nucleic acid molecules, wherein this recombinant nucleic acid molecules comprises first polynucleotide of the non-Listera signal peptide of (a) coding, (b) be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide, wherein this recombinant nucleic acid molecules coding comprises the fusion rotein of this non-Listera signal peptide and this polypeptide.The present invention also provides the expression cassette that comprises this recombinant nucleic acid molecules, and wherein this expression cassette further comprises the promoter of first and second polynucleotide of this recombinant nucleic acid molecules that is operably connected.The carrier that comprises this recombinant nucleic acid molecules and/or this expression cassette also is provided.In addition, also provide the Listera that comprises this recombinant nucleic acid molecules and/or this expression cassette to belong to antibacterial.Also provide and comprised that this Listera belongs to the pharmaceutical composition of antibacterial, immunogenic composition and vaccine.Also provide and used this Listera compositions of this Listera (or comprise) to come the method for induce immune response and/or prevention or treatment host disease (for example, disease).
Further in the aspect, the invention provides the reorganization Listera that comprises recombinant nucleic acid molecules and belong to antibacterial, wherein this recombinant nucleic acid molecules comprises first polynucleotide of the non-Listera signal peptide of (a) coding, (b) be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide, wherein this recombinant nucleic acid molecules coding comprises the fusion rotein of this non-Listera signal peptide and this polypeptide.Also provide the pharmaceutical composition that comprises this Listera, immunogenic composition and vaccine.Also provide and used this Listera compositions of this Listera (or comprise) to come the method for induce immune response and/or prevention or treatment host disease (for example, disease).
Again in the one side, the invention provides the Listera that comprises expression cassette and (for example belong to antibacterial, from the Listeria monocytogenes kind), this expression cassette comprises first polynucleotide of the non-Listera signal peptide of encoding, the coded polypeptide that is arranged in the translation frame identical with first polynucleotide (for example, the promoter of second polynucleotide antigen) and be operably connected first and second polynucleotide.This expression cassette coding comprises the fusion rotein of this non-Listera signal peptide and this polypeptide.In some embodiments, first and/or second polynucleotide are that codon is optimized for the expression in Listera.Also provide the pharmaceutical composition that comprises this Listera, immunogenic composition and vaccine.In addition, the invention provides this reorganization Listera of use and belong to the method that antibacterial comes induce immune response and/or prevention or treatment host's disease (for example, disease).
The present invention also provides recombinant nucleic acid molecules, first polynucleotide that comprise (a) coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, wherein this recombinant nucleic acid molecules coding comprises by the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant, wherein, in the protein chimera, polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge or are positioned at autolysin or its catalytic activity fragment or catalytic activity variant.The carrier and the antibacterial that comprise this recombinant nucleic acid molecules and/or expression cassette also are provided, and the pharmaceutical composition that comprises this antibacterial, immunogenic composition and vaccine.Also provide the compositions of using this antibacterial or comprising this antibacterial to come the method for induce immune response and/or treatment host's disease such as disease.
On the other hand, the invention provides recombinant nucleic acid molecules, wherein at least two discrete non-Listera polypeptide of this nucleic acid molecule encoding.The present invention further provides and comprise this recombinant nucleic acid and further comprise the expression cassette of promoter, wherein promoter this recombinant nucleic acid molecules that is operably connected.The carrier that comprises this recombinant nucleic acid molecules and/or expression cassette further is provided.In addition, also provide the reorganization Listera that comprises this recombinant nucleic acid molecules (and/or this expression cassette) to belong to antibacterial.Also provide the pharmaceutical composition that comprises this Listera, immunogenic composition and vaccine.In addition, also provide and use this Listera compositions of this Listera (or comprise) to come the method for induce immune response and/or prevention or treatment host disease (for example, disease).
On the other hand, the invention provides the reorganization Listera that comprises the polycistronic expression box and belong to antibacterial, wherein at least two discrete non-Listera polypeptide of this polycistronic expression box coding.Also provide the pharmaceutical composition that comprises this Listera, immunogenic composition and vaccine.In addition, also provide and use this Listera compositions of this Listera (or comprise) to come the method for induce immune response and/or prevention or treatment host disease (for example, disease).
In other aspects, the invention provides recombinant nucleic acid molecules, first polynucleotide that comprise (a) coded signal peptide, (b) coding secretory protein or its segmental second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the 3rd polynucleotide of coding secretory protein or its segmental heterologous polypeptide, wherein the 3rd polynucleotide are arranged in and first and second translation frame that polynucleotide are identical, wherein this recombinant nucleic acid molecules coding comprises signal peptide, polypeptide by the 3rd polynucleotide encoding, with secretory protein or its segmental protein chimera, and wherein, in the protein chimera,, or be positioned at secretory protein or its fragment by polypeptide and the secretory protein or the fusion of its fragment of the 3rd polynucleotide encoding.Also provide and comprised that this recombinant nucleic acid molecules also further comprises first of this recombinant nucleic acid molecules that is operably connected, the expression cassette of the promoter of second and the 3rd polynucleotide.The carrier and the antibacterial that comprise this recombinant nucleic acid molecules and/or expression cassette also are provided, and the pharmaceutical composition that comprises this antibacterial, immunogenic composition and vaccine.Also provide the compositions of using this antibacterial or comprising this antibacterial to come the method for induce immune response and/or prevention or treatment host disease.
In some embodiments, induce the host that the method for antigenic immunne response is comprised effective dose (for example is included in this, in above-mentioned either side, or in following detailed Description Of The Invention or embodiment) compositions of described recombinant bacteria gives the host, wherein by recombinant nucleic acid molecules in this antibacterial, the polypeptide of expression cassette and/or vector encoded comprises antigen.In some embodiments, the method for prevention or treatment host's disease such as disease comprises that the compositions that comprises recombinant bacteria described herein with effective dose gives the host.
(for example the present invention further provides at this, in above-mentioned either side, or in following detailed Description Of The Invention or embodiment) described recombinant bacteria induces the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein by recombinant nucleic acid molecules in this antibacterial, the polypeptide of expression cassette and/or vector encoded comprises antigen.In some embodiments, this antigen is heterologous antigen.The present invention also provides the purposes of recombinant bacteria described herein in the medicine of making prevention or treatment host's disease (for example, disorders such as cancers or infectious disease).The present invention further provides recombinant bacteria described herein and be used to induce the host to antigenic immunne response, wherein by recombinant nucleic acid molecules in this antibacterial, the polypeptide of expression cassette and/or vector encoded comprises antigen.The present invention further provides recombinant bacteria described herein and be used for prevention or treatment host's disease (as disease).
In the other aspect, the invention provides in host bacteria and to express and the modification method of secretion heterogenous protein.
Also provide preparation to comprise the method for the antibacterial of above-mentioned each recombinant nucleic acid molecules and expression cassette.The method of using this antibacterial to produce vaccine also is provided.
The present invention further provides coded signal peptide and/or antigenic multiple polynucleotide, comprised that for the expression in Listeria monocytogenes be the optimized polynucleotide of codon.
Accompanying drawing
Fig. 1 has shown the hly promoter comparison of Listeria monocytogenes DP-L4056 (SEQ ID NO:1) (bottom sequence) and EGD bacterial strain (SEQ ID NO:2) (top sequence).
Fig. 2 has shown that coding comprises LLO signal peptide, the sequence of the polynucleotide of LLO PEST sequence and the antigenic fusion rotein of total length people EphA2 (SEQ ID NO:3).
Fig. 3 has shown by the sequence of the fusion rotein of polynucleotide encoding shown in Figure 2 (SEQ IDNO:4).
Fig. 4 has shown the natural nucleus glycoside acid sequence (SEQID NO:5) of coding people EphA2 ectodomain (EX2).
Fig. 5 has shown for the expression in Listeria monocytogenes it has been the nucleotide sequence (SEQ ID NO:6) of the optimized coding of codon people EphA2 ectodomain.
Fig. 6 has shown the aminoacid sequence (SEQ IDNO:7) of people EphA2 ectodomain (EX2).
Fig. 7 has shown that coding comprises LLO signal peptide, the optimized polynucleotide sequence of non-codon of the fusion rotein of LLO PEST sequence and people EphA2 ectodomain (SEQ ID NO:8).
Fig. 8 has shown the sequence (SEQID NO:9) by the fusion rotein of the coding of the coded sequence shown in Fig. 7.
Fig. 9 has shown and has comprised that hly promoter and coding comprise the LLO signal peptide, the Expression of Fusion Protein box of LLO PEST sequence and people EphA2 ectodomain (SEQ ID NO:10).In this sequence, the sequence of coding people EphA2 ectodomain is that codon is optimized for the expression in Listeria monocytogenes.
Figure 10 has shown by the expression cassette amino acid sequence coded of Fig. 9 (SEQ ID NO:11).
Figure 11 has shown and has comprised that hly promoter and coding comprise the LLO signal peptide, the Expression of Fusion Protein box of LLO PEST sequence and people EphA2 ectodomain (SEQ ID NO:12).In this sequence, coding LLO signal peptide, the sequence of LLO PEST sequence and people EphA2 ectodomain all has been that codon is optimized for the expression in Listeria monocytogenes.
Figure 12 has shown by the expression cassette amino acid sequence coded of Figure 11 (SEQ ID NO:13).
Figure 13 has shown and has comprised that hly promoter and coding comprise the Expression of Fusion Protein box (SEQ ID NO:14) of phoD Tat signal peptide and people EphA2 ectodomain.In this sequence, the sequence of coding phoD Tat signal peptide and people EphA2 ectodomain has been that codon is optimized for the expression in Listeria monocytogenes.
Figure 14 has shown by the expression cassette amino acid sequence coded of Figure 13 (SEQ ID NO:15).
Figure 15 has shown the natural nucleus glycoside acid sequence (SEQID NO:16) of coding people EphA2 born of the same parents intracellular domain (CO).
Figure 16 has shown for the expression in Listeria monocytogenes it has been the nucleotide sequence (SEQ ID NO:17) of the optimized coding of codon people EphA2 born of the same parents intracellular domain.
Figure 17 has shown the aminoacid sequence (SEQ IDNO:18) of people EphA2 born of the same parents intracellular domain (EX2).
Figure 18 has shown that coding comprises LLO signal peptide, the optimized polynucleotide sequence of non-codon (SEQ IDNO:19) of the fusion rotein of LLO PEST sequence and people EphA2 born of the same parents intracellular domain.
Figure 19 has shown the sequence (SEQ ID NO:20) by the fusion rotein of the coding of the coded sequence shown in Figure 18.
Figure 20 has shown and has comprised that hly promoter and coding comprise the LLO signal peptide, the Expression of Fusion Protein box of LLO PEST sequence and people EphA2 born of the same parents intracellular domain (SEQ ID NO:21).In this sequence, the sequence of coding people EphA2 born of the same parents intracellular domain is that codon is optimized for the expression in Listeria monocytogenes.
Figure 21 has shown by the expression cassette amino acid sequence coded of Figure 20 (SEQ ID NO:22).
Figure 22 has shown and has comprised that hly promoter and coding comprise the LLO signal peptide, the Expression of Fusion Protein box of LLO PEST sequence and people EphA2 born of the same parents intracellular domain (SEQ ID NO:23).In this sequence, coding LLO signal peptide, the sequence of LLO PEST sequence and people EphA2 born of the same parents intracellular domain all has been that codon is optimized for the expression in Listeria monocytogenes.
Figure 23 has shown by the expression cassette amino acid sequence coded of Figure 22 (SEQ ID NO:24).
Figure 24 has shown and has comprised that hly promoter and coding comprise the Expression of Fusion Protein box (SEQ ID NO:25) of phoD Tat signal peptide and people EphA2 born of the same parents intracellular domain.In this sequence, the sequence of coding phoD Tat signal peptide and people Eph2A born of the same parents intracellular domain has been that codon is optimized for the expression in the mononuclear cell Listera.
Figure 25 has shown by the expression cassette amino acid sequence coded of Figure 24 (SEQ ID NO:26).
Figure 26 has shown and has comprised that hly promoter and coding comprise the optimized expression cassette of codon (SEQ ID NO:27) of LLO signal peptide and the antigenic fusion rotein of NY-ESO-1.Coded signal peptide and antigenic sequence are that codon is optimized for the expression in Listeria monocytogenes.
Figure 27 has shown by the expression cassette amino acid sequence coded of Figure 26 (SEQ ID NO:28).
Figure 28 has shown the polynucleotide (SEQ ID NO:29) of the hly promoter that comprises the optimized Usp45 signal coding sequence of the codon that is operably connected.
Figure 29 has shown the polynucleotide (SEQ ID NO:30) of the hly promoter that comprises the natural coded sequence of p60 signal peptide that is operably connected.
Figure 30 has shown the polynucleotide (SEQ ID NO:31) of the hly promoter that comprises the optimized p60 signal coding sequence of the codon that is operably connected.
Figure 31 has shown the sequence (SEQ ID NO:32) of hlyP-p60 genetic fragment.
Figure 32 has shown (comprising Figure 32 A, 32B and 32C) sequence (SEQID NO:33) of pAM401-MCS, and pAM401-MCS is the pAM401 plasmid that comprises multiple clone site (MCS) from the pPL2 carrier.
Figure 33 has shown for the expression in Listeria monocytogenes it has been the coded sequence (SEQ ID NO:34) of the optimized people's mesothelium of codon element (mesothelium element).
Figure 34 has shown the aminoacid sequence (SEQ ID NO:35) of people's mesothelium element.
Figure 36 has shown the coded sequence (SEQ ID NO:36) of Mus mesothelium element, and it has been that codon is optimized for the expression in Listeria monocytogenes.
Figure 37 has shown the western blot analysis of the secretory protein of the reorganization Listera that comes the natural Eph2A CO of own coding domain sequence.
Figure 38 has shown the western blot analysis of the secretory protein of the reorganization Listera that comes the natural or codon optimization LLO secA1 signal peptide that own coding and codon optimization EphA2 EX2 domain sequence merge.
Figure 39 has shown the western blot analysis of the secretory protein of the reorganization Listera that comes natural or codon optimization LLO secA1 signal peptide or codon optimization Tat signal peptide that own coding and codon optimization EphA2 CO domain sequence merge.
Figure 40 has shown the western blot analysis with the lysate of 48 hours 293 cells behind the pCDNA4 plasmid DNA transfection of the natural EphA2 sequence of coding total length.
Figure 41 has shown that reorganization Listera immunity with coding OVA.AH1 or OVA.AH1-A5 has the diagram that the Balb/C mice of CT26.24 (huEphA2+) lung tumor can long-term surviving.
Figure 42 is the diagram that has improved the Balb/C mice survival that has CT26.24 (huEphA2+) lung tumor when having shown the reorganization Listera immunity of the codon optimization secA1 signal peptide that merges with coding and codon optimization EphA2 EX2 domain sequence.
Figure 43 has shown that reorganization Listera immunity with coding EphA2 CO domain has the diagram that the Balb/C mice of CT26.24 (huEphA2+) lung tumor can long-term surviving.
Figure 44 has shown with the reorganization Listera of coding EphA2CO domain but the plasmid DNA immunity of coding total length EphA2 of no use has the diagram that the Balb/C mice of CT26.24 (huEphA2+) lung tumor can long-term surviving.
Figure 45 has shown that the Listera of expressing hEphA2 causes the diagram of EphA2 specific C D8+T cellular response.
Figure 46 has shown that CD4+ and CD8+T cellular response help to express the diagram of antitumor efficacy of hEphA2 orientation of the Listera of hEphA2.
Figure 47 has shown the sequence (SEQ ID NO:38) of hly promoter of the Bacillus anthracis that is operably connected (B.anthracis) the protective antigen signal peptide of monocyte Listeria monocytogenes strain 10403S, is that codon is optimized for the secretion in Listeria monocytogenes.Comprise six other nucleotide corresponding to Bam HI restriction enzyme recognition site (5 '-GGATCC-3 ') at the c-terminus of signal peptide sequence, any selected coded sequence helps to be operably connected in frame.5 ' end of hly promoter comprises single KpnI restriction enzyme recognition site.
Figure 48 has shown effective expression and the secretion from the total length human tumor antigen of reorganization Listera.Figure 48 A has shown the plain expression/secretion of mesothelium, uses the construct that comprises with the plain LLO signal peptide that merges of the people's mesothelium that uses natural codon.Figure 48 B has shown the expression/secretion of mesothelium element, uses to comprise and the construct that for expression in Listera is the various signal peptides of the optimized people's mesothelium element of codon fusion.Figure 48 C has shown expression/secretion of NY-ESO-1, uses the construct that comprises the codon optimization LLO signal peptide that merges with the optimized NY-ESO-1 of the plain codon of people's mesothelium.
Figure 49 has shown the coded sequence (SEQ ID NO:39) of phEphA2 KD.
Figure 50 has shown the sub-fragment of Mlu I (SEQ ID NO:40) of the optimized people EphA2 of the codon that comprises the actA-plcB intergenic region.
Figure 51 has shown the terminal amino acid whose sequence of p60 (SEQ IDNO:41) of hly promoter-70N-.
Figure 52 has shown the sub-fragment of KpnI-BamHI (SEQ ID NO:42) of plasmid pPL2-hlyP-Np60 CodOp (1-77).
Figure 53 has shown the sub-fragment of KpnI-BamHI (SEQ ID NO:43) of plasmid pPL2-hlyP-Np60 CodOp (1-77)-mesothelium element.
Figure 54 has shown the sub-fragment of KpnI-BamHI (SEQ ID NO:44) of the plain Δ SP/ of plasmid pPL2-hlyP-Np60 CodOp (1-77)-mesothelium Δ GPI.
Figure 55 has shown from antigenic expression that comprises the chimeric reorganization Listera of antigen-bacterioprotein and excretory western blot analysis.
Figure 56 has shown the expressed protein engram analysis from EphA2 born of the same parents' intracellular domain (ICD) of bicistronic mRNA information.
Figure 57 has shown that the conduct of proof in the different bacterium part and the N-of various codon optimization signal peptides hold the function that merges to express and excretory western blot analysis based on the Mus mesothelium of plasmid is plain: excretory protein (Figure 57 A); Cell wall (Figure 57 B); And cell lysate (Figure 57 C).
Figure 58 has shown in the Listeria monocytogenes expression and the excretory western blot analysis based on chromosomal people's mesothelium element.Shown the plain expressed protein engram analysis of mesothelium in the various bacterial cells part and from the result of the Listera of the mesothelium element of signal sequence expression shown in contrast Listera (the mesothelium element of not encoding) and the coding.
Figure 59 A and 59B have shown that heterologous antigen (AH1-A5) is by the diagram of Listera vaccine delivery to MHC I classpath.The Listera vaccine comprises Listera (Figure 59 A) of expressing p60-AH1-A5 protein chimera (being embedded in the AH1-A5 among the p60) or the Listera (Figure 59 B) of expressing the fusion rotein that comprises LLO signal peptide and AH1-A5.
Figure 60 A and 60B have shown the diagram of sending of the antibacterial specific antigen of Listera vaccine mediation to MHC I classpath, wherein this vaccine comprises Listera (Figure 60 A) of expressing p60-AH1-A5 protein chimera (being embedded in the AH1-A5 among the p60) or the Listera (Figure 60 B) of expressing the fusion rotein that comprises LLO signal peptide and AH1-A5, and wherein add based on the test peptides in the mensuration of cell and be no test peptides (not stimulating) (Figure 60 A), LLO
91-99(Figure 60 A), no test peptides (Figure 60 B) or p60
217-225(Figure 60 B).
The diagram of the therapeutic efficiency that Figure 61 is the Listera that shown expressing human mesothelium element in the vaccinated animal that has a tumor, wherein with the tumor cell through engineering approaches with expressing human mesothelium element.
Figure 62 is the diagram that has shown that in the mice that has tumor of the Listera of having inoculated expressing human mesothelium element lung tumor tuberosity level reduces, wherein with the tumor cell through engineering approaches with expressing human mesothelium element.
Figure 63 is the diagram that has shown the comparative study of using CT.26 parent target cell, that is, cell does not have through engineering approaches with expressing human mesothelium element, and the antitumor efficacy that has proved Lm-Meso vaccination is that the mesothelium element is specific.
Figure 64 has shown to have inoculated the diagram that the Listera of expressing codon optimization people mesothelium element has reduced gross tumor volume.
Figure 65 has shown the immunogenic ELISPOT result of experiment that shows the plain bacterial strain of Listera Δ actA/ Δ inlB-h mesothelium, and the nucleic acid of people's mesothelium element of wherein encoding has been integrated in the genome of Listera.
Detailed Description Of The Invention
I. introduction
The invention provides multiple polynucleotides, comprise for expressing at bacterium such as Listera and/or secrete polypeptide comprises recombinant nucleic acid molecules, expression cassette and the expression vector of heterologous polypeptide (for example, antigen and/or mammalian proteins). In some embodiments, these polynucleotides can be used for improving expression and/or the secretion of bacterium polypeptide. Some expression cassettes comprise the coded sequence of the optimized polypeptide of codon and/or signal peptide. In addition, some expression cassettes that are used for bacterium comprise be derived from other bacterial origins and/or from the signal peptide sequence of multiple different secretory pathways. The bacterium that comprises expression cassette also is provided, and the composition that comprises this bacterium, such as vaccine. The method of using these polynucleotides, bacterium and composition to come induce immune response and/or prevention or treatment host's illness such as disease (for example, cancer) also is provided.
The present invention is based in part on following discovery: the codon optimization of signal peptide sequence can improve expression and/or the secretion (especially with the codon optimization of this heterologous polypeptide be combined) of heterologous polypeptide (such as antigen) in recombinant bacteria in the expression cassette, even signal peptide sequence be bacterium intrinsic in (referring to, for example, following embodiment 19 and 27). In addition, have been found that from the signal peptide sequence of non--secA1 secretory pathway and/or from the signal peptide sequence in non-Listera source also can be used for realizing heterologous polypeptide the effective expression of Listera and/or secretion (referring to, for example, following embodiment 19,27 and 30). The present invention also part based on following other discovery: the codon optimization of heterologous polypeptide coded sequence can improve the expression of heterologous polypeptide in the Listera and/or secretion (referring to, for example, following embodiment 19). Shown that also expression that heterologous protein that the optimization by expression cassette obtains improves and/or secretion cause comprising the immunogenicity (referring to, for example, following embodiment 20) of raising of the bacterium of optimization expression cassette. In addition, having shown that coding comprises that the chimeric expression cassette of protein that is embedded in the heterologous antigen in the autolysin can be used for realizing the effective expression of heterologous antigen in the Listera and secretion (referring to, for example, following embodiment 29). Shown that also autolysin protein chimera is immunogenic (referring to, for example, following embodiment 31A). In addition, also shown the Listera that comprises the optimized expression cassette of codon and/or comprised that the expression cassette of non-Listera signal peptide is immunogenic in mouse model, reduce gross tumor volume and improved survival (referring to, for example, following embodiment 31B-E).
Therefore, on the one hand, the invention provides recombinant nucleic acid molecules, first polynucleotides that comprise the code signal peptide, wherein first polynucleotides are that codon is optimized for the expression in bacterium, and second polynucleotides of coded polypeptide (for example, antigen), wherein second polynucleotides is arranged in the translation frame identical with first polynucleotides, and wherein recombinant nucleic acid molecules coding comprises the fusion of signal peptide and polypeptide. In some embodiments, second polynucleotides also are codon optimized (being often used in expressing in the bacterium with first polynucleotides same type). In some embodiments, first polynucleotides or first and second polynucleotides are at Listerial, bacillus (Bacillus), Yersinia pestis (Yersinia pestis), Salmonella (Salmonella), Shigella (Shigella), Brucella (Brucella), the expression in mycobacterium or the Escherichia coli (E.coli) is that codon is optimized. In some embodiments, polynucleotides are that codon is optimized for the expression in Listerial such as monocyte Listeria monocytogenes. In some embodiments, be (or comprising) antigen by the polypeptide of second polynucleotide encoding, in some cases, it can be abacterial antigen. For example, in some embodiments, antigen is tumor associated antigen or is derived from such tumor associated antigen. For example, in some embodiments, antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA. For example, in some embodiments, antigen is mesothelin, or the antigen fragment of mesothelin or antigenic variant. In some other embodiments, antigen is NY-ESO-1, or the antigen fragment of NY-ESO-1 or antigenic variant. In some embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, signal peptide is bacterium (Listera Listera or non-). In some embodiments, be that bacterium is intrinsic by the signal peptide of optimized first polynucleotide encoding of codon. In other embodiments, be external source by the signal peptide of optimized first polynucleotide encoding of codon to bacterium. In some embodiments; signal peptide is the secA1 signal peptide; such as the LLO signal peptide from monocyte Listeria monocytogenes, from the Usp45 signal peptide of Lactococcus lactis (Lactococcus lactis), or from the protective antigens signal peptide of Bacillus anthracis. In some embodiments, signal peptide is the secA2 signal peptide. For example, signal peptide can be the p60 signal peptide from monocyte Listeria monocytogenes. In addition, recombinant nucleic acid molecules randomly comprises the polynucleotide sequence of coding p60 or its fragment, be arranged in and first and second translation frame that polynucleotides are identical, wherein second polynucleotides are in the 3rd polynucleotides or between first and the 3rd polynucleotides. In the other embodiments, signal peptide is the Tat signal peptide, such as bacillus subtilis (B.subtilis) Tat signal peptide (for example, PhoD). The present invention also provides and has comprised recombinant nucleic acid molecules and further comprise and (for example be operably connected recombinant nucleic acid molecules, the expression cassette that connects the promoter of first and second polynucleotides (and the 3rd polynucleotides, if present)). The expression vector and the recombinant bacteria (for example, Listera) that comprise this expression cassette also are provided, and the pharmaceutical composition that comprises this bacterium, immunogenic composition and vaccine. The method of coming induce immune response and/or prevention or treatment illness such as disease with this bacterium or the composition that comprises this bacterium also is provided. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the second aspect, the invention provides recombinant nucleic acid molecules, first polynucleotides that comprise the signal peptide that (a) coding bacterium is intrinsic, wherein first polynucleotides are that codon is optimized for the expression in this bacterium, (b) second of coded polypeptide polynucleotides, wherein second polynucleotides is arranged in the translation frame identical with first polynucleotides, and wherein the recombinant nucleic acid molecules coding comprises the fusion of signal peptide and polypeptide. In some embodiments, be allos by polypeptide and the signal peptide of second polynucleotide encoding. In some embodiments, second polynucleotides and first polynucleotides are allos. In some embodiments, polypeptide is that its intrinsic bacterium is external source to signal peptide. In some embodiments, being allos by polypeptide and the signal peptide of second polynucleotide encoding, is external source to bacterium, or both. In some embodiments, the bacterium that produces signal peptide is intracellular bacteria. In some embodiments, bacterium is selected from Listerial, bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacterium and Escherichia coli. In some embodiments, bacterium is Listerial bacterium (for example, monocyte Listeria monocytogenes). In some embodiments, second polynucleotides is that codon is optimized for the expression in bacterium. In some embodiments, the codon optimization of first and/or second polynucleotides has improved the expression of coded fusion in bacterium and/or the secretion from bacterium (with respect to the optimized sequence of non-codon). In some embodiments, comprise antigen by the polypeptide of second polynucleotide encoding. Polypeptide by second polynucleotide encoding is antigen. In some embodiments, antigen is non-bacterial antigens. In some embodiments, antigen is tumor associated antigen or comprises the antigen that is derived from tumor associated antigen. In some embodiments, antigen is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA. For example, in some embodiments, antigen is mesothelin, or its antigen fragment or variant, or NY-ESO-1, or its antigen fragment or variant. In some alternate embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, signal peptide is the secA1 signal peptide LLO signal peptide of monocyte Listeria monocytogenes (for example, from). In some embodiments, signal peptide is secA2 signal peptide (for example, the p60 signal peptide of monocyte Listeria monocytogenes). The expression cassette of promoter that comprises this recombinant nucleic acid molecules and further comprise first and second polynucleotides of the recombinant nucleic acid molecules that is operably connected also is provided. The expression vector that comprises this expression cassette also is provided. The recombinant bacteria that comprises this recombinant nucleic acid molecules also is provided, and wherein first polynucleotides are that codon is optimized for the expression in recombinant bacteria. In some embodiments, recombinant bacteria is intracellular bacteria. In some embodiments, recombinant bacteria is selected from Listerial, bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacterium and Escherichia coli. In some embodiments, bacterium is restructuring Listerial bacterium (for example, the monocyte Listeria monocytogenes of restructuring). The immunogenic composition that comprises this recombinant bacteria also is provided, and wherein the polypeptide by second polynucleotide encoding is antigen. Also provide and induced the host to the method for the immune response of antigen, comprised that the composition that comprises recombinant bacteria with effective dose gives the patient, wherein the polypeptide by second polynucleotide encoding is (or comprising) antigen. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the third aspect, the invention provides comprise recombinant nucleic acid molecules the Listerial bacterium (for example, monocyte Listeria monocytogenes), wherein this recombinant nucleic acid molecules comprises first polynucleotides of (a) code signal peptide, wherein first polynucleotides are that codon is optimized for the expression in the Listerial bacterium, (b) second of coded polypeptide polynucleotides, wherein second polynucleotides is arranged in the translation frame identical with first polynucleotides, and wherein this recombinant nucleic acid molecules coding comprises the fusion of signal peptide and polypeptide. In some embodiments, be allos by polypeptide and the signal peptide of second polynucleotide encoding. In some embodiments, recombinant nucleic acid molecules is the part of expression cassette that further comprises the promoter of be operably connected first and second polynucleotides. In other words, in some embodiments, this restructuring Listerial bacterium comprises the expression cassette that contains this recombinant nucleic acid molecules, and wherein this expression cassette further comprises the promoter of first and second polynucleotides of the recombinant nucleic acid molecules that is operably connected. In some embodiments, expression cassette is the polycistronic expression box. In some embodiments, second polynucleotides is that codon is optimized for the expression in the Listerial bacterium. In some embodiments, the codon optimization of first and/or second polynucleotides has improved expression and/or the secretion from Listerial bacterium (with respect to non-codon optimized sequence) of coded fusion in the Listerial bacterium. In some embodiments, be external source (that is being allos with the Listerial bacterium) by the polypeptide of second polynucleotide encoding to the Listerial bacterium. In some embodiments, comprise antigen (for example, non-Listera antigen or non-bacterial antigens) by the polypeptide of second polynucleotide encoding. In some embodiments, be antigen by the polypeptide of second polynucleotide encoding. In some embodiments, antigen is tumor associated antigen or is derived from tumor associated antigen. In some embodiments, antigen is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA. For example, in some embodiments, antigen is mesothelin, or its antigen fragment or antigenic variant. In some embodiments, antigen is people's mesothelin. In some embodiments, antigen is people's mesothelin of its signal peptide of disappearance and GPI link field. In some alternate embodiments, antigen is NY-SEO-1, or its antigen fragment or antigenic variant. In some alternate embodiments, antigen is infectious disease antigen or the antigen that is derived from infectious disease antigen. In some embodiments, signal peptide is non-Listera. In some embodiments, signal peptide is bacterium. In some embodiments, signal peptide is external source to the Listerial bacterium. In other embodiments, signal peptide is that Listera is intrinsic. In some embodiments, signal peptide is secA1 signal peptide (for example, from the LLO signal peptide of monocyte Listeria monocytogenes, from the Usp45 signal peptide of Lactococcus lactis, with from the protective antigens signal peptide of Bacillus anthracis). In some embodiments, signal peptide is the secA2 signal peptide p60 signal peptide of monocyte Listeria monocytogenes (for example, from). In some embodiments, signal peptide is Tat signal peptide (for example, from bacillus subtilis PhoD signal peptide). In some embodiments, the Listerial bacterium is attenuation. For example, the Listera attenuation cell and intercellular diffusion be can be used for, non-phagocytic cell or propagation entered. In some embodiments, restructuring Listerial bacterium is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ actA Δ inlB). In some embodiments, restructuring Listerial bacterium lacks functional ActA, Internalin B, or ActA and Internalin B. In some embodiments, the nucleic acid of recombinant bacteria is by being modified with nucleic acid target compound (for example, psoralen compound) reaction. The present invention also provides the pharmaceutical composition that comprises restructuring Listerial bacterium and pharmacological-acceptable carrier, and the immunogenic composition that comprises restructuring Listerial bacterium, and wherein the polypeptide by second polynucleotide encoding is antigen. The present invention also provides the vaccine that comprises restructuring Listerial bacterium. Also provide and induced the host to the method for the immune response of antigen, comprised that the composition that comprises this recombinant bacteria with effective dose gives the host, wherein the polypeptide by second polynucleotide encoding is (or comprising) antigen. The method of prevention or treatment host's illness (for example, disorders such as cancers or infectious disease) also is provided, has comprised that the composition that comprises this restructuring Listerial bacterium with effective dose gives the host. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the fourth aspect, the invention provides recombinant nucleic acid molecules, (for example comprise first polynucleotides of the non-secA1 bacterium signal peptide of encoding and coded polypeptide, antigen) second polynucleotides, wherein second polynucleotides is arranged in the translation frame identical with first polynucleotides, and wherein this recombinant nucleic acid molecules coding comprises the fusion of signal peptide and polypeptide. In some embodiments, first polynucleotides and/or second polynucleotides are that codon is optimized for the expression in the particular type bacterium. In some embodiments, the codon optimization of first and/or second polynucleotides has improved the expression of fusion in bacterium and/or the secretion from bacterium (with respect to the optimized sequence of non-codon). In some embodiments, first polynucleotides and/or second polynucleotides are at Listerial, bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, the expression in mycobacterium or the Escherichia coli are that codon is optimized. In some embodiments, polynucleotides are that codon is optimized for the expression in Listerial such as monocyte Listeria monocytogenes. In some embodiments, be that the optimized bacterium of codon is intrinsic by the signal peptide of optimized first polynucleotide encoding of codon. In some embodiments, first polynucleotides of code signal peptide and second polynucleotides are allos. In some embodiments, be allos by polypeptide and the signal peptide of second polynucleotide encoding. In some embodiments, comprise antigen by the polypeptide of second polynucleotide encoding. In some embodiments, be antigen by the polypeptide of second polynucleotide encoding, in some cases, it can be non-bacterial antigens. In some embodiments, antigen is tumor associated antigen or is derived from such tumor associated antigen. For example, in some cases, antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA. For example, in some embodiments, antigen is mesothelin, or the antigen fragment of mesothelin or antigenic variant. In some other embodiments, antigen is NY-ESO-1, or the antigen fragment of NY-SEO-1 or antigenic variant. In some embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, be Listera by the signal peptide of first polynucleotide encoding of recombinant nucleic acid molecules. In other embodiments, signal peptide is non-Listera. In some embodiments, signal peptide is derived from gram-positive bacterium. In some embodiments, signal peptide is derived from and belongs to bacillus, the bacterium of staphylococcus (Staphylococcus) or lactococcus. In some embodiments, signal peptide is the secA2 signal peptide. For example, signal peptide can be the p60 signal peptide from monocyte Listeria monocytogenes. In addition, recombinant nucleic acid molecules randomly comprises the 3rd polynucleotides of coding p60 or its fragment, be arranged in and first and second translation frame that polynucleotides are identical, wherein second polynucleotides are in the 3rd polynucleotides or between first and the 3rd polynucleotides. In the other embodiments, signal peptide is the Tat signal peptide, such as bacillus subtilis Tat signal peptide (for example, bacillus subtilis PhoD signal peptide). The present invention also provides the expression cassette of promoter that comprises recombinant nucleic acid molecules and further comprise first and second polynucleotides of the recombinant nucleic acid molecules that is operably connected. The expression vector and the bacterium that comprise this expression cassette and/or recombinant nucleic acid molecules also are provided, and the pharmaceutical composition that comprises this bacterium, immunogenic composition and vaccine. In some embodiments, the recombinant bacteria that comprises this expression cassette or recombinant nucleic acid molecules is intracellular bacteria. In some embodiments, bacterium is to be selected from Listerial, bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacterium or colibacillary bacterium. In some embodiments, bacterium is Listerial bacterium (for example, the member of monocyte Listeria monocytogenes kind). In some embodiments, be external source (that is being allos with bacterium) by the polypeptide of second polynucleotide encoding to bacterium. The method of coming induce immune response and/or prevention or treatment host's illness (for example, disease) with this bacterium or the composition that comprises this bacterium also is provided. In some embodiments, illness is cancer. In other embodiments, illness is infectious disease. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
On the other hand, the invention provides the restructuring Listerial bacterium that comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotides of the non-secA1 bacterium signal peptide of (a) coding, (b) second of coded polypeptide polynucleotides, wherein second polynucleotides is arranged in the translation frame identical with first polynucleotides, wherein the recombinant nucleic acid molecules coding comprises the fusion of signal peptide and polypeptide, in some embodiments, by the polypeptide of second polynucleotide encoding and signal peptide be allos or be external source to bacterium, or both. In some embodiments, the Listerial bacterium belongs to the monocyte Listeria monocytogenes kind. In some embodiments, recombinant nucleic acid molecules is the part of expression cassette, and this expression cassette further comprises the promoter of be operably connected first and second polynucleotides. In other words, in some embodiments, restructuring Listerial bacterium comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein this expression cassette further comprises the promoter of first and second polynucleotides of the recombinant nucleic acid molecules that is operably connected. In some embodiments, expression cassette is the polycistronic expression box. In some embodiments, first polynucleotides, second polynucleotides, or first is that codon is optimized with second polynucleotides for the expression in Listerial bacterium (for example, monocyte Listeria monocytogenes). In some embodiments, the codon optimization of first and/or second polynucleotides has improved expression and/or the secretion from this bacterium (with respect to non-codon optimized sequence) of fusion in this bacterium. In some embodiments, be allos each other by first and second polynucleotides. In some embodiments, be allos each other by the polypeptide of second polynucleotide encoding and signal peptide. In some embodiments, be external source (that is being allos with the Listerial bacterium) by the polypeptide of second polynucleotide encoding to the Listerial bacterium. In some embodiments, comprise antigen by the polypeptide of second polynucleotide encoding. In some embodiments, be antigen (for example, non-Listera or non-bacterial antigens) by the polypeptide of second polynucleotide encoding. In some embodiments, antigen is tumor associated antigen or is derived from tumor associated antigen. In some embodiments, antigen is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA. For example, in some embodiments, antigen is mesothelin, or its antigen fragment or antigenic variant. In some embodiments, antigen is people's mesothelin. In some embodiments, antigen is people's mesothelin of its signal peptide of disappearance and GPI link field. In some alternate embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, signal peptide is non-Listera. In some embodiments, non-secA1 signal peptide is the Listera signal peptide. In other embodiments, non-secA1 signal peptide is non-Listera signal peptide. In some embodiments, signal peptide is the secA2 signal peptide p60 signal peptide of monocyte Listeria monocytogenes (for example, from). In some embodiments, the recombinant nucleic acid molecules that comprises the secA2 signal peptide (for example further comprises coding secA2 autolysin, p60 or-acetylmuramic acid enzyme) or its fragment is (for example, the catalytic activity fragment) the 3rd polynucleotides, be arranged in and first and second translation frame that polynucleotides are identical, wherein second polynucleotides are in the 3rd polynucleotides of recombinant nucleic acid molecules or between first and the 3rd polynucleotides. In some embodiments, second polynucleotides is positioned at the 3rd polynucleotides. In some embodiments, signal peptide is the Tat signal peptide. In some embodiments, signal peptide is the Tat signal peptide that is derived from bacillus subtilis (for example, from bacillus subtilis PhoD signal peptide). In some embodiments, Listera is attenuation. For example, the Listera attenuation can be used for cell and intercellular diffusion, enter in the non-phagocytic cell or propagation. In some embodiments, the restructuring Listera is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ act Δ AinlB). In some embodiments, restructuring Listerial bacterium is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ actA Δ inlB). In some embodiments, the nucleic acid of recombinant bacteria is by being modified with nucleic acid target compound (for example, psoralen compound) reaction. The present invention also provides and has comprised the pharmaceutical composition that can accept carrier on restructuring Listerial bacterium and the materia medica. The present invention also provides the immunogenic composition that comprises recombinant bacteria, and wherein the polypeptide by second polynucleotide encoding is antigen. The present invention also provides the vaccine that comprises restructuring Listerial bacterium. Also provide and induced the host to the method for the immune response of antigen, comprised that the composition that contains recombinant bacteria with effective dose gives the host, wherein the polypeptide by second polynucleotide encoding is (or comprising) antigen. The method of prevention or treatment host's illness (for example, disorders such as cancers or infectious disease) also is provided, has comprised that the composition that contains restructuring Listerial bacterium with effective dose gives the host. Also provide bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
On the other hand, the invention provides and comprise that coding Listerial bacterium allogenic polypeptide is (such as antigen, such as cancer antigen or non-Listera antigen) the recombinant nucleic acid molecules of polynucleotides, wherein polynucleotides are that codon is optimized for the expression in Listera. In some embodiments, the codon optimization of polynucleotides has improved expression and/or the secretion from Listerial bacterium (with respect to non-codon optimized sequence) of polypeptide in the Listerial bacterium. In some embodiments, allogenic polypeptide comprises antigen. In some embodiments, allogenic polypeptide is antigen. In some embodiments, antigen is non-bacterial antigens. For example, in some embodiments, antigen is tumor associated antigen or is derived from such tumor associated antigen. In some embodiments, polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA. In some embodiments, antigen is mesothelin, or the antigen fragment of mesothelin or antigenic variant. In some other embodiments, antigen is NY-ESO-1, or the antigen fragment of NY-ESO-1 or antigenic variant. In some embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, recombinant nucleic acid molecules comprises that further coding is arranged in the polynucleotides of the signal peptide of the translation frame identical with allogenic polypeptide, so that the recombinant nucleic acid molecules coding comprises the fusion of signal peptide and allogenic polypeptide. In some embodiments, the polynucleotides of code signal peptide (it is can yes or no natural to Listera) are that codon is optimized for the expression in monocyte Listeria monocytogenes. The present invention further provides the expression cassette of promoter that comprises recombinant nucleic acid molecules and further comprise first and second polynucleotides of the recombinant nucleic acid molecules that is operably connected. The carrier that comprises recombinant nucleic acid molecules and/or expression cassette (for example, expression vector) also is provided. The present invention also provides the restructuring Listerial that comprises recombinant nucleic acid molecules and/or expression cassette bacterium. In some embodiments, the Listerial bacterium belongs to the monocyte Listeria monocytogenes kind. The pharmaceutical composition, immunogenic composition and the vaccine that comprise restructuring Listerial bacterium also are provided. The present invention further provides and induced the host to the method for the immune response of antigen, comprised that the composition that contains restructuring Listerial bacterium with effective dose gives the patient, wherein polypeptide is (or comprising) antigen. In addition, the invention provides the method for coming induce immune response and/or prevention or treatment illness (for example, disease) with restructuring Listerial bacterium. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein allogenic polypeptide comprises antigen.
On the other hand, the invention provides the restructuring Listerial bacterium that comprises expression cassette, wherein expression cassette comprises that coding Listerial bacterium allogenic polypeptide is (such as antigen, such as cancer antigen or non-Listera antigen) polynucleotides and the promoter of polynucleotides of the encoding exogenous polypeptide that is operably connected, wherein polynucleotides are that codon is optimized for the expression in Listera. In some embodiments, the Listerial bacterium belongs to the monocyte Listeria monocytogenes kind. In some embodiments, the codon optimization of polynucleotides has improved expression and/or polypeptide the secretion (with respect to non-codon optimized sequence) from Listerial bacterium of polypeptide in the Listerial bacterium. In some embodiments, allogenic polypeptide comprises antigen. In some embodiments, allogenic polypeptide is antigen, and in some cases, it can be non-bacterial antigens. For example, in some embodiments, antigen is tumor associated antigen or is derived from such tumor associated antigen. For example, in some embodiments, polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA. In some embodiments, antigen is mesothelin, or the antigen fragment of mesothelin or antigenic variant. In some other embodiments, antigen is NY-ESO-1, or the antigen fragment of NY-ESO-1 or antigenic variant. In some embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, expression cassette further comprises the polynucleotides promoter that is operably connected and that be arranged in the code signal peptide of the translation frame identical with allogenic polypeptide, so that the expression cassette coding comprises the fusion of signal peptide and allogenic polypeptide. In some embodiments, the polynucleotides of code signal peptide (it is can yes or no natural for Listera) are that codon is optimized for the expression in monocyte Listeria monocytogenes. The pharmaceutical composition, immunogenic composition and the vaccine that comprise restructuring Listerial bacterium also are provided. The present invention further provides and induced the host to the method for the immune response of antigen, comprised that the composition that contains restructuring Listerial bacterium with effective dose gives the patient. In addition, the invention provides the method for coming induce immune response and/or prevention or treatment illness (for example, disease) with this restructuring Listerial bacterium. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein allogenic polypeptide comprises antigen.
In the other aspect, the invention provides the restructuring Listerial bacterium (for example monocyte Listeria monocytogenes) that comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotides of the non-Listera signal peptide of (a) coding; (b) be arranged in second polynucleotides of the coded polypeptide of the translation frame identical with first polynucleotides, wherein the recombinant nucleic acid molecules coding comprises the recombinant protein of non-Listera signal peptide and polypeptide. In some embodiments, recombinant nucleic acid molecules is positioned at the expression cassette of the promoter that further comprises be operably connected first and second polynucleotides. Therefore, in some embodiments, restructuring Listerial bacterium comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein expression cassette further comprises the promoter of first and second polynucleotides of the recombinant nucleic acid molecules that is operably connected. In some embodiments, this expression cassette is polycistronic expression box (for example, bicistronic mRNA expression cassette). In some embodiments, first polynucleotides, second polynucleotides, or first is that codon is optimized with second polynucleotides for the expression in Listera (for example, monocyte Listeria monocytogenes). In some embodiments, the codon optimization of first and/or second polynucleotides has improved expression and/or fusion the secretion (with respect to non-codon optimized sequence) from bacterium of fusion in bacterium. In some embodiments, first and second polynucleotides are allos each other. In some embodiments, be allos each other by the polypeptide of second polynucleotide encoding and signal peptide. In some embodiments, be external source (that is being allos with the Listerial bacterium) by the polypeptide of second polynucleotide encoding to the Listerial bacterium. In some embodiments, comprise antigen (for example, non-Listera antigen) by the polypeptide of second polynucleotide encoding. In some embodiments, be antigen by the polypeptide of second polynucleotide encoding. In some embodiments, antigen is tumor associated antigen or is derived from tumor associated antigen. In some embodiments, antigen is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA. For example, in some embodiments, antigen is mesothelin, or its antigen fragment or antigenic variant. In some embodiments, antigen is people's mesothelin. In some embodiments, antigen is people's mesothelin of its signal peptide of disappearance and GPI link field. In some alternate embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, signal peptide is bacterium. In some embodiments, signal peptide is derived from intracellular bacteria. In some embodiments, signal peptide is derived from gram-positive bacterium. In some embodiments, signal peptide is from belonging to bacillus, the bacterium of staphylococcus or lactococcus (for example, Bacillus anthracis, bacillus subtilis, staphylococcus aureus or Lactococcus lactis). In some embodiments, signal peptide is seaA1 signal peptide (for example, from the Usp45 signal peptide of Lactococcus lactis or from the protective antigens signal peptide of Bacillus anthracis). In some embodiments, signal peptide is the secA2 signal peptide. In some embodiments, signal peptide is Tat signal peptide (for example, from bacillus subtilis PhoD signal peptide). In some embodiments, the Listerial bacterium is attenuation. For example, in some embodiments, the Listera attenuation is used for cell and intercellular diffusion, enters in the non-phagocytic cell or propagation. In some embodiments, restructuring Listerial bacterium is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ actA Δ inlB). In some embodiments, the restructuring Listera is to lack functional ActA, Internalin B, or ActA and Internalin B. In some embodiments, the nucleic acid of recombinant bacteria is by being modified with nucleic acid target compound (for example, psoralen compound) reaction. The present invention also provides and has comprised the pharmaceutical composition that can accept carrier on restructuring Listerial bacterium and the materia medica. The present invention also provides the immunogenic composition that comprises this recombinant bacteria, and wherein the polypeptide by second polynucleotide encoding is antigen. The present invention also provides the vaccine that comprises restructuring Listerial bacterium. Also provide and induced the host to the method for the immune response of antigen, comprised that the composition that contains restructuring Listerial bacterium with effective dose gives the host, wherein the polypeptide by second polynucleotide encoding is (or comprising) antigen. The method of prevention or treatment host illness (for example disease, such as cancer or infectious disease) also is provided, has comprised that the composition that contains restructuring Listerial bacterium with effective dose gives the host. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
Again in the one side, the invention provides comprise expression cassette restructuring Listerial bacterium (for example, from the monocyte Listeria monocytogenes kind), this expression cassette comprises first polynucleotides of the non-Listera signal peptide of encoding, be arranged in second polynucleotides of the coded polypeptide of the translation frame identical with first polynucleotides, and the promoter of be operably connected first and second polynucleotides. This expression cassette coding comprises the fusion of non-Listera signal peptide and polypeptide. In some embodiments, Listerial bacterium attenuation is used for cell and intercellular diffusion, enters in the non-phagocytic cell or propagation. In some embodiments, first polynucleotides, second polynucleotides, or first is that codon is optimized with second polynucleotides for the expression in Listera. In some embodiments, the codon optimization of first and/or second polynucleotides has improved expression and/or the secretion from this bacterium (with respect to non-codon optimized sequence) of coded fusion in this bacterium. In some embodiments, first polynucleotides and/or second polynucleotides are that codon is optimized for the expression in monocyte Listeria monocytogenes. In some embodiments, comprise antigen by the polypeptide of second polynucleotide encoding. In some embodiments, be antigen by the polypeptide of second polynucleotide encoding, in some cases, it is non-bacterial antigens. For example, in some embodiments, antigen is tumor associated antigen or is derived from such tumor associated antigen. For example, in some embodiments, antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from K-Ras, H-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA. For example, in some embodiments, antigen is mesothelin, or mesothelin antigen fragment or antigenic variant. In some other embodiments, antigen is NY-ESO-1, or the antigen fragment of NY-ESO-1 or antigenic variant. In some embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In the preferred embodiment, signal peptide is bacterium. In some embodiments, signal peptide is from belonging to bacillus, the bacterium of staphylococcus or lactococcus. For example, in some embodiments, signal peptide is from Bacillus anthracis, bacillus subtilis, staphylococcus aureus or Lactococcus lactis. In some embodiments, signal peptide is the secA1 signal peptide, such as the Usp45 signal peptide from Lactococcus lactis, or from the protective antigens signal peptide of Bacillus anthracis. In some embodiments, signal peptide is the secA2 signal peptide. In the other embodiments, signal peptide is the Tat signal peptide, such as bacillus subtilis Tat signal peptide (for example, PhoD). The pharmaceutical composition, immunogenic composition and the vaccine that comprise restructuring Listerial bacterium described herein also are provided. In addition, the invention provides the method for coming induce immune response and/or prevention or treatment illness such as disease with restructuring Listerial bacterium. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
The present invention further provides recombinant nucleic acid molecules, comprised first polynucleotides of (a) coding bacterium autolysin or its catalytic activity fragment or catalytic activity variant; (b) second of coded polypeptide polynucleotides, wherein second polynucleotides is positioned at the translation frame identical with first polynucleotides, wherein the recombinant nucleic acid molecules coding comprises by the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant, wherein in the protein chimera, polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or are positioned at autolysin or its catalytic activity fragment or catalytic activity variant. In some embodiments, first polynucleotide encoding bacterium autolysin. In some embodiments, the protein chimera has the catalytic activity the same with autolysin. In some embodiments, the bacterium autolysin is from intracellular bacteria (for example, Listera). In some embodiments, the bacterium autolysin is the Listera autolysin. In some embodiments, second polynucleotides of coded polypeptide are positioned at first polynucleotides of coding autolysin or its catalytic activity fragment or catalytic activity variant, and encode polypeptide wherein of recombinant nucleic acid molecules is positioned at the protein chimera (that is, polypeptide is embedded in autolysin or its catalytic activity fragment or the variant) of autolysin or its catalytic activity fragment or catalytic activity variant. In some alternate embodiments, second polynucleotides are positioned at outside first polynucleotides of coding autolysin or its catalytic activity fragment or catalytic activity variant, and the recombinant nucleic acid molecules wherein protein chimera of polypeptide and autolysin or its catalytic activity fragment or the fusion of catalytic activity variant of encoding. In some embodiments, polypeptide and autolysin are allos. In some embodiments, first polynucleotides and second polynucleotides are allos each other. In some embodiments, recombinant nucleic acid molecules comprises that further (c) is arranged in the 3rd polynucleotides of the code signal peptide of the translation frame identical with first and second polynucleotides, wherein the recombinant nucleic acid molecules coding comprises signal peptide, by the polypeptide of second polynucleotide encoding, and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant. In some embodiments, signal peptide is secA2 signal peptide (such as p60). In some embodiments, signal peptide is actually the signal peptide (for example, signal peptide be p60 and autolysin be p60) relevant with autolysin. In some embodiments, autolysin is secA2 dependence autolysin. In some embodiments, autolysin is peptide glycan hydrolase (for example,-acetylmuramic acid enzyme or p60). In some embodiments, comprise antigen by the polypeptide of second polynucleotide encoding. In some embodiments, polypeptide is antigen (for example, tumor associated antigen is derived from the antigen of tumor associated antigen, infectious disease antigen, or be derived from the antigen of infectious disease antigen). In some embodiments, antigen is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA. For example, in some embodiments, antigen is mesothelin, or its antigen fragment or antigenic variant. In some embodiments, antigen is people's mesothelin. In some embodiments, antigen is people's mesothelin of its signal peptide of disappearance and GPI anchor. The present invention also provides the expression cassette of promoter that comprises recombinant nucleic acid molecules and further comprise first and second polynucleotides of the recombinant nucleic acid molecules that is operably connected, and the expression vector that comprises this expression cassette. The present invention further provides the recombinant bacteria that comprises recombinant nucleic acid molecules. In some embodiments, recombinant bacteria is intracellular bacteria, such as Listerial bacterium (for example, monocyte Listeria monocytogenes). In some embodiments, be external source by the polypeptide of second polynucleotide encoding to recombinant bacteria. The pharmaceutical composition that comprises (a) recombinant bacteria and (b) can accept carrier on the materia medica also is provided. In addition, also provide the immunogenic composition that comprises recombinant bacteria, wherein the polypeptide by second polynucleotide encoding is antigen. The vaccine that comprises recombinant bacteria also is provided, and wherein the polypeptide by second polynucleotide encoding is antigen. The present invention also provides and has induced the host to the method for the immune response of antigen, comprises that the composition that contains recombinant bacteria with effective dose gives the host, and wherein the polypeptide by second polynucleotide encoding is (or comprising) antigen. The method of prevention or treatment host illness also is provided, has comprised that the composition that contains recombinant bacteria with effective dose gives the host. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the one side, the invention provides the restructuring Listerial bacterium that comprises the polycistronic expression box again, wherein at least two discrete non-Listera polypeptide of polycistronic expression box coding. For example, in some embodiments, expression cassette comprises first polynucleotides of first non-Listera polypeptide of encoding, second polynucleotides of second non-Listera polypeptide of coding, and the promoter of be operably connected first and second polynucleotides. In some embodiments, expression cassette further comprises the intergenic sequence between first and second polynucleotides. In some embodiments, the polycistronic expression box is the bicistronic mRNA expression cassette, two discrete non-Listera polypeptide of its coding. In some embodiments, restructuring Listerial bacterium belongs to the monocyte Listeria monocytogenes kind. In some embodiments, at least one non-Listera polypeptide of being encoded by the polycistronic expression box comprises antigen. In some embodiments, at least two non-Listera polypeptide comprise the fragment of same antigen separately. In some embodiments, antigen is tumor associated antigen or is derived from tumor associated antigen. For example, in some embodiments, antigen is to be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA, or be derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA. In some embodiments, antigen is mesothelin, or its antigen fragment or antigenic variant. In some embodiments, antigen is people's mesothelin. In some embodiments, antigen is people's mesothelin of its signal peptide of disappearance and GPI anchor. In some embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen. In some embodiments, at least one non-Listera polypeptide of being encoded by the polycistronic expression box comprises signal peptide (Listera signal peptide or non-Listera signal peptide). In some embodiments, signal peptide is the secA1 signal peptide. In some embodiments, signal peptide is the secA2 signal peptide. In other embodiments, signal peptide is the Tat signal peptide. In some embodiments, expression cassette comprises the polynucleotides of code signal peptide, and wherein the polynucleotides of code signal peptide are that codon is optimized for the expression in Listera. The present invention also provides the pharmaceutical composition that comprises (a) restructuring Listerial bacterium and (b) can accept carrier on the materia medica. The immunogenic composition that comprises restructuring Listerial bacterium also is provided. The vaccine that comprises restructuring Listerial bacterium also is provided. Also provide and induced the host to the method for the immune response of antigen, comprised that the composition that contains restructuring Listerial bacterium with effective dose gives the host, wherein at least one non-Listera polypeptide comprises antigen. The method of prevention or treatment host illness also is provided, has comprised that the composition that contains restructuring Listerial bacterium with effective dose gives the host. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in manufacturing, wherein at least one the non-Listera polypeptide by polycistronic expression box coding comprises antigen.
On the other hand, the invention provides recombinant nucleic acid molecules, first polynucleotide molecule that comprises (a) code signal peptide, (b) second polynucleotides of coding secretory protein or its fragment, wherein second polynucleotides is positioned at the translation frame identical with first polynucleotides, (c) the 3rd of the polypeptide of coding and secretory protein or its fragment allos the polynucleotides, wherein the 3rd polynucleotides are positioned at and first and second translation frame that polynucleotides are identical, wherein the recombinant nucleic acid molecules coding comprises signal peptide, by the polypeptide of the 3rd polynucleotide encoding and the protein chimera of secretory protein or its fragment, and wherein in the protein chimera, by polypeptide and secretory protein or its segment composition of the 3rd polynucleotide encoding, or be positioned at secretory protein or its fragment. In some embodiments, secretory protein is natural secretory protein (that is the protein of, secreting from its n cell). In some embodiments, in recombinant nucleic acid molecules, the 3rd polynucleotides are positioned at second polynucleotides, and in the protein chimera by recombinant nucleic acid molecules coding, are positioned at secretory protein or its fragment by the polypeptide of the 3rd polynucleotide encoding. In some embodiments, in recombinant nucleic acid molecules, the 3rd polynucleotides are positioned at outside second polynucleotides, and in the protein chimera, by polypeptide and secretory protein or its segment composition of the 3rd polynucleotide encoding. Also provide and comprised recombinant nucleic acid molecules and further comprise first of the recombinant nucleic acid molecules that is operably connected, the expression cassette of the promoter of second and the 3rd polynucleotides. In some embodiments, comprise antigen by the polypeptide of second polynucleotide encoding. In some embodiments, be antigen by the polypeptide of second polynucleotide encoding. For example, in some embodiments, antigen is tumor associated antigen or is derived from tumor associated antigen and (for example, is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA, or be derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA). In some embodiments, antigen is mesothelin, or its antigen fragment or antigenic variant. For example, in some embodiments, antigen is people's mesothelin or the people's mesothelin that has lacked its signal peptide and GPI anchor. In the alternate embodiment, antigen is infectious disease antigen or is derived from infectious disease antigen. The expression vector that comprises expression cassette also is provided. The recombinant bacteria that comprises recombinant nucleic acid molecules also is provided. Also providing restructuring Listerial bacterium (for example, monocyte Listeria monocytogenes), and in some embodiments, is external source by the polypeptide of the 3rd nucleotide coding to the Listerial bacterium. The present invention also provides the immunogenic composition that comprises this recombinant bacteria, and wherein the polypeptide by the 3rd polynucleotide encoding is antigen. Also provide and induced the host to the method for the immune response of antigen, comprised that the composition that contains this recombinant bacteria with effective dose gives the host, wherein the polypeptide by the 3rd polynucleotide encoding is (or comprising) antigen. The pharmaceutical composition and the vaccine that comprise this bacterium also are provided, and with this recombinant bacteria or comprise that the composition of this bacterium prevents or treat the method for host's illness. Also provide this bacterium to induce the host to the purposes in the medicine of the immune response of antigen in preparation, wherein the polypeptide by the 3rd polynucleotide encoding comprises antigen.
In the other aspect, the invention provides improving one's methods of in host bacteria expression and secretion heterologous protein. The present invention also provides the method that improves the expression and secretion of foreign protei in bacterium. The present invention further provides preparation recombinant nucleic acid molecules described herein, expression cassette, the method for expression vector and recombinant bacteria.
The present invention also provides and has been used for the optimization heterologous polynucleotide at the multiple polynucleotides of the expression of bacterium such as Listera.
Be to be understood that the Ma Kushi group, Ma Kushi claim or by the embodiment described in " or set of symbols ", comprise the embodiment that each separates, the combination of the embodiment that each separates, and by each all embodiments of separating invention that form or that comprise them, unless stipulate clearly or by context in addition.
Further describing and other embodiments of the present invention and aspect of the above-mentioned aspect of the present invention and embodiment below is provided.
II. recombinant nucleic acid molecules
The invention provides the multiple polynucleotide that are used in antibacterial such as Listera expression polynucleotide such as heterologous polynucleotide.For example, provide the recombinant nucleic acid molecules that comprises the signal peptide polypeptide of signal peptide (or comprise) coded sequence and polypeptide such as the new combination of heterologous antigen coded sequence.The recombinant nucleic acid molecules that comprises codon optimization polynucleotide sequence is provided.In some embodiments, these recombinant nucleic acid molecules are allogenic, because they comprise the polynucleotide (that is, polynucleotide sequence) that are not natural discovery, it is bonded to each other as the part of identical nucleic acid molecule.In some embodiments, recombinant nucleic acid molecules is isolating.In some embodiments, recombinant nucleic acid molecules is positioned at the expression cassette of antibacterial, expression vector, plasmid DNA, and/or even the sequence of the genomic DNA of antibacterial (inserting the back) in.In some embodiments, recombinant nucleic acid molecules provides polypeptide expression and/or secretion that (for example, heterologous polypeptide) improves in antibacterial.
In some embodiments, recombinant nucleic acid molecules is DNA.In some embodiments, recombinant nucleic acid molecules is RNA.In some embodiments, recombinant nucleic acid is a strand.In other embodiments, recombinant nucleic acid is double-stranded.
In some embodiments, recombinant nucleic acid molecules encoding fusion protein described herein as comprise signal peptide and another polypeptide as with the fusion rotein of the allogenic polypeptide of signal peptide.In some embodiments, signal peptide is the antibacterial signal peptide.The polypeptide fractions that is to be understood that described fusion rotein is passable, but need not and must directly merge each other.In some embodiments, the polypeptide fractions of fusion rotein can be separated on peptide sequence by one or more aminoacid sequences that interleave.In some embodiments, another polypeptide right and wrong antibacterial, for example, mammiferous or viral.
For example, in the one side, the invention provides recombinant nucleic acid molecules, comprise first polynucleotide of (a) coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial; (b) second polynucleotide of coded polypeptide (for example, antigen), wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In the other embodiments, second polynucleotide (coded polypeptide such as antigenic polynucleotide) also are that codon is optimized for the expression in antibacterial.Wherein first and/or second polynucleotide are that the optimized antibacterial of codon should be wherein to expect the antibacterial of placing the recombinant nucleic acid molecules type.
On the other hand, the invention provides recombinant nucleic acid molecules, comprise (a) coding antibacterial first polynucleotide of inherent signal peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, be allogenic by second polynucleotide and first polynucleotide.In some embodiments, polypeptide and signal peptide are that its inherent antibacterial is allogenic (that is being external source to antibacterial).In some embodiments, being allogenic by the polypeptide and the signal peptide of second polynucleotide encoding, is external source to antibacterial, or both.In some embodiments, the antibacterial that produces signal peptide is an intracellular bacteria.In some embodiments, antibacterial is selected from Listera and belongs to bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria and escherichia coli.In some embodiments, it is inherent that signal peptide is that Listera belongs to antibacterial.In some embodiments, it is inherent that signal peptide is that the Listera that belongs to the Listeria monocytogenes kind belongs to antibacterial.In some embodiments, second polynucleotide is that codon is optimized for the expression in antibacterial.
On the other hand, the invention provides recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotide of (a) coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression that belongs at Listera in the antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, it is inherent that signal peptide is that Listera belongs to antibacterial.In other embodiments, it is external source that signal peptide belongs to antibacterial to Listera.In some embodiments, signal peptide be allogenic by the polypeptide of second polynucleotide encoding.In some embodiments, be allogenic by the polypeptide and the Listera of second polynucleotide encoding.In some embodiments, Listera belongs to antibacterial and belongs to the Listeria monocytogenes kind.
The present invention also provides recombinant nucleic acid molecules, comprise that the coding Listera (for example belongs to the antibacterial allogenic polypeptide, cancer or non-Listera infectious disease antigen) polynucleotide, wherein the polynucleotide of encoding exogenous polypeptide are that codon is optimized for the expression that belongs at Listera in the antibacterial.
On the other hand, the invention provides recombinant nucleic acid molecules, first polynucleotide that comprise the non-secA1 antibacterial signal peptide of (a) coding, (b) coded polypeptide such as antigenic second polynucleotide, wherein second polynucleotide is positioned at the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, non-secA1 antibacterial signal peptide is secA2 signal peptide or Tat signal peptide.In some embodiments, first polynucleotide of the non-secA1 signal peptide of encoding are that codon is optimized for the expression in the antibacterial (for example, Listera) of the recombinant nucleic acid molecules of expection placement therein.In some embodiments, coded polypeptide such as antigenic second polynucleotide are that codon is optimized for the expression in the antibacterial of the recombinant nucleic acid molecules of expection placement therein.In some embodiments, be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, be external source to the antibacterial of wherein waiting to mix or mixed recombinant nucleic acid molecules by the polypeptide of second polynucleotide encoding.In some embodiments, by the polypeptide of second polynucleotide encoding to the antibacterial of wherein waiting to mix or mixed recombinant nucleic acid molecules be external source and also be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.
The present invention further provides recombinant nucleic acid molecules, first polynucleotide that comprise the non-secA1 antibacterial signal peptide of encoding, coded polypeptide (for example, heterologous protein and/or antigen) second polynucleotide, with coding SecA2 autolysin or its segmental the 3rd polynucleotide, it is positioned at and first and second translation frame that polynucleotide are identical, and wherein second polynucleotide are in the 3rd polynucleotide or between first and the 3rd polynucleotide.In some embodiments, the recombinant nucleic acid molecules coding comprises signal peptide, the fusion rotein of polypeptide and autolysin.In some embodiments, the fragment of autolysin has the catalytic activity the same with autolysin.In some embodiments, autolysin is from intracellular bacteria.In some embodiments, autolysin is the Peptidoglycan hydrolytic enzyme.In some embodiments, the antibacterial autolysin is the Listera autolysin.In some embodiments, autolysin is p60.In some embodiments, autolysin is the-acetylmuramic acid enzyme.
The present invention also provides recombinant nucleic acid molecules, and wherein recombinant nucleic acid molecules comprises first polynucleotide of the non-Listera signal peptide of (a) coding; (b) second of coded polypeptide polynucleotide, it is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.In some embodiments, non-Listera signal peptide be allogenic by the polypeptide of second polynucleotide encoding.In some embodiments, first polynucleotide, second polynucleotide, or first is that codon is optimized with second polynucleotide for the expression that belongs at Listera in the antibacterial.
The present invention also provides recombinant nucleic acid molecules, first polynucleotide that comprise (a) coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises by the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant, wherein, in the protein chimera, polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or are positioned at autolysin or its catalytic activity fragment or catalytic activity variant.In some embodiments, second polynucleotide is positioned at first polynucleotide, and recombinant nucleic acid molecules coding wherein is positioned at autolysin or its catalytic activity fragment or catalytic activity by the polypeptide of second polynucleotide encoding and becomes intravital protein chimera.In some embodiments, second polynucleotide is positioned at outside first polynucleotide, and the recombinant nucleic acid molecules coding is wherein by the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or the fusion of its catalytic activity variant.In some embodiments, first polynucleotide encoding autolysin.In some embodiments, recombinant nucleic acid molecules further comprises the 3rd polynucleotide of (c) coded signal peptide, it is arranged in and first and second translation frame that polynucleotide are identical, wherein the recombinant nucleic acid molecules coding comprises signal peptide, by the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant.In some embodiments, be allogenic by the polypeptide and the autolysin of second polynucleotide encoding.In some embodiments, the fragment of autolysin is at least about 30, at least about 40, and at least about 50, or at least about 100 amino acid longs.In some embodiments, autolysin is from intracellular bacteria.In some embodiments, the antibacterial autolysin is the Listera autolysin.The catalytic activity variant of autolysin comprises because of one or more replacements, and disappearance is added, and/or inserts and be different from the variant of original autolysin.In some embodiments, autolysin is the Peptidoglycan hydrolytic enzyme.In some embodiments, autolysin is p60.Some embodiments, autolysin are the-acetylmuramic acid enzymes.
Can identify and characterize other autolysin by the receptor of signal recognition particle body, this is technology well known by persons skilled in the art (referring to, for example, Lenz etc. (2003) Proc.Natl.Acad.Sci.USA 100:12432-12437).The receptor of signal recognition particle body can also be used to measure given autolysin fragment and/or whether variant has the activity the same with autolysin.Can also use this technology to evaluate the specified protein chimera and whether have the catalytic activity the same with autolysin.
In some embodiments, the catalytic activity fragment and/or the variant of autolysin have at least about 10% as autolysin, at least about 30%, and at least about 50%, at least about 75%, at least about 90%, or at least about the catalytic activity of 95% natural autolysin.
In some embodiments, the protein chimera has the catalytic activity the same with autolysin.In some embodiments, the protein chimera has at least about 10% as autolysin, at least about 30%, and at least about 50%, at least about 75%, at least about 90%, or at least about the catalytic activity of 95% natural autolysin.
Another option that is used for the heterologous protein expression is to use wherein the albumen " support " of " in the frame " functional insertion heterologous protein.In the said composition, insert in the scaffolding protein and run through scaffolding protein with whole gene or corresponding to the gene element of for example I class MHC or II class MHC epitope.Scaffolding protein can be the bacterioprotein (as Listera albumen, as LLO or p60) of highly expressing, but in another embodiment, can be highly to express at it, stability, secretion, and/or the heterologous protein of (shortage) immunogenicity selection.The representative example of scaffolding protein is a chicken egg white, or other people protein, as betaglobulin or albumin.
The present invention also provides recombinant nucleic acid molecules, first polynucleotide that comprise (a) coded signal peptide, (b) coding secretory protein or its segmental second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the 3rd polynucleotide of coding and secretory protein or the allogenic polypeptide of its fragment, wherein the 3rd polynucleotide are arranged in and first and second translation frame that polynucleotide are identical, wherein the recombinant nucleic acid molecules coding comprises signal peptide, polypeptide by second polynucleotide encoding, with secretory protein or its segmental protein chimera, and wherein in the protein chimera, polypeptide and secretory protein or its fragment merge, or are positioned at secretory protein or its fragment.In some embodiments, second polynucleotide encoding secretory protein.In some embodiments, secretory protein is from the excretory albumen of its n cell.In some embodiments, the 3rd polynucleotide are arranged in second polynucleotide of recombinant nucleic acid molecules, and in by recombinant nucleic acid molecules encoded protein matter chimera, are positioned at secretory protein or its fragment by the polypeptide of the 3rd polynucleotide encoding.In some embodiments, the 3rd polynucleotide are arranged in outside second polynucleotide of nucleic acid molecules, and in the protein chimera, by polypeptide and the secretory protein or the fusion of its fragment of the 3rd polynucleotide encoding.In some embodiments, secretory protein is an ovalbumin.In some embodiments, use the ovalbumin of clipped form.In some embodiments, secretory protein is p60.In some embodiments, secretory protein is the-acetylmuramic acid enzyme.In some embodiments, signal peptide is usually the signal peptide relevant with secretory protein.In some embodiments, signal peptide and secretory protein are allogenic.In some embodiments, the fragment of secretory protein is at least about 30, at least about 40, and at least about 50, or at least about 100 amino acid longs.
In some embodiments, recombinant nucleic acid molecules, expression cassette or expression vector comprise the coded sequence of antibacterial allogenic polypeptide, it is embedded in the part or entire coded sequence of antibacterial inner height expressed protein.In some embodiments, highly the sequence of Biao Daing is that wherein to wait to express the antibacterial of this sequence inherent.In other embodiments, highly the sequence of Biao Daing is not that wherein to wait to express the antibacterial of this sequence inherent, but enough expression still are provided.
On the other hand, the invention provides recombinant nucleic acid molecules, wherein at least two discrete non-Listera polypeptide of this nucleic acid molecule encoding.In some embodiments, the polynucleotide of the non-Listera polypeptide of encoding are that codon is optimized for the expression that belongs at Listera in the antibacterial.
Preparation comprises that above-mentioned those the method for recombinant nucleic acid molecules is known to a person of ordinary skill in the art.For example, can be by preparing recombinant nucleic acid molecules making on its dna synthesizer that overlaps each other synthetic long oligonucleotide and carry out the double-stranded DNA that extension and/or PCR produce aequum then.Can come cutting double-stranded DNA and be inserted in the required expression or cloning vehicle with restriction endonuclease.Can check order to examine and obtain correct sequence.In addition, as limiting examples, one or more parts of recombinant nucleic acid molecules can obtain from the plasmid that contains this part.Can carry out the PCR of plasmid relevant portion and/or the restriction enzyme digestion of plasmid relevant portion and remove, then connect and/or PCR makes relevant polynucleotide in conjunction with to produce required recombinant nucleic acid molecules.Such technology is a standard in this area.Also can use the standard clone technology to insert recombinant nucleic acid sequence in the plasmid and in host cell such as antibacterial, duplicate this recombinant nucleic acid.Can separate this recombinant nucleic acid from host cell then.
The present invention also provides and has used any recombinant nucleic acid molecules described herein to produce the recombinant bacteria method of (for example, the reorganization Listera belongs to antibacterial).In some embodiments, the method for using recombinant nucleic acid molecules described herein to make recombinant bacteria comprises to be introduced recombinant nucleic acid molecules in the antibacterial.In some embodiments, with recombinant nucleic acid molecule integrates to the genome of antibacterial.In some other embodiments, on the plasmid of recombinant nucleic acid molecules in mixing antibacterial.Some embodiments are mixed recombinant nucleic acid molecules in the antibacterial by engaging.Can realize entering the introducing of antibacterial by any standard technique as known in the art.For example, can transduce (transfection) or be converted recombinant nucleic acid molecules is incorporated in the antibacterial by engaging.
III. signal peptide
In some embodiments, recombinant nucleic acid molecules of the present invention, expression cassette and/or vector encoded comprise signal peptide and are suitable for expressing in host cell such as antibacterial or excretory fusion rotein or protein chimera from host cell such as antibacterial.Therefore, in some embodiments, recombinant nucleic acid molecules of the present invention, expression cassette and/or carrier comprise the polynucleotide of coded signal peptide.
Term " signal peptide " and " signal sequence " can be used alternatingly at this.In some embodiments, signal peptide helps lend some impetus to the polypeptide that merges with signal peptide and passes cell (for example, bacterial cell transporting of) cell membrane makes polypeptide secrete from cell.Therefore, in some embodiments, signal peptide is " secreting signal peptide " or " secretion sequence ".In some embodiments, signal peptide is positioned at the N-end for the treatment of secrete polypeptide.
In some embodiments, the sequence of coded signal peptide is positioned at recombinant nucleic acid molecules or expression cassette in recombinant nucleic acid molecules or the expression cassette, makes the encoded signals peptide realize the secretion of polypeptide from required host cell (for example, antibacterial) with its fusion.In some embodiments, in recombinant nucleic acid molecules or expression cassette, the polynucleotide of coded signal peptide are positioned at 5 ' the end frame interior (directly or by interleaving polynucleotide separating) that coding is treated the polynucleotide of secrete polypeptide (for example, comprising antigenic polypeptide).
In some embodiments, as by recombinant nucleic acid molecules, at least a other the peptide sequence in the signal peptide of the fusion rotein of expression cassette and/or expression vector codes and/or a protein chimera part and fusion rotein and/or the protein chimera is allogenic.In some embodiments, by recombinant nucleic acid molecules, the signal peptide of expression cassette and/or expression vector codes is to wherein waiting to introduce or introduced recombinant nucleic acid molecules, and the antibacterial of expression cassette and/or expression vector is allogenic (that is external source).In some embodiments, signal peptide is a recombinant nucleic acid molecules wherein to be introduced, and the antibacterial of expression cassette and/or expression vector is inherent.
In some embodiments, the polynucleotide of coded signal peptide are that codon is optimized for the expression in antibacterial (for example, Listera is as Listeria monocytogenes).In some embodiments, be that the optimized polynucleotide of codon are external sources to antibacterial for specific bacteria.In other embodiments, be that the optimized polynucleotide of codon are that antibacterial is inherent for specific bacteria.
Multiple signal peptide is as known in the art.In addition, can be used for the various algorithms and the software program of prediction signal peptide sequence, as " SignalP " algorithm, be that this area is obtainable.For example, referring to Antelmann etc., Genome Res., 11:1484-502 (2001); Menne etc., Bioinformatics, 16:741-2 (2000); Nielsen etc., Protein Eng., 10:1-6 (1997); Zhang etc., Protein Sci., 13:2819-24 (2004); Bendtsen etc., J.Mol.Biol., 340:783-95 (2004) (about Signal P3.0); Hiller etc., Nucleic Acids Res., 32:W375-9 (2004); Schneider etc., Proteomics 4:1571-80 (2004); Chou, Curr.Protein Pept.Sci., 3:615-22 (2002); Shah etc., Bioinformatics, 19:1985-96 (2003); With Yuan etc., Biochem.Biophys.Res.Commun.312:1278-83 (2003).
In some embodiments, signal peptide is procaryotic.In some embodiments, signal peptide is Eukaryotic.The eukaryotic signal peptide is used for being described in Humphreys etc. in the proteinic purposes of expression in escherichia coli, Protein Expression and Purification (protein expression and purification), 20:252-264 (2000).
In some embodiments, signal peptide is the antibacterial signal peptide, and in some embodiments, signal peptide is non-Listera signal peptide.In some embodiments, signal peptide is the Listera signal peptide.In some embodiments, signal peptide is derived from gram-positive bacterium.In some embodiments, signal peptide is derived from intracellular bacteria.
In some embodiments, recombinant nucleic acid molecules, signal peptide (for example, non-secA1 antibacterial signal peptide) used in expression cassette or the expression vector is derived from Listera.In some embodiments, this signal peptide is derived from Listeria monocytogenes.In some embodiments, signal peptide is the signal peptide from Listeria monocytogenes.In some embodiments, signal peptide is not to be derived from Listera, belongs to Listera and belongs to antibacterial beyond the antibacterial but be derived from.In some embodiments, the antibacterial signal peptide is derived from bacillus.In some embodiments, the antibacterial signal peptide is derived from bacillus subtilis.In some embodiments, the antibacterial signal peptide is derived from the antibacterial that belongs to staphylococcus.In some embodiments, the antibacterial signal peptide is derived from lactococcus bacteria.In some embodiments, the antibacterial signal peptide is derived from bacillus, staphylococcus or lactococcus bacteria.In some embodiments, the antibacterial signal peptide is to be derived from Bacillus anthracis, bacillus subtilis, the signal peptide of staphylococcus aureus or lactococcus lactis.In some embodiments, the antibacterial signal peptide is the signal peptide that is derived from Bacillus anthracis.In some embodiments, the antibacterial signal peptide is the signal peptide that is derived from bacillus subtilis.In some embodiments, the antibacterial signal peptide is the signal peptide that is derived from lactococcus lactis.In some embodiments, the antibacterial signal peptide is the signal peptide from staphylococcus aureus.
In some embodiments of polynucleotide described herein, being derived from the signal peptide of organism such as antibacterial, is identical with the signal peptide sequence of the natural generation that obtains from organism.In other embodiments, by recombinant nucleic acid molecules, the signal peptide sequence of expression cassette and/or expression vector codes is derived from the signal peptide sequence of natural generation, i.e. the fragment of the signal peptide sequence of natural generation and/or variant, and wherein this fragment or variant still play signal peptide.Variant comprises because of one or more replacements, and disappearance is added and/or inserted and be different from the polypeptide of original series.For example, in some embodiments, comprise one or more conservative sudden changes by the signal peptide of polynucleotide encoding.It is known to a person of ordinary skill in the art that possible conserved amino acid changes.The additional information that changes about conserved amino acid, referring to, for example, the following IV part of Xiang Shuing.
The signal peptide (that is, the fragment of another kind of signal peptide and/or variant) that is derived from another kind of signal peptide preferably is equal to the primary signal peptide basically.The influence of for example, the change that the ability that is derived from the super signal peptide effect of signal peptide of another kind of signal peptide should not be subjected to the primary signal peptide sequence is carried out basically (disappearance, sudden change etc.).In some embodiments, deutero-signal peptide is at least about 70%, at least about 80%, and at least about 90%, or can the same signal peptide that plays a part with the natural signals peptide sequence at least about 95%.In some embodiments, this signal peptide and primary signal peptide have at least about 70%, at least about 80%, and at least about 90%, or at least about 95% aminoacid homogeneity.In some embodiments, the optimal change of carrying out in signal peptide sequence is that conserved amino acid is replaced.The fragment of signal peptide preferably primary signal peptide length at least about 80% or at least about 90%.
In some embodiments, by recombinant nucleic acid molecules, the signal peptide of the polynucleotide encoding in expression cassette or the expression vector is the secA1 signal peptide, secA2 signal peptide or double arginine transhipment (Tat) signal peptide.In some embodiments, signal peptide is the secA1 signal peptide.In some embodiments, signal peptide right and wrong secA1 signal peptide.In some embodiments, signal peptide is the secA2 signal peptide.In some embodiments, signal peptide is double arginine transhipment (Tat) signal peptide.In some embodiments, these secA1, secA2 or Tat signal peptide are derived from Listera.In some embodiments, these secA1, secA2 or Tat signal peptide are non-Listeras.For example, in some embodiments, secA1, secA2 or Tat signal peptide are derived from the antibacterial that belongs to one of subordinate: bacillus, staphylococcus or Lactococcus.
Antibacterial utilizes different protein secreting approach, comprises secA1, secA2 or two-arginine transport (Tat).Utilizing which kind of approach mainly is that type by the signal peptide sequence of albumen N-end before being positioned at decides.Most of secretory protein utilizes the Sec approach, and wherein protein is transported by the protein S ec hole that is embedded in bacterial membrane with not folding conformation.On the contrary, utilize the protein of Tat approach with folding conformation secretion.Coding can heredity be gone up coding and required heterologous protein coded sequence frame endomixis corresponding to the nucleotide sequence of any signal peptide in these protein secreting approach.Signal peptide is preferably in its carboxyl terminal and comprises the signal peptidase cleavage site and be used for the desired protein of vacuum is released into born of the same parents' external environment (Sharkov and Cai.2002 J.Biol.Chem.277:5796-5803; Nielsen etc., 1997 Protein Engineering10:1-6; With, www.cbs.dtu.dk/services/SignalP).
Signal peptide used in the polynucleotide of the present invention not only can be derived from different secretory pathways, and can be derived from different antibacterial genus.Signal peptide has common structure structure usually, has charged N-end (N-domain), hydrophobic core zone (H-domain) and pluripolarity C-stub area (C-domain), however they do not demonstrate sequence conservation.In some embodiments, the C-domain of signal peptide carries I type signal peptidase (SpaseI) cleavage site, has consensus sequence A-X-A, in the position with respect to cleavage site-1 and-3.The signal peptide that has average 28 residues by the excretory protein of sec approach.SecA2 protein secreting approach at first is found in Listeria monocytogenes; In the secA2 likeness in form thing (paralogue) mutant be characterized as coarse bacterium colony phenotype on the agar culture medium and in mice virulence phenotype (Lenz and Portnoy, 2002 Mol.Microbiol.45:1043-1056 of attenuation; With Lenz etc., 2003 PNAS 100:1432-12437).And have the tripartite structure similar by the relevant signal peptide of the excretory protein of Ta t approach to the Sec signal peptide, but it is characterized in that having RR-motif (R-R-X-#-#, wherein # is a hydrophobic residue), be positioned at N-domain/H-domain border.Long 14 aminoacid of antibacterial Tat signal peptide average specific sec signal peptide.Bacillus subtilis secretion protein groups (secretome) can comprise nearly 69 protein of inferring that utilize the Tat secretory pathway, and wherein 14 comprise Spase I cleavage site (Jongbloed etc., 2002 J Biol.Chem.277:44068-44078; Thalsma etc., 2000 Microbiol.Mol.Biol.Rev.64:515-547).
Shown in the following table 1 is the limiting examples of signal peptide, these signal peptides can be used for having the fusion compositions (comprising protein chimera compositions) of selected other polypeptide such as heterologous polypeptide, cause coded protein to secrete from antibacterial.
Some example signal peptides of table 1.
| Secretory pathway | Signal peptide aminoacid sequence (NH 2-CO 2) ? | The signal peptidase site (with ' the expression cleavage site) | Gene | Belong to/kind |
| ?secA1 ? | MKKIMLVFITLILVSLPIAQQ TEAKD(SEQ?ID?NO：45) | TEA’KD (SEQ?ID?NO：54) | hly(LLO) ? | Listeria monocytogenes |
| MKKKIISAILMSTVILSAAAP LSGVYADT (SEQ?ID?NO：46) | VYA’DT (SEQ?ID?NO：55) ? | Usp45 ? ? | Lactococcus lactis | |
| MKKRKVLIPLMALSTILVSST GNLEVIQAEV (SEQ?ID?NO：47) | IQA’EV (SEQ?ID?NO：56) ? | Pag (protective antigen) | Bacillus anthracis | |
| ?secA2 ? | MNMKKATIAATAGIAVTAFAA PTIASAST | ASA’ST (SEQ?ID?NO：57) | Iap invades relevant | Listeria monocytogenes |
| (SEQ?ID?NO：48) | Albumen p60 | |||
| MQKTRKERILEALQEEKKNKK SKKFKTGATIAGVTAIATSI TVPGIEVIVSADE(SEQ?ID NO：49) | VSA’DE (SEQ?ID?NO：58) ? ? | NamA lmo 2691 (autolysin) | Listeria monocytogenes | |
| MKKLKMASCALVAGLMFSGLT PNAFAED (SEQ?ID?NO：50) | AFA’ED (SEQ?ID?NO：59) ? | ? *BA_0281 (NLP/P60 family) | Bacillus anthracis | |
| MAKKFNYKLPSMVALTLVGSA VTAHQVQAAE (SEQ?ID?NO：51) | VQA’AE (SEQ?ID?NO：60) ? | ? *Atl (autolysin) | Staphylococcus aureus | |
| Tat ? ? ? | MTDKKSENQTEKTETKENKGM TRREMLKLSAVAGTGIAVGA TGLGTILNVVDQVDKALT (SEQ?ID?NO：52) | DKA’LT (SEQ?ID?NO：61) ? ? | lmo0367 ? ? ? | Listeria monocytogenes |
| MAYDSRFDEWVQKLKEESFQN NTFDRRKFIQGAGKIAGLSL GLTIAQSVGAFG(SEQ?ID NO：53) | VGA’FG (SEQ?ID?NO：62) ? ? | PhoD (alkali phosphatase) | Bacillus subtilis |
* by the excretory antibacterial autolysin of sec approach (not determining secA1 or secA2).
Therefore, in some embodiments, the sequential coding secA1 signal peptide of coded signal peptide.The example of SecA1 signal peptide is Listera lysin O (LLO) signal peptide from Listeria monocytogenes.In some embodiments, comprise the recombinant nucleic acid molecules of polynucleotide of coding LLO signal peptide or the polynucleotide sequence that expression cassette further comprises coding LLO PEST sequence.Other examples that are applicable to secA1 signal peptide of the present invention comprise from Usp45 gene in the lactococcus lactis (referring to above table 1 and following embodiment 12) with from the signal peptide of Pag (protective antigen) gene of Bacillus anthracis.Therefore, in some embodiments, signal peptide is the protective antigen signal peptide from Bacillus anthracis.In some other the embodiment, signal peptide is except from the secA1 signal peptide the protectiveness signal peptide of Bacillus anthracis.Another example of secA1 signal peptide is the SpsB signal peptide (Sharkov etc., J.of Biological Chemistry, 277:5796-5803 (2002)) from staphylococcus aureus.
In some alternate embodiments, will merge in allogeneic coding sequence and the signal peptide heredity by the identification of secA2 pathway protein secretion complex.Cause identifying in the infectious gram-positive bacterium of serious or fatal people complementary SecA likeness in form thing (SecA2) at nine kinds.SecA2 is that Listera belongs to (Braunstein etc., Mol.Microbiol.48:453-64 (2003) that the secretion of the protein group of the discharge of Mycobacterium and Streptococcus (secretory protein group) subclass is required; Bensing etc., Mol.Microbiol., 44:1081-94 (2002); Lenz etc., Mol.Microbil., 45:1043-1056 (2002); With Braunstein etc., J.Bacteriology, 183:6979-6990 (2001)).Listeria monocytogenes SecA2 by with antibacterial smooth-dependency of coarse variation is identified, and the sudden change among the secA2 has reduced the virulence of Listeria monocytogenes and mycobacterium tuberculosis.
For example, Listera protein p60 is by the excretory Peptidoglycan autolysin of secA2 approach.For example, secA2 signal peptide and the signal peptidase cleavage site from p60 can connect by amino terminal upward hereditary and desirable proteins (for example, antigen) encoding gene.In one embodiment, translated from the expression cassette in the antibacterial by the preceding protein that secA2 signal peptide and signal peptidase-antigen fusant are formed, transport by the gram-positive cell wall, wherein the heterologous protein of vacuum is released in born of the same parents' external environment.
Perhaps, heterologous sequence can " in the frame " mix in the p60, makes heterologous protein secrete out with the form of chimeric p60-heterologous protein.For example, can heterologous protein coded sequence in the frame be inserted among the p60 at the abutment between signal peptidase cleavage site and the ripe p60 protein.In this embodiment, chimeric protein keeps suitable secA2 secretion signal, and keeps its autolysin activity, this means that heterologous protein secretes as the non-revenue passenger of p60.Can enter at any heterologous antigen of naming a person for a particular job in keeping proteinic secretion of p60 and the active p60 of autolysin in the frame of p60 and mix through engineering approaches.The case description of part expression cassette that is suitable for inserting required antigen or other heterologous polypeptide coded sequences is in following embodiment 13.
In some embodiments, the fusion rotein of being encoded by recombinant nucleic acid molecules is the chimera that comprises the bacterioprotein (except required heterologous protein such as antigen) with specific desired characteristic.In some embodiments, chimera comprises hydrolytic enzyme.In some embodiments, the recombinant nucleic acid molecules coding comprises the p60 chimera of endopeptidase p60 (the Peptidoglycan hydrolytic enzyme of bacterium for degrading cell wall).In some embodiments, fusion rotein by the recombinant nucleic acid molecules coding comprises the Listeria monocytogenes hydrolytic enzyme, for example, p60 (referring to, for example, Genbank accession number no.NP_464110) or-acetylmuramic acid enzyme (NamA) (Genbank accession number no.NP_466213), the two all is the excretory protein of secA2 dependency of degradation of cell wall.Such specified protein chimera compositions not only utilizes bacterioprotein to secrete needed chaperone, and utilizes the activity that can help its excretory bacterioprotein.The particular proteins chimera is made up of the accurate layout of heterologous protein coded sequence and Listeria monocytogenes hydrolytic enzyme, cause the effective expression of heterologous protein and secretion (referring to, for example, following specific embodiment, embodiment 29).Therefore, in some embodiments, the signal peptide as a fusion rotein part of being encoded by recombinant nucleic acid molecules is the p60 signal peptide.In some embodiments, the signal peptide as a fused protein part of being encoded by recombinant nucleic acid molecules is the NamA signal peptide.
In some embodiments, recombinant nucleic acid molecules comprises coding p60 albumen or its segmental the 3rd polynucleotide sequence, it is positioned in the translation frame identical with second polynucleotide of first polynucleotide of the p60 signal peptide of encoding and the another kind of polypeptide of encoding (for example, antigen).Thereby recombinant nucleic acid molecules coding comprises signal peptide, by polypeptide (for example, antigen) and p60 protein or its segmental fusion rotein of second polynucleotide encoding.In such embodiment, second polynucleotide are preferably placed in the 3rd nucleotide or between first and the 3rd polynucleotide.
In some embodiments, the secA2 signal peptide is the secA2 signal peptide that is derived from Listera.For example, in some embodiments, signal peptide is that the secA2 signal peptide is as p60 signal peptide or-acetylmuramic acid enzyme (NamA) signal peptide from Listeria monocytogenes.In addition, secA2 not in the presence of, Listeria monocytogenes protein (the Lenz etc. that do not secrete other have been identified, Mol.Microbiology 45:1043-1056 (2002)), and in some embodiments, can use the polynucleotide of coding from these proteinic signal peptides.In addition, the secA2 signal peptide from the antibacterial except that Listera can be used for expression and the secretion of heterologous protein from reorganization Listera or other antibacterials.For example, as an illustration property but non-limiting instance can be used for recombinant nucleic acid molecules and/or expression cassette from the secA2 signal peptide of Bacillus anthracis.In other embodiments, use secA2 signal peptide from staphylococcus aureus.Referring to table 1.Also in other antibacterials, identified by the excretory protein of SecA2 approach (referring to, for example, Braunstein etc., Mol.Microbiol., 48:453-64 (2003) and Bensing etc., Mol.Microbiol.44:1081-94 (2002)).
Can identify by excretory other protein of secA2 approach.In the various bacteria kind, identify the SecA2 congener (referring to, for example, Lenz etc., Mol.Microbiology45:1043-1056 (2002) and Braunstein etc., J.Bacteriology, 183:6979-6990 (2001)).Use well known to a person skilled in the art that technology relatively can identify other secA2 congener by further sequence.In case identify congener, can from the bacterium living beings body, lack this congener and produce Δ secA2 mutant.The supernatant protein of wild type and mutant bacterial culture can carry out TCA-precipitation and by any proteomic techniques analysis known in the art to determine which protein is excretory by wild-type bacterium rather than Δ secA2 mutant.For example, can analyze excretory protein by SDS-PAGE and silver dyeing.Can more resulting bring evaluation SecA2 do not take place in the presence of not excretory those protein (referring to, for example, Lenz etc., Mol.Microbiology45:1043-1056 (2002)).Can analyze these proteinic N-terminal sequences (for example, using the algorithm of predicted signal peptide cleavage site) then and determine the secA2 signal peptide sequence that this protein is used.Also can carry out holding order-checking to come the sequence of identification signal peptide by the N-of the Edman of automatization degraded.
In the alternate embodiment, the polypeptide (for example, allogeneic polypeptide sequence) that merges with the signal peptide of discerning by Tat pathway protein secretion complex in the polynucleotide encoding heredity.Antibacterial comprises that Listera belongs to several and utilizes the Tat secretory pathway, is used to secrete protein folding in antibacterial.For example, harmless Listera (Listeria innocua) protein Y wbN has the Tat motif of inferring at its amino terminal, and therefore utilize the Tat approach to be used for secretion (Genbank accession number No.NP_469731[gi|16799463|ref|NP_469731.1|, conservative putative protein matter (harmless Listera) with bacillus subtilis YwbN protein similar], be hereby incorporated by).The albumen that another kind contains the Tat signal peptide is that (Genbank accession number No.NP_463897[gi|16802412|ref|NP_463897.1| is with the conservative putative protein matter of bacillus subtilis YwbN protein similar (Listeria monocytogenes EGD (e)]) for YwbN albumen from monocyte Listeria monocytogenes strain EGD (e).For example, the YwbN signal peptide with can heredity from the signal peptidase cleavage site of YwbN on be connected the amino terminal of desirable proteins (for example, antigen) encoding gene.In the said composition, the preceding albumen that the expression cassette translation in antibacterial is made up of Tat signal peptide and signal peptidase-antigen fusant transports by the gram-positive cell wall, and wherein real heterologous protein is released in born of the same parents' external environment.Prediction will be secreted the another kind of protein that comes out by the Tat approach from harmless Listera be 3-oxo acyl group-acyl carrier protein synzyme (Genbank accession number No.NP_471636[gi|16801368|ref|NP_471636.1, similar to 3-oxo acyl group-acyl group-carrier protein synzyme (harmless Listera)]).Coding is secreted the signal sequence in any protein of coming out from these predictions by the Tat secretory pathway from Listera polynucleotide can be used for polynucleotide described herein, in expression cassette and/or the expression vector.
Tat signal sequence from other antibacterials also can be used as signal peptide, includes, but not limited to the phoD from bacillus subtilis.Example from the Tat signal peptide of bacillus subtilis as phoD, is described in Jongbloed etc., J.of Biological Chemistry, 277:44068-44078 (2002); Jongbloed etc., J.of Biological Chemistry, 275:41350-41357 (2000), Pop etc., J.of Biological Chemistry, 277:3268-3273 (2002); Van Dijl etc., J.of Biotechnology 98:243-254 (2002); With Tjalsma etc., Microbiology and Molecular BiologyReviews, 64:515-547 (2000), all these are incorporated herein by reference with its integral body at this.Predicted and will comprise those sequences with following Genbank/Emb1 accession number: CAB15017[gi|2635523|emb|CAB15017.1|, to the bi-component sensor histidine kinase by excretory other protein in bacillus subtilis, identified of Tat approach) (YtsA) (bacillus subtilis) similar]; CAB12056[gi|2632548|emb|CAB12056.1| phosphodiesterase/alkali phosphatase D (bacillus subtilis)]; CAB12081[gi12632573|emb|CAB12081.1, similar to the protein (bacillus subtilis) of supposition]; CAB13278[gi12633776|emb|CAB13278.1, similar to the protein (bacillus subtilis) of supposition]; The CAB14172[gi|2634674|emb|CAB14172.1| methyl is quinone how: cytochrome C oxidoreductase (ferrum-sulfur subunit)) (bacillus subtilis)]; CAB15089[gi|2635595|emb|CAB15089.11yubF (bacillus subtilis)]; (29d～similar to the protein (bacillus subtilis) of supposition, wherein all sequences are hereby incorporated by the gene title alternative with CAB15852[gi|2636361|emb|CAB15852.1|: ipa.Therefore, in some embodiments, be the Tat signal peptide that is derived from bacillus subtilis by the signal peptide of the polynucleotide encoding in recombinant nucleic acid molecules and/or the expression cassette.Ochsner etc., PNAS provides the information of relevant Tat signal peptide from Pseudomonas aeruginosa (Pseudomonas aeruginosa) among the 99:8312-8317 (2002).And, be described in Dilks etc., J.of Bacteriology from the Tat signal peptides of multiple other antibacterials, 185:1478-1483 (2003) and Berks etc., Molecular Microbiology, 35:260-274 (2000), both are incorporated herein by reference with its integral body at this.
Can from Sec type signal peptide, identify and distinguish other Tat signal peptide by its " two-arginine " consensus motif.As mentioned above, with have the structure that be divided into three parts similar by the relevant signal peptide of the excretory protein of Tat approach to the Sec signal peptide, but it is characterized in that having the RR-motif (R-R-X-#-#, wherein # is a hydrophobic residue) that is positioned at N-domain/H-domain border.Usually the Tat signal peptide also is longer than Sec type signal peptide and hydrophobicity less than Sec type signal peptide.Referring to, Berks etc. for example, Adv.Microb.Physiol., 47:187-254 (2003) and Berks etc., Mol.Microbiol.35:260-74 (2000).
In addition, be similar to above-mentioned those and be used to identify that the technology by excretory novel protein of SecA2 approach and corresponding SecA2 signal peptide thereof also can be used for identifying by excretory novel protein of Tat approach and signal peptide thereof.List of references Jongbloed etc., J.Biological Chem., 277:44068-44078 (2002) provide the example that can be used for identifying by the technology of passing through two-identical bacteria types expressed protein of the excretory protein of arginine transport approach.
IV. polypeptide
Recombinant nucleic acid molecules described herein, and expression cassette described herein or expression vector, any required polypeptide can be used to encode.Particularly, recombinant nucleic acid molecules, expression cassette and expression vector are used in and express heterologous polypeptide in the antibacterial.
(depend on used recombinant nucleic acid molecules, expression cassette or expression vector) in some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or expression vector is expressed as the part with fusion rotein of signal peptide.In other embodiments, by recombinant nucleic acid molecules, the polypeptide of expression cassette or expression vector codes is expressed as discrete polypeptide.Still in other embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette or expression vector is as not comprising that the part of the fusion rotein of signal peptide expresses.Still in other embodiments, by recombinant nucleic acid molecules of the present invention, the polypeptide of the polynucleotide encoding of expression cassette or expression vector is expressed as the part that polypeptide wherein is embedded in the fusion rotein (being also referred to as the protein chimera at this) in another peptide sequence.
Therefore, be to be understood that by recombinant nucleic acid molecules of the present invention, the polynucleotide encoding of expression cassette or expression vector can pass through recombinant nucleic acid molecules at each listed polypeptide of these (following and other places), expression cassette or expression vector (merge with signal peptide and/or other polypeptide as fusion rotein, or in other polypeptide) or discrete polypeptide express, this depends on used specific recombinant nucleic acid molecules, expression cassette or expression vector.For example, in some embodiments, comprise that the recombinant nucleic acid molecules of the polynucleotide of coding for antigens CEA will be encoded CEA as the fusion rotein with signal peptide.
In some embodiments, polypeptide is a recombinant nucleic acid molecules, the part of the fusion rotein of expression cassette or expression vector codes, and with the signal peptide of fusion rotein be allogenic.In some embodiments, polypeptide is positioned in another peptide sequence allogenic with it (for example, secretory protein or autolysin or its fragment or variant).
In some embodiments, polypeptide is antibacterial (Listera or a non-Listera).In some embodiments, polypeptide right and wrong antibacterial.In some embodiments, be the mammal polypeptide by the polypeptide of polynucleotide encoding.For example, polypeptide can be corresponding to the peptide sequence (that is human polypeptides) of philtrum discovery.In some embodiments, polypeptide is a Listera.In some embodiments, polypeptide is non-Listera.In some embodiments, polypeptide is to wherein waiting to introduce or introduce recombinant nucleic acid molecules, and the antibacterial of expression cassette and/or expression vector is not natural (that is external source).
In some embodiments, the polynucleotide of coded polypeptide are that codon is optimized for the expression in antibacterial.In some embodiments, the polynucleotide of coded polypeptide are that complete codon is optimized for the expression in antibacterial.In some embodiments, be external source (that is being allogenic) to antibacterial with antibacterial by the polypeptide of codon optimization polynucleotide encoding.
Term " polypeptide " can be used alternatingly with " peptide " and " protein " at this, and for the length of wherein contained aminoacid sequence or size without limits.Yet usually, polypeptide comprises at least about 6 aminoacid.In some embodiments, polypeptide comprises at least about 9, at least about 12, and at least about 20, at least about 30, or at least about 50 aminoacid.In some embodiments, polypeptide comprises at least about 100 aminoacid.In some embodiments, polypeptide is a proteinic ad hoc structure territory (for example, ectodomain, born of the same parents' intracellular domain, catalyst structure domain or a binding structural domain).In some embodiments, polypeptide comprises complete (that is total length) protein.
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or expression vector comprises antigen or protein, and the alleviating property treatment to disease is provided.In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or expression vector has provided antigen or the protein to the alleviating property treatment of disease.In some embodiments, encoded polypeptide is therapeutic protein (or comprising therapeutic protein).
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or carrier comprises antigen (for example, any antigen described herein).In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or carrier is an antigen.In some embodiments, antigen is bacterial antigens.In some embodiments, antigen is the antigen of non-Listera antibacterial.Yet in some embodiments, antigen is the antigen of non-Listera.In other embodiments, antigen is non-bacterial antigens.In some embodiments, antigen is mammiferous antigen.In some embodiments, antigen is people's antigen.In some embodiments, polypeptide is the antigen that (or comprising) contains one or more immunogenicity epitopes.In some embodiments, antigen comprises one or more MHC I class epitopes.In other embodiments, antigen comprises MHC II class epitope.In some embodiments, epitope is CD4+T-cellular antigens decision position.In other embodiments, epitope is CD8+T-cellular antigens decision position.
The polynucleotide of coding for antigens (for example are not limited to any accurate nucleotide sequence, encode natural generation, the antigenic nucleotide sequence of total length), can be any sequence that is coded in the polypeptide that when giving individuality, is enough to cause required immunne response in antibacterial of the present invention or the compositions.Term " antigen " also is interpreted as the fragment that comprises big antigen protein as used in this, as long as this fragment is antigenic (that is, immunogenic).In addition, in some embodiments, by recombinant nucleic acid molecules, the antigen of the polynucleotide encoding of expression cassette or expression vector can be the variant of the antigen sequence of natural generation.(same, for coding other, the polynucleotide of non-antigen protein, the given proteic polynucleotide sequence of encoding can change, as long as the desired protein of expressing provides required effect (for example, remission effect) when giving individuality).
Being derived from another kind of antigenic antigen comprises as the segmental antigen of another kind of antigenic antigenicity (that is, immunogenicity), another kind of antigenic antigenicity variant, or the antigenicity variant of another kind of antigen fragment.Antigenic variant comprises because of one or more replacements, and disappearance is added and/or inserted and be different from the antigen of original antigen.
Antigen fragment can have any length, but modal be at least about 6 aminoacid, at least about 9 aminoacid, at least about 12 aminoacid, at least about 20 aminoacid, at least about 30 aminoacid, at least about 50 aminoacid, or at least about 100 aminoacid.Antigenic antigenicity fragment comprises that at least one is from antigenic epitope.In some embodiments, epitope is a MHC I class epitope.In other embodiments, epitope is a MHC II class epitope.In some embodiments, epitope is CD4+T-cellular antigens decision position.In other embodiments, epitope is CD8+T-cellular antigens decision position.
Multiple algorithm and the software kit that is used for predicted protein matter endoantigen zone (comprising epitope) is that those skilled in the art are obtainable.For example, can be used to select the algorithm in conjunction with the epitope of MHC I class and II quasi-molecule is that the public is obtainable.For example, the public obtainable " SYFPEITHI " algorithm can be used to predict MHC-binding peptide (Rammensee etc. (1999) Immunogenetics 50:213-9).For other examples of the obtainable algorithm of the public, referring to following list of references: Parker etc., (1994) J.Immunol 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 etc. (2004) Bioinformatics20:1388-97; Donnes etc. (2002) BMC Bioinformatics 3:25; Bhasin etc. (2004) Vaccine 22:3195-204; Guan etc. (2003) Nucleic Acids Res31:3621-4; Reche etc. (2002) Hum.Immunol.63:701-9; Schirle etc. (2001) J.Immunol Methods 257:1-16; Nussbaum etc. (2001) Immunogenetics (2001) 53:87-94; Lu etc. (2000) Cancer Res.60:5223-7.Can also referring to, for example, Vector NTI Suite (Informax, Inc, Bethesda, MD), GCG Wisconsin Package (Accelrys, Inc., San Diego, CA), Welling etc. (1985) FEBS Lett.188:215-218, Parker etc. (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 (1993) Pept.Res.6:183-190.The prediction that some algorithms discussed in the above listed list of references in this paragraph or software kit relate to MHC I class and/or II class binding peptide or epitope, other relate to the evaluation of the cleavage site of albuminous body, also have some to relate to based on hydrophilic antigenic prediction.
In case identified and thought the candidate antigens fragment of the epitope that contains at least one required character, can introduce the polynucleotide sequence of this sequence of coding in the expression cassette and introduce in Listera vaccine carrier or other bacterial vaccine carriers.Then by evaluating the immunogenicity that Listera of expressing this antigen fragment or the immunne response that other antibacterials produced can confirm this antigen fragment.Standard immunoassay is measured as ELISPOT and is measured, and intracellular cytokine dyeing (ICS) is measured, and cytotoxic T-cytoactive mensuration etc. can be used to examine selected antigen fragment and keep required immunogenicity.Following embodiment provide the example that these types measure (referring to, for example, embodiment 21).In addition, also can use antitumor efficacy that the method described in following examples evaluates Listera and/or bacterial vaccine (for example, in mice, implant the segmental CT26 Mus of antigen expressed colon cell, then also observe) with respect to contrast and/or the antigenic effect of total length to tumor size, transfer, survival etc. with candidate vaccine inoculation mice.
In addition, be used to identify that the big data base of containing epitope and/or MHC part information of antigen fragment is that the public is obtainable.Referring to, for example, Brusic etc. (1998) NucleicAcid Res.26:368-371; Schonbach etc. (2002) Nucleic Acids Research30:226-9; With (2003) Bioinformatics 19:665-666 such as Bhasin; With (1999) Immunogenetics 50:213-9 such as Rammensee.
The aminoacid sequence of antigenic variant and original antigen have 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% homogeneity.
In some embodiments, antigenic variant be have at least about 80% with the conservative variant of the homogeneity of original antigen, the replacement between antigenic variant and the original antigen sequence is that conserved amino acid is replaced.Think that following replacement is that conserved amino acid is replaced: valine, isoleucine and leucine substitute alanine; Lysine, glutamine or agedoite place of arginine; Glutamine, histidine, lysine or arginine substitute agedoite; Glutamic for aspartic acids; Serine substitutes cysteine; Agedoite substitutes glutamine; Aspartic acid substitutes glutamic acid; Proline or alanine substitute glycine; Agedoite, glutamine, lysine or arginine alternate sets propylhomoserin; Leucine, valine, methionine, alanine, phenylalanine or nor-leucine substitute isoleucine; Nor-leucine, isoleucine, valine, methionine, alanine or phenylalanine substitute leucine; Arginine, glutamine or agedoite are replaced lysine; Leucine, phenylalanine or isoleucine substitute methionine; Leucine, valine, isoleucine, alanine or tyrosine substitute phenylalanine; Alanine substitutes proline; Threonine substitutes serine; Serine substitutes threonine; Tyrosine or phenylalanine substitute tryptophan; Tryptophan, phenylalanine, threonine or serine substitute tyrosine; Isoleucine, leucine, methionine, phenylalanine, alanine or nor-leucine substitute valine.In some embodiments, antigenic variant be have at least about 90% with the conservative variant of the homogeneity of original antigen.
In some embodiments, be derived from first-class substantially another antigen that is same as of another antigenic antigen.Have at least about 70% amino acid sequence identity and keep immunogenicity at least about 70% original antigen if be derived from another antigenic antigen and original antigen, this antigen is first-class substantially so is same as the original antigen that it is derived from.In some embodiments, antigen that is equal to basically and original antigen have at least about 80%, at least about 90%, and at least about 95%, or at least about 98% amino acid sequence identity.In some embodiments, the antigen that is equal to basically includes only conservative the replacement with respect to original antigen.In some embodiments, the antigen that is equal to is basically kept at least about 80%, at least about 90%, or at least about the immunogenicity of 95% original antigen.For determine specific derive immunogenicity of antigens and and the immunogenicity of original antigen compare to determine whether deutero-antigen is equal to basically with original antigen, people can test deutero-and primary antigen simultaneously in any of panimmunity originality algoscopy well known by persons skilled in the art.For example, express the original antigen or the antigenic Listera of deriving as described herein can the preparation.Can following measurement those express the ability that different antigenic Listeras produce immunne response, promptly, inoculate mice with Listera, use ELISPOT to measure then, intracellular cytokine dyeing (UCS) is measured, and the standard technique of cytotoxic T-cytoactive mensuration etc. is evaluated immunogenic response.Following embodiment provide these type algoscopys example (referring to, for example, embodiment 21).
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or carrier comprises antigen.In some embodiments, antigen is selected from tumor associated antigen, is derived from the polypeptide of tumor associated antigen, infectious disease antigen and be derived from the antigenic polypeptide of infectious disease.
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or carrier comprises tumor associated antigen or comprises the antigen that is derived from tumor associated antigen.In some embodiments, polypeptide comprises tumor associated antigen.In some embodiments, encoded polypeptides comprises that more than a kind of antigen this antigen is tumor associated antigen or the antigen that is derived from tumor associated antigen.For example, in some embodiments, encoded polypeptides comprises mesothelium element (or its antigen fragment or antigenic variant) and K-Ras, 12-K-Ras or PSCA (or K-Ras, the antigen fragment of 12-K-Ras or PSCA or antigenic variant).
In some embodiments, by recombinant nucleic acid molecules, the antigen of the polynucleotide encoding of expression cassette and/or expression vector is tumor associated antigen or the antigen that is derived from tumor associated antigen.In some embodiments, antigen is tumor associated antigen.
In some embodiments, recombinant nucleic acid molecules, the polynucleotide encoding antigen in expression cassette and/or the expression vector (or coding comprises antigenic polypeptide), this antigen is different from tumor associated antigen, but is derived from the antigen of tumor associated antigen.For example, in some embodiments, by recombinant nucleic acid molecules, the antigen of the polynucleotide encoding of expression cassette and/or expression vector can comprise the fragment of tumor associated antigen, the variant of tumor associated antigen, or the segmental variant of tumor associated antigen.In the certain situation, antigen as tumor antigen, when its aminoacid sequence is different from the endogenous aminoacid sequence of host slightly, can be induced more significant immunne response in the vaccine.In other situations, the immunne response of deutero-antigen induction is remarkable not as the inductive immunne response of original antigen, still, for example, because the less heterogenous expression of the deutero-antigen of size in the Listera vaccine carrier is more convenient.In some embodiments, the tumor associated antigen variant, or the aminoacid sequence of tumor associated antigen fragment variant has one or more aminoacid to be different from tumor associated antigen or its corresponding segmental aminoacid sequence.The antigen that is derived from tumor associated antigen comprises at least one can induce required immunne response when these antigenic polynucleotide of coding are expressed in the host epitope sequence.
Therefore, in some embodiments, recombinant nucleic acid molecules, the polynucleotide encoding polypeptide in expression cassette or the carrier, this polypeptide comprises the antigen that is derived from tumor associated antigen, wherein this antigen comprises the antigen fragment of at least one tumor associated antigen.In some embodiments, recombinant nucleic acid molecules, the polynucleotide encoding in expression cassette or the carrier is derived from the antigen of tumor associated antigen, and wherein this antigen comprises the antigen fragment of at least one tumor associated antigen.Antigen fragment comprises the epitope of at least one tumor associated antigen.In some embodiments, being derived from another antigenic antigen is another antigenic antigenicity (that is immunogenicity) fragment or antigenic variant.In some embodiments, antigen is another antigenic fragment.In some embodiments, antigen is another antigenic antigenic variant.
Identified massive tumor related antigen (Renkvist etc., Cancer Immunol Innumother 50:3-15 (2001)) by the T cell recognition.These tumor associated antigens can be differentiation antigen (for example, PSMA, tryrosinase, gp100), (for example, PAP PSA), grows antigen to tissure specific antigen, tumor correlated virus antigen (for example, HPV 16E7), carcinoma of testis antigen (for example, MAGE, BAGE, NY-SEO-1), embryonal antigen (for example, CEA, α-Jia Taidanbai), and oncoprotein antigen (for example, Ras, p53), the proteantigen of overexpression (for example, ErbB2 (Her2/Neu), MUC1), or the proteantigen of sudden change.Tumor associated antigen by the heterologous nucleic acid sequence coding includes, but not limited to 707-AP, annexin II, AFP, ART-4, BAGE, beta-catenin is white/m, BCL-2, bcr-abl, bcr-abl p190, bcr-abl p210, BRCA-1, BRCA-2, CAMEL, CAP-1, CASP-8, CDC27/m, CDK-4/m, CEA (Huang etc., Exper Rev.Vaccines (2002) 1:49-63), CT9, CT10, Cyp-B, Dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), EphA2 (Zantek etc., Cell Growth Differ. (1999) 10:629-38; Carles-Kinch etc., Cancer Res. (2002) 62:2840-7), ELF2M, EphA2 (Zantek etc., Cell Growth Differ. (1999) 10:629-38; Carles-Kinch etc., 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, gp100, HAGE, HER2/neu, HLA-A*0201-R170I, HPV-E7, H-Ras, HSP70-2M, HST-2, hTERT, hTRT, iCE, apoptotic inhibitor (for example, survivin), KIAA0205, K-Ras, 12-K-Ras (K-Ras) with codon 12 sudden changes, LAGE, LAGE-1, LDLR/FUT, MAGE-1, 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, MC 1R, MDM-2, mesothelium element, myosin/m, MUC1, MUC2, MUM-1, MUM-2, MUM-3, newly-and poly A polymerase, NA88-A, N-Ras, NY-ESO-1, NY-ESO-1a (CAG-3), PAGE-4, PAP, protease 3 (PR3) (Molldrem etc., Blood (1996) 88:2450-7; Molldrem etc., Blood (1997) 90:2529-34), P15, p190, Pml/RARa, PRAME, PSA, PSM, PSMA, RAGE, RAS, RCASI, RU1, RU2, SAGE, SART-1, SART-2, SART-3, SP17, SPAS-1, TEL/AML1, TPI/m, tryrosinase, TARP, TRP-1 (gp75), TRP-2, TRP-2/INT2, WT-1, perhaps, the NY-ESO-ORF2 of translation and CAMEL protein are derived from NY-ESO-1 and LAGE-1 gene.
In some embodiments, by recombinant nucleic acid molecules, the antigen of the polynucleotide encoding in expression cassette and/or the carrier comprises any tumor associated antigen that can cause tumour-specific immune response, comprises antigen still to be identified.In some embodiments, recombinant nucleic acid molecules, the polynucleotide encoding in expression cassette and/or the carrier is more than a tumor associated antigen.
In some embodiments, antigen is mesothelium element (Argani etc., Clin Cancer Res.7 (12): 3862-8 (2001)), Spl7 (Lim etc., Blood 97 (5): 1508-10 (2001)), gp100 (Kawa kami etc., Proc.Natl.Acad.Sci.USA 91:6458 (1994)), PAGE-4 (Brinkmann etc., Cancer Res.59 (7): 1445-8 (1999)), TARP (Wolfgang etc., Proc.Natl.Acad.Sci.USA 97 (17): 9437-42 (2000)), EphA2 (Tatsumi etc., Cancer Res.63 (15): 4481-9 (2003)), PR3 (Muller-Berat etc., Clin.Immunol.I mmunopath.70 (1): 51-9 (1994)), prostate stem cell antigen (PSCA) (Reiter etc., Proc.Natl.Acad.Sci.USA, 95:1735-40 (1998); Kiessling etc., Int.J.Cancer, 102:390-7 (2002)), or SPAS-1 (U.S. Patent Application Publication No.2002/0150588).
In embodiments more of the present invention, the antigen of being encoded by recombinant nucleic acid molecules or expression cassette is CEA.In other embodiments, antigen is antigen fragment and/or the antigenic variant of CEA.CEA (comprises 90% colorectal carcinoma, gastric cancer and cancer of pancreas, 70% nonsmall-cell lung cancer in suitable people's tumor of vast scale, and 50% breast carcinoma) adhesive glycoprotein between the 180-kDA theca cell of overexpression in, (Hammarstrom, Semin.Cancer Biol., 9:67-81).After deliberation the panimmunity therapeutic agent as the simulation CEA anti-idiotype monoclonal antibody (Foon etc., Clin.Cancer Res., 87:982-90 (1995), or the recombined vaccinia virus vaccination (Tsang etc. of CEA are expressed in use, J.Natl.Cancer Inst., 87:982-90 (1995)), yet, unfortunately, only obtain limited success.However, researcher has been identified the epitope by the HLA*0201-restriction of the T cell line identification that produces, CAP-1 (CEA 605-613) from the patient of inoculation.DC inoculation patient with this epitope pulse fails to induce clinical response (Morse etc., Clin.CancerRes., 5:1331-8 (1999)).Recently, identify the CEA605-613 peptide agonists, had the irregular variation (CAP1-6D) that 610 aspartic acids are replaced to agedoite in the position.Although this aminoacid is replaced the MHC binding affinity that does not change this peptide, the raising that the use of the peptide part (APL) of change has still caused external CEA-specificity cell toxicity T lymphocyte (CTL) to produce.The CAP1-6D-specific CTL is kept their identification and ability (Zaremba etc., Cancer Res., the 57:4570-7 (1997) of the tumor cell of natural CEA are expressed in cracking; Salazar etc., Int.J.Cancer, 85:829-38 (2000)).Fong etc. have proved inducing of CEA-specific immunity among the colon cancer patient of the DC inoculation of using the Flt3-part expansion of hatching with this APL.Inspiring ground, among 12 patients 2 have experienced noticeable and peptide-MHC tetramer after vaccination
+The T cell induce relevant tumour regression (Fong etc., Proc.Natl.Acad.Sci.USA., 98:8809-14 (2001)).
In another embodiment, antigen is proteinase-3 or is derived from proteinase-3.For example, in the embodiment, antigen comprises HLA-A2.1-restriction peptide PR1 (aa 169-177; VLQELNVTV (SEQ ID NO:63)).Information about proteinase-3 and/or PR1 epitope can obtain in following list of references: U.S. Patent No. 5,180,819, Molldrem etc., Blood, 90:2529-2534 (1997); Molldrem etc., Cancer Research, 59:2675-2681 (1999); Molkdrem etc., Nature Medicine, 6:1018-1023 (2000); With Molldrem etc., Oncogene, 21:8668-8673 (2002).
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette and/or the expression vector comprises and is selected from K-Ras, H-Ras, N-Ras, N-Ras, 12-K-Ras, mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP and CEA, or comprise being derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigenic antigen of TARP and CEA.
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette and/or the carrier comprises and is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA.In some embodiments, polypeptide comprises K-Ras.In some embodiments, polypeptide comprises H-Ras.In some embodiments, polypeptide comprises N-Ras.In some embodiments, polypeptide comprises K-Ras.In some embodiments, polypeptide comprises mesothelium element (for example, people's mesothelium element).In some embodiments, polypeptide comprises PSCA.In some embodiments, polypeptide comprises NY-ESO-1.In some embodiments, polypeptide comprises WT-1.In some embodiments, polypeptide comprises survivin.In some embodiments, polypeptide comprises gp100.In some embodiments, polypeptide comprises PAP.In some embodiments, polypeptide comprises protease 3.In some embodiments, polypeptide comprises SPAS-1.In some embodiments, polypeptide comprises SP-17.In some embodiments, polypeptide comprises PAGE-4.In some embodiments, polypeptide comprises TARP.In some embodiments, polypeptide comprises CEA.
In some embodiments, by recombinant nucleic acid molecules, the antigen of the polynucleotide encoding in expression cassette and/or the carrier is to be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA.In some embodiments, antigen is K-Ras.In some embodiments, antigen is H-Ras.In some embodiments, antigen is N-Ras.In some embodiments, antigen is K-Ras.In some embodiments, antigen is the mesothelium element.In some embodiments, antigen is PSCA.In some embodiments, antigen is NY-ESO-1.In some embodiments, antigen is WT-1.In some embodiments, antigen is survivin.In some embodiments, antigen is gp100.In some embodiments, antigen is PAP.In some embodiments, antigen is protease 3.In some embodiments, antigen is SPAS-1.In some embodiments, antigen is SP-17.In some embodiments, antigen is PAGE-4.In some embodiments, antigen is TARP.In some embodiments, antigen is CEA.In some embodiments, antigen is people's mesothelium element.
In some embodiments, antigen is the mesothelium element, SPAS-1, and protease 3, EphA2, SP-17, gp100, PAGE-4, TARP or CEA, or be derived from antigen a kind of in those protein.In some embodiments, antigen is the mesothelium element or is derived from the mesothelium element.In other embodiments, antigen is EphA2 or the antigen that is derived from EphA2.In some embodiments, by recombinant nucleic acid molecules described herein, the antigen of the polynucleotide encoding in expression cassette or the expression vector is not Epha 2 (or be derived from Epha 2 antigen).In some embodiments, antigen is the tumor associated antigen except that Epha 2.In some embodiments, antigen is derived from the tumor associated antigen except that Epha 2.In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette and/or the expression vector comprises the antigen except that Epha 2.In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette and/or the expression vector comprises except that Epha 2 or is derived from antigen Epha 2 antigens.
In some embodiments, recombinant nucleic acid molecules, the polynucleotide encoding polypeptide in expression cassette and/or the expression vector, this polypeptide comprise and are derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA.In some embodiments, polypeptide comprises the antigen that is derived from K-Ras.In some embodiments, polypeptide comprises the antigen that is derived from H-Ras.In some embodiments, polypeptide comprises the antigen that is derived from N-Ras.In some embodiments, polypeptide comprises the antigen that is derived from 12-K-Ras.In some embodiments, polypeptide comprises the antigen that is derived from the mesothelium element.In some embodiments, polypeptide comprises the antigen that is derived from PSCA.In some embodiments, polypeptide comprises the antigen that is derived from NY-ESO-1.In some embodiments, polypeptide comprises the antigen that is derived from WT-1.In some embodiments, polypeptide comprises the antigen that is derived from survivin.In some embodiments, polypeptide comprises the antigen that is derived from gp100.In some embodiments, polypeptide comprises the antigen that is derived from PAP.In some embodiments, polypeptide comprises the antigen that is derived from protease 3.In some embodiments, polypeptide comprises the antigen that is derived from SPAS-1.In some embodiments, polypeptide comprises the antigen that is derived from SP-17.In some embodiments, polypeptide comprises the antigen that is derived from PAGE-4.In some embodiments, polypeptide comprises the antigen that is derived from TARP.In some embodiments, polypeptide comprises the antigen that is derived from CEA.
In some embodiments, recombinant nucleic acid molecules, the polynucleotide encoding in expression cassette and/or the expression vector is derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP or CEA.In some embodiments, antigen is derived from K-Ras.In some embodiments, antigen is derived from H-Ras.In some embodiments, antigen is derived from N-Ras.In some embodiments, antigen is derived from 12-K-Ras.In some embodiments, antigen is the antigen that is derived from the mesothelium element.In some embodiments, antigen is the antigen that is derived from PSCA.In some embodiments, antigen is the antigen that is derived from NY-ESO-1.In some embodiments, antigen is the antigen that is derived from WT-1.In some embodiments, antigen is the antigen that is derived from survivin.In some embodiments, antigen is the antigen that is derived from gp100.In some embodiments, antigen is the antigen that is derived from PAP.In some embodiments, antigen is the antigen that is derived from protease 3.In some embodiments, antigen is the antigen that is derived from SPAS-1.In some embodiments, antigen is the antigen that is derived from SP-17.In some embodiments, antigen is the antigen that is derived from PAGE-4.In some embodiments, antigen is the antigen that is derived from TARP.In some embodiments, antigen is the antigen that is derived from CEA.
In some embodiments, antigen is the mesothelium element, or its antigen fragment or antigenic variant.Therefore, in some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette and/or the carrier comprises the mesothelium element, or its antigen fragment or antigenic variant.In some embodiments, be the mesothelium element by the polypeptide of polynucleotide encoding, or its antigen fragment or antigenic variant.
In some embodiments, antigen is the mesothelium element (for example, people's mesothelium element) that lacked of the plain signal peptide of mesothelium and/or GPI (glycosyl-phosphatidyl inositol) anchor wherein.Therefore, in some embodiments, comprise the mesothelium element that plain signal peptide of mesothelium wherein and/or GPI anchor have lacked by the polypeptide of polynucleotide encoding.In some embodiments, be the mesothelium element that lacked of the plain signal peptide of mesothelium and/or GPI anchor wherein by the polypeptide of polynucleotide encoding.In some embodiments, be people's mesothelium element of having lacked of the plain signal peptide of mesothelium and/or GPI anchor wherein by the polypeptide of polynucleotide encoding.In some embodiments, be people's mesothelium element of having lacked of the plain signal peptide of mesothelium and GPI anchor both wherein by the polypeptide of polynucleotide encoding.
In some embodiments, antigen is NY-ESO-1, or its antigen fragment or antigenic variant.Therefore, in some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette or the carrier comprises NY-ESO-1 antigen, or its antigen fragment or antigenic variant.In some embodiments, polypeptide is a NY-ESO-1 antigen, or its antigen fragment or antigenic variant.
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette or the carrier comprises the antigen fragment of at least one tumor associated antigen, for example, and human psca (PSCA; Genbank Acc.No.AF043498), people's testis antigen (NY-ESO-1; Genbank Acc.No.NM_001327), hCEA (CEA; Genbank Acc.No.M29540), people's mesothelium element (Genbank Acc.No.U40434), people's survivin (Genbank Acc.No.U75285), human protease 3 (Genbank No.X55668), people K-Ras (Genbank Acc.Nos.M54969﹠amp; P01116), people H-Ras (Genbank Acc.No.P01112), people N-Ras (Genbank Acc.No.P01111), and 12-K-Ras (K-Ras that comprises the Gly12Asp sudden change) (referring to, for example, GenbankAcc.No.K00654).In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette or the expression vector comprises the antigen fragment with at least one conservative amino acid whose tumor associated antigen of replacing.In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette or the expression vector comprises the antigen fragment of the amino acid residue with at least one disappearance.In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding in expression cassette or the expression vector comprises the combination that is derived from more than the antigen sequence of one type tumor associated antigen, for example, is derived from the combination of the antigen fragment of the plain and Ras of mesothelium.
Prediction is that the example in antigenic tumor antigen zone comprises following: the aminoacid 25-35 of the PSCA aminoacid sequence among the Genbank Acc.No.AF043498; 70-80; And 90-118; The aminoacid 40-55 of NY-ESO-1 among the Genbank Acc.No.NM_001327,75-85,100-115 and 128-146; The aminoacid 70-75 of the CEA aminoacid sequence of Genbank Acc.No.M29540,150-155,205-225,330-340 and 510-520; The aminoacid 90-110 of the plain peptide sequence of the mesothelium of Genbank Acc.No.U40434,140-150,205-225,280-310,390-410,420-425 and 550-575; The amino acid/11 2-20 of the survival peptide sequence of Genbank Acc.No.U75285,30-40,45-55,65-82,90-95,102-115 and 115-130; The amino acid/11 0-20 of the aminoacid sequence of the protease 3 of finding among the Genbank Acc.No.X55668,30-35,65-75,110-120 and 160-170; The amino acid/11 0-20 of Genbank Acc.No.P01117 or M54968 (people K-Ras), 30-50,55-75,85-110,115-135,145-155 and 160-185; The amino acid/11 0-20 of Genbank Acc.No.P01112 (people H-Ras), 25-30,35-45,50-70,90-110,115-135 and 145-175; The amino acid/11 0-20 of Genbank Acc.No.P01111 (people N-Ras), 25-45,50-75,85-110,115-135,140-155 and 160-180; And 25 aminoacid of 12-k-Ras (being disclosed in the sequence among the Genbank Acc.No.K00654).Mark and draw by Hopp-Woods and Welling antigenicity and to predict these antigen zones.
In some embodiments, the discrete polypeptide of polypeptide conduct by polynucleotide encoding of the present invention, as fusion rotein with selected signal peptide, or inserted the protein chimera of another polypeptide as polypeptide wherein, be the polypeptide that comprises one or more following people's mesotheliums element peptides: SLLFLLFSL (aminoacid 20-28; (SEQ ID NO:64)); VLPLTVAEV (aminoacid 530-538; (SEQ ID NO:65)); ELAVALAQK (aminoacid 83-92; (SEQID NO:66)); ALQGGGPPY (aminoacid 225-234; (SEQ ID NO:67)); FYPGYLCSL (aminoacid 435-444; (SEQ ID NO:68)); And LYPKARLAF (aminoacid 475-484; (SEQ ID NO:69)).For example, in some embodiments, be to comprise people's mesothelium element (antigenicity) fragments one or more in these peptides by the antigen of polynucleotide encoding of the present invention.Can in the open WO 2004/006837 of PCT, find about the plain peptide sequences of these mesotheliums and with other information that the medical science related immune is replied dependency.
Perhaps, recombinant nucleic acid molecules, the polynucleotide in expression cassette or the expression vector autoimmune disease specific antigen polypeptide of autoimmune disease specific antigen (or comprise) of can encoding.In the cell-mediated autoimmune disease of T, the T cell causes autoimmune disease to the response of self antigen.Can cause the specific T-cells that autoimmune is replied by targeting with antigenic type used in the vaccine therapy autoimmune disease of the present invention.For example, antigen can be the part of TXi Baoshouti, that is, are specific idiotypes to those T cells that cause autoimmune to be replied, wherein introduce antigen in the vaccine of the present invention and will cause those T cells that causes autoimmune to be replied are had antigen-specific immune responses.Eliminating those T cells is the treatment mechanism of alleviating autoimmune disease.Another kind of probability is that the polynucleotide of coding for antigens are introduced in the recombinant nucleic acid molecules, the immunne response that the specific b cells that this antigen will cause targeting in autoimmune disease the antibody or the targeting of self antigen generation to be secreted this antibody is cloned.For example, the polynucleotide of coding idiotypicantigen can be introduced in the recombinant nucleic acid molecules, it will cause the anti-idiotype immunne response of such B cell and/or the reaction of the self antigen in antibody and the autoimmune disease.Include, but not limited to rheumatoid arthritis with the medicable autoimmune disease of the vaccine that comprises the antibacterial that contains expression cassette of the present invention and recombinant nucleic acid molecules, multiple sclerosis, Crohn disease, lupus, myasthenia gravis, vitiligo, scleroderma, psoriasis, pemphigus vulgaris, fibromyalgia, colitis and diabetes.Can adopt similar methods to treat allergy, wherein introduce the antigen targeting T cell in the vaccine antibacterial, B cell or effectively regulate allergic antibody.In some autoimmune diseases such as the psoriasis, disease causes the growth of super proliferative cell, and antigenic expression that also can targeting.Should consider such antigen that will cause to the immunne response of super proliferative cell.
In some embodiments, antigen is the antigen of the unique disease relative protein white matter of targeting structure.One of example of this situation is to use aforesaid idiotypicantigen targeting antibodies, B cell or T cell.Another kind of probability is the particular protein structure that targeting is produced by specified disease.The example of this situation is to introduce the antigen that will produce proteinic immunne response, and this protein causes at disease such as Alzheimer's disease, observed amyloid speckle in Creutz Fil spy-Jacob disease (CJD) and the mad cow disease (BSE).Although this method may only be used for the minimizing that speckle forms, it may provide medicable vaccine in the disease situation as CJD.This disease is that the infection form by prion protein causes.In some embodiments, the antigen of polynucleotide encoding prion protein infectious form of the present invention makes and can eliminate, alleviates or control the infectious albumen that causes CJD by the immunne response of vaccine generation.
In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or expression vector comprises infectious disease antigen or is derived from the antigenic antigen of infectious disease.In some embodiments, polypeptide comprises infectious disease antigen.In some other embodiments, polypeptide comprises and is derived from the antigenic antigen of infectious disease.In some embodiments, by recombinant nucleic acid molecules, the polypeptide of the polynucleotide encoding of expression cassette and/or expression vector is infectious disease antigen or is derived from the antigenic antigen of infectious disease.In some embodiments, by recombinant nucleic acid molecules, the polypeptide of expression cassette and/or expression vector codes is an infectious disease antigen.In some embodiments, by recombinant nucleic acid molecules, the peptide source of expression cassette and/or expression vector codes is from infectious disease antigen.
In other embodiments of the present invention, antigen is derived from human or animal's pathogen.Pathogen is virus alternatively, antibacterial, fungus or protozoacide.For example, antigen can be virus or fungus or bacterial antigens.In one embodiment, by recombinant nucleic acid molecules, the antigen that is derived from pathogen of expression cassette and/or expression vector codes is the protein that is produced by pathogen, or is derived from the protein that is produced by pathogen.For example, in some embodiments, by recombinant nucleic acid molecules, the polypeptide of expression cassette and/or expression vector codes is proteinic fragment and/or the variant that is produced by pathogen.
For example, in some embodiments, antigen is derived from the human immunodeficiency virus (as gp120, gp160, gp41, gag antigen such as p24gag and p55gag, and the pol that is derived from HIV, env, tat, vif, rev, nef, vpr, the protein in vpu and LTR zone), feline immunodeficiency virus, or human or animal's hepatitis virus.For example, in some embodiments, antigen is gp120.In one embodiment, antigen be derived from 1 type and herpes simplex types 2 virus (HSV) (as gD, gB, gH, early protein such as ICP27 immediately), from cytomegalovirus (as gB and gH), from inferior Pneumovirinae (metapneumovirus), from Epstein-Barr virus or from varicella zoster virus (as gpI, II or III) (referring to, for example, Chee etc. (1990) Cytomegaloviruses (J.K.McDougall, editor, Springer Verlag, pp.125-169; McGeoch etc. (1988) J.Gen.Virol.69:1531-1574; U.S. Patent No. 5,171,568; Baer etc., (1984) Nature 310:207-211; With (1986) J.Gen.Virol.67:1759-1816. such as Davison)
In another embodiment, antigen is derived from hepatitis virus such as hepatitis B virus (for example, hepatitis B surface antigen), hepatitis A virus (HAV), hepatitis C virus, hepatitis, hepatitis E virus, or hepatitis G virus.Referring to, for example, WO 89/04669; WO 90/11089 and WO 90/14436.Hepatitis antigen can be the surface, core or other relevant antigen.Several virus proteins of HCV genome encoding comprise E1 and E2.Referring to, for example, Houghton etc., Hepatology14:381-388 (1991).
It is the virus that the antigen of virus antigen randomly is derived from following arbitrary Viraceae: Picornaviridae (for example, poliovirus, rhinovirus etc.); Caliciviridae; Togavirus section (for example, rubella virus, dengue virus etc.); Flaviviridae; Coronaviridae; Arc reovirus virus section (for example, rotavirus etc.); Birnavirus section; Rhabdoviridae (for example, rabies virus, etc.); Orthomyxoviridae family (for example, first, second, influenza virus C, etc.); Filamentous form virus section; Paramyxoviridae (for example, mumps virus, Measles virus, respiratory syncytial virus, parainfluenza virus, etc.); The Bu Niluoao Viraceae; Arenaviridae; Retroviridae (for example, HTLV-I; HTLV-11; HIV-1 (be also referred to as HTLV-111, LAV, ARV, hTLR, etc.)), comprising but be not limited to from separator HIVI11b HIVSF2, HTVLAV, HIVLAI, HIVMN; HIV-1CM235, HIV-1; HIV-2; Simian immunodeficiency virus (SIV); Human papillomavirus, Cicadae passes encephalitis; Deng antigen.Referring to, for example, Virology, the 3rd edition (W.K.Joklik edits, 1988); FundamentalVirology, the 3rd edition (B.N.Fields, D.M.Knipe and P.M.Howley edit, and 1996), these and other viruses have been described.In one embodiment, antigen is Flu-HA (Morgan etc., J.Immunol.160:643 (1998)).
In some alternate embodiments, antigen is derived from bacterial pathogens, as Mycobacterium, bacillus, Yersinia, Salmonella, eisseria (Neisseria), Borrelia (Borrelia) (for example, OspA or OspB or derivatives thereof), chlamydiaceae (Chlamydia) or Boulder Salmonella (Bordetella) are (for example, P.69, PT and FHA), or be derived from parasite such as plasmodium or bow slurry worm.In one embodiment, antigen be derived from mycobacterium tuberculosis (Mycobacterium tuberculosis) (for example, ESAT-6,85A, 85B, 85C, 72F), Bacillus anthracis (for example, PA) or Yersinia pestis (for example, F1, V).In addition, can cause the pathogen acquisition of disease or produce the antigen that is applicable to the present invention that these diseases comprise from known, but be not limited to diphtheria, pertussis, tetanus, pulmonary tuberculosis, bacterial pneumonia or fungal pneumonia, otitis media, gonorrhea, cholera, typhoid fever, meningitis, monocytosis, pestilence, shigellosis or salmonellosis, legionnaires disease, Lyme disease, leprosy, malaria, ancylostomiasis, onchocerciasis, schistosomicide, african trypanosomiasis, leishmaniasis, giardiasis, amebiasis, filaricide, Borelia and trichonematosis.More again antigen can obtain or generation from unconventional pathogen, described unconventional pathogen such as Kuru disease, CJ disease (CJD), pruritus disease, the pathogen of heritability ermine encephalopathy and chronic wasting disease, or from the albumen infectious particles as the Protein virus relevant with bovine spongiform encephalopathy.
Still in other embodiments, antigen is from neurodegenerative disease (as Alzheimer's disease), and related biotic factor obtains or generation in the outbreak of metabolic disease (as type i diabetes) and drug dependence (as nicotine addiction) or the progress.Perhaps, the antigen of being encoded by recombinant nucleic acid molecules is used for pain management, and antigen is other related factors in pain receptor or the pain signal conduction.
In some embodiments, antigen is human protein or is derived from human protein.In other embodiments, antigen is non-human protein or is derived from non-human protein's matter (its fragment and/or variant).In some embodiments, be from non-human animal's protein or be derived from non-human animal's protein by the antigen part of the fusion rotein of expression cassette coding.For example, even antigen will be expressed in being used for the vaccine based on Listera of human body, in some embodiments, antigen also can be Mus mesothelium element or be derived from Mus mesothelium element.
V. codon optimization
In some embodiments, the one or more polynucleotide (that is polynucleotide sequence) in the recombinant nucleic acid molecules, expression cassette and/or expression vector are codon optimized (with respect to natural coded sequences).In some embodiments, the polynucleotide of the coded signal peptide in the recombinant nucleic acid molecules described herein (and/or expression cassette and/or expression vector) are that codon is optimized for the expression in antibacterial.In some embodiments, polypeptide such as antigen or the proteic polynucleotide of other treatment beyond the coded signal peptide are that codon is optimized for the expression in antibacterial.In some embodiments, another polynucleotide with the polypeptide of signal peptide fusion of the polynucleotide of coded signal peptide and coding are that codon is optimized for the expression in antibacterial.In some embodiments, coding is that codon is optimized as the polynucleotide of the secretory protein (or its fragment) of support or the polynucleotide of coding autolysin (or its fragment or variant).
The codon more commonly used than original password of natural coded sequence substitutes if at least one codon of the natural coded sequence of polynucleotide has wherein been waited to express the organism (" target organism ") of coded sequence, comprises that then the polynucleotide of coded sequence are " codon is optimized ".For example, preferential codon of expressing substitutes in the certain detail strain of non-bacterial antigens if at least one is wherein waited to express from the codon of natural bacteria polynucleotide sequence, and then coding treats that the polynucleotide of the non-bacterial antigens that reach at certain detail strain invading the exterior are that codon is optimized.As another example, substituting if at least one codon in the polynucleotide sequence more is usually used in this amino acid whose codon by Listeria monocytogenes than the codon in the primitive man sequence, will be that codon is optimized for the antigenic polynucleotide of coding human cancer of the part of expression cassette in the reorganization Listeria monocytogenes then.Equally, substitute if at least one codon in the polynucleotide sequence of coded signal peptide more is usually used in this amino acid whose codon by Listeria monocytogenes than the codon in original (natural) sequence, then will comprise that the polynucleotide of the coding Listeria monocytogenes natural signals peptide (as the LLO signal peptide from Listeria monocytogenes) of the part of the antigenic Expression of Fusion Protein box of human cancer are that codon is optimized for coding in the reorganization Listeria monocytogenes.In some embodiments, replaced at least one codon is alternate by target organism be most commonly used to the to encode codon of same amino acid in the codon optimization sequence.
In some embodiments, the organism codon more commonly used than original password of natural coded sequence that at least two codons of the natural coded sequence of polynucleotide have wherein been waited to express coded sequence substitutes.In some embodiments, the natural coded sequence of polynucleotide at least about 5 codons, at least about 10 codons, or the organism of wherein having been waited to express coded sequence at least about 20 codons codon more commonly used than original password of natural coded sequence substitutes.
In some embodiments, the codon at least about 10% in the codon optimization polynucleotide is substituted by the codon of target organism (than original password of native sequences) (or the most frequently used) more commonly used.In some embodiments, the codon at least about 25% in the codon optimization polynucleotide is substituted by the codon of target organism (or the most frequently used) more commonly used.In other embodiments, the codon at least about 50% in the codon optimization polynucleotide is substituted by the codon of target organism (or the most frequently used) more commonly used.Still in other embodiments, the codon at least about 75% in the codon optimization polynucleotide is substituted by the codon of target organism (or the most frequently used) more commonly used.
Those skilled in the art broad research the codon of different organisms preferentially select.For example, referring to Sharp etc., Nucleic Acids Res., 15:1281-95 (1987) and Uchijima etc., The Journal of Immunology, 161:5594-9 (1998).As a result, the codon option table of multiple organism is that the public is obtainable.For example, can be on the internet with www.kazusa.or.jp/codon/ and the codon option table that on the obtainable website of other public, finds multiple organism.(referring to, for example, Nakamura etc., (2000) Nucleic Acids Research 28:292.) from the sub-option table of the example password of www.kazusa.or.jp/codon/, codon option table (the http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi of Listeria monocytogenes? species=Listeria+monocytogenes+[gbbtc]), in following table 2A, reproduce for convenience.Following table 2B, 2C, 2D, Bacillus anthracis also is provided respectively among 2E and the 2F, mycobacterium tuberculosis, Salmonella typhimurium (Salmonella typhimurium), the sub-option table of example password of Mycobacterium bovis (Mycobacterium bovis) BCG and Fei Shi shigella (Shigellaflexneri).
Table 2A: the codon option table (from www.kazusa.or.jp/codon/) of Listeria monocytogenes
Listeria monocytogenes: 3262CDS ' s (1029006 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
UUU?29.4(30274) UCU?13.2(13586) UAU?22.9(23604) UGU?3.8(3960)
UUC?14.1(14486) UCC?6.5(6714) UAC?10.7(11055) UGC?1.9(1972)
UUA?36.8(37821) UCA?10.4(10751) UAA?2.2(2307) UGA?0.6(583)
UUG?12.3(12704) UCG?6.1(6278) UAG?0.4(372) UGG?9.3(9580)
CUU?21.0(21567) CCU?8.4(8622) CAU?12.0(12332) CGU?12.6(12930)
CUC?5.4(5598) CCC?1.7(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)
CUG?5.0(5120) CCG?7.0(7219) CAG?5.1(5234) CGG?2.8(2884)
AUU?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)
AUA?9.4(9642) ACA?26.5(27318) AAA?61.6(63379) AGA?6.9(7111)
AUG?25.9(26651) ACG?12.9(13285) AAG?10.4(10734) AGG?1.2(1254)
GUU?26.4(27202) GCU?24.3(24978) GAU?39.8(40953) GGU?24.2(24871)
GUC?8.7(8990) GCC?8.4(8612) GAC?14.3(14751) GGC?14.2(14581)
GUA?21.6(22247) GCA?28.6(29401) GAA?60.4(62167) GGA?19.1(19612)
GUG?13.1(13518) GCG?16.6(17077) GAG?13.1(13507) GGG?8.7(9003)
Table 2B: the codon option table (from www.kazusa.or.jp/codon/) of Bacillus anthracis
Bacillus anthracis [gbbct]: 312CDS ' s (90023 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
UUU?32.4(2916) UCU?17.2(1547) UAU?31.9(2876) UGU?5.1(455)
UUC?10.4(934) UCC?5.0(453) UAC?9.5(853) UGC?1.8(164)
UUA?43.7(3931) UCA?14.8(1330) UAA?2.2(199) UGA?0.5(47)
CUG?11.4(1024) UCG?4.2(375) UAG?0.7(66) UGG?9.3(835)
CUU?14.4(1300) CCU?10.7(967) CAU?15.5(1392) CGU?9.8(883)
CUC?3.7(335) CCC?2.7(242) CAC?4.2(379) CGC?2.5(223)
CUA?12.4(1117) CCA?17.8(1599) CAA?32.3(2912) CGA?6.3(569)
CUG?4.4(392) CCG?5.9(534) CAG?9.5(859) CGG?2.0(179)
AUU?44.5(4009) ACU?21.0(1890) AAU?44.0(3959) AGU?17.4(1565)
AUC?11.9(1072) ACC?5.0(453) AAC?14.1(1268) AGC?5.2(467)
AUA?22.7(2042) ACA?26.8(2414) AAA?64.3(5786) AGA?13.7(1236)
AUG?23.3(2098) ACG?9.4(844) AAG?22.7(2047) AGG?4.1(368)
GUU?20.3(1824) GCU?17.8(1598) GAU?39.3(3536) GGU?17.9(1611)
GUC?4.6(414) GCC?4.1(372) GAC?9.0(811) GGC?5.8(524)
GUA?26.4(2374) GCA?23.5(2117) GAA?53.9(4855) GGA?24.5(2203)
GUG?10.8(973) GCG?7.9(709) GAG?17.9(1614) GGG?12.0(1083)
The 3rd alphabetical GC25.51% of coding GC 34.55% first alphabetical GC44.99% second letter GC 33.16%
Table 2C: the codon option table (from www.kazusa.or.jp/codon/) of mycobacterium tuberculosis
Mycobacterium tuberculosis [gbbct]: 363CDS ' s (131426 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
UUU?5.4(709) UCU?2.0(265) UAU?6.0(788) UGU?2.5(326)
UUC?25.6(3359) UCC?11.4(1499) UAC?17.6(2307) UGC?5.6(738)
UUA?1.8(231) UCA?4.3(571) UAA?0.4(52) UGA?1.5(201)
UUG?14.8(1945) UCG?19.2(2522) UAG?0.8(103) UGG?17.9(2352)
CUU?5.9(778) CCU?3.9(511) CAU?5.4(711) CGU?8.0(1048)
CUC?17.7(2329) CCC?18.3(2411) CAC?14.7(1928) CGC?26.7(3508)
CUA?4.0(521) CCA?6.4(843) CAA?7.8(1030) CGA?5.8(764)
CUG?45.9(6032) CCG?33.2(4359) CAG?24.2(3176) CGG?21.1(2772)
AUU?7.6(993) ACU?4.1(545) AAU?4.8(637) AGU?4.0(531)
AUC?32.7(4300) ACC?36.0(4735) AAC?26.3(3451) AGC?15.0(1976)
AUA?2.1(282) ACA?4.7(616) AAA?5.8(761) AGA?1.5(192)
AUG?19.7(2591) ACG?16.4(2158) AAG?26.5(3485) AGG?3.3(429)
GUU?8.3(1095) GCU?11.2(1473) GAU?15.6(2046) GGU?18.7(2455)
GUC?32.3(4249) GCC?51.5(6769) GAC?44.6(5858) GGC?48.6(6383)
GUA?4.7(622) GCA?12.4(1625) GAA?16.8(2211) GGA?9.0(1183)
GUG?35.7(4687) GCG?41.7(5482) GAG?35.8(4702) GGG?16.9(2215)
The 3rd alphabetical GC79.95% of coding first alphabetical GC65.27% second letter GC48.28% of GC64.43%
Table 2D: the codon option table of Salmonella typhimurium (from www.kazu sa.or.jp/codon/)
Salmonella typhimurium [gbbct]: 1322CDS ' s (416065 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
UUU?21.7(9041) UCU?8.5(3518) UAU?16.5(6853) UGU?4.6(1920)
UUC?15.1(6265) UCC?10.6(4430) UAC?11.6(4826) UGC?6.1(2524)
UUA?13.6(5650) UCA?7.9(3286) UAA?1.8(731) UGA?1.1(465)
UUG?12.1(5025) UCG?9.4(3924) UAG?0.3(121) UGG?14.1(5851)
CUU?12.1(5038) CCU?7.9(3290) CAU?12.1(5047) CGU?18.1(7542)
CUC?10.6(4396) CCC?7.0(2921) CAC?9.2(3818) CGC?20.8(8659)
CUA?4.7(1958) CCA?6.5(2712) CAA?12.8(5315) CGA?4.1(1695)
CUG?49.3(20508) CCG?22.7(9463) CAG?30.8(12803) CGG?7.2(3004)
AUU?28.1(11700) ACU?8.2(3401) AAU?19.5(8107) AGU?8.6(3569)
AUC?23.9(9941) ACC?24.0(9980) AAC?21.4(8920) AGC?18.0(7485)
AUA?6.7(2771) ACA?8.0(3316) AAA?33.0(13740) AGA?3.2(1348)
AUG?26.1(10842) ACG?18.6(7743) AAG?12.4(5151) AGG?2.3(959)
GUU?16.4(6831) GCU?14.4(5985) GAU?32.9(13700) GGU?18.1(7541)
GUC?17.7(7367) GCC?27.5(11462) GAC?21.5(8949) GGC?33.0(13730)
GUA?11.9(4935) GCA?14.8(6156) GAA?36.1(15021) GGA?9.1(3788)
GUG?24.3(10092) GCG?37.0(15387) GAG?20.9(8715) GGG?11.6(4834)
The 3rd alphabetical GC57.71% of coding first alphabetical GC58.32% second letter GC41.31% of GC52.45%
Table 2E: the codon option table (from www.kazusa.or.jp/codon/) of Mycobacterium bovis BGG
Mycobacterium bovis BGG[gbbct]: 51CDS ' s (16528 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
UUU?4.7(77) UCU?1.9(31) UAU?6.6(109) UGU?2.0(33)
UUC?27.4(453) UCC?11.4(189) UAC?17.0(281) UGC?6.7(110)
UUA?1.6(26) UCA?4.5(74) UAA?0.9(15) UGA?1.3(22)
UUG?14.7(243) UCG?20.8(343) UAG?0.8(14) UGG?14.3(237)
CUU?5.6(92) CCU?2.9(48) CAU?4.9(81) CGU?9.4(155)
CUC?14.8(244) CCC?16.3(270) CAC?17.2(285) CGC?33.8(559)
CUA?5.1(85) CCA?5.1(84) CAA?7.3(120) CGA?7.1(118)
CUG?51.5(852) CCG?31.0(512) CAG?25.5(421) CGG?26.7(441)
AUU?6.1(100) ACU?3.1(51) AAU?4.8(80) AGU?2.8(46)
AUC?39.6(654) ACC?36.8(609) AAC?22.3(369) AGC?14.5(240)
AUA?2.2(37) ACA?4.4(73) AAA?6.2(102) AGA?1.1(19)
AUG?20.2(334) ACG?17.4(288) AAG?24.5(405) AGG?3.8(62)
GUU?7.8(129) GCU?9.6(158) GAU?13.4(222) GGU?26.9(280)
GUC?30.1(497) GCC?54.3(898) GAC?45.6(754) GGC?42.6(704)
GUA?4.1(67) GCA?12.5(206) GAA?16.5(273) GGA?7.3(120)
GUG?37.6(621) GCG?41.7(689) GAG?32.7(541) GGG?16.7(276)
The 3rd alphabetical GC81.04% of coding first alphabetical GC65.36% second letter GC48.07% of GC64.82%
Table 2F: the codon option table (from www.kazusa.or.jp/codon/) of Fei Shi shigella
Fei Shi shigella [gbbct]: 706CDS ' s (180312 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
UUU?25.8(4658) UCU?16.6(2986) UAU?21.9(3945) UGU?6.9(1252)
UUC?15.1(2714) UCC 9.5(1717) UAC?11.0(1992) UGC?5.6(1011)
UUA?20.8(3756) UCA?15.6(2821) UAA?2.0(362) UGA?1.4(254)
UUG?13.4(2424) UCG 6.9(1241) UAG?0.5(91) UGG?13.1(2357)
CUU?17.6(3169) CCU 9.2(1656) CAU?15.1(2725) CGU?15.0(2707)
CUC?10.4(1878) CCC 5.9(1072) CAC?8.2(1472) CGC?12.6(2269)
CUA?7.2(1295) CCA 9.7(1744) CAA?15.9(2861) CGA?5.8(1046)
CUG?33.5(6045) CCG?12.2(2199) CAG?23.6(4255) CGG?9.0(1627)
AUU?30.0(5417) ACU?13.8(2480) AAU?33.5(6044) AGU?15.3(2764)
AUC?16.7(3018) ACC?13.4(2413) AAC?18.6(3348) AGC?12.7(2281)
AUA?18.9(3402) ACA?16.2(2930) AAA?41.6(7507) AGA?10.3(1865)
AUG?23.3(4198) ACG?10.0(1809) AAG?16.4(2961) AGG?5.7(1029)
GUU?19.8(3576) GCU?19.6(3527) GAU?34.0(6123) GGU?19.2(3468)
GUC?11.8(2126) GCC?18.5(3338) GAC?16.3(2939) GGC?15.3(2754)
GUA?13.1(2370) GCA?22.2(4009) GAA?37.5(6763) GGA?15.1(2727)
GUG?16.1(2910) GCG?15.2(2732) GAG?21.7(3913) GGG?10.9(1970)
The 3rd alphabetical GC43.32% of coding first alphabetical GC51.72% second letter GC38.85% of GC44.63%
In embodiments more of the present invention, in the codon optimization coded sequence at least about 10%, at least about 25%, at least about 50% or be this used in the target organism amino acid whose most preferably codon at least about 75% codon.In other embodiments, 100% codon is this amino acid whose most preferably codon (that is, this sequence is " codon is optimized fully ") used in the target organism in the codon optimization coded sequence.For example, among the embodiment shown below, all codons that are characterized by the optimized sequence of codon all are the most frequently used codons of target organism; Yet any codon replacement that forms the codon more commonly used than original (natural) sequence can be thought " codon is optimized ".Shown in the following table 3 in the Listeria monocytogenes each amino acid whose best codon has been selected.
Table 3: the best codon option table in the Listeria monocytogenes
| Aminoacid | The single-letter code | Best Listera codon |
| Alanine | A | GCA |
| Arginine | R | CGU |
| Agedoite | N | AAU |
| Aspartic acid | D | GAU |
| Cysteine | C | UGU |
| Glutamine | Q | CAA |
| Glutamic acid | E | GAA |
| Glycine | G | GGU |
| Histidine | H | CAU |
| Isoleucine | I | AUU |
| Leucine | L | UUA |
| Lysine | K | AAA |
| Methionine | M | AUG |
| Phenylalanine | F | UUU |
| Proline | P | CCA |
| Serine | S | AGU |
| Threonine | T | ACA |
| Tryptophan | W | UGG |
| Tyrosine | Y | UAU |
| Valine | V | GUU |
In some embodiments, codon optimization polynucleotide encoding signal peptide.In some embodiments, signal peptide is that the optimized antibacterial of codon is an external source to sequence wherein.In other embodiments, signal peptide is that the optimized antibacterial of codon is natural to sequence wherein.For example; in some embodiments; the optimized polynucleotide encoding signal peptide of codon; these signal peptides are selected from the LLO signal peptide of Listeria monocytogenes; the USP45 signal peptide of lactococcus lactis; the protective antigen signal peptide of Bacillus anthracis, the PhoD signal peptide Tat signal peptide of the p60 signal peptide of Listeria monocytogenes and bacillus subtilis.In some embodiments, the signal peptide of the optimized polynucleotide encoding of codon except that the protective antigen signal peptide of anthrax bacillus cereus.In some embodiments, the polynucleotide of coded signal peptide are that codon is optimized for the expression in Listeria monocytogenes.
In some embodiments, the optimized polynucleotide encoding of codon has been (non-signal peptide) protein of the optimized antibacterial external source of codon to polynucleotide sequence wherein.In some embodiments, the optimized polynucleotide encoding of codon comprises antigenic polypeptide.For example, in some embodiments, the optimized polynucleotide encoding of codon comprises antigenic polypeptide, and this antigen is tumor associated antigen or the antigen that is derived from tumor associated antigen.
In some embodiments, the codon optimization of the polynucleotide of coded signal peptide and/or other polypeptide, with respect to the polypeptide that does not have the optimized polynucleotide of codon to improve to comprise signal peptide and/or other polypeptide of correspondence (as fusion rotein, protein chimera and/or by recombinant nucleic acid molecules, the allogenic polypeptide of expression cassette or expression vector codes) expression in antibacterial.In some embodiments, the codon optimization of polynucleotide has improved to be expressed at least about 2 times, at least about 5 times, and at least about 10 times, or at least about 20 times (with respect to not having for the optimized polynucleotide of codon of correspondence).In some embodiments, the codon optimization of the polynucleotide of coded signal peptide and/or other polypeptide, there are not optimized polynucleotide of codon with respect to correspondence, improved the secretion of polypeptide (as fusion rotein, protein chimera and/or allogenic polypeptide) from antibacterial that comprises signal peptide and/or other polypeptide.In some embodiments, the codon optimization has improved secretion at least about 2 times, at least about 5 times, and at least about 10 times, or at least about 20 times (with respect to correspondences do not have optimized polynucleotide of codon).In some embodiments, improved simultaneously and expressed and excretory level.Use those standard techniques of this area such as the western blot analysis of various Related Bacteria culture components can easily evaluate expression and/or excretory level.
VI. expression cassette
The present invention also provides expression cassette.For example, in some embodiments, the invention provides the expression cassette that comprises arbitrary recombinant nucleic acid molecules described herein and further comprise the promoter sequence of coded sequence in the recombinant nucleic acid molecules that is operably connected (for example, second polynucleotide of first polynucleotide of coded signal peptide and another polypeptide of coding).In some embodiments, expression cassette is isolating.In some other embodiments, expression cassette is contained in the expression vector, and this expression vector can be isolatingly maybe can be contained in the antibacterial.Still in the more embodiment, expression cassette is arranged in the chromosomal DNA of antibacterial.For example, in some embodiments, expression cassette has been integrated in the genome of antibacterial.In some embodiments, the expression cassette that is integrated in the bacterial genomes comprises one or more elements from genomic DNA.For example, in some embodiments, the a certain site of recombinant nucleic acid molecules insertion bacterial genomes DNA (for example, by site-specific integration or homologous recombination), make and be operably connected promoter in the genomic DNA Already in of this recombinant nucleic acid therefore produce the new expression cassette that is integrated in the genomic DNA.In some other the embodiment, expression cassette is as the complete unit integration (for example, by site-specific integration or homologous recombination) to genomic DNA that comprises promoter and recombinant nucleic acid molecules.
In some embodiments, the design expression cassette is used at the antibacterial express polypeptide.In some embodiments, the design expression cassette is used for expressing heterologous polypeptide such as heterologous antigen on antibacterial.In some embodiments, expression and/or secretion that expression cassette provides polypeptide to improve.
Usually, expression cassette comprises following orderly element: the polynucleotide of (1) promoter and (2) coded polypeptide.In some embodiments, expression cassette comprises following element: (1) promoter; (2) polynucleotide of coded signal peptide; (3) polynucleotide of coded polypeptide (for example, heterologous protein).Still in other embodiments, expression cassette comprises following element: (1) procaryotic promoter; (2) SD sequence; (3) polynucleotide of coded signal peptide; (4) polynucleotide of coded polypeptide (as heterologous protein).In some embodiments, expression cassette comprises more than a promoter.
In some embodiments, expression cassette can also comprise the transcription terminator that inserts the terminal downstream of the sub-C-of the translation stop codon relevant with heterologous polypeptide.For example, in some embodiments, transcription terminator can use in being designed for the construct of stable integration to the bacterial chromosome.Although do not need, with transcription terminator as final orderly element be contained in can prevent in the allogeneic gene expression box since connect read to transcribe cause contiguous gene is expressed the polarity effect of regulating.Therefore, in some embodiments, the proper sequence element with not relying on the tanscription termination of rho that promotion well known by persons skilled in the art relies on rho can place the heterologous protein expression cassette.
On the one hand, the invention provides the expression cassette that comprises following composition: (a) first polynucleotide of coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial; (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise the fusion rotein of signal peptide and polypeptide.
On the other hand, the invention provides expression cassette, first polynucleotide that comprise (a) coding antibacterial natural signals peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the be operably connected promoter of first and second polynucleotide of expression cassette, wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, be allogenic by second polynucleotide and first polynucleotide.In some embodiments, polypeptide is that natural antibacterial is allogenic (that is being external source to antibacterial) to signal peptide.In some embodiments, the antibacterial that produces signal peptide is an intracellular bacteria.In some embodiments, antibacterial is selected from Listera and belongs to bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria and escherichia coli.In some embodiments, antibacterial is the antibacterial (for example, Listeria monocytogenes) that Listera belongs to.In some embodiments, second polynucleotide is that codon is optimized for the expression in antibacterial.
On the other hand, the invention provides expression cassette, wherein expression cassette comprises first polynucleotide of (a) coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression that belongs at Listera in the antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the be operably connected promoter of first and second polynucleotide of expression cassette, wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, expression cassette is the polycistronic expression box.In some embodiments, second polynucleotide is that codon is optimized for the expression that belongs at Listera in the antibacterial.In some embodiments, by the polypeptide of second polynucleotide encoding Listera being belonged to antibacterial is external source (that is it is allogenic, Listera being belonged to antibacterial).In some embodiments, be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, expression cassette comprises more than a promoter.
On the other hand, the invention provides expression cassette, comprise first polynucleotide of the non--secA1 antibacterial signal peptide of (a) coding; (b) be positioned at second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise the fusion rotein of signal peptide and polypeptide.In some embodiments, first polynucleotide and/or second polynucleotide are that codon is optimized for the expression in antibacterial, described antibacterial such as Listera belong to, bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria or escherichia coli.In some embodiments, polynucleotide are that codon is optimized for the expression in Listera such as Listeria monocytogenes.In some embodiments, be natural to the optimized antibacterial of codon by the signal peptide of optimized first polynucleotide encoding of codon.In some embodiments, first polynucleotide of coded signal peptide and second polynucleotide are allogenic.In some embodiments, be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, expression cassette is the polycistronic expression box.In some embodiments, first polynucleotide, second polynucleotide, or first is that codon is optimized with second polynucleotide for the expression that belongs at Listera in the antibacterial (for example, Listeria monocytogenes).In some embodiments, first and second polynucleotide are allogenic each other.In some embodiments, be allogenic each other by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, by the polypeptide of second polynucleotide encoding Listera being belonged to antibacterial is external source (that is it is allogenic, Listera being belonged to antibacterial).In some embodiments, expression cassette comprises more than a promoter.
The present invention also provides the expression cassette that comprises following composition: (a) polynucleotide of coding Listera allogenic polypeptide, and wherein polynucleotide are that codon is optimized for the expression in Listera; (b) promoter, the polynucleotide of the encoding exogenous that is operably connected polypeptide.In some embodiments, be antigen (for example, referring to the antigenic description of above possibilities) by the expression cassette encoded polypeptides.In some embodiments, expression cassette further comprises the polynucleotide of coded signal peptide.The polynucleotide of the coded signal peptide promoter that also is operably connected makes expression cassette express the fusion rotein that comprises allogenic polypeptide and signal peptide.The polynucleotide that are applicable to the coded signal peptide in the expression cassette include, but not limited to described above those.In some embodiments, the polynucleotide that are included in the coded signal peptide in the expression cassette are that codon is optimized for the expression in antibacterial such as aforesaid Listera (for example, Listeria monocytogenes).
The present invention also provides the expression cassette that comprises following composition: (a) first polynucleotide of the non-Listera signal peptide of coding; (b) be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.In some embodiments, expression cassette is the polycistronic expression box.In some embodiments, first polynucleotide, second polynucleotide, or first is that codon is optimized with second polynucleotide for the expression in Listera (for example, Listeria monocytogenes).In some embodiments, first and second polynucleotide are allogenic each other.In some embodiments, be allogenic each other by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, the antibacterial that Listera is belonged to by the polypeptide of second polynucleotide encoding is external source (that is, the antibacterial that Listera is belonged to is allogenic).In some embodiments, expression cassette comprises more than a promoter.
The present invention further provides expression cassette, wherein expression cassette comprises first polynucleotide of (a) coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant; (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises by the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant, wherein in the protein chimera, polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or insert in autolysin or its catalytic activity fragment or the catalytic activity variant.In some embodiments, the protein chimera has the catalytic activity the same with autolysin.In some embodiments, polypeptide and autolysin are allogenic.In some embodiments, the antibacterial autolysin is from intracellular bacteria (for example, Listera).In some embodiments, second polynucleotide of coded polypeptide are inserted in first polynucleotide of coding autolysin or its catalytic activity fragment or catalytic activity variant, and the expression cassette polypeptide of encoding wherein inserts autolysin or its catalytic activity fragment or catalytic activity and becomes intravital protein chimera (that is, polypeptide is embedded in autolysin or its catalytic activity fragment or the catalytic activity variant).In the alternate embodiment, second polynucleotide are positioned at outside first polynucleotide of coding autolysin or its catalytic activity fragment or catalytic activity variant, and the expression cassette wherein protein chimera of polypeptide and autolysin or its catalytic activity fragment or the fusion of catalytic activity variant of encoding.In some embodiments.Polypeptide and autolysin are allogenic.In some embodiments, first polynucleotide and second polynucleotide are allogenic each other.In some embodiments, autolysin is a SecA2-dependency autolysin.In some embodiments, autolysin is Peptidoglycan hydrolytic enzyme (for example,-acetylmuramic acid enzyme or p60).In some embodiments, expression cassette further comprises the polynucleotide of coded signal peptide (for example, usually relevant with autolysin signal peptide or with the allogenic signal peptide of this signal peptide).For example, in some embodiments, expression cassette coding comprises the p60 signal peptide, p60 protein (or its catalytic activity fragment or catalytic activity variant) and be embedded in the p60 sequence and the protein chimera allogenic polypeptide of p60.In some embodiments, be non-Listera polypeptide by the polypeptide of second polynucleotide encoding.
On the other hand, the invention provides expression cassette, first polynucleotide that comprise (a) coded signal peptide, (b) coding secretory protein or its segmental second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the 3rd polynucleotide of coding and secretory protein or the allogenic polypeptide of its fragment, wherein the 3rd polynucleotide are arranged in and first and second translation frame that polynucleotide are identical, (d) be operably connected first, the promoter of second and the 3rd polynucleotide, wherein the recombinant nucleic acid molecules coding comprises signal peptide, polypeptide and secretory protein or its segmental protein chimera by second polynucleotide encoding, and wherein in the protein chimera, by polypeptide and the secretory protein or the fusion of its fragment of the 3rd polynucleotide encoding, or be positioned at secretory protein or its fragment.
In some embodiments, the promoter in the expression cassette described herein (or recombinant nucleic acid molecules described herein) is a promoter in prokaryote.For example, promoter in prokaryote can be the Listera promoter.In some embodiments, the Listera promoter is the hly promoter.In some embodiments, promoter is prfA-dependency promoter (for example, an actA promoter).In some embodiments, promoter is constitutive promoter (for example, a p60 promoter).In some embodiments, the expression cassette that comprises recombinant nucleic acid molecules described herein comprises hly, actA or the p60 promoter of the polynucleotide of the recombinant nucleic acid molecules that is operably connected.The host bacteria those skilled in the art that consider the desired use of expression cassette and will place expression cassette can easily identify other prokaryote and/or the Listera promoter that is applicable in the expression cassette.
For example, be applicable to that the multiple mycobacteria promoter in the interior recombinant expression cassettes of mycobacteria and other antibacterials is known.These promoteres comprise Mycobacterium bovis BCG promoter HSP60 and HSP70, also comprise such promoter such as mycobactin promoter, the α of mycobacterium tuberculosis and BCG-antigen promoter and 45KDa antigen promoter, the superoxide dismutase promoter, MBP-70, mycobacteria asd promoter, mycobacteria 14kDa and 12kDa antigen promoter, mycobacteriophage promoter such as Bxb1, Bxb2 and Bxb3 promoter, L1 and L5 promoter, D29 promoter and TM4 promoter (referring to, for example, U.S. Patent No. 6,566,121).Be applicable to that the promoter in the Bacillus anthracis includes, but not limited to the pagA promoter, α-Dian Fenmei promoter (Pamy) and Pntr (referring to, for example, Ga t etc., Infect.Immun., 71; 801-13 (2003)).Be applicable to that the promoter in recombinant salmonella expression cassette and the vaccine also is known and comprises the nirB promoter, the osmC promoter, P (pagC) and P (tac) (referring to, Bumann for example, Infect.Immun.69:7493-500 (2001); Wang etc., Vaccine, 17:1-12 (1999); McSorley etc., Infect.Immun.65:171-8 (1997)).Multiple escherichia coli promoter also is that those of ordinary skills are known.
In some embodiments, used promoter is a constitutive promoter in the expression cassette described herein.In other embodiments, used promoter is an inducible promoter in the expression cassette described herein.Endogenous molecule (for example, protein) with the antibacterial that wherein will use expression cassette can be induced inducible promoter.Perhaps, can be with wherein using the heterologous molecule (for example, micromolecule or protein) of the antibacterial of expression cassette to induce inducible promoter.Multiple inducible promoter is known to a person of ordinary skill in the art.
In some embodiments of expression cassette, 3 ' of promoter-end is a polypurine SD sequence, and promptly 30S ribosomal subunit (via 16S rRNA) is connected with heterologous gene rna transcription thing and translates and starts needed element.The SD sequence has following consensus sequence: 5 '-NAGGAGGU-N5-10-AUG (start codon)-3 ' (SEQ ID NO:85) usually.There is the variation of polypurine SD sequence.Especially, the Listera hly gene of coding Listera lysin O (LLO) has following SD sequence: A
AGGAGAGTGAAACCCATG (SEQ IDNO:70) (underlined is the SD sequence, overstriking be translation initiation codon).
Being used for the structure of the expression cassette of antibacterial, even being specifically designed to the structure of the expression cassette in the recombinant bacteria vaccine, is known in the art.For example, the production of various bacteria expression cassette and/or recombinant bacteria vaccine and the description of use can be found in below with reference to document, wherein every piece is incorporated herein by reference with its integral body at this: Horwitz etc., Proc.Natl.Acad.Sci.USA, 97:13853-8 (2000); Garmory etc., J Drug Target, 11:471-9 (2003); Kang etc., FEMS Immunol.Med.Microbiol., 37:99-104 (2003); Garmory etc., Vaccine, 21:3051-7 (2003); Kang etc., Infect.Immun., 1739-49 (2002); Russman etc., J.Immunol., 167:357-65 (2001); Harth etc., Microbiology, 150:2143-51 (2004); Varaldo etc., Infect.Immun., 72:3336-43 (2004); Goonetilleke etc., J.Immunol., 171:1602-9 (2003); Uno-Furuta etc., Vaccine, 21:3149-56 (2003); Biet etc., Infect.Immun., 71:2933-7 (2003); Bao etc., Infect.Immun., 71:1656-61 (2003); Kawahara etc., Clin.Immunol., 105:326-31 (2002); Anderson etc., Vaccine, 18:2193-202 (2000); Bumann, Infect.Immun., 69:7493-500 (2001); Wang etc., Vaccine, 17:1-12 (1999); McSorley etc., Infect.Immun., 65:171-8 (1997); Gat etc., Infect.Immun., 71:801-13 (2003); U.S. Patent No. 5,504,005; U.S. Patent No. 5,830,702; U.S. Patent No. 6,051,237; U.S. Patent Publication No.2002/0025323; U.S. Patent Publication No.2003/0202985; WO 04/062597; U.S. Patent No. 6,566,121; With U.S. Patent No. 6,270,776.
In some embodiments, it is desirable to make up the bicistronic mRNA that utilizes allogeneic coding sequence, the expression cassette that polycistron (being also referred to as multiple cistron) is expressed.Such expression cassette can utilize, for example, and the single promoter of two or more absolute coding sequences that are operably connected.These coded sequences can, for example, corresponding to independent gene or can be corresponding to the required and/or selected subfragrnent of whole appointment gene.In this back one example, gene can contain the sequence of coding hydrophobic transmembrane domain, and this domain can suppress the effective secretion from Listera potentially.Therefore, it may be ideal the coded sequence of this gene and hydrophobic domains being separated; In this case, then two subfragrnents are expressed as bicistronic mRNA information.The 30S ribosomal subunit rests on the polycistron RNA information after using polycistronic expression to need the release of first coded sequence translation termination and 50s ribosomal subunit, " connect and read " RNA information subsequently to next start codon, the 50s ribosomal subunit combines with the 30s ribosomal subunit that combines RNA in this process, and starts translation once more.
Listeria monocytogenes, the same with other antibacterials, utilize the polycistronic expression of its genome repertoire.For example, can be used to make up the polycistronic expression box from the intergenic region of the Listeria monocytogenes of selected polycistronic message and be used to express selected heterologous protein from reorganization Listera kind.For example, from the several prfA-dependency virulence factors of polycistronic message expression from Listeria monocytogenes.For example, Listeria monocytogenes ActA and PlcB protein are expressed as bicistronic mRNA information.Below shown DNA sequence (5 '-3 ') corresponding to Listeria monocytogenes actA-plcB intergenic sequence:
5’-TAAAAACACAGAACGAAAGAAAAAGTGAGGTGAATGA-3’(SEQ?IDNO：71)
(the SD sequence that has shown the plcB translation initiation with runic.3 nucleotide correspondences of sequence are in Ochre termination codon).For the limiting examples of two-cistron expression vector, be used for the bicistronic mRNA hEphA2 expression vector of Listeria monocytogenes, referring to following embodiment 28.
Perhaps, other known intergenic regions or composition sequence can be used for making up the polycistronic expression box that is used for Listera or other antibacterials.Should prevent to cause the structure of the intergenic region of substantive secondary RNA structure, avoid the tanscription termination of unwanted mechanism by not relying on rho.
Importantly, the secretion of any or all of translated protein of expressing from polycistronic message connects each coding region on the essential function of signal peptide if desired.In some embodiments, these signal peptides differ from one another.
Therefore, in some embodiments, the expression cassette that is used for Listera or other antibacterials described herein is polycistronic (for example, bicistronic mRNA).Two or more polypeptide by bicistronic mRNA or the discrete polypeptide of polycistronic expression box coding conduct.In some embodiments, bicistronic mRNA or polycistronic expression box comprise the intergenic sequence (for example, from bicistronic mRNA or polycistron gene) between these two polypeptid coding sequences.In some embodiments, intergenic sequence comprises and promotes ribosome to enter the sequence with translation initiation.In some embodiments, intergenic sequence comprises the SD sequence.In some embodiments, intergenic sequence is a Listeria monocytogenes actA-plcB intergenic sequence.Usually, intergenic sequence is positioned between the polynucleotide sequence of the polynucleotide sequence of first polypeptide of coding (or comprise first polypeptide and signal peptide first fusion rotein) and second polypeptide of encoding second fusion rotein of second polypeptide and signal peptide (or comprise).
Therefore, in the one side, the invention provides the expression cassette that comprises following composition: first polynucleotide of first polypeptide of (a) encoding; (b) second polynucleotide of second polypeptide of coding; (c) intergenic sequence between first and second polynucleotide; (f) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding is as first and second polypeptide of two discrete polypeptide.In some embodiments, first and second polypeptide are the polypeptide that is selected from (for example, in above IV part) described any polypeptide at this.In some embodiments, at least one comprises antigen in first or second polypeptide.In some embodiments, first and second polynucleotide comprise (similar and different) fragment of same antigen separately.In some embodiments, antigen is tumor associated antigen or is derived from tumor associated antigen.
The present invention further provides the expression cassette that comprises following composition: first polynucleotide of first signal peptide of (a) encoding; (b) encode second polynucleotide of first (non-signal) polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the 3rd polynucleotide of second signal peptide of coding; (d) the 4th polynucleotide of second (non-signal) peptide of coding, wherein the 4th polynucleotide are arranged in and the 3rd the translation frame that polynucleotide are identical; (e) intergenic sequence (being usually located between second polynucleotide and the 3rd polynucleotide); (f) first polynucleotide that are operably connected, second polynucleotide, the promoter of the 3rd polynucleotide and the 4th polynucleotide makes the expression cassette coding comprise first fusion rotein of first signal peptide and first polypeptide and second fusion rotein that comprises second signal peptide and second polypeptide.In some embodiments, one or more polynucleotide of coded signal peptide are that codon is optimized for the expression in antibacterial.In some embodiments, the 3rd and/or the 4th polynucleotide are codon optimized (preferably except the codon optimizations of the polynucleotide of coded signal peptide) for the expression in antibacterial.In some embodiments, first and/or second signal peptide right and wrong-secA1 antibacterial signal peptide.In some embodiments, intergenic sequence is a Listeria monocytogenes actA-plcB intergenic sequence.In some embodiments, second and the 3rd polypeptide are the polypeptide that is selected from (for example, in above IV part) described any polypeptide at this, as comprise antigenic polypeptide.In some embodiments, first and second polypeptide are the polypeptide that is selected from (for example, in above IV part) described any polypeptide at this.In some embodiments, at least one comprises antigen in first or second polypeptide.In some embodiments, first and second polypeptide comprise the fragment of same antigen separately.In some embodiments, antigen is tumor associated antigen or is derived from tumor associated antigen.
For example, the invention provides the polycistronic expression box that is used for expressing heterologous polypeptide, wherein at least two discrete non-Listera polypeptide of expression cassette coding at Listera.In some embodiments, the polycistronic expression box is the bicistronic mRNA expression cassette, its two discrete non-Listera polypeptide of encoding.In some embodiments, expression cassette comprises following: first polynucleotide of first non-Listera polypeptide of (a) encoding; (b) second polynucleotide of second non-Listera polypeptide of coding; (c) intergenic sequence between first and second polynucleotide; (d) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding is as first and second polypeptide of two discrete polypeptide.If expression cassette is the polycistronic expression box of coding as three polypeptide of discrete polypeptide, then expression cassette will comprise the 3rd polynucleotide and second intergenic sequence between second and the 3rd polynucleotide of the promoter that is operably connected.In some embodiments, at least one non-Listera polypeptide comprises antigen.In some embodiments, at least two non-Listera polypeptide comprise the fragment of same antigen separately.
In some embodiments, expression cassette comprises following: first polynucleotide of first signal peptide of (a) encoding; (b) encode second polynucleotide of first (non-signal) non-Listera polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the 3rd polynucleotide of second signal peptide of coding; (d) the 4th polynucleotide of second (non-signal) non-Listera polypeptide of coding, wherein the 4th polynucleotide are arranged in and the 3rd the translation frame that polynucleotide are identical; (e) intergenic sequence between second polynucleotide and the 3rd polynucleotide; (f) first polynucleotide that are operably connected, second polynucleotide, the promoter of the 3rd polynucleotide and the 4th polynucleotide makes the expression cassette coding comprise first fusion rotein of first signal peptide and first polypeptide and second fusion rotein that comprises second signal peptide and second polypeptide.In some embodiments, at least one non-Listera polypeptide is an antigen.In some embodiments, each fragment of same antigen naturally of at least two non-Listera polypeptide.
The present invention also provides and has used arbitrary expression cassette described herein to produce the method for recombinant bacteria (for example, the antibacterial of reorganization Listera genus).In some embodiments, the method for using expression cassette described herein to prepare recombinant bacteria comprises to be introduced expression cassette in the antibacterial.In some embodiments, expression cassette is integrated in the genome of antibacterial.In some other embodiments, expression cassette is to be positioned on the plasmid of introducing antibacterial.In some embodiments, carry out the introducing that expression cassette enters antibacterial by engaging.The introducing that expression cassette enters in the antibacterial can realize by any standard technique known in the art.For example, by engaging, transduce (transfection) or be converted and carry out the introducing that expression cassette enters antibacterial.
VII. carrier
The present invention further provides carrier, as expression vector, it comprises arbitrary expression cassette described herein and/or recombinant nucleic acid molecules.In some embodiments, carrier is a plasmid, and in some embodiments, carrier is linear.In some embodiments, carrier is cyclic.In some embodiments, carrier is to integrate or homologous recombination vector.In some embodiments, carrier is pAM401.In some embodiments, carrier is pPL2.In some embodiments, carrier is isolating.
As mentioned above, in some embodiments, expression cassette described herein is contained in the expression vector.In some embodiments, carrier is a plasmid.In other embodiments, carrier is linear.In the alternate embodiment, use expression vector expression cassette to be inserted (that is, integrating) to the genomic DNA of antibacterial.In some embodiments, expression vector is linear.In other embodiments, expression vector is cyclic.
Be applicable to that the expression vector in antibacterial such as the Listera is well known by persons skilled in the art.Exist multiple suitable carriers to be suitable for use as plasmid construction body skeleton and be used to assemble expression cassette.Expression based on needs polynucleotide (that is the polynucleotide of coding heterologous antigen) is to select certain plasmid construct skeleton from bacterial chromosome or from extrachromosomal episome.
In some embodiments, the integration vector that use contains the expression cassette of Listera phage intergrase is finished mixing in the bacterial chromosome that expression cassette (and/or recombinant nucleic acid molecules) enters Listeria monocytogenes (Listera), and the sequence-specific that described intergrase catalytic carrier enters in the Listera chromosome is integrated.For example, the integration vector that is called pPL1 and pPL2 is carried out stable single copy integration of the heterologous protein expression cassette in the bacterial genomes harmless area, and has been described in (Lauer etc., 2002, J.Bacteriol.184:4177-4178 in the document; U.S. Patent Publication No.20030203472).Integration vector is stable as the plasmid in the escherichia coli and passes through to engage and introduce in the required Listera background.Each carrier lacks the specific origin of replication of Listera and the phage intergrase of encoding, and is stable when making carrier only in being integrated into the chromosome phage attachment site.Begin with required plasmid construction body, the process that produces the reorganization Listera bacterial strain of expressing desired protein approximately needs week age.PPL1 and pPL2 integration vector are separately based on U153 and PSA Listera phage.The pPL1 carrier is integrated in the open reading frame of comK gene, and pPL2 integrates in the tRNAArg gene, makes the native sequences of gene be restored when successfully integrating in such a way, therefore keeps the complete of its natural expressive function.PPL1 and pPL2 integration vector comprise the multiple clone site sequence, so that help to contain the plasmid of recombinant nucleic acid molecules or the structure of expression cassette such as heterologous protein expression cassette.Some instantiations that use the pPL2 integration vector have been described among following examples 2 and the embodiment 3.
Perhaps, finish expression cassette (and/or recombinant nucleic acid molecules) by allele switching method well known by persons skilled in the art and enter mixing in the Listera chromosome.Especially, wherein do not need to introduce the compositions of the proteic gene of coding antibiotic resistance as the part of the construct that contains expression cassette, the allele switching method is ideal.For example, the pKSV7 carrier (Camilli etc., Mol.Microbiol. (1993) 8,143-157), contain temperature sensitivity Listera Gram-positive origin of replication, use it for the recombinant clone of under non-permissive temperature, selecting to represent the pKSV7 plasmid to the Listera chromosome of recombinating.PKSV7 allele exchange plasmid vector contains the multiple clone site sequence, so that help to contain the structure of the plasmid of protein expression box and chloramphenicol resistance gene.In order to insert in the Listera chromosome, the chromosomal DNA sequence that the expression cassette construct can the about 1kb of side joint corresponding to required integration exact position.According to the standard method of gram-positive bacterium electroporation, pKSV7-expression cassette plasmid can be introduced in the required bacterial isolates by electroporation.The non-limiting example of using the pSKV7 carrier to realize the method for allele exchange is provided among the following embodiment 16.
In other embodiments, may wish express polypeptide from stable plasmid episome (comprising the fusion rotein that contains polypeptide).Keeping the plasmid episome by going down to posterity of many generations needs proteic coexpression, and this albumen gives selection advantage for the antibacterial that contains plasmid.As limiting examples, can be for example chlorampenicol resistant albumen of antibiotic in conjunction with polypeptide from the albumen of plasmid coexpression, maybe can be the bacterioprotein that also can give selection advantage (from the albumen of the chromosomal expression of wild-type bacterium).The limiting examples of bacterioprotein comprises the synthetic needed enzyme of purine or amino acid bio (use lack defined media that related amino acid or other must precursor macromolecule select), or give the needed transcription factor (Gunn etc. of gene expression of selection advantage in the external or body, 2001, J.Immuol.167:6471-6479).As limiting examples, pAM401 is that suitable plasmid is used for the episome expression (Wirth etc., 1986J.Bacteriol 165:831-836) that selected polypeptide belongs to different gram-positive bacteriums.For further describing of the example that uses pAM401, referring to following embodiment 3 and 13.
For example, can realize that with the integration vector of expression cassette that contains the phage intergrase expression cassette enters the mixing of bacterial chromosome of Bacillus anthracis, the sequence-specific that described intergrase catalytic carrier enters in the Bacillus anthracis chromosome is integrated.The intergrase of Bacillus anthracis phage and attachment site can be used to the integration vector of deriving, required antigen presentation box is introduced in the vaccine combination, as non-limiting instance, the intergrase of Bacillus anthracis temperate phage w-α and the attachment site Bacillus anthracis specificity integration vector (McCloy that is used to derive, E.W.1951, Studies on a lysogenic Bacillusus strain (to the research of molten source Bacillus strain) I.A bacteriophage specific for Bacillususanthracis (Bacillus anthracis is had specific phage) J.Hyg.49:114-125).
Perhaps, can finish the antigen presentation box by allele switching method well known by persons skilled in the art and enter mixing in the Bacillus anthracis chromosome.Referring to, for example, Gat etc., Infect.Immun, 71; 801-13 (2003).Particularly, wherein do not need to introduce the composition of the proteic gene of coding antibiotic resistance as the part of the construct that contains expression cassette, the allele switching method is ideal.For example, the pKSV7 carrier (Camilli etc., Mol.Microbiol. (1993) 8,143-157), contain temperature sensitivity Listera Gram-positive origin of replication, use it for the recombinant clone of under non-permissive temperature, selecting to represent the pKSV7 plasmid to bacterial chromosome of recombinating.PKSV7 allele exchange plasmid vector contains the multiple clone site sequence, so that help to contain the structure of the plasmid of protein expression box and chloramphenicol resistance gene.In order to insert in the Bacillus anthracis chromosome, the chromosomal DNA sequence that the expression cassette construct can the about 1kb of side joint corresponding to required integration exact position.According to the standard method of gram-positive bacterium electroporation, pKSV7-expression cassette plasmid can be introduced in the required bacterial isolates by electroporation.U.S. Patent Application Serial 10/883,559 provides the limiting examples that realizes the allele switching method in Bacillus anthracis, is incorporated herein by reference with its integral body at this.Especially, use the allele exchange of pKSV7 carrier can be used for the Bacillus anthracis bacterial strain with the required antigen presentation box of any desired location adding in bacterial chromosome.
The allele switching method of above-mentioned use pKSV7 can be widely used in using in gram-positive bacterium, in addition, is used for the multiple expression vector of antibacterial, comprises the recombinant bacteria carrier, is that those of ordinary skills are known.Example comprises below with reference to those carriers described in the document, and wherein every piece is incorporated herein by reference with its integral body at this: Horwitz etc., Proc.Natl.Acad.Sci.USA, 97:13853-8 (2000); Garmory etc., J:Drug Target, 11:471-9 (2003); Kang etc., FEMS Immunol.Med.Microbiol., 37:99-104 (2003); Garmory etc., Vaccine, 21:3051-7 (2003); Kang etc., Infect.Immun., 1739-49 (2002); Russman etc., J.Immunol., 167:357-65 (2001); Harth etc., Microbiology, 150:2143-51 (2004); Varaldo etc., Infect.Immun., 72:3336-43 (2004); Goonetilleke etc., J:Immunol., 171:1602-9 (2003); Uno-Furuta etc., Vaccine, 21:3149-56 (2003); Biet etc., Infect.Immun., 71:2933-7 (2003); Bao etc., Infect.Immun., 71:1656-61 (2003); Kawahara etc., Clin.Immunol, 105:326-31 (2002); Anderson etc., Vaccine, 18:2193-202 (2000); Bumann, Infect.Immun., 69:7493-500 (2001); Wang etc., Vaccine, 17:1-12 (1999); McSorley etc., Infect Immun., 65:171-8 (1997); Gat etc., Infect.Immun., 71:801-13 (2003); U.S. Patent No. 5,504,005; U.S. Patent No. 5,830,702; U.S. Patent No. 6,051,237; U.S. Patent Publication No.2002/0025323; U.S. Patent Publication No.2003/0202985; WO 04/062597; U.S. Patent No. 6,566,121; With U.S. Patent No. 6,270,776.
The present invention further provides the expression vector that comprises expression cassette, this expression cassette comprises following: first polynucleotide of first signal peptide of (a) encoding; (b) encode second polynucleotide of first polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) intergenic sequence; (d) the 3rd polynucleotide of second signal peptide of coding; (e) the 4th polynucleotide of second polypeptide of coding, wherein the 4th polynucleotide are arranged in and the 3rd the translation frame that nucleotide is identical; (f) first polynucleotide that are operably connected, second polynucleotide, the 3rd polynucleotide, the promoter of the 4th polynucleotide and intergenic sequence makes the expression cassette coding comprise first fusion rotein of first signal peptide and first polypeptide and second fusion rotein that comprises second signal peptide and second polypeptide.
The present invention further provides and used any expression vector described herein to produce the method for recombinant bacteria (for example, the antibacterial of reorganization Listera genus).In some embodiments, the method for using expression vector described herein to prepare recombinant bacteria comprises to be introduced expression vector in the antibacterial.
VIII. antibacterial and other host cells
The present invention further provides and comprised recombinant nucleic acid molecules described herein, the host cell of expression cassette and/or carrier (referring to, for example, the above summary of the invention and the I of detailed Description Of The Invention, II, VI and VII part and following specific embodiment).In some embodiments, cell is procaryotic.In some embodiments, cell is Eukaryotic.In some embodiments, cell is mammiferous.In some embodiments, cell is an antigen-presenting cell, as dendritic cell.In some embodiments, cell is a bacterial cell.In some embodiments, host cell is isolating.
For example, the invention provides and comprise recombinant nucleic acid molecules described herein, the antibacterial of expression cassette and/or carrier (referring to, for example, the above summary of the invention and the I of detailed Description Of The Invention, II, VI and VII part and following specific embodiment).The antibacterial that comprises these polynucleotide alternatively is called " recombinant bacteria " at this, and comprises recombinant nucleic acid molecules described herein, and the antibacterial of expression cassette or carrier alternatively is called " recombinant bacteria " at this.In some embodiments, comprise recombinant nucleic acid molecules, the antibacterial of expression cassette and/or expression vector is isolating.In some embodiments, comprise recombinant nucleic acid molecules, the recombinant bacteria of expression cassette and/or expression vector is expressed by wherein contained recombinant nucleic acid molecules, the polypeptide of expression cassette and/or expression vector codes or fusion rotein.In some embodiments, recombinant bacteria is secreted by wherein contained recombinant nucleic acid molecules, the polypeptide of expression cassette and/or expression vector codes or fusion rotein.In some embodiments, recombinant bacteria expression and secrete polypeptide and/or fusion rotein present in an amount at least sufficient to antibacterial (or contain antibacterial compositions) is being produced immunne response when giving host (for example, people patient) in the host.
In some embodiments, antibacterial is selected from gram-positive bacterium, gram negative bacteria, intracellular bacteria and mycobacteria.In some embodiments, antibacterial is a gram-positive bacterium.In embodiments more of the present invention, antibacterial is intracellular bacteria (for example, facultative intracellular bacteria).In some embodiments, antibacterial belongs to Listera and belongs to.In other embodiments, antibacterial is the member of Listeria monocytogenes kind.In some other embodiments, antibacterial is Yi Shi Listera (Listeria ivanovii), the member that Si Shi Listera (Listeriaseeligerii) or harmless Listera (Listeria innocuai) are planted.In some embodiments, antibacterial is the member of bacillus.In another embodiment, antibacterial is a Bacillus anthracis.Still in another embodiment, antibacterial is a Yersinia pestis.In other embodiments of the present invention, antibacterial is from Salmonella.In some embodiments, antibacterial is a Salmonella typhimurium.In some embodiments, antibacterial belongs to Shigella.For example, in some embodiments, antibacterial is the Fei Shi shigella.In some embodiments, antibacterial is the member of Brucella.In the alternate embodiment, antibacterial is a mycobacteria.Mycobacteria randomly is the member of mycobacterium tuberculosis kind, and in some embodiments, antibacterial is bacillus calmette-guerin vaccine (BCG).In some embodiments, antibacterial is escherichia coli, for example, and modified escherichia coli of expressing Listera lysin O (LLO).Therefore, in some embodiments, comprise recombinant nucleic acid molecules described herein, the antibacterial of expression cassette and/or carrier is selected from Listera and belongs to Bacillus anthracis, Yersinia pestis, Salmonella and mycobacteria.In some other embodiments, comprise recombinant nucleic acid molecules described herein, the antibacterial of expression cassette and/or carrier is selected from Listera and belongs to, bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria and escherichia coli.
In some embodiments, by inserting recombinant nucleic acid molecules described herein, expression cassette and/or carrier (are for example modified, referring to the above summary of the invention and the I of detailed Description Of The Invention, II, VI and VII part and following embodiment) with express polypeptide, and at least some embodiments, the antibacterial of the present invention of secrete polypeptide is a wild-type bacterium.For example, in some embodiments, recombinant bacteria is to comprise recombinant nucleic acid molecules, and the wild type Listera of expression cassette and/or carrier belongs to antibacterial, as Listeria monocytogenes.Yet in embodiments more of the present invention, the antibacterial that comprises expression cassette and/or carrier is the mutant of antibacterial.In some embodiments, antibacterial is an attenuation.In some embodiments, antibacterial is the attenuation mutant of antibacterial.Wherein the mutant that lacked of gene " xyz " alternatively is called Δ xyz or xyz at this
-Or xyz deletion mutant.For example, wherein the bacterial isolates that lacked of urvA gene alternatively is called the urvA mutant at this, Δ urvA or urvA
-In addition, those skilled in the art will appreciate that specific mutant or bacterial strain are called mutant or the bacterial strain that " xyz " mutant or " xyz " bacterial strain are meant that sometimes xyz gene has wherein lacked.
Comprise that the sudden change in the mutant bacterial of expression cassette and/or expression vector can be the sudden change of any kind.For example, sudden change can constitute point mutation, and frameshift mutation inserts, partly or entirely the disappearance of gene.In addition, modify in some embodiments of bacterial strain, the part of bacterial genomes is substituted by heterologous polynucleotide.In some embodiments, sudden change is abiogenous.In other embodiments, sudden change is the result of artificial mutation method.Still in other embodiments, the sudden change in the bacterial genomes is engineered result.
In some embodiments, comprise said recombinant nucleic acid molecules, the antibacterial of any of expression cassette and/or carrier is that attenuation (with respect to wild-type bacterium) is used for cell and intercellular diffusion, enters non-phagocytic cell or propagation.Can modify the antibacterial attenuation by sudden change or by other.In some embodiments, comprise said any recombinant nucleic acid molecules, the antibacterial of expression cassette and/or expression vector be attenuation be used for cell and intercellular diffusion (for example, Listeria monocytogenes actA mutant).In some embodiments, comprise said recombinant nucleic acid molecules, the antibacterial of any of expression cassette and/or expression vector be attenuation be used to enter non-phagocytic cell (for example, Listeria monocytogenes internalin mutant is as the inlB deletion mutant).In some embodiments, comprise said recombinant nucleic acid molecules, the antibacterial of any of expression cassette and/or expression vector is the propagation that is used for of attenuation.In some embodiments, antibacterial is being used for cell and intercellular diffusion and entering non-phagocytic cell of attenuation.
In some embodiments, will comprise that the antibacterial attenuation of expression cassette described herein and/or expression vector is used for cell and intercellular diffusion.In some embodiments, antibacterial (for example, Listera) lacks (with respect to not mutated body or wild-type bacterium) or its equivalent (depending on organism).In some embodiments, antibacterial comprises one or more actA sudden changes.For example, antibacterial (for example, Listera) can be the actA deletion mutant.ActA is by the actin polymerization enzyme of actA gene code (Kock etc., Cell, 68:521-531 (1992); Genbank accession number no.AL591974, nts 9456-11389).The actin polymerization pheron participates in host F-actin belongs to antibacterial at Listera the raising and polymerization an of limit.Polymerization of actin subsequently and dissolving cause Listera to spread all over cytosolic propelling and enter in the contiguous cell.This mobility can make antibacterial directly spread and can further not be exposed in born of the same parents' external environment at cell and iuntercellular, and the defence of therefore escaping the host is as producing antibody.In some embodiments, the Listera of attenuation randomly comprises the sudden change of internalin gene such as inlB and actA.The Listera bacterial strain of the embodiment of the present invention is an attenuation for entering non-phagocytic cell, and is attenuation for cell and intercellular diffusion.
In some embodiments, with respect to the antibacterial (for example, wild-type bacterium) that does not have the attenuation sudden change, the ability that attenuated bacteria is used for cell and iuntercellular diffusion reduces at least about 10%, at least about 25%, and at least about 50%, at least about 75%, or at least about 90%.In some embodiments, with respect to the antibacterial that does not have the attenuation sudden change, the ability that attenuated bacteria is used for cell and iuntercellular diffusion reduces at least about 25%.In some embodiments, with respect to the antibacterial that does not have the attenuation sudden change, the ability that attenuated bacteria is used for cell and iuntercellular diffusion reduces at least about 50%.
Measuring antibacterial such as Listera belongs to antibacterial whether be used for that cell and iuntercellular spread is that the external test method of attenuation is that those of ordinary skills are known.For example, can measure the diameter of the plaque that in a period of time, forms after selected cultured cell monolayer infects.As before at Sun, A., A.Camilli and D.A.Portnoy, 1990, Isolation of Listeriamonocytogenes small-plaque mutants defective for intracellulargrowth and ce11-to-cell spread (spreading separating of the little plaque type mutant of defective Listeria monocytogenes with cell and iuntercellular) for growth in the born of the same parents, carry out plaque assay in the L2 cell monolayer described in the Infect.Immun.58:3770-3778, use improved measuring method, as be described in Skoble, J., D.A.Portnoy and M.D.Welch.2000, Three regions within ActA promote Arp2/3complex-mediated actin nucleation and Listeria monocytogenesmotility (three districts in the ActA promote the actin nucleation and the Listeria monocytogenes motion of Arp2/3 complex-mediation), J.Cell Biol.150:527-538.In brief, the L2 cell grows to converge in six hole tissue culture wares and uses bacterial infection 1h then.After the infection, be warmed to 40 ℃ by the DME that contains 0.8% agarose, the culture medium of hyclone (for example, 2%) and desired concn gentamycin composition covers cell.Gentamicin concentration appreciable impact plaque size in the culture medium, be that the Listera bacterial strain of selecting carries out cell and iuntercellular diffusion energy force measurement (Glomski, I J., M.M.Gedde, A.W.Tsang, J.A.Swanson, and D.A.Portnoy, 2002, J.Cell Biol.156:1029-1038).For example, monolayer infected back 3 days, was the culture medium of the gentamycin of 50 μ g/ml when covering with containing concentration, compared with the wild type Listera, and the plaque size with Listera bacterial strain of cell and iuntercellular diffusion defect phenotype reduces at least 50%.On the other hand, when the agarose with culture medium+only contain 5 μ g/ml gentamycins covered the monolayer that infects, having cell was similar to the Listera bacterial strain of iuntercellular diffusion defect phenotype and the plaque size between the wild type Listera.Therefore, can contain gentamicin concentration in the culture medium of agarose by change and measure selected bacterial strain carries out cell and iuntercellular diffusion in the infection cell monolayer with respect to the wild type Listera relative ability.Randomly, contain dimethyl diaminophenazine chloride (GIBCO BRL by adding in back 48 hours in infection; 1: 250 dilution factor in DME+ agarose culture medium) culture medium covers the observation and the measurement that can help the plaque diameter.In addition, can in the monolayer that is derived from other primary cells or successive cell, carry out plaque assay.HepG2 cell for example, the deutero-cell line of hepatocyte, or the primary human hepatocyte can be used to estimate the ability that selected Listera mutant carries out cell and iuntercellular diffusion, compares with the wild type Listera.In some embodiments, the Listera that comprises sudden change or make the Listera attenuation be used for other modifications of cell and iuntercellular diffusion produces " needle point " plaque when high concentration gentamycin (about 50 μ g/ml).
In some embodiments, the antibacterial that comprises expression cassette described herein and/or expression vector is to be attenuated to be used for the mutant bacterial that nucleic acid repairs (with respect to wild type as there not being the antibacterial of attenuation gene mutation).For example, in some embodiments.Antibacterial is at least one DNA repairase disappearance (for example, Listeria monocytogenes uvrAB mutant).In some embodiments, antibacterial is PhrB, UvrA, UvrB, UvrC, UvrD and/or RecA, or (genus and the kind that depend on antibacterial) of one of their equivalent disappearance.In some embodiments, antibacterial is UvrA, UvrB and/or UvrC disappearance.In some embodiments, antibacterial comprises phrB, uvrA, uvrB, uvrC, the attenuation sudden change of uvrD and/or recA gene.In some embodiments, antibacterial comprises uvrA, one or more sudden change in uvrB and/or the uvrC gene.In some embodiments, antibacterial is to lack UvrA on the function, UvrB and/or UvrC's.In some embodiments, antibacterial is disappearance UvrA and UvrB on the function.In some embodiments, antibacterial is the uvrAB deletion mutant.In some embodiments, antibacterial is a Δ uvrA Δ actA mutant.In some embodiments, be attenuated be used for the antibacterial nucleic acid reparation and/or that at least one DNA repairase be disappearance nucleic acid by being modified (referring to following) with nucleic acid target compound reaction.Nucleic acid is repaired mutant, as Δ uvrAB Listeria monocytogenes mutant, and the method for preparing this mutant, be described in detail among the U.S. Patent Publication No.2004/0197343 (referring to, for example, the embodiment 7 of U.S.2004/0197343).
In some embodiments, with respect to the antibacterial (for example, wild-type bacterium) that does not have the attenuation sudden change, the ability that attenuated bacteria is used for the nucleic acid reparation reduces at least about 10%, at least about 25%, and at least about 50%, at least about 75%, or at least about 90%.In some embodiments, with respect to the antibacterial that does not have the attenuation sudden change, the ability that attenuated bacteria is used for the nucleic acid reparation reduces at least about 25%.In some embodiments, with respect to the antibacterial that does not have the attenuation sudden change, the ability that attenuated bacteria is used for the nucleic acid reparation reduces at least about 50%.
Can obtain to exist in the bacterial isolates affirmation of specific sudden change by the known several different methods of those of ordinary skills.For example, can be with strain gene group relevant portion clone and order-checking.Perhaps, can use the coding and the paired primer of disappearance or other sudden change adjacent domains to identify specific sudden change by PCR.Southern blotting technique also can be used for the change in the bacterial detection genome.And, can use the standard technique of this area such as western blotting to analyze specific protein and whether be expressed by this bacterial strain.More also can obtain to contain in the required gene confirmation of the bacterial strain of sudden change by the phenotype of this bacterial strain and the phenotype of reporting before.For example, can use following algoscopy to measure the existence of nucleotide excision reparation sudden change as uvrAB disappearance, promptly use the ability of nucleotide excision reparation (NER) method bacteria tested repair of nucleic acids and compare this ability with respect to wild-type bacterium.Such functional examination method is known in the art.For example, can measurement ring butane dimer the excision of excision or UV-inductive (6-4) product measure NER enzyme in the mutant shortage (referring to, for example, Franklin etc., Proc.Natl.Acad.Sci.USA, 81:3821-3824 (1984))).Perhaps, can survive and measure the shortage that nucleic acid is repaired.For example, antibacterial is carried out psoralen/UVA handle, measure they are compared propagation and/or survive with wild type ability then.
In some embodiments, antibacterial be attenuation be used to enter non-phagocytic cell (with respect to not mutated or wild-type bacterium).In some embodiments, antibacterial (for example, Listera) lacks for one or more internalin (or equivalent).In some embodiments, antibacterial lacks for internalin A.In some embodiments, antibacterial lacks for internalin B.In some embodiments, antibacterial comprises the sudden change of inlA.In some embodiments, antibacterial comprises the sudden change of inlB.In some embodiments, antibacterial comprises the sudden change of actA and inlB.In some embodiments, antibacterial lacks functional ActA and internalin B.In some embodiments, antibacterial is the two deletion mutants of Δ actA Δ inlB.In some embodiments, antibacterial lacks for ActA and internalin B.
In some embodiments, with respect to the antibacterial (for example, wild-type bacterium) that does not have the attenuation sudden change, attenuated bacteria enters nonphagocytic ability to be reduced at least about 10%, at least about 25%, and at least about 50%, at least about 75%, or at least about 90%.In some embodiments, with respect to the antibacterial that does not have the attenuation sudden change, attenuated bacteria enters nonphagocytic ability to be reduced at least about 25%.In some embodiments, with respect to the antibacterial that does not have the attenuation sudden change, attenuated bacteria enters nonphagocytic ability to be reduced at least about 50%.In some embodiments, with respect to the antibacterial that does not have the attenuation sudden change, attenuated bacteria enters nonphagocytic ability to be reduced at least about 75%.
In some embodiments, the antibacterial of attenuation, as the Listera bacterial strain of sudden change, the non-phagocytic cell that is used to enter more than a type is not an attenuation.For example, it may be attenuation that the bacterial strain of attenuation is used to enter hepatocyte, is not attenuation but be used to enter epithelial cell.As another example, it may be attenuation that attenuated strain is used to enter epithelial cell, is not attenuation but be used to enter hepatocyte.It should also be understood that and be used for specific modification antibacterial to enter nonphagocytic attenuation be to make the result who specifies gene mutation, for example deletion mutation, this gene code itself and specific cells acceptor interaction go into to invade protein, and the result helps nonphagocytic infection.For example, Listera Δ inlB mutant strain be used to enter the non-phagocytic cell of expressing C-MET HGFr (c-met) comprise hepatocyte cell line (for example, HepG2) and the primary human hepatocyte be attenuation.
In some embodiments, be attenuation even antibacterial (for example, the Listera of sudden change) is used to enter non-phagocytic cell, Listera still can be by phagocyte as dendritic cell and/or macrophage are taken at least.In one embodiment, attenuated bacteria enters cytophagous ability not to be had because of to the change that bacterial strain carried out, reduce as the sudden change of invasion (that is, after change, keep bacterial strain by about 95% or the ability taken in of the phagocyte that records more).In other embodiments, attenuated bacteria enters cytophagous ability to be reduced and not to be higher than approximately 10%, is not higher than approximately 25%, is not higher than approximately 50%, or is not higher than about 75%.
In embodiments more of the present invention, the attenuation that antibacterial enters (for example, Listera belongs to antibacterial) the non-phagocytic cell ability is that twice is reduced to higher levels of decay.In some embodiments, antibacterial enters decaying at least about 0.3log of non-phagocytic cell ability, about 1log, and about 2log, about 3log, about 4log, about 5log, or at least about 6log.In some embodiments, decay to about 0.3 to＞0.8log, about 2 to＞8log, about 4 to＞8log, about 6 to＞8log, about 0.3-8log, and about 0.3-7log, and about 0.3-6log, and about 0.3-5log, and about 0.3-4log, and about 0.3-3log, and about 0.3-2log, and about 0.3-1log.In some embodiments, decay to about 1 to＞8log, 1-7log, 1-6log, and about 2-6log, and about 2-5log, and about 3-5log.
In Listeria monocytogenes, identified a plurality of internalin (Boland etc., Clinical Microbiology Reviews, 2001,14:584-640).These internalin include, but are not limited to InlA, InlB, InlC, InlC2, InlD, InlE, InlF, InlG and InlH (Dramsi etc., Infection and Immunity, 65:1615-1625 (1997); Raffelsbauer etc., Mol.Gen.Genet.260:144-158 (1988)).Reported these proteic gene orders of coding before.For example, the sequence of inlA and inlB has been reported in Gaillard etc., Cell, and 65:1127-1141 (1991) and GenBank accession number are M67471.At Glaser etc., Science has identified other members' of coding internalin associated protein family gene in the webpage table 2 (www.sciencemag.org/cgi/content/full/294/5543/849/DC1) of augmenting the webpage material of 294:849-852 (2001), comprise lmo0327, lmo0331, lmo0514, lmo0610, lmo0732, lmo1136, lmo1289, lmo2396, lmo0171, lmo0333, lmo0801, lmo1290, lmo2026 and lmo2821.(internalin associated protein each member's of family sequence can be at monocyte Listeria monocytogenes strain EGD genome, GenBank accession number no.AL591824, and/or monocyte Listeria monocytogenes strain EGD-e genome, find among the GenBank accession number no.NC_003210.Indicated the position of various internalin related genes among the Glaser etc.).
InlA (internalin A) (Gaillard etc., Cell, 65:1127-1141 (1991); GenBank accession number no.NC_003210) instructs those absorptions of epithelial cell such as intestinal to Listera.
InlB (internalin B) (Gaillard etc., Cell, 65:1127-1141 (1991); GenBank accession number no.AL591975 (monocyte Listeria monocytogenes strain EGD, complete genome group, fragment 3/12, inlB gene regions: nts.97008-98963); With GenBank accession number no.NC_003210 (monocyte Listeria monocytogenes strain EGD, complete genome, the inlB gene regions: nts.457008-458963), wherein each is incorporated herein by reference with its integral body at this) instruct hepatocyte or epithelial cell as the absorption of the vascular epithelial cell of the brain microvasculature that comprises blood brain barrier to Listera.(about more descriptions of internalin B, referring to Ireton etc., J.of Biological Chemistry, 274:17025-17032 (1999); Dramsi etc., Molecular Microbiology 16:251-261 (1995); Mansell etc., J.of Biological Chemistry, 276:43597-43603 (2001); With Bierne etc., J.of Cell Science 115:3357-3367 (2002), wherein every piece is incorporated herein by reference with its integral body at this).
In some embodiments, antibacterial is ActA, internalin B, or ActA and internalin B shortage.In some embodiments, antibacterial is functional ActA, internalin B, or ActA and internalin B disappearance.In some embodiments, antibacterial is functional ActA disappearance.In some embodiments, antibacterial is functional internalin B disappearance.In some embodiments, antibacterial is functional ActA and internalin B disappearance.
Whether measuring antibacterial (for example, sudden change Listera bacterial strain), to be used to enter non-phagocytic cell be that the external test method of attenuation is that those of ordinary skills are known.For example, Dramsi etc., Molecular Microbiology 16:251-261 (1995) and Gaillard etc., Cell65:1127-1141 (1991) have described and have screened the algoscopy that the sudden change monocyte Listeria monocytogenes strain enters the ability of some cell line.For example, whether for the non-phagocytic cell that enter particular type be attenuation in order to measure the Listera with specific modification if belonging to antibacterial, the Listera of measuring attenuation belongs to antibacterial and enters the nonphagocytic ability of particular type and belong to antibacterial with the identical Listera that does not change and enter nonphagocytic ability and compare.Equally, in order to measure whether the Listera bacterial strain with specific sudden change is attenuation for the non-phagocytic cell that enters particular type, mensuration sudden change Listera bacterial strain enters the nonphagocytic ability of particular type and enters nonphagocytic ability with the Listera bacterial strain that does not have sudden change compares.In addition, also can obtain bacterial strain by the phenotype of bacterial strain is compared with the phenotype of the internalin B mutant of reporting before is the confirmation that internalin B lacks.
In some embodiments, can measure the attenuation of antibacterial to host's biological effect according to Listera.By measuring the LD in mice or other vertebratess
50Can measure the pathogenicity of bacterial isolates.LD
50Be to cause 50% vertebrates death need be injected into the consumption or the dosage of vertebrate Listera.The antibacterial and the LD that does not have specific change antibacterial that can relatively have specific change (for example, sudden change)
50Value is used as the tolerance of attenuation level.For example, if there is not the bacterial isolates of specific sudden change to have 10
3The LD of antibacterial
50Have 10 with bacterial isolates with specific sudden change
5The LD of antibacterial
50, bacterial strain attenuation then makes LD
50Improve 100 times or 2log.
Perhaps, can be in the external attenuation degree of more directly measuring antibacterial (for example, Listera) infection non-phagocytic cell ability.For example, the Listera of change infects non-phagocytic cell such as hepatocellular ability, can infect cytophagous ability with the Listera of non-change or wild type Listera and compare.In such mensuration, usually one limited period (for example in the external adding non-phagocytic cell of Listera that will change or non-change, one hour), use the solution washing cell that contains gentamycin to kill the outer antibacterial of any born of the same parents then, lysis is coated with then measures titre.Such mensuration example can find in U.S. Patent Publication No.2004/0228877.
As other example, the degree that can also come the quantitative measurement attenuation by the degree or the serum liver enzyme level of other biological effect such as lesion tissue.In clinical laboratory, measure the alanine aminotransferase (ALT) in the mice serum of having injected Listera (or other antibacterials), aspartate aminotransferase (AST), albumin and bilirubin level.For the Listera that has or do not have specific change/sudden change, can carry out the comparison of these effects in mice or other vertebratess, be used as measuring a kind of method of Listera attenuation.Also can measure the attenuation of Listera by histopathology.Can be from infecting vertebrate various tissue such as liver, the amount of the Listera of obtaining in spleen and the nervous system also can be as the tolerance of attenuation level, by these values in the vertebrates of relatively having injected sudden change and not mutated Listera.For example, the amount of the time dependent Listera that can obtain from infected tissue such as liver or spleen can be as the tolerance of attenuation, by these values in the mice of relatively having injected sudden change and not mutated Listera.
Therefore, can measure the attenuation of Listera according to the bacterial load in the special selected organs of mice, known mice is the target of wild type Listera.For example, by counting on the BHI agar culture medium coating liver or spleen homogenate (at H
2Homogenize among the O+0.2%NP40) dilution and the bacterium colony (colony-forming units that forms; CFU) measure the attenuation of Listera.For example, can comprise intravenous by any approach, intraperitoneal behind intramuscular and the subcutaneous Listera that changes, is measured the cfu of in a period of time liver or spleen.In addition, can measure after Listera and the competitive assessment of indices method by as described and the administration drug resistance in the liver and spleen (or any other selected organs) in a period of time, wild type Listera (or any other Listera bacterial strain of selecting) is compared.
For safe and effective vaccine is provided, the attenuation degree that non-phagocytic cell is taken in attenuated bacteria does not need absolute attenuation.In some embodiments, the attenuation degree is that toxic reduction is enough to prevention or alleviates signs of toxicity to non-fatal level.
In embodiments more of the present invention, comprise recombinant nucleic acid molecules described herein, the antibacterial of expression cassette and/or expression vector is the mutant of Listera.In the other embodiments, antibacterial is the attenuation mutant of Listeria monocytogenes.The attenuation mutant case description of multiple Listera is in U.S. Patent application No.60/446,051 (application on February 6th, 2003), 60/449,153 (applications on February 21st, 2003), 60/511,719 (applications on October 15th, 2003), 60/511,919 (application on October 15th, 2003), 60/511,869 (applications on October 15th, 2003), 60/541,515 (application on February 2nd, 2004) and 10/883,599 (application on June 30th, 2004), and U.S. Patent Publication No.2004/0197343 and US 2004/0228877, wherein every piece is incorporated herein by reference with its integral body at this.The mutant of Listera also is described among the international application No.PCT/US2004/23881 of application on July 23rd, 2004, is incorporated herein by reference with its integral body at this.For example, the antibacterial that comprises expression cassette and/or carrier randomly is to lack ActA or internalin B, or the mutant of the Listeria monocytogenes of ActA and internalin B.In some embodiments, antibacterial is the mutant of Listeria monocytogenes, that is, and and actA
-(for example, and DP-L4029 (be described in Skoble etc., J.of Cell Biology, the DP-L3078 bacterial strain among the 150:527-537 (2000) is incorporated herein by reference with its integral body at this, as at (Lauer etc., J.Bacteriol.184:4177 (2002); U.S. Patent application No.2003/0203472) spontaneous recovery of its phage described in), actA
-InlB
-(for example, DP-L4029 inlB, on October 3rd, 2003,, be preserved in U.S. typical case culture Culture Center (ATCC) according to the regulation of the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose, 10801 University Blvd., Manassas (Manassas), Virginia (Virginia) 20110-2209, the U.S. (Unitited States ofAmercian), and preserving number is PTA-5562), actA
-UvrAB
-(for example, DP-L4029uvrAB, on October 3rd, 2003, the regulation according to the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose was preserved in American type culture collection (ATCC), 10801 University Blvd., Manassas (Manassas), Virginia (Virginia) 20110-2209, the U.S. (Unitited States ofAmercian), and preserving number is PTA-5563), or actA
-InlB
-UvrAB
-In some embodiments, it is the two deletion mutants of Δ actA Δ inlB that the Listera of attenuation belongs to antibacterial (for example, Listeria monocytogenes).
Can pass through traditional method of mutagenesis, obtain mutant bacteria as mutation chemical drugs or radiation and then select mutant.Those skilled in the art can also obtain mutant bacteria by recombinant DNA technology.For example, use Camilli etc., the pKSV7 carrier described in the Molecular Micro.8:143-157 (1993) be applicable to that about the heterogenous expression box being introduced the allele switching method described in the bacterial genomes producing mutant comprises deletion mutant with above.(Camilli etc. (1993) are incorporated herein by reference with its integral body at this).A representative embodiment using the pKSV7 carrier to produce Listeria monocytogenes internalin B mutant is provided among the following embodiment 24.Perhaps, can use Biswas etc., the gene substitution experimental program described in the J.Bacteriol.175:3628-3635 (1993).Other similar methods are that those of ordinary skills are known.
The structure of various bacteria mutant is described in U.S. Patent Application Serial 10/883,599, and among U.S. Patent Publication No.2004/0197343 and the U.S. Patent Publication No.2004/0228877, wherein every piece is incorporated herein by reference with its integral body at this.
In embodiments more of the present invention, comprise recombinant nucleic acid molecules, the antibacterial of expression cassette and/or expression vector is the mutant of Bacillus anthracis.In some embodiments, antibacterial is the Sterne bacterial strain.In some embodiments, antibacterial is the Ames bacterial strain.In some embodiments, the Bacillus anthracis bacterial strain is the uvrAB mutant.In some embodiments, the Bacillus anthracis bacterial strain is the uvrC mutant.In some embodiments, the Bacillus anthracis mutant is the recA mutant.In some embodiments, antibacterial is Δ uvrAB mutant (the Bacillus anthracis Sterne Δ uvrAB mutant for example of Bacillus anthracis, on February 20th, 2004, regulation according to the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose, be preserved in American type culture collection (ATCC), 108011 University Blvd., Manassas (Manassas), Virginia (Virginia) 20110-2209, the U.S. (Unitited States of Amercian), preserving number is PTA-5825).
It is well known by persons skilled in the art changing the genomic method of Bacillus anthracis.A kind of method that produces sudden change in Bacillus anthracis is by using the allele exchange of allele exchange carrier well known by persons skilled in the art.Representative allele exchange plasmid is pKSV7, is described in Camilli etc., Molecular Microbiology, 8:143-147 (1993).As the first step that produces the sudden change Bacillus anthracis, lack waiting or the genome area that suddenlys change and about 1000bp of Bacillus anthracis genome upstream and downstream carry out pcr amplification, be cloned into then in the pKSV7 plasmid vector (or similar carrier).(bacillus specificity or Bacillus anthracis specificity temperature (ts) replicon can substitute the Listera ts replicon that is present in the pKSV7 allele exchange plasmid vector).Restriction endonuclease recognition site in the zone of waiting to lack or suddenling change can be used for lacking the required part of this zone targeting gene.Perhaps, the part that can remove targeting gene in this zone also uses the sequence that contains required sudden change or other changes to substitute.Before or after being cloned into allele exchange plasmid, the genomic zone of the Bacillus anthracis of being increased can change, and for example, uses the combination of restriction endonuclease or restriction endonuclease and synthetic gene sequence.In some embodiments, sequence can be changed into pcr amplification and be cloned among the pKSV7 then.In the alternate embodiment, at first amplicon is inserted in another plasmid, change then, excision is also inserted among the pKSV7.Perhaps, pcr amplification is directly inserted in the pKSV7 plasmid, change then, for example, use conventional restriction endonuclease.The pKSV7 plasmid that will contain the sequence that changes is then introduced in the Bacillus anthracis.This can be undertaken by electroporation.Then in the presence of the chloromycetin under permissive temperature on culture medium selecting bacteria.Then change integration in the single cross that the selection of going down to posterity by a plurality of generations is used for entering bacterial chromosome under non-permissive temperature in the presence of the chloromycetin.At last, bacterium colony carries out going down to posterity of a plurality of generations and obtains plasmid excision and eliminate (double crossing over) under permissive temperature in not containing antibiotic culture medium.U.S. Provisional Application series number 60/584,886 and 60/599,522 and U.S. Patent Publication No.2004/0197343 is incorporated herein by reference with its integral body at this, and the other description about dissimilar Bacillus anthracis mutation constructions is provided.
In embodiments more of the present invention, comprise recombinant nucleic acid molecules, the antibacterial of expression cassette and/or expression vector is the antibacterial that has changed, and making antibacterial be used to breed is (with respect to the antibacterial of non-change) of attenuation.Preferably, although change, the antibacterial of change is still kept enough gene expression doses.For example, in some embodiments, the influence that gene expression dose is not changed basically makes that antigenic expression is enough to stimulate antigenic immunne response when the antibacterial with antigen expressed gives the host.In some embodiments, the nucleic acid of antibacterial is by being modified with the reaction of nucleic acid target compound.In some embodiments, the nucleic acid of the antibacterial of change by being modified with the nucleic acid target compound reaction of direct and nucleic acid reaction, makes the propagation of antibacterial decay.In some embodiments, the nucleic acid target compound is nucleic acid alkylating agent (alkylator), as Beta-alanine, and N-(acridine-9-yl), two (2-chloroethyl) amino of 2-[] ethyl ester.In some embodiments, activate the nucleic acid target compound by radiation such as UVA irradiation.In some embodiments, the antibacterial of having used the psoralen compound treatment.For example, in some embodiments, used psoralen, as 4 '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen (" S-59 ") and UVA optical processing change antibacterial.In some embodiments, by shining the nucleic acid of modifying antibacterial with psoralen compound treatment and UVA.Use the nucleic acid target compound to change antibacterial and be described in following each U.S. Patent application or publication with the method that its attenuation is used for breeding, wherein each is incorporated herein by reference with its integral body at this: 60/446,051 (application on February 6th, 2003), 60/449,153 (applications on February 21st, 2003), 60/490,089 (application on July 24th, 2003), 60/511,869 (applications on October 15th, 2003), 60/541,515 (application on February 2nd, 2004) 10/883,599 (application on June 30th, 2004) and US2004/0197343.Antibacterial that changes and uses thereof also is described among the international application No.PCT/US2004/23881 of application on July 23rd, 2004, is incorporated herein by reference with its integral body at this.
For example, for the processing of Δ actA Δ uvrAB Listeria monocytogenes, in some embodiments, can be at (pact) OD
600Add the S-59 psoralen in=0.5 the logarithmic (log) phase culture to 200nM, when the optical density of culture reaches 1, then use 6J/m
2The deactivation of UVA light.In the bacteria growth process of Listera actA/uvrAB bacterial strain, by changing the concentration of S-59, UVA dosage, the time optimization deactivation condition that the time of contact S-59 and change are handled before UVA handles.With parent's Listera bacterial strain with comparing.On BHI (brain heart infusion) agar plate, there is not the deactivation (log-kills) that the ability that forms bacterium colony is measured Listera by antibacterial.In addition, those skilled in the art can use the 35S-pulse chase experiment to confirm that the expression of the heterologous protein of Listera of S-59/UVA deactivation and virulence factor such as LLO and p60 measures the synthetic and secretion of new expressed protein after the S-59/UVA deactivation.Use
35The LLO of S-metabolic marker and the expression of p60 can conventional determinings.Can
35The S-methionine exists down hatches the Li Siteshi actA/uvrAB of S-59/UVA deactivation 1 hour.Can measure whole cell lysate and the sedimentary heterologous protein of inoculum TCA, the antigen presentation of endogenous LLO and p60 and secretion.LLO, p60 and heterologous protein monoclonal antibody specific can be used for immunoprecipitation and verify after the deactivation continuous expression and secretion from the reorganization Listera.
In some embodiments, the antibacterial that attenuation is used to breed be used for the nucleic acid reparation also be attenuation and/or be to lack at least one DNA repairase.For example, in some embodiments, its amplifying nucleic acid has been the uvrAB deletion mutant by the antibacterial that nucleic acid target compound such as psoralen (handling in conjunction with UVA) are modified.
In some embodiments, bacterial multiplication is decayed at least about 0.3log, and at least about 1log, about 2log, about 3log, about 4log, about 6log, or at least about 8log.In another embodiment, bacterial multiplication decay is at least about 0.3 to＞10log, and about 2 to＞10log, about 4 to＞10log, about 6 to＞10log, about 0.3-8log, about 0.3-6log, about 0.3-5log, about 1-5log, or about 2-5log.In some embodiments, antibacterial to antigenic be expressed as bacterial nucleic acid wherein do not have adorned antibacterial to antigenic expression at least about 10%, about 25%, about 50%, about 75%, or at least about 90%.
In some embodiments, the nucleic acid of antibacterial is not as yet by being modified with the reaction of nucleic acid target compound.In some embodiments, recombinant bacteria is for propagation not decay as yet.In some embodiments, recombinant bacteria is for the not decay of nucleic acid repair ability.In some embodiments, recombinant bacteria is not to lack at least one DNA repairase.
In some embodiments, by recombinant nucleic acid molecules contained in the recombinant bacteria, the signal peptide of first polynucleotide encoding in expression cassette and/or the expression vector is natural to recombinant bacteria.In some embodiments, the polynucleotide of coding recombinant bacteria natural signals peptide have been that codon is optimized for the expression in recombinant bacteria.In some embodiments, polynucleotide have been that complete codon is optimized.In some embodiments, by recombinant nucleic acid molecules contained in the recombinant bacteria, the signal peptide of first polynucleotide encoding in expression cassette and/or the expression vector is an external source to host's recombinant bacteria.In some embodiments, the polynucleotide of coding host recombinant bacteria external source signal peptide have been that codon is optimized for the expression in recombinant bacteria.
In some embodiments, the recombinant nucleic acid molecules in the recombinant bacteria, second polynucleotide in expression cassette and/or the expression vector have been that codon is optimized for the expression in recombinant bacteria.In some embodiments, second polynucleotide has been that complete codon is optimized for the expression in recombinant bacteria.In some embodiments, first in the recombinant bacteria and second polynucleotide have been that codon is optimized for the expression in recombinant bacteria.In some embodiments, first in the recombinant bacteria and second polynucleotide have been that complete codon is optimized for the expression in recombinant bacteria.
On the one hand, the invention provides the antibacterial that comprises expression cassette, wherein expression cassette comprises first polynucleotide of (a) coded signal peptide, and wherein first polynucleotide are that codon is optimized for the expression in antibacterial; (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises the fusion rotein of signal peptide and polypeptide.As at this, for example, described in the part III, in some embodiments, coded signal peptide is derived from antibacterial.In some embodiments, be derived from and comprise the antibacterial same genus of expression cassette and/or the antibacterial of kind by the antibacterial signal peptide of expression cassette coding.In some embodiments, signal peptide is natural to host's recombinant bacteria.In other embodiments, the antibacterial signal peptide of being encoded by expression cassette is derived from the antibacterial that does not belong to together and/or plant with the antibacterial that comprises expression cassette.In some embodiments, signal peptide is an external source to host's recombinant bacteria.In some embodiments, signal peptide is secA1, secA2 or Tat signal peptide.In some embodiments, be allogenic (that is external source) to antibacterial by the polypeptide of second polynucleotide encoding.
On the other hand, the invention provides the antibacterial that comprises recombinant nucleic acid molecules, first polynucleotide that comprise (a) coding antibacterial natural signals peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, antibacterial is an intracellular bacteria.In some embodiments, recombinant nucleic acid molecules is the part of expression cassette, and this expression cassette further comprises the promoter of be operably connected first and second polynucleotide.In some embodiments, antibacterial is selected from Listera and belongs to bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria and escherichia coli.In some embodiments, antibacterial is Listera (for example a, Listeria monocytogenes).
On the other hand, (for example the invention provides antibacterial that the reorganization Listera that comprises recombinant nucleic acid molecules belongs to, Listeria monocytogenes), wherein recombinant nucleic acid molecules comprises first polynucleotide of (a) coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression that belongs at Listera in the antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, recombinant nucleic acid molecules is the part of expression cassette, and this expression cassette further comprises the promoter of be operably connected first and second polynucleotide.In some embodiments, second polynucleotide is that codon is optimized for the expression that belongs at Listera in the antibacterial.In some embodiments, be external source (that is it is allogenic, Listera being belonged to antibacterial) to the antibacterial that Listera belongs to by the polypeptide of second polynucleotide encoding.In some embodiments, it is attenuation that Listera belongs to antibacterial.For example, the Listera attenuation can be used for cell and intercellular diffusion, enter non-phagocytic cell or propagation.In some embodiments, it is to lack ActA that the reorganization Listera belongs to antibacterial, (for example, the two deletion mutants of Δ actA Δ inlB) of Internalin B or ActA and Internalin B.In some embodiments, by reacting the nucleic acid of having modified recombinant bacteria with nucleic acid target compound (for example, psoralen chemical compound).
On the other hand, the invention provides the antibacterial that comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotide of the non-secA1 antibacterial signal peptide of encoding, and coded polypeptide (for example, antigen) second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, recombinant nucleic acid molecules is the part of expression cassette, and this expression cassette further comprises the promoter of be operably connected first and second polynucleotide.In some embodiments, antibacterial is to be selected from Listera to belong to bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria or colibacillary antibacterial.In some embodiments, be external source (that is being allogenic) to antibacterial to antibacterial by the polypeptide of second polynucleotide encoding.
On the other hand, the invention provides the antibacterial that comprises expression cassette, wherein expression cassette comprises first polynucleotide of the non-secA1 antibacterial signal peptide of (a) coding; (b) be arranged in second polynucleotide of the coded polypeptide (for example, to the allogenic polypeptide of antibacterial) of identical translation frame with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises the fusion rotein of signal peptide and polypeptide.As at this, for example described in the above part III, in some embodiments, non-secA1 antibacterial signal peptide is the secA2 signal peptide.In some other embodiments, non-secA1 antibacterial signal peptide is the Tat signal peptide.In some embodiments, be derived from and comprise the antibacterial same genus of expression cassette and/or the antibacterial of kind by the antibacterial signal peptide of expression cassette coding.In other embodiments, the antibacterial signal peptide of being encoded by expression cassette is derived from the antibacterial that does not belong to together and/or plant with the antibacterial that comprises expression cassette.
On the other hand, the invention provides the antibacterial of the reorganization Listera genus that comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotide of the non-secA1 antibacterial signal peptide of (a) coding, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, recombinant nucleic acid molecules is the part of expression cassette, and this expression cassette further comprises the promoter of be operably connected first and second polynucleotide.In some embodiments, the antibacterial that Listera belongs to is an attenuation.In some embodiments, the antibacterial that Listera belongs to is a Listeria monocytogenes.For example, the Listera attenuation can be used for cell and intercellular diffusion, enter non-phagocytic cell or propagation.In some embodiments, the antibacterial that the reorganization Listera belongs to is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ actA Δ inlB).In some embodiments, by reacting the nucleic acid of having modified recombinant bacteria with nucleic acid target compound (for example, psoralen chemical compound).
On the other hand, the invention provides the antibacterial of the reorganization Listera genus that comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises that the coding Listera belongs to the polynucleotide of antibacterial allogenic polypeptide, and wherein polynucleotide are that codon is optimized for the expression in Listera.
Again in the one side, the invention provides the antibacterial of the reorganization Listera genus that comprises expression cassette, wherein expression cassette comprises following composition: (a) the coding Listera belongs to the polynucleotide of antibacterial allogenic polypeptide, and wherein polynucleotide are that codon is optimized for the expression in Listera; (b) promoter, the polynucleotide of the encoding exogenous that is operably connected polypeptide.In addition, in some embodiments, Listera is a Listeria monocytogenes.In other embodiments, the antibacterial that Listera belongs to belongs to the Yi Shi Listera, Si Shi Listera or harmless Listera kind.In some embodiments, the antibacterial that Listera belongs to is the attenuated strain that aforesaid Listera belongs to antibacterial.
In the other aspect, (for example the invention provides antibacterial that the reorganization Listera that comprises recombinant nucleic acid molecules belongs to, Listeria monocytogenes), wherein recombinant nucleic acid molecules comprise (a) coding non-Listera signal peptide first polynucleotide; (b) be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.In some embodiments, the antibacterial that Listera belongs to is an attenuation.For example, the Listera attenuation can be used for cell and intercellular diffusion, enter non-phagocytic cell or propagation.In some embodiments, the antibacterial that the reorganization Listera belongs to is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ actA Δ inlB).In some embodiments, by reacting the nucleic acid of having modified recombinant bacteria with nucleic acid target compound (for example, psoralen chemical compound).
Again in the one side, (for example the invention provides antibacterial that the reorganization Listera that comprises expression cassette belongs to, from the Listeria monocytogenes kind), this expression cassette comprises first polynucleotide of the non-Listera signal peptide of encoding, the coded polypeptide that is arranged in the translation frame identical with first polynucleotide (for example, the promoter of second polynucleotide non-Listera polypeptide) and be operably connected first and second polynucleotide.The expression cassette coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.In some embodiments, the antibacterial that Listera belongs to is used for cell and intercellular diffusion, and entering non-phagocytic cell or propagation is attenuation.In some embodiments, the antibacterial that the reorganization Listera belongs to is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ actA Δ inlB).In some embodiments, by reacting the nucleic acid of having modified recombinant bacteria with nucleic acid target compound (for example, psoralen chemical compound).In some embodiments, first polynucleotide, second polynucleotide, or first is that codon is optimized with second nucleotide for the expression in Listera.In some embodiments, first polynucleotide and/or second polynucleotide are that codon is optimized for the expression in Listeria monocytogenes.In some embodiments, being antigen by the polypeptide of second polynucleotide encoding, in some cases, can be non-bacterial antigens.For example, in some embodiments, polypeptide is tumor associated antigen or is derived from such tumor associated antigen.For example, in some embodiments, polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or be derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, TARP or CEA.For example, in some embodiments, polypeptide is the mesothelium element, or the fragment of mesothelium element or variant.In some other embodiments, polypeptide is NY-ESO-1, or the fragment of NY-ESO-1 or variant.In some embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen.In the preferred embodiment, signal peptide is an antibacterial.In some embodiments, signal peptide is from belonging to bacillus, the antibacterial of staphylococcus or Lactococcus.For example, in some embodiments, signal peptide is from Bacillus anthracis, bacillus subtilis, staphylococcus aureus or lactococcus lactis.In some embodiments, signal peptide is the secA1 signal peptide, as from USP 45 signal peptides of lactococcus lactis or from the protective antigen signal peptide of Bacillus anthracis.In some embodiments, signal peptide is the secA2 signal peptide.Still in the other embodiments, signal peptide is the Tat signal peptide, as bacillus subtilis Tat signal peptide (for example, PhoD).
The present invention further provides the recombinant bacteria that comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises: (a) first polynucleotide of coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant; (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises by the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant, wherein, in the protein chimera, polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or are positioned at autolysin or its catalytic activity fragment or catalytic activity variant.In some embodiments, recombinant bacteria is an intracellular bacteria, as the antibacterial (for example, Listeria monocytogenes) of Listera genus.In some embodiments, be external source to recombinant bacteria by the polypeptide of second polynucleotide encoding.
In the one side, the invention provides the antibacterial of the reorganization Listera genus that comprises the polycistronic expression box, wherein at least two discrete non-Listera polypeptide of polycistronic expression box coding again.For example, in some embodiments, expression cassette comprises first polynucleotide of first non-Listera polypeptide of encoding, second polynucleotide of second non-Listera polypeptide of coding and the promoter of be operably connected first and second polynucleotide.In some embodiments, the antibacterial that the reorganization Listera belongs to belongs to the Listeria monocytogenes kind.In some embodiments, first and/or second non-Listera polypeptide comprise antigen (or its fragment).
In some embodiments, the invention provides the recombinant bacteria (for example, Listera) that comprises expression cassette, this expression cassette comprises following composition: first polynucleotide of first signal peptide of (a) encoding; (b) encode second polynucleotide of first polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) intergenic sequence; (d) the 3rd polynucleotide of second signal peptide of coding; (e) the 4th polynucleotide of second polypeptide of coding, wherein the 4th polynucleotide are arranged in and the 3rd the translation frame that polynucleotide are identical; (f) first polynucleotide that are operably connected, second polynucleotide, the 3rd polynucleotide, the promoter of the 4th polynucleotide and intergenic sequence makes the expression cassette coding comprise first fusion rotein of first signal peptide and first polypeptide and second fusion rotein that comprises second signal peptide and second polypeptide.In some embodiments, one or more polynucleotide of coded signal peptide are that codon is optimized for the expression in antibacterial.In some embodiments, the 3rd and/or the 4th polynucleotide are that codon is optimized for the expression in antibacterial.In some embodiments, first and/or second polypeptide are allogenic (for example, heterologous antigens) to recombinant bacteria.In some embodiments, first and/or second signal peptide right and wrong secA1 antibacterial signal peptide.First and/or second signal peptide are natural or external source to recombinant bacteria.In some embodiments, recombinant bacteria is that the antibacterial that belongs to of Listera and first and/or second signal peptide are non-Listeras.In some embodiments, intergenic sequence is a Listeria monocytogenes actA-plcB intergenic sequence.In some embodiments, antibacterial is a Listeria monocytogenes.
In other aspects, the invention provides the antibacterial that comprises recombinant nucleic acid molecules, this recombinant nucleic acid molecules comprises first polynucleotide of (a) coded signal peptide, (b) coding secretory protein or its segmental second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the 3rd polynucleotide of coding and secretory protein or the allogenic polypeptide of its fragment, wherein the 3rd polynucleotide are arranged in and first and second translation frame that polynucleotide are identical, and wherein the recombinant nucleic acid molecules coding comprises signal peptide, polypeptide and secretory protein or its segmental protein chimera by second polynucleotide encoding, and wherein in the protein chimera, polypeptide and secretory protein or its fragment merge, or are positioned at secretory protein or its fragment.In some embodiments, antibacterial is the antibacterial that Listera belongs to.In some embodiments, antibacterial is the antibacterial that Listera belongs to, and is external source by the polypeptide of the 3rd polynucleotide encoding to this Listera.In some embodiments, antibacterial is a Listeria monocytogenes.
In some embodiments (for example, in some embodiments of above-mentioned each aspect), contained expression cassette is integrated in the bacterial genomes in the antibacterial.In other embodiments, contained expression cassette is on the plasmid in antibacterial in the antibacterial.
Usually, used promoter is the expression cassette that is suitable for carrying out with selected specific bacteria host heterogenous expression in the expression cassette.Art technology ordinary person can distinguish easily which promoter is suitable for using in which antibacterial.In some embodiments, promoter is the antibacterial promoter.In the other embodiments, the promoter in the antibacterial on the expression cassette is the promoter from the antibacterial of the antibacterial same genus that belongs to and contain this expression cassette.In other embodiments, the promoter in the antibacterial on the expression cassette is from the promoter that belongs to the antibacterial that contains this expression cassette antibacterial mutually of the same race.For example, belong to the Listeria monocytogenes kind if contain the antibacterial of expression cassette, used promoter randomly is derived from Listera gene such as hly on the expression cassette so.In other embodiments, promoter is constitutive promoter (for example, p60 promoter) or prfA-dependency promoter (for example, actA promoter).In addition, as mentioned above, in some embodiments, the promoter of expression cassette is a constitutive promoter.In other embodiments, also as mentioned above, the promoter of expression cassette is an inducible promoter.
In some embodiments (for example, in some embodiments of above-mentioned each aspect), by the expression cassette encoded polypeptides in the antibacterial or comprise that the fusion rotein of this polypeptide is antigen or other protein with therapeutic value, for example, as with described in the IV of top.In some embodiments, polypeptide or comprise that the protein of this polypeptide secretes from antibacterial.In some embodiments, the polypeptide that comes out from bacterial expression and/or secretion is allogenic to antibacterial.
Therefore, in some embodiments, the invention provides the reorganization Listera that comprises expression cassette, wherein expression cassette comprises first polynucleotide of (a) coding antibacterial (Listera or non-Listera) signal peptide, and wherein first polynucleotide are that codon is optimized for the expression in Listera; (b) antigenic second polynucleotide of the non-Listera of coding, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises signal peptide and antigenic fusion rotein.Further in the embodiment, Listera is a monocyte Listeria monocytogenes strain, as actA
-InlB
-Bacterial strain.In some embodiments, expression cassette has been integrated in the genome of reorganization Listera.In some embodiments, second polynucleotide is that codon is optimized for the expression in Listera.
The present invention also provides the Listera that comprises expression cassette, and wherein expression cassette comprises first polynucleotide of (a) coding secA2 or Tat antibacterial signal peptide; (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises signal peptide and antigenic fusion rotein.In some embodiments, the antibacterial signal peptide is a Listera.In other embodiments, the antibacterial signal peptide is non-Listera.Further in the embodiment, Listera is the bacterial strain of Listeria monocytogenes, as actA
-InlB
-Bacterial strain.In some embodiments, expression cassette has been integrated in the genome of reorganization Listera.In some embodiments, the polynucleotide of coded signal peptide (even signal peptide is the Listera signal peptide) and/or the polynucleotide of coding for antigens are that codon is optimized for the expression in Listera.
Further in the embodiment, the invention provides the reorganization Listera that comprises expression cassette, wherein expression cassette comprises following composition: (a) the antigenic polynucleotide of the non-Listera of coding, and wherein polynucleotide are that codon is optimized for the expression in Listera; (b) promoter connects the polynucleotide of encoding exogenous polypeptide joinably.In some embodiments, expression cassette further comprises the polynucleotide of coding antibacterial signal peptide, and it also is that codon is optimized for the expression in Listera.In one embodiment, the antibacterial signal peptide is a Listera.In another embodiment, the antibacterial signal peptide is non-Listera.In some embodiments, the antibacterial signal peptide is the secA1 signal peptide, secA2 signal peptide, or Tat signal peptide.Further in the embodiment, Listera is the bacterial strain of Listeria monocytogenes, as actA
-InlB
-Bacterial strain.In some embodiments, expression cassette has been integrated in the genome of reorganization Listera.
Again in the embodiment, the invention provides the antibacterial that the reorganization Listera belongs to, comprise first polynucleotide of (a) coding antibacterial (Listera or non-Listera) signal peptide, wherein first polynucleotide are that codon is optimized for the expression in Listera; (b) coding non-Listera antigenic second polynucleotide, wherein second polynucleotide also are that codon is optimized and be arranged in the translation frame identical with first polynucleotide for the expression in Listera; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises signal peptide and antigenic fusion rotein.In some embodiments, the antibacterial that Listera belongs to belongs to the Listeria monocytogenes kind.In some embodiments, the antibacterial that Listera belongs to is the actA of Listeria monocytogenes
-InlB
-Mutant.
The antibacterial such as the Listera that comprise more than a said expression cassette have been the present invention further provides.In the specific composition, given proteinic molecular weight can suppress it from the expression of recombinant bacteria as the reorganization Listera.A kind of method that addresses this problem is with " cutting apart " on the gene molecule of coding target protein and will merges with its excretory sequence (for example, secA1, secA2 or Tat element) from antibacterial of execution on each partitioning portion function.A kind of method is the reorganization Listera that generates each partitioning portion of expression of heterologous genes separately.Perhaps, each component (also comprise and be used for excretory appropriate members) of the gene cut apart on the molecule is introduced the intergenic region that runs through bacterial chromosome, use method well known in the art, for example exchange by allele.Another example is to express the gene of cutting apart on the molecule as single polycistronic message.According to this composition, being interspersed between the protein coding sequence of the gene of cutting apart on the molecule will be the SD ribosome binding sequence, so that restart protein synthesis according to polycistronic message.
In other aspects, the invention provides and improve expression and/or the excretory method of heterologous polypeptide in recombinant bacteria such as Listera.Polynucleotide described herein, any in expression cassette and/or the expression vector may be used in these methods.For example, the invention provides and improve expression and/or the excretory method of heterologous polypeptide in Listera that merges with signal peptide, comprise with the polypeptid coding sequence on the expression cassette signal coding sequence of expression cassette, or both codon optimizations.Expression and/or the excretory method of heterologous polypeptide in Listera that the present invention also provides raising and signal peptide to merge, comprise use from non-Listera source and/or from the signal peptide of secretory pathway beyond the secA1.
The present invention also provides the production recombinant bacteria, and (for example, the antibacterial that the reorganization Listera belongs to) method comprises that with recombinant nucleic acid molecules described herein expression cassette and/or expression vector are introduced in the antibacterial and produced recombinant bacteria.For example, in some embodiments, recombinant nucleic acid molecules comprises first polynucleotide of (a) coding antibacterial natural signals peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide, this recombinant nucleic acid molecules is introduced in the antibacterial produced recombinant bacteria.In some embodiments, recombinant nucleic acid molecules comprises first polynucleotide of the non--secA1 antibacterial signal peptide of (a) coding, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide, this recombinant nucleic acid molecules is introduced in the antibacterial produced recombinant bacteria.In some embodiments, introducing the recombinant nucleic acid molecules of producing recombinant bacteria in the antibacterial is such recombinant nucleic acid molecules, wherein this recombinant nucleic acid molecules comprises first polynucleotide of the non-Listera signal peptide of (a) coding, (b) be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.In some embodiments, the recombinant nucleic acid molecules that is used to produce recombinant bacteria is the recombinant nucleic acid molecules that comprises following composition: (a) first polynucleotide of coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, recombinant nucleic acid molecules coded protein chimera wherein, wherein non-Listera polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or insert in autolysin or its catalytic activity fragment or the catalytic activity variant.In some other embodiments, the method that the reorganization Listera belongs to antibacterial of producing is provided, comprise the polycistronic expression box is introduced the antibacterial that produces reorganization Listera genus in the antibacterial of Listera genus, wherein at least two discrete non-Listera polypeptide of expression cassette coding.
IX. pharmaceutical composition, immunogenic composition and/or vaccine combination
The present invention also provides multiple different compositions such as pharmaceutical composition, immunogenic composition and vaccine.These compositionss comprise arbitrary recombinant bacteria described herein (referring to, for example, above summary of the invention, other places of the part I of detailed Description Of The Invention and VIII and description comprise following embodiment).In some embodiments, compositions is isolating.
For example, the invention provides the pharmaceutical composition that comprises following composition: (i) acceptable carrier on the materia medica; Recombinant bacteria (ii) described herein.
For example, the invention provides the pharmaceutical composition that comprises following composition: (i) acceptable carrier on the materia medica; The recombinant bacteria that (ii) comprises expression cassette, this expression cassette comprises first polynucleotide of coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, second polynucleotide of coded polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, with the promoter of be operably connected first and second polynucleotide, make the expression cassette coding comprise the fusion rotein of signal peptide and polypeptide.
The present invention also provides and has comprised acceptable carrier on (i) materia medica; The pharmaceutical composition that (ii) comprises the recombinant bacteria of expression cassette, wherein expression cassette comprises first polynucleotide of the non-secA1 antibacterial signal peptide of encoding, be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide, with the promoter of be operably connected first and second polynucleotide, make the expression cassette coding comprise the fusion rotein of signal peptide and polypeptide.
The present invention further provides and comprised acceptable carrier on (i) materia medica; Comprise that (ii) the reorganization Listera of expression cassette belongs to the pharmaceutical composition of antibacterial, wherein expression cassette comprises following composition: (a) polynucleotide of coding Listera allogenic polypeptide, and wherein polynucleotide are that codon is optimized for the expression in Listera; (b) promoter, the polynucleotide of the encoding exogenous that is operably connected polypeptide.
The present invention also provides pharmaceutical composition, comprises acceptable carrier on (i) materia medica; The reorganization Listera that (ii) comprises expression cassette belongs to antibacterial, and this expression cassette comprises first polynucleotide of the non-Listera signal peptide of (a) coding; (b) be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.
As used in this, " carrier " comprises any He all solvents, disperse medium, excipient, coating, diluent, antifungal, isotonic agent and absorption delay agent, buffer agent, carrier solution, suspension, colloid etc.Acceptable carrier is known to a person of ordinary skill in the art on the materia medica, and comprises any material, during the combination of this material and active component, makes active component keep biological activity and does not react with patient's immune system.For example, acceptable carrier includes, but not limited to water on the materia medica, buffered saline solution (for example, 0.9% saline), emulsion such as oil/aqueous emulsion and various types of wetting agent.Possible carrier includes, but not limited to oil (for example, mineral oil), glucose solution, glycerite, Chalk, starch, salt, glycerol and gelatin.
Although the known any suitable carrier of those of ordinary skills all can be used in the pharmaceutical composition, the type of carrier will change according to administering mode.Compositions of the present invention can be prepared and be used for any suitable administering mode, for example comprise, the part, oral, nose, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.In some embodiments, for parenterai administration, as subcutaneous injection, carrier comprises water, saline, ethanol, fat, wax or buffer agent.In some embodiments, any of above carrier or solid carrier, as mannitol, lactose, starch, magnesium stearate, saccharin sodium, Talcum, cellulose, glucose, sucrose and magnesium carbonate may be used to oral administration.
By known conventional method prepare the compositions that comprises these carriers (referring to, for example, Remington ' s Pharmaceutical Sciences, the 18th edition, A.Gennaro edits, Mack Publishing Co., Easton, PA, 1990; And Remington, The Scienceand Practice of Pharmacy, the 20th edition, Mack Publishing, 2000).
Except pharmaceutical composition, also provide immunogenic composition.For example, the invention provides comprise said recombinant bacteria (referring to, for example, above summary of the invention, other places of the part I of detailed Description Of The Invention and VIII and description comprise following embodiment) immunogenic composition.In some embodiments, immunogenic composition comprises recombinant bacteria, wherein by the peptide sequence of the part of recombinant bacteria polypeptide expressed, as discrete protein, part as fusion rotein, or be embedded in the protein chimera and (depend on used recombinant nucleic acid molecules or expression cassette), be antigen or comprise antigen.In other words, in some embodiments, immunogenic composition comprises recombinant bacteria, and this recombinant bacteria comprises that coding comprises the recombinant nucleic acid molecules or the expression cassette of antigenic polypeptide.Suitable antigen includes, but not limited to said any of (for example, in top IV) in those.In some embodiments, the recombinant bacteria in the immunogenic composition is expressed and is comprised antigenic polypeptide, to be enough to induce the level to antigenic immunne response when compositions being given host (for example mammal such as people).In some embodiments, the immunne response that immunogenic composition stimulates is a cell-mediated immune responses.In some embodiments, the immunne response that immunogenic composition stimulates is a humoral immunoresponse(HI).In some embodiments, the immunne response that immunogenic composition stimulates comprises body fluid and cell-mediated immune responses.
For example, on the one hand, the invention provides the immunogenic composition that comprises recombinant bacteria, wherein antibacterial comprises expression cassette, expression cassette comprises following composition: (a) first polynucleotide of coded signal peptide, and wherein first polynucleotide are that codon is optimized for the expression in antibacterial; (b) second of coding for antigens polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
On the other hand, the invention provides the immunogenic composition that comprises recombinant bacteria, wherein antibacterial comprises expression cassette, and expression cassette comprises following composition: (a) first polynucleotide of the non-secA1 antibacterial signal peptide of coding; (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
Again in the one side, the invention provides and comprise that the reorganization Listera belongs to the immunogenic composition of antibacterial, the Listera of wherein recombinating belongs to antibacterial and comprises expression cassette, and wherein expression cassette comprises following composition: (a) the non-Listera antigen of coding and be the optimized polynucleotide of codon for expressing in Listera; (b) promoter, the polynucleotide of the coding for antigens that is operably connected.
The present invention also provides and has comprised that the reorganization Listera that contains expression cassette belongs to the immunogenic composition of antibacterial, and this expression cassette comprises: (a) first polynucleotide of the non-Listera signal peptide of coding; (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises non-Listera signal peptide and antigenic fusion rotein.
On the other hand, the invention provides the immunogenic composition (or vaccine) that comprises the recombinant bacteria that contains recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotide of (a) coding antibacterial natural signals peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, (b) coding comprises second polynucleotide of antigenic polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
On the other hand, the invention provides the immunogenic composition (or vaccine) that comprises reorganization Listera antibacterial, wherein recombinant bacteria comprises recombinant nucleic acid molecules, this recombinant nucleic acid molecules comprises first polynucleotide of (a) coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression in Listera, (b) coding comprises second polynucleotide of antigenic polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
On the other hand, the invention provides the immunogenic composition (or vaccine) that comprises the recombinant bacteria that contains recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotide of the non-secA1 antibacterial signal peptide of encoding, comprise second polynucleotide of antigenic polypeptide with coding, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
Again in the one side, the invention provides and comprise that the reorganization Listera that contains recombinant nucleic acid molecules belongs to the immunogenic composition (or vaccine) of antibacterial, wherein recombinant nucleic acid molecules comprises first polynucleotide of the non-secA1 antibacterial signal peptide of (a) coding, (b) coding is allogenic or be second polynucleotide of the polypeptide of external source to antibacterial with signal peptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.In some embodiments, the polypeptide of first polynucleotide encoding comprises antigen.
On the other hand, the invention provides and comprise that the reorganization Listera belongs to the immunogenic composition (or vaccine) of antibacterial, the Listera of wherein recombinating belongs to antibacterial and comprises recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises the polynucleotide of coding Listera allogenic polypeptide, and wherein the polynucleotide of encoding exogenous polypeptide are that codon is optimized for the expression in Listera.In some embodiments, allogenic polypeptide comprises antigen.
On the other hand, the invention provides and comprise that the reorganization Listera belongs to the immunogenic composition (or vaccine) of antibacterial, wherein recombinant bacteria comprises recombinant nucleic acid molecules, this recombinant nucleic acid molecules comprises first polynucleotide of the non-Listera signal peptide of (a) coding, (b) coding comprises second polynucleotide of antigenic polypeptide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein recombinant nucleic acid molecules coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.
The present invention also provides the immunogenic composition that comprises recombinant bacteria (or vaccine), wherein recombinant bacteria comprises nucleic acid molecules, this nucleic acid molecules comprises first polynucleotide of (a) coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant, wherein in the protein chimera, polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or are positioned at autolysin or its catalytic activity fragment or catalytic activity variant.In some embodiments, the polypeptide of second polynucleotide encoding comprises antigen.
On the other hand, the invention provides and comprise that the reorganization Listera belongs to the immunogenic composition (or vaccine) of antibacterial, the Listera of wherein recombinating belongs to antibacterial and comprises recombinant nucleic acid molecules, at least two discrete non-Listera polypeptide of recombinant nucleic acid molecules coding wherein, wherein at least one comprises antigen.
In other aspects, the invention provides the immunogenic composition (or vaccine) that comprises recombinant bacteria, this recombinant bacteria comprises recombinant nucleic acid molecules, this recombinant nucleic acid molecules comprises first polynucleotide of (a) coded signal peptide, (b) coding secretory protein or its segmental second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, (c) the 3rd polynucleotide of coding and secretory protein or the allogenic polypeptide of its fragment, wherein the 3rd polynucleotide are arranged in and first and second translation frame that polynucleotide are identical, wherein the recombinant nucleic acid molecules coding comprises signal peptide, the polypeptide of the 3rd polynucleotide encoding and secretory protein or its segmental protein chimera, and wherein in the protein chimera, the polypeptide of the 3rd polynucleotide encoding and secretory protein or its fragment merge, or are positioned at secretory protein or its fragment.In some embodiments, the heterologous polypeptide of the 3rd polynucleotide encoding comprises antigen.
Can be by the test recombinant bacteria external or whether can be used for (or as vaccine) in the immunogenic composition at the recombinant bacteria (and/or particular expression box) that the ability of model system internal stimulus immunne response is measured particular form.
Whether can measuring these immune cell responses by method in the external and body, to measure the immunne response of specific recombinant bacteria (and/or particular expression box) effective.A kind of probability is that measurement target albumen or antigen are by being presenting of delivery cell with recombinant bacteria group antigens mixed.Recombinant bacteria can with suitable antigen-presenting cell or cell line for example dendritic cell mix, and can measure the antigen presentation of dendritic cell to Recognition Protein or antigenic T cell.If with enough horizontal expression albumen or antigen, then will being processed into fragments of peptides and being by dendritic cell, recombinant bacteria passs T cell in MHC I class or II class environment.In order to detect the albumen or the antigenic purpose of presenting, can use response specific protein or antigenic T cell clone or T cell line.The T cell also can be the T quadroma, wherein makes T cell infinite multiplication by merging with cancerous cell line.Such T quadroma, T cell clone or T cell line can comprise CD8+ or CD4+T cell.According to the approach of process antigen, dendritic cell can be passs CD8+ or CD4+T cell.Antigen in the CD8+T cell recognition MHC I class environment, and the antigen in the CD4+ identification MHC II class environment.By the TXi Baoshouti specific recognition by the antigenic stimulus of presenting the T cell, cause can quantitative measurement (for example, use the ELISA algoscopy, the ELISPOT algoscopy, or intracellular cytokine dyeing (ICS)) some albumen such as IL-2, tumor necrosis factor-alpha (TNF-α), or the generation of interferon-(INF-γ).These are the technology also understood specifically among the well known in the art and following embodiment (referring to, for example, following embodiment 21).
Perhaps, activated reporter gene in the time of can designing hybridoma and be included in the antigenic stimulus T quadroma of presenting is as tilactase.Can easily measure the increase that beta galactosidase produces by its activity to substrate such as chlorophenol red-beta galactose glycosides, it causes the change of color.Color change can be used as the indicator that specific antigen presents and directly measures.
Measure that method is that those of ordinary skills are known in the external and body of other of antigen presentation of recombinant bacteria vaccine of the present invention.Also can directly measure the expression of recombinant bacteria to specific heterologous antigen.For example, radiolabeled aminoacid can be added in the cell mass, and measure the radioactivity amount of mixing in the specified protein.Can for example pass through gel electrophoresis or capillary electrophoresis, and the quantitative measurement radioactivity amount be measured the expression of specific protein with by the synthetic Separation of Proteins of cell mass.Perhaps, can express does not have radioactive albumen and observes by the whole bag of tricks, as the ELISA algoscopy or by gel electrophoresis and western blot analysis, uses enzyme len antibody or fluorescent-labeled antibody to detect.
How following embodiment 21 provides in those of ordinary skills' known technology some are used to measure more immunogenic concrete representative embodiment.For example, the Elispot algoscopy, intracellular cytokine dyeing algoscopy (ICS), the measurement of the cytokine-expressing of the splenocyte of stimulation, and external and test cells in vivo toxicity T cytoactive all is the immunogenic technology that is used to measure well known by persons skilled in the art.Provide the representativeness of antigenic these technology of use pattern to describe among the embodiment 21.Representational algoscopy has also been described among following examples 31A and the 31E.
In addition, can then measure the therapeutic efficiency that survival or tumor growth come more directly to measure vaccine combination by giving animal model such as mouse model with immunogenic composition or vaccine.For example, can and excite (for example, using tumor or pathogen) back to measure survival in administration.Referring to, for example, the algoscopy described in following examples 20 and the 31B-D.
At first change tumor cell, make it express target antigen or pattern antigen, be used to test the immunogenic composition of expression specific antigen or the immunogenic mouse model of vaccine with producing in the target antigens expressed tumor cell implantation mice then.After (in order to test the prevention effects of candidate set compound) or tumor cell are implanted mice before the tumor cell implantation (in order to test the therapeutic efficiency of candidate set compound), can be with comprising the candidate's immunogenic composition or the vaccination mice of expressing the recombinant bacteria that contains target antigen or the antigenic polypeptide of pattern.
For example, can use the standard technique transfection CT26 mice mouse colon cancer cell of this area with the suitable carrier that comprises required antigen of coding or the antigenic expression cassette of pattern.Standard technique such as flow cytometry and Western blotting can be used for identifying with enough levels then comes antigen expressed or the antigenic clone of pattern to be used for the mensuration of immunogenicity and/or effect.
Perhaps, can test the candidate set compound of the recombinant bacteria that comprises antigen expressed, this antigen corresponding to or be derived from the endogenous antigen of tumor cell line (for example, the endogenous retrovirus gp70 of CT26 mice mouse colon cancer cell tumor antigen AH1, or irregular epitope AH1-A5).In such mensuration, tumor cell can be implanted in the animal model and do not need further to change to express other antigen.Can test then and comprise antigenic candidate vaccine.
As described, also provide the vaccine combination that comprises said antibacterial.For example, the invention provides comprise said recombinant bacteria (referring to, for example, at above summary of the invention, other places of detailed Description Of The Invention part I and VIII and description, comprise the recombinant bacteria described in the following embodiment) vaccine, wherein as discrete protein, as the part of fusion rotein, or to be embedded in the peptide sequence that (depends on used recombinant nucleic acid molecules or expression cassette) in the protein chimera by the part of recombinant bacteria polypeptide expressed be antigen.Suitable antigen comprises said any of (for example, in above part IV) in those.
On the one hand, the invention provides the vaccine that comprises antibacterial, wherein antibacterial comprises the expression cassette that contains following composition: (a) first polynucleotide of coded signal peptide, and wherein first polynucleotide are that codon is optimized for the expression in antibacterial; (b) second of coding for antigens polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
On the other hand, the invention provides the vaccine that comprises antibacterial, wherein antibacterial comprises the expression cassette that contains following composition: (a) first polynucleotide of the non-secA1 antibacterial signal peptide of coding; (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
Again in the one side, the invention provides and comprise that the reorganization Listera that contains nucleic acid molecules belongs to the vaccine of antibacterial, wherein nucleic acid molecules comprises following composition: (a) the non-Listera antigen of coding and be the optimized polynucleotide of codon for the expression in Listera; (b) promoter, the polynucleotide of the coding for antigens that is operably connected.
On the other hand, the invention provides and comprise that the reorganization Listera that contains expression cassette belongs to the vaccine of antibacterial, this expression cassette comprises: (a) polynucleotide of the non-Listera signal peptide of coding; (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises non-Listera signal peptide and antigenic fusion rotein.
In some embodiments, vaccine combination comprises the antigen-presenting cell (APC) that has infected with arbitrary recombinant bacteria described herein.In some embodiments, vaccine (or immunogenic composition or pharmaceutical composition) does not comprise antigen-presenting cell (that is, vaccine or compositions are based on vaccine or the compositions of antibacterial, are not based on vaccine or the compositions of APC).
The medication that is suitable for administration of vaccines compositions (with pharmaceutical composition and immunogenic composition) is known in the art, and comprise oral, intravenous, Intradermal, intraperitoneal, intramuscular, in the lymph, intranasal and subcutaneous route of administration.
Bacterin preparation is known in the art, and can comprise many additives in some embodiments, as antiseptic (for example, thimerosal, the 2-phenyl phenol), stabilizing agent, adjuvant are (for example, aluminium hydroxide, aluminum phosphate, cytokine), antibiotic is (for example, neomycin, streptomycin) and other materials.In some embodiments, add stabilizing agent such as lactose or monosodium glutamate (MSG) and come the stabilization of vaccines preparation, handle as temperature change or lyophilizing to resist multiple condition.In some embodiments, bacterin preparation can also comprise suspension liquid or diluent such as sterilized water, saline or isotonic buffer saline (for example, being buffered to the phosphate of physiological pH).Vaccine can also contain a small amount of residual substance from production process.
For example, in some embodiments, with vaccine combination lyophilizing (that is lyophilization).Before administration, can with freeze dried preparation and sterile solution (for example, citrate-bicarbonate buffer agent, buffered water, 0.4% saline, etc.) merge.
In some embodiments, vaccine combination may further include other one-tenth known in the art and assigns to improve immunne response to vaccine, as adjuvant or costimulatory molecules.Except shown in above those, possible adjuvant comprises chemotactic factor and bacterial nucleic acid sequence, as CpG.In some embodiments, vaccine comprises the antibody of raising to the immunne response of vaccine, as CTLA4.In some embodiments, vaccine combination of the present invention randomly comprises costimulatory molecules, and this costimulatory molecules comprises that one or more are selected from GM-CSF, IL-2, IL-12, IL-14, IL-15, IL-18, B7.1, the factor of B7.2 and B7-DC.Other costimulatory moleculeses are that those of ordinary skills are known.
In other aspects, the invention provides the vaccine that improves the Listera comprise antigen expressed or the method for immunogenic composition.Any polynucleotide described herein, expression cassette and/or expression vector may be used in these methods.For example, the invention provides to improve and comprise that Listera belongs to the method for vaccine or the immunogenic composition of antibacterial, wherein Listera belongs to the heterologous antigen of bacterial expression and signal peptide fusion, comprise with the polypeptid coding sequence on the expression cassette signal coding sequence of expression cassette or both codon optimizations.The invention provides to improve and comprise that Listera belongs to the method for vaccine or the immunogenic composition of antibacterial, wherein Listera belongs to the heterologous antigen that bacterial expression and signal peptide merge, comprise use from non-Listera source and/or from the signal peptide of the secretory pathway except that secA1.
The method of production vaccine of the present invention also is provided.For example, in the embodiment, production comprises that recombinant bacteria (for example, the reorganization Listera belongs to antibacterial) the method for vaccine comprise recombinant nucleic acid molecules described herein, expression cassette or expression vector are introduced in the antibacterial, wherein recombinant nucleic acid molecules, expression cassette or expression vector codes antigen.For example, in some embodiments, recombinant nucleic acid molecules comprises first polynucleotide of (a) coding antibacterial natural signals peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial, (b) second of coding for antigens polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises signal peptide and antigenic fusion rotein, this recombinant nucleic acid molecules introduced in the antibacterial produced vaccine.In some embodiments, recombinant nucleic acid molecules comprises first polynucleotide of the non-secA1 antibacterial signal peptide of (a) coding, (b) second of coding for antigens polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises signal peptide and antigenic fusion rotein, this recombinant nucleic acid molecules is introduced in the antibacterial produced vaccine.In some embodiments, introducing the recombinant nucleic acid molecules of producing vaccine in the antibacterial is such recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises first polynucleotide of the non-Listera signal peptide of (a) coding, (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises non-Listera signal peptide and antigenic fusion rotein.In some embodiments, the recombinant nucleic acid molecules that is used to produce vaccine is such recombinant nucleic acid molecules, first polynucleotide that comprise (a) coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant, (b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, recombinant nucleic acid molecules coded protein chimera wherein, wherein non-Listera polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or insert in autolysin or its catalytic activity fragment or the catalytic activity variant.In some embodiments, provide to produce and comprised that the reorganization Listera belongs to the method for the vaccine of antibacterial, it comprises that the polycistronic expression box is introduced Listera to be belonged to and produce vaccine in the antibacterial, at least two discrete non-Listera polypeptide of polycistronic expression box coding wherein, wherein at least one polypeptide is an antigen.
Also provide and comprised any recombinant nucleic acid molecules of the present invention, expression cassette, carrier, the medicine box of antibacterial and/or compositions.
X. using method
Provide and used recombinant bacteria described herein or pharmaceutical composition, immunogenic composition or vaccine combination induce immune response, and/or the several different methods of prevention or treatment host disease.In some embodiments, the disease for the treatment of or preventing is a disease.In some embodiments, disease is a cancer.In some embodiments, disease is an infectious disease.In addition, recombinant bacteria can also be used for producing and separating heterologous protein, as mammalian proteins.
As used in this, " treatment (treatment) " of (about disease or disease) or " treatment (treating) " are to obtain useful or required effect, comprise and the method for preferred clinical effectiveness.For the purposes of the present invention, useful or required effect about disease comprises, but be not limited to, following one or more: the disease of improvement and disease association, cure diseases, the order of severity that palliates a disease, the progress that postpones disease, alleviate the symptom of one or more and disease association, improve the patient's who suffers from disease quality of life, and/or prolong survival.Equally, for the purposes of the present invention, include, but not limited to following one or more about the useful or required effect of disease: improve disease, cure disease, alleviate the order of severity of disease, delay the progress of disease, alleviate one or more symptoms relevant with disease, improve the quality of life of suffering from the disease patient, and/or prolong survival.For example, those are used for the treatment of compositions described herein in the embodiment of cancer, useful or required effect comprises, but be not limited to, following one or more: the hypertrophy (or destruction) tumor cell or the cancerous cell that reduce tumor cell or cancerous cell, reduce the transfer of the tumor cell of finding in the cancer, dwindle the size of tumor, alleviate the symptom that causes by cancer, improve the patient's who suffers from cancer quality of life, the dosage of the other drug that reduction treatment disease needs delays the progress of cancer, and/or prolongs cancer patient's survival.
As used in this; term " prevention " disease or " protection host " avoid disease (can be used alternatingly at this) and comprise; but be not limited to; following one or more: stop; postpone, hinder, slow down; block and/or delay the outbreak or the progress of disease, the progress of stable disease and/or delay advancing of disease.Term " prevention " disease or " protection patient " are avoided disease (can be used alternatingly at this) and are included, but not limited to following one or more: stop; postpone, hinder, slow down; block and/or delay the outbreak or the progress of disease, stablize the development of the progress and/or the delay disease of disease.The time period of this prevention can be the time of different length, depends on the historical and/or individuality to be treated of disease or disease.For example; when the design vaccine prevented or resists the infectious disease that is caused by pathogen, term " prevention " disease or " protection host " avoided disease and include, but are not limited to; following one or more: stop; postpone, hinder, slow down; block and/or delay the infection of host disease substance; the progress of host disease pathogen infection, or with outbreak or the progress of pathogen to the relevant disease of host's infection, and/or the stable and progress of pathogen to the relevant disease of host's infection.And for example, when vaccine was anti-cancer vaccine, term " prevention " disease or " protection host " avoided disease and comprise; but be not limited to following one or more: stop, postponing, hinder; slow down, block and/or delay the development or the transfer of cancer, the progress of cancer, or the recurrence of cancer.
On the one hand, the invention provides the method for host of inducing to antigenic immunne response, comprise with the recombinant bacteria described herein of effective dose or effective dose comprise said recombinant bacteria (referring to, for example, above summary of the invention, the part I of detailed Description Of The Invention, VIII and IX, or following embodiment) compositions (for example, pharmaceutical composition, immunogenic composition or vaccine) gives the host.In some embodiments, by the recombinant nucleic acid molecules in the recombinant bacteria, the polypeptide of expression cassette and/or expression vector codes comprises antigen, or comprises antigenic fusion rotein or protein chimera.
For example, on the one hand, the invention provides the method for host of inducing to antigenic immunne response, comprise that the compositions that comprises recombinant bacteria with effective dose gives the host, wherein recombinant bacteria comprises the expression cassette that contains following composition: (a) first polynucleotide of coded signal peptide, and wherein first polynucleotide are that codon is optimized for the expression in antibacterial; (b) second of coding for antigens polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
On the other hand, the invention provides the method for host of inducing to antigenic immunne response, comprise that the compositions that will contain the recombinant bacteria of expression cassette comprising of effective dose gives the host, wherein expression cassette comprises following composition: (a) first polynucleotide of the non-secA1 antibacterial signal peptide of coding; (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
Again in the one side, the invention provides the method for host of inducing to the antigenic immunne response of non-Listera, comprise that the compositions that the reorganization Listera that will contain nucleic acid molecules comprising of effective dose belongs to antibacterial gives the host, wherein nucleic acid molecules comprises following composition: (a) the non-Listera antigen of coding and be the optimized polynucleotide of codon for the expression in Listera; (b) promoter, the polynucleotide of the coding for antigens that is operably connected.
On the other hand, the invention provides the method for host of inducing to antigenic immunne response, comprise that the reorganization Listera that comprises expression cassette with effective dose belongs to antibacterial and gives the host, this expression cassette comprises following composition: (a) first polynucleotide of the non-Listera signal peptide of coding; (b) be arranged in second polynucleotide of the coding for antigens of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises non-Listera signal peptide and antigenic fusion rotein.
In some embodiments of the method for induce immune response described herein, with pharmaceutical composition, the form of immunogenic composition and/or vaccine combination gives antibacterial.
In some embodiments, immunne response is that MHC I para-immunity is replied.In other embodiments, immunne response is that MHC II para-immunity is replied.Still in other embodiments, be MHC I class and two kinds of immunne response of MHC II class by giving the inductive immunne response of antibacterial or compositions.Therefore, in some embodiments, immunne response comprises the CD4+T cellular response.In some embodiments, immunne response comprises the CD8+T cellular response.In some embodiments, immunne response comprises CD4+T cellular response and CD8+T cellular response.In some embodiments, immunne response comprises B-cellular response and/or T-cellular response.Can use the known method of those of ordinary skills, measure the B-cellular response at antigenic antibody titer by measuring.In some embodiments, be humoral immunoresponse(HI) by the inductive immunne response of compositions described herein.In other embodiments, inductive immunne response is a cellullar immunologic response.In some embodiments, immunne response comprises cell and two kinds of immunne response of body fluid.In some embodiments, immunne response is an antigenic specificity.In some embodiments, immunne response is the T-cellular response of antigenic specificity.
Except the method that induce immune response is provided, the present invention also provides the method for prevention or treatment host (for example, patient such as people patient) disease.In some embodiments, this disease is a disease.This method comprises the recombinant bacteria described herein with effective dose, or comprise said recombinant bacteria (referring to, for example, summary of the invention, above detailed Description Of The Invention part I, VIII and IX, or following embodiment) compositions give the host.In some embodiments, disease is a cancer.In some embodiments, disease is an infectious disease.
For example, on the one hand, the invention provides the method for prevention or treatment host disease (or disease), comprise that the compositions that comprises antibacterial with effective dose gives the host, wherein antibacterial comprises the expression cassette that contains following composition: (a) first polynucleotide of coded signal peptide, and wherein first polynucleotide are that codon is optimized for the expression in antibacterial; (b) coded polypeptide () second polynucleotide for example, antigen and/or therapeutic mammalian proteins, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
On the other hand, the invention provides the method for prevention or treatment host disease (or disease), comprise that the compositions that contains recombinant bacteria with effective dose gives the host, wherein antibacterial comprises expression cassette, and wherein expression cassette comprises following composition: (a) first polynucleotide of the non-secA1 antibacterial signal peptide of coding; (b) be arranged in second polynucleotide of the coded polypeptide (for example, antigen and mammalian proteins) of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide makes the expression cassette coding comprise signal peptide and antigenic fusion rotein.
Again in the one side, the invention provides the method for prevention or treatment host disease (or disease), comprise that the compositions that the reorganization Listera that will contain nucleic acid molecules comprising of effective dose belongs to antibacterial gives the host, wherein nucleic acid molecules comprises following composition: (a) coding non-Listera polypeptide (for example, antigen and/or therapeutic mammalian proteins) and be the optimized polynucleotide of codon for the expression in Listera; (b) promoter, the polynucleotide of the coding for antigens that is operably connected.
On the other hand, the invention provides the method for prevention or treatment host disease (or disease), comprise that the compositions that the reorganization Listera that will contain expression cassette comprising of effective dose belongs to antibacterial gives the host, this expression cassette comprises: (a) first polynucleotide of the non-Listera signal peptide of coding; (b) be arranged in second polynucleotide of the coded polypeptide (for example, antigen and/or therapeutic mammalian proteins) of the translation frame identical with first polynucleotide; (c) the be operably connected promoter of first and second polynucleotide, wherein the expression cassette coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.
In some embodiments, disease is a cancer.In some embodiments, when disease to be treated or prevention was cancer, disease was a melanoma, breast carcinoma, cancer of pancreas, hepatocarcinoma, colon cancer, colorectal carcinoma, pulmonary carcinoma, the brain cancer, carcinoma of testis, ovarian cancer, squamous cell carcinoma, gastrointestinal cancer, cervical cancer, renal carcinoma, thyroid carcinoma or carcinoma of prostate.In some embodiments, cancer is a melanoma.In some embodiments, cancer is a cancer of pancreas.In some embodiments, cancer is a colon cancer.In some embodiments, cancer is a carcinoma of prostate.In some embodiments, cancer is metastatic.
In other embodiments, disease is an autoimmune disease.Still in other embodiments, disease is an infectious disease or by pathogen such as virus, antibacterial, the another kind of disease that fungus or protozoacide cause.In some embodiments, disease is an infectious disease.
In some embodiments, the use of recombinant bacteria comprises that the cell that recombinant bacteria is delivered to the individual immunity system prevents or treats the cancer of existence or is delivered to risk factor such as individuality that environmental exposure and/or familial factor improve in prevention or the treatment cancer.In other embodiments, the use of recombinant bacteria comprises recombinant bacteria is delivered to and has excised tumor or suffered from cancer in the past but the individuality of remission at present in prevention or the treatment cancer.
In some embodiments, will comprise that the compositions of recombinant bacteria described herein gives the CD4+T cellular response that the patient has caused the host.In some other embodiments, will comprise that the compositions of recombinant bacteria described herein gives the CD8+T cellular response that the host has caused the host.In some embodiments, will comprise that the compositions of recombinant bacteria described herein gives CD4+T cellular response and the CD8+T cellular response that the host has caused the host.
Can estimate the effect of vaccine in the individual for example mice or other combination treatment diseases.Think that mouse model is to be used for estimating at the model of people's effect and to can be used for assessment and limit vaccine of the present invention.Mouse model is used for proving the potential of vaccine at the effectiveness of any individuality.Can estimate vaccine the prevention of antagonism specified disease or the ability of therapeutic effect are provided.For example, in the case of infectious disease, can use the of the present invention suitable vaccination a group mice of aequum, wherein recombinant bacteria is expressed the infectious disease related antigen.Can use infectant infecting mouse relevant and test that anti-infective is protected subsequently with vaccine antigen.Can observe the progress of infectious disease with respect to control group (non-inoculation or only inoculated carrier or do not contained the antibacterial of suitable antigen).
In the situation of cancer vaccine, can utilize tumor models, wherein can be before having inoculated the compositions that comprises the antibacterial of the present invention that contains required tumor antigen (treatment model) or (prophylaxis model) afterwards, the tumor cell line of expressing required tumor antigen is injected in a group mice.Inoculation contain tumor antigen recombinant bacteria can with not inoculation, inoculated carrier or inoculated the control group of expressing irrelevant antigenic recombinant bacteria and compared.Can estimate the effectiveness (for example, embodiment 31D) of vaccine in such model over time over time or according to survival colony behind the tumor injection according to gross tumor volume behind the tumor injection.In one embodiment, the gross tumor volume of having inoculated in the mice of the compositions that contains recombinant bacteria is littler by about 5%, about 10% than the gross tumor volume of not inoculating or inoculated carrier or express in the mice of irrelevant antigenic antibacterial, about 25%, about 50%, about 75%, about 90% or about 100%.In another embodiment, at least about 10, about 17 after tumor is implanted mice, or about this species diversity of observing gross tumor volume in 24 days.In one embodiment, the half survival time of mice of having inoculated the compositions that comprises recombinant bacteria is longer at least about 2 than not inoculating or inoculated carrier or expressing the mice of irrelevant antigenic antibacterial, and about 5, about 7 or at least about 10 days.
Host in the said method (that is, the patient) is any vertebrates, and preferred mammal comprises domestic animal, and mutation animal and primates comprise the people.In some embodiments, the host is a mammal.In some embodiments, the host is the people.
Can be by any suitable method, as Intradermal, subcutaneous, intraperitoneal, intravenous, intramuscular, in the lymph, oral or intranasal, and by sending recombinant bacteria and the compositions that comprises this bacterial strain for any given pernicious or infectious disease or the appropriate any approach of other diseases.
Can give the host in turn or separately with comprising the compositions while of recombinant bacteria and immunostimulant.The example of immunostimulant includes, but not limited to IL-2, IL-12, GMCSF, IL-15, B7.1, B7.2 and B7-DC and IL-14.Other examples of stimulant are provided among the above part IX.
As used in this, " effective dose " of antibacterial or compositions (as pharmaceutical composition or immunogenic composition) is the amount that is enough to realize useful or required effect.For preventative purposes, useful or required effect comprises as eliminating or reduce the risk of disease disease, the order of severity that palliates a disease, or the effect of delay seizure of disease, the biochemistry that comprises disease, tissue and/or behavior symptom, the middle pathology phenotype that presents in its complication and the disease progression process.For therapeutic use, useful or required effect comprises that clinical effectiveness is as suppressing or oppressive disease, reduce the symptom (biochemistry that one or more are caused by disease, tissue and/or behavior symptom), comprise the middle pathology phenotype that presents in its complication and the disease progression process, improve the quality of life of suffering from the disease patient, reduce the dosage of the required other drug of treatment disease, improve the effect of another kind of medicine, postpone the progress of disease and/or prolongation patient's survival.Can give effective dose with form in single or divided doses.For the purposes of the present invention, medicine, the effective dose of chemical compound or pharmaceutical composition are the amounts that is enough to directly or indirectly realize preventative or therapeutic treatment.As understanding in the clinical setting, medicine, the effective dose of chemical compound or pharmaceutical composition can or cannot be in conjunction with another kind of medicine, and chemical compound or pharmaceutical composition obtain.Therefore, in the situation of one or more therapeutic agents of administration, can consider effective dose,, then can think to have given single kind of medicament with effective dose if, may or obtain required result in conjunction with a kind of and multiple other medicaments.
In some embodiments, for the treatment for cancer treatment, effective dose comprises and will cause the amount of required immunne response, and the wherein immunne response growth of target tumor of having slowed down has reduced the tumor size, or preferably eliminated tumor fully.Can come the administration of repetition vaccine with suitable interval, and can be simultaneously in a plurality of different parts administrations of the individuality of inoculation.In some embodiments, for the prophylactic treatment of cancer, effective dose comprises and will cause the dosage of protective immune response, makes the individual probability that produces cancer significantly reduce.Vaccination regimen can comprise single dose, or can repeat in suitable interval, until the immunne response of setting up protectiveness.
In some embodiments, the individuality that the therapeutic treatment of individual cancer can start from being diagnosed as cancer is as initial therapy, or can use in conjunction with other treatment.For example, in order to reduce or eliminate the tumor of any remnants in the individuality, or reduce the risk of cancer return, surgical resection tumor or can use vaccine therapy with the individuality of radiotherapy or chemotherapeutic treatment.In some embodiments, the prophylactic treatment of individual cancer starts from because environmental condition or inherited genetic factors infect the individuality that the risk of some cancer improves.
The dosage that gives host's pharmaceutical composition or vaccine will be according to host's species, host's size and host's disease or disease and change.The dosage of compositions also will depend on the administration number of times and the route of administration of compositions.Select definite dosage by independent doctor according to patient to be treated.
In some embodiments, pharmaceutical compositions for use in the method, immunogenic composition or vaccine comprise and contain recombinant nucleic acid molecules described herein, the recombinant bacteria of expression cassette and/or expression vector.In some embodiments, recombinant bacteria is an antibacterial that change and/or sudden change, as U.S. Patent Application Serial 10/883,599, denomination of invention is " microorganism of the free living of change; vaccine combination and using method thereof ", Thomas W.Dubensky, Jr etc., application on June 30th, 2004, described in U.S. Patent Publication No.2004/0228877 and the U.S. Patent Publication No.2004/0197343 those, wherein every piece is incorporated herein by reference with its integral body at this.In some embodiments, comprise that the antibacterial such change and/or that suddenly change or the pharmaceutical composition of any other recombinant bacterias described herein or the single dose of vaccine comprise about 10
2To about 10
12The bacterium living beings body.In another embodiment, single dose comprises about 10
5To about 10
11The bacterium living beings body.In another embodiment, single dose comprises about 10
6To about 10
11The bacterium living beings body.In the embodiment, the single dose of pharmaceutical composition or vaccine comprises about 10 again
7To about 10
10The bacterium living beings body.In the embodiment, the single dose of pharmaceutical composition or vaccine comprises about 10 again
7To 10
9The bacterium living beings body.
In some embodiments, single dose comprises at least about 1 * 10
2The bacterium living beings body.In some embodiments, the single dose of compositions comprises at least about 1 * 10
5Organism.In another embodiment, the single dose of compositions or vaccine comprises at least about 1 * 10
6The bacterium living beings body.In the embodiment, the single dose of compositions or vaccine comprises at least about 1 * 10 again
7The bacterium living beings body.
In some embodiments, comprise reorganization described herein, the pharmaceutical composition of antibacterial change and/or sudden change, the single dose of immunogenic composition or vaccine comprise about 1CFU/kg extremely about 1 * 10
10CFU/kg (CFU=colony-forming units).In some embodiments, the single dose of compositions comprises that about 10CFU/kg is to about 1 * 10
9CFU/kg.In another embodiment, the single dose of compositions or vaccine comprises about 1 * 10
2CFU/kg is to about 1 * 10
8CFU/kg.In the embodiment, the single dose of compositions or vaccine comprises about 1 * 10 again
3CFU/kg is to about 1 * 10
8CFU/kg.In the embodiment, the single dose of compositions or vaccine comprises about 1 * 10 again
4CFU/kg is to about 1 * 10
7CFU/kg.In some embodiments, the single dose of compositions comprises at least about 1CFU/kg.In some embodiments, the single dose of compositions or vaccine comprises at least about 10CFU/kg.In another embodiment.The single dose of compositions or vaccine comprises at least about 1 * 10
2CFU/kg.In the embodiment, the single dose of compositions or vaccine comprises at least about 1 * 10 again
3CFU/kg.In the embodiment, the single dose of compositions or vaccine comprises at least about 1 * 10 again
4CFU/kg.
In some embodiments, can use the LD of method known to those skilled in the art from another kind of host such as mice for suitable (that is, effective) dosage of a kind of host such as people
50Data extrapolate out.
In some embodiments, pharmaceutical composition, immunogenic composition or vaccine comprise that the antigen-presenting cell that the recombinant bacteria of expression cassette and/or expression vector infects is as dendritic cell with comprising recombinant nucleic acid molecules described herein.In some embodiments, antibacterial has been changed and/or has been mutant such as U.S. Patent Application Serial 10/883,599, application on June 30th, 2004, with described in U.S. Patent Publication No.2004/0228877 and the US2004/0197343 those, wherein every piece is incorporated herein by reference with its integral body at this.For example, such vaccine based on antigen-presenting cell has been described below: international application No.PCT/US2004/23881, denomination of invention is " antigen-presenting cell vaccine and a using method thereof ", Thomas W.Dubensky, Jr etc., application in July 23 in 2004; U.S. Patent Application Serial 10/883,599, application on June 30th, 2004; U.S. Patent Publication No.2004/0228877; With U.S. Patent Publication No.US2004/0197343, wherein every piece is incorporated herein by reference with its integral body at this.In some embodiments, based on comprising about 1 * 10 comprising of antigen-presenting cell as said those the single dosage of vaccine of antibacterial
3To about 1 * 10
10Antigen-presenting cell.In some embodiments, the single dosage of vaccine comprises about 1 * 10
5To about 1 * 10
9Antigen-presenting cell.In some embodiments, the single dosage of vaccine comprises about 1 * 10
7To about 1 * 10
9Antigen-presenting cell.
In some embodiments, the multiple dosing of preferred dose unit is in one day or in the process in a week or month or a year or several years.In some embodiments, give dosage unit every day and continue many days, or how all administration is weekly.
The present invention further provides any recombinant bacteria described herein (promptly, comprise recombinant nucleic acid molecules described herein, any antibacterial of expression cassette or carrier) induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein by the recombinant nucleic acid molecules in the antibacterial, the polypeptide of expression cassette and/or vector encoded comprises antigen.In some embodiments, antigen is heterologous antigen.The present invention also provides the purposes of recombinant bacteria described herein in the medicine of making prevention or treatment host's disease (for example, disorders such as cancers or infectious disease).The present invention further provides recombinant bacteria described herein and be used to induce the host to antigenic immunne response, wherein by the recombinant nucleic acid molecules in the antibacterial, the polypeptide of expression cassette and/or vector encoded comprises antigen.The present invention further provides recombinant bacteria described herein and be used for prevention or treatment host's disease (as disease).
The present invention also provides the method for MHC I class antigen presentation on the inducing antigen presenting cell or the antigen presentation of MHC II class, comprises antibacterial described herein is contacted with antigen-presenting cell.
The present invention further provides the method for host of inducing to antigenic immunne response, may further comprise the steps:, recombinant bacteria described herein is contacted with antigen-presenting cell from the host (a) in appropriate condition be enough to antigen loaded and be under the time of delivery cell; (b) give the host with antigen-presenting cell.
Recombinant nucleic acid molecules, other of expression cassette and antibacterial may purposes be that those of ordinary skills can be cognitive.For example, recombinant nucleic acid molecules described herein, expression cassette, carrier can be used for producing and separating heterologous polypeptide with recombinant bacteria (with other host cells).Therefore, in the selectable aspect, the invention provides the method for express polypeptide in antibacterial, comprise that (a) will (for example, by transfection, transform or engage) in expression cassette described herein or the carrier introducing antibacterial; (b) antibacterial is grown being suitable under the condition of protein expression in culture.In another selectable aspect, the invention provides the method for producing isolated polypeptide, comprise the steps: that (a) will (for example, by transfection, transform or engage) in expression cassette described herein or the carrier introducing antibacterial; (b) antibacterial is grown being suitable under the condition of protein expression in cell culture; (c) protein isolate from the bacterial cell culture.Transform, the appropriate method of transfection and joint is known to a person of ordinary skill in the art, and it also is like this cultivating with making bacterial growth and separate excretory or non-secretory proteic method from cell culture.
Embodiment
Embodiment below providing illustrates but does not limit the present invention.
The preparation of embodiment 1. examples sudden change Listera bacterial strain
The Listera bacterial strain is derived from 10403S (Bishop etc., J.Immunol.139:2005 (1987)).The method of use generally acknowledging by SOE-PCR and allele exchange produce have shown in the Listera bacterial strain (Camilli, etc., Mol.Microbiol.8:143 (1993)) of in-frame deletion of gene.Mutant LLO L461T (DP-L4017) is described in Glomski etc., among the J.Cell.Biol.156:1029 (2002), is hereby incorporated by.ActA-mutant (DP-L4029) is to be described in Skoble etc., J.of CellBiology, and the DP-L3078 bacterial strain among the 150:527-537 (2000) is incorporated herein by reference with its integral body at this, the spontaneous recovery of its prophage.(prophage spontaneous recovery is described in (Lauer etc., J.Bacteriol.184:4177 (2002); U.S. Patent Publication No.2003/0203472)).ActA
-UvrAB
-The structure of bacterial strain is described in the U.S. Provisional Application 60/446,051, on February 6th, 2003 application, and as L4029/uvrAB (referring to, for example, the embodiment 7 of this application), and U.S. Patent Publication No.2004/0197343.DP-L4029uvrAB (Listeria monocytogenes actA
-/ uvrAB
-Two deletion mutants) on October 3rd, 2003, regulation according to the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose, be preserved in American type culture collection (ATCC), 10801 University Blvd., Manassas (Manassas), Virginia (Virginia) 20110-2209, the U.S. (Unitited States of Amercian), and preserving number is PTA-5563.Other descriptions about the sudden change Listera below are provided in application or the publication, and wherein every piece is incorporated herein by reference with its integral body at this: U.S. Patent Publication No.2004/0228877; U.S. Patent Publication No.US2004/0197343; PCT international application No.PCT/US2004/23881, application on July 23rd, 2004; With U.S. Patent Application Serial 10/883,599, application on June 30th, 2004.In addition, the two deletion mutants of example Listeria monocytogenes Δ actA Δ inIB are on October 3rd, 2003, regulation according to the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose, be preserved in American type culture collection (ATCC), 10801 University Blvd., Manassas (Manassas), Virginia (Virginia) 20110-2209, the U.S. (Unitited States of Amercian), and preserving number is PTA-5562.
Provide the gene that lacks in the Listeria monocytogenes to produce a non-limiting example of the method for attenuation mutant in following examples 24.
Make the sudden change Listera bacterial strain of the pattern antigen ovalbumin (OVA) of expressing clipped form, from the immunodominant antigen decision position (SPSYVYHQF (SEQ ID NO:72)) that is called AH1 of mice colorectal carcinoma (CT26); With the epitope AH1-A5 (SPSYAYHQF (SEQ ID NO:73) that changes; Slansky etc., Immunity, 13:529-538 (2000)).PPL2 integration vector (Lauer etc., J Bacteriol.184:4177 (2002); U.S. Patent Publication No.2003/0203472) is used to produce OVA and the AH1-A5/OVA reorganization Listera bacterial strain that contains the single copy that is integrated into the harmless site of Listera genome.
A. express the structure of OVA Listera (DP-L4056)
At first make that LLO by the hemolysin disappearance that merges with truncate OVA forms and be contained in antigen presentation box (pPL2/LLO-OVA) in the pPL2 integration vector.By at PSA (from the phage of ScottA) attachment site tRNA
Arg-attBB ' introduces pPL2/LLO-OVA among the monocyte Listeria monocytogenes strain DP-L4056 of curing and produces Listera-OVA vaccine strains.
Use the increase LLO of hemolysin disappearance of PCR, use following template and primer:
Source: DP-L4056 genomic DNA
Primer:
Forward (KpnI-LLO nts.1257-1276):
5′-CTCT
GGTACCTCCTTTGATTAGTATATTC(SEQ?ID?NO：74)
(T
m: LLO-specificity: 52 ℃.Generally: 80 ℃)
Oppositely (BamHI-XhoI-LLO nts.2811-2792):
5′-CAAT
GGATCCCTCGAGATCATAATTTACTTCATCCC(SEQ?ID?NO：75)
(T
m: LLO-specificity: 52 ℃.Generally: 102 ℃)
Also use the increase OVA of truncate of PCR, use following template and primer:
Source: from the colibacillary pDP3616 plasmid DNA of DP-E3616 (Higgins etc., Mol.Molbiol.31:1631-1641 (1999))
Primer:
Forward (Xhol-NcoI OVA cDNA nts.174-186):
5′-ATTT
CTCGAGT
CCATGGGGGGTTCTCATCATC(SEQ?ID?NO：76)
(T
m: OVA-specificity: 60 ℃.Generally: 88 ℃)
Oppositely (XhoI-NotI-HindIII):
5′-GGTG
CTCGAGT
GCGGCCGCAAGCTT(SEQ?ID?NO：77)
(T
m: generally: 82 ℃)
An experimental program finishing building process comprises at first and cuts the LLO amplicon with KpnI and BamHI, and the KpnI/BamHI carrier is inserted in the pPL2 carrier (pPL2-LLO).Then with XhoI and NotI cutting OVA amplicon and insert among the pPL2-LLO with the XhoI/NotI cutting.(remarks: the pPL2 carrier does not contain any XhoI site; PPD-3616 contains an XhoI site, is used for the design of OVA reverse primer).Confirm construct pPL2/LLO-OVA by restriction analysis (KpnI-LLO-XhoI-OVA-NotI) and order-checking.By transforming plasmid pPL2/LLO-OVA is introduced in the escherichia coli, then introduce and be integrated into (DP-L4056) in the Listera, (or introduce another kind of required Listera bacterial strain, according to Lauer etc. is described fully as inlB by engaging
-Mutant or inlB
-ActA
-Two deletion mutants).
B. express the structure of the Listera bacterial strain of AH1/OVA or AHI-A5/OVA.
Have this antigenic insertion fragment in order to make the Listera of expressing AH1/OVA or AH1-A5/OVA antigen sequence, at first to make, be connected to then among the carrier pPL2/LLO-OVA (as above making) from oligonucleotide.
Following oligonucleotide is used to prepare AH1 or AH1-A5 inserts fragment:
The AH1 epitope inserts fragment (ClaI-PstI compatible termini):
Cochain oligomer (on the AH1):
5′-CGATTCCCCTAGTTATGTTTACCACCAATTTGCTGCA(SEQ?ID?NO：78)
Following chain oligomer (under the AH1):
5′-GCAAATTGGTGGTAAACATAACTAGGGGAAT(SEQ?ID?NO：79)
The AHI-A5 epitope inserts fragment (ClaI-AvaII compatible termini):
The sequence of AH1-A5 epitope is SPSYAYHQF (SEQ ID NO:73) (5 '-AGT CCA AGT TAT GCA TAT CAT CAA TTT-3 ' (SEQ ID NO:80)).
On: 5 '-CGATAGTCCAAGTTATGCATATCATCAATTTGC (SEQ ID NO:81)
Down: 5 '-GTCGCAAATTGATGATATGCATAACTTGGACTAT (SEQ ID NO:82)
With the oligonucleotide of given epitope with etc. mixed in molar ratio, 95 ℃ of heating 5 minutes.Oligonucleotide mixture is slowly cooled off.Then annealed oligonucleotide is connected with the pPL2-LLO/OVA plasmid that makes with the relevant limit enzymic digestion the mol ratio with 200 to 1.Verify the characteristic of novel constructs by restriction analysis and/or order-checking.
By transforming plasmid introduced escherichia coli then, then to introduce and be integrated in the Listera (DP0L4056) by engaging, described according to Lauer etc. fully, or introduce in the another kind of required Listera bacterial strain, as actA
-Mutant (DP-L4029), LLO L461T bacterial strain (DP-L4017), or actA
-/ uvrAB
-Bacterial strain (DP-L4029uvrAB).
The structure of embodiment 3. Listera polynucleotide and expression cassette element
A. cloning vehicle
With selected heterologous antigen expression cassette molecule construction body insert pPL2 (Lauer etc., JBacteriol.2002), or pAM401 (Wirth etc., J Bacteriol.
165: 831-836), modified with the polyclone sequence that contains pPL2 (the AatII small fragment 171bp), inserts between the XbaI and NruI recognition site of passivation, in the tetracycline resistance gene (pAM401-MCS, Figure 32).Usually, with between the unique KpnI and BamII site in hly promoter and slotting pPL2 of (selecting) signal peptide sequence or the pAM401-MCS plasmid vector.Between unique BamHI and SacI site that selected EphA2 gene is (sometimes modified to contain N-end and C-end epitope label subsequently; Referring to following description) be cloned in these constructs.The method of using those skilled in the art to use always will be introduced in the selected monocyte Listeria monocytogenes strain of also handling with lysozyme by electroporation based on the molecule construction body of pAM401-MCS plasmid vector.Verify the plasmid structure of expecting in the Listera transfectant by restriction enzyme analysis by DNA isolation from the bacterium colony that forms at the BHI agar plate that contains chloromycetin (10 μ g/ml).Be used for measuring heterologous protein expression and secretion with the various reorganization Listeras that transform based on the heterologous protein expression cassette of pAM401-MCS, as described below.
To mix the tRNA in the selected Listera strain gene group based on the heterologous protein expression cassette construct of pPL2
ArgIn the gene, according to described method [Lauer etc., JBacteriol.184,4177-4186 (2002)] before.In brief, at first pPL2 heterologous protein expression cassette construct plasmid is introduced among the e. coli host bacteria strain SM10 (Simon etc., Bio/Technology 1:784-791 (1983)) by electroporation or by chemical method.Subsequently, will be transferred to the selected Listera bacterial strain from the SM10 that transforms based on the plasmid of pPL2 by engaging.After on the drug selectivity BHI agar plate that contains 7.5 every ml of μ g chloromycetin and the every ml of 200 μ g streptomycins as described, hatching, come the selected bacterium colony of purification for 3 times by on the flat board of same composition, going down to posterity.In order to verify of the integration of pPL2 carrier at phage attachment site, select single bacterium colony and by PCR screening, use the primer of forward primer NC16 (5 '-gtcaaaacatacgctcttatc-3 ' (SEQ ID NO:94)) and reverse primer PL95 (5 '-acataatcagtccaaagtagatgc-3 ' (SEQ ID NO:95)) right.Mix tRNA in the selected Listera strain gene group based on the plasmid of pPL2
ArgSelected bacterium colony in the gene has produced the diagnostic DNA amplicon of 499bp.
B. promoter
The heterologous protein expression cassette contains prfA-dependency hly promoter, and it drives the gene transcription of coding Listera lysin O (LLO), and is activated in the microenvironment of infection cell.From monocyte Listeria monocytogenes strain DP-L4056 amplification of nucleotide 205586-206000 (414bp), use primer shown below right by PCR.The zone of amplification comprises 28 aminoacid of hly promoter and LLO, comprises secA1 signal peptide (referring to above) and PEST domain.This regional expected sequence for monocyte Listeria monocytogenes strain EGD can find (accession number: gi|16802048|ref|NC_003210.1|[16802048]) in GenBank.Primer used in the PCR reaction is as follows:
Primer is right:
Forward (KpnI-LLO nts.1257-1276):
5′-CTCT
GGTACCTCCTTTGATTAGTATATTC(SEQ?ID?NO：74)
Oppositely (Bam HI-LLO nts.):
5′-CTCT
GGATCCATCCGCGTGTTTCTTTTCG(SEQ?ID?NO：84)
(leukorrhagia line be the restriction endonuclease recognition site.)
With 442bp pcr amplification be cloned into plasmid vector pCR-XL-TOPO (Invitrogen, Carlsbad, CA) in, according to the description of manufacturer.Measure the nucleotide sequence of Listera specificity base among the pCR-XL-TOPO-hly promoter plasmid clone.Compare with the EGD bacterial strain, monocyte Listeria monocytogenes strain DP-L4056 contains the prfA box both sides in the hly promoter eight nucleotide bases and changes.Shown the hly promoter comparison of Listeria monocytogenes DP-L4056 and EGD bacterial strain among following Fig. 1.
By from pCR-XL-TOPO-hly promoter plasmid clone, discharge 422bp DNA with KpnI and BamHI digestion corresponding to hly promoter and secA1 LLO signal peptide, and be cloned into pPL2 plasmid vector (Lauer etc., 2002J.Bact.) in, according to well known to a person skilled in the art conventional method.This plasmid is called pPL2-hlyP (natural).
The C.SD sequence
3 ' end in promoter contains polypurine SD sequence, and 30S ribosomal subunit (by 16S rRNA) is connected with heterologous gene rna transcription thing and starts translates needed sequence.The SD sequence has following consensus sequence: 5 '-NAGGAGGU-N usually
5-10-AUG (start codon)-3 ' (SEQ ID NO:85).There is the variation of polypurine SD sequence.Especially, the Listera hly gene of coding Listera lysin O (LLO) has following SD sequence: A
AGGAGAGTGAAACCCATG (SEQ ID NO:70) (leukorrhagia line be the SD sequence, and runic is translation initiation codon).
Polynucleotide
Show coding and the antigenic expression cassette sequence of total length people EphA2 that secA1 signal peptide (LLO signal peptide) merges among Fig. 2, added LLO PEST sequence.Shown aminoacid sequence among Fig. 3 by the fusion rotein of this expression cassette coding.
The codon optimization of embodiment 5. people EphA2 ectodomains (EX2)
The sequence of coding people's EphA2 ectodomain (aminoacid 25-526) has been that codon is optimized for the expression in Listeria monocytogenes.The natural nucleus glycoside acid sequence that has shown coding people EphA2 ectodomain among Fig. 4.Shown the nucleotide sequence that is used in the best codon selection of Listera among Fig. 5.The aminoacid sequence that has shown people EphA2 ectodomain among Fig. 6.
The polynucleotide of fusion rotein (EX2)
A. there are not the optimized polynucleotide of codon
Shown the polynucleotide sequence of coding among Fig. 7, added LLO PEST sequence with the antigenic ectodomain of people EphA2 of secA1 signal peptide (LLO signal peptide) fusion.Shown aminoacid sequence among Fig. 8 by the fusion rotein of this expression cassette coding.
B. the expression cassette that has the optimized people EphA2 of codon ectodomain
Shown the expression cassette sequence of coding among Fig. 9 with the people EphA2 antigen ectodomain of secA1 signal peptide (LLO signal peptide) fusion, add LLO PEST sequence, the sequence of the EphA2 ectodomain of wherein encoding is that codon is optimized for the expression in Listeria monocytogenes.Shown aminoacid sequence among Figure 10 by the fusion rotein of this expression cassette coding.
C. have optimized secA1 signal peptide of codon and the optimized people EphA2 of codon
The expression cassette of ectodomain
Shown the expression cassette sequence of coding among Figure 11 with the people EphA2 antigen ectodomain of secA1 signal peptide (LLO signal peptide), add LLO PEST sequence, the sequence of wherein encode EphA2 ectodomain and signal peptide and PEST sequence all are that codon is optimized for the expression in Listeria monocytogenes.Shown aminoacid sequence among Figure 12 by the fusion rotein of this expression cassette coding.
The codon optimization expression cassette of the fusion rotein of ectodomain (EX2)
Shown the expression cassette sequence of coding with the EphA2 antigen ectodomain of Tat signal peptide (bacillus subtilis phoD) fusion among Figure 13, the sequence of wherein encode EphA2 ectodomain and signal peptide all is that codon is optimized for the expression in Listeria monocytogenes.Shown aminoacid sequence among Figure 14 by the fusion rotein of this expression cassette coding.
The codon optimization of embodiment 8. people EphA2 born of the same parents intracellular domain (CO)
The sequence of coding people's EphA2 born of the same parents' intracellular domain (aminoacid 558-975) has been that codon is optimized for the expression in Listeria monocytogenes.The natural nucleus glycoside acid sequence that has shown coding people EphA2 ectodomain among Figure 15.Shown the nucleotide sequence that is used in the best codon selection of Listera among Figure 16.The aminoacid sequence that has shown people EphA2 ectodomain among Figure 17.
The polynucleotide of fusion rotein (CO)
A. there are not the optimized polynucleotide of codon
Shown the polynucleotide sequence of coding among Figure 18, added LLO PEST sequence with the people EphA2 antigen born of the same parents intracellular domain of secA1 signal peptide (LLO) fusion.Shown aminoacid sequence among Figure 19 by the fusion rotein of this expression cassette coding.
B. the expression cassette that has the optimized people EphA2 born of the same parents of codon intracellular domain
Shown the expression cassette sequence of coding among Figure 20 with the huEphA2 antigen born of the same parents intracellular domain of secA1 signal peptide (LLO signal peptide) fusion, add LLO PEST sequence, the sequence of the EphA2 born of the same parents' intracellular domain of wherein encoding is that codon is optimized for the expression in Listeria monocytogenes.Shown aminoacid sequence among Figure 21 by the fusion rotein of this expression cassette coding.
C. the expression cassette that has codon optimization secA1 signal peptide and codon optimization people EphA2 born of the same parents intracellular domain
Shown the expression cassette sequence of coding among Figure 22 with the EphA2 antigen born of the same parents intracellular domain of secA1 signal peptide (LLO signal peptide) fusion, add LLO PEST sequence, the sequence of wherein encode EphA2 born of the same parents' intracellular domain and signal peptide and PEST sequence all are that codon is optimized for the expression in Listeria monocytogenes.Shown aminoacid sequence among Figure 23 by the fusion rotein of this expression cassette coding.
The codon optimization expression cassette of the fusion rotein in structure territory (CO)
Shown the expression cassette sequence of coding with the EphA2 antigen born of the same parents intracellular domain of Tat signal peptide (bacillus subtilis phoD) fusion among Figure 24, the sequence of wherein encode EphA2 born of the same parents' intracellular domain and signal peptide all is that codon is optimized for the expression in Listeria monocytogenes.Shown aminoacid sequence among Figure 25 by the fusion rotein of this expression cassette coding.
The optimization expression cassette
The design expression cassette is used at Listeria monocytogenes expressing human carcinoma of testis antigen NY-ESO-1 (GenBank accession number No.NM_001327).Shown the expression cassette sequence of coding among Figure 26, added LLO PEST sequence with the NY-ESO-1 of secA1 signal peptide (LLO) fusion.The sequence of coding for antigens and signal peptide is that codon is optimized for the expression in Listeria monocytogenes in the expression cassette.Shown aminoacid sequence among Figure 27 by the fusion rotein of this expression cassette coding.
Embodiment 12. codings and non-Listera secA1 signal peptide (lactococcus lactis usp45)
The antigenic codon optimization expression cassette that merges
Use non-Listera secA1 signal peptide to design expression cassette to be used for expressing heterologous antigen at Listeria monocytogenes.Aminoacid sequence (Steidler etc. have below been shown from the usp45 signal peptide of lactococcus lactis, Nature Biotechnology, 21:785-9 (2003)), its natural coded sequence and for the optimized coded sequence of the expression in Listeria monocytogenes.
Aminoacid sequence:
MKKKIISAILMSTVILSAAAPLSGVYA′DT(SEQ?ID?NO：46)
Signal peptidase recognition site: VYA-DT (SEQ ID NO:55)
The natural nucleus glycoside acid sequence:
5′ATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCTGACACA3′(SEQ?ID?NO：86)
For the optimized codon of the expression in Listeria monocytogenes:
5′ATGAAAAAAAAAATTATTAGTGCAATTTTAATGAGTACAGTTATTTTAAGTGCAGCAGCACCATTAAGTGGTGTTTATGCAGATACA3′(SEQ?ID?NO：87)
The sequence that has shown the part expression cassette of the Listeria monocytogenes hly promoter that comprises the codon optimization Usp45 signal coding sequence that is operably connected among Figure 28.This sequence can comprise Usp45 signal peptide and required antigenic fusion rotein in conjunction with expressing with codon optimization or the optimized antigen sequence of non-codon.
Antigenic codon the best that embodiment 13. codings and secA2 signal peptide (p60) merge
Change expression cassette and carrier
A. the design of codon optimization expression cassette
Use the secA2 secretory pathway to design expression cassette in Listeria monocytogenes, to express heterologous antigen.The aminoacid sequence that has below shown the p60 signal peptide of Listeria monocytogenes, its natural coded sequence and for the optimized coded sequence of the expression in Listeria monocytogenes.
Aminoacid sequence:
MNMKKATIAATAGIAVTAFAAPTIASA′ST(SEQ?ID?NO：48)
Signal peptidase recognition site: ASA-ST (SEQ ID NO:57)
The natural nucleus glycoside acid sequence:
5′ATGAATATGAAAAAAGCAACTATCGCGGCTACAGCTGGGATTGCGGTAACAGCATTTGCTGCGCCAACAATCGCATCCGCAAGCACT3′(SEQ?ID?NO：90)
For the optimized codon of the expression in Listeria monocytogenes:
5′ATGAATATGAAAAAAGCAACAATTGCAGCAACAGCAGGTATTGCAGTTACAGCATTTGCAGCACCAACAATTGCAAGTGCAAGTACA?3′(SEQ?ID?NO：91)
The sequence that has shown the part expression cassette of the Listeria monocytogenes hly promoter that comprises the natural coded sequence of p60 signal peptide that is operably connected among Figure 29.The sequence that has shown the part expression cassette of the Listeria monocytogenes hly promoter that comprises the p60 signal peptide codon optimization coded sequence that is operably connected among Figure 30.
The structure of B.pPL2-hlypro_p60
All right construction expression box is wherein with in one or more sites of inserting in the antigen encoding sequence frame in the p60 gene coded sequence.The description of the part expression cassette structure that is used for insertion antigen sequence in p60 sequence inside casing has below been described.This part expression cassette contains the hly promoter.
Use Pfx or Vent polymerase to carry out first independent PCR reaction, use following primer and pPL2-hlyP-OVA (identical) as first template with the pPL2/LLO-OVA described in the above embodiment 2A:
pPL2-5F：5′-GACGTCAATACGACTCACTATAG(SEQ?ID?NO：92)
p60-hlyP-237R：5′-CTTTTTTCATATTCATGGGTTTCACTCTCCTTCTAC(SEQ?ID?NO：93)
The size of resultant amplicon is 285bp.
Also use Pfx or Vent polymerase to carry out first independent PCR reaction, use following primer and pCR-TOPO-p60 as second template.(carrier pCR-TOPO-p60 is from deriving from Invitrogen, Carlsbad, and the pCR-TOPO carrier of California makes, and has wherein inserted the genome p60 sequence of Listeria monocytogenes.The p60 coded sequence in any other many obtainable selectable sources can be used as alternate template).Primer used in this PCR reaction is as follows:
hlyP-p60-1F：5′-AAGGAGAGTGAAACCCATGAATATGAAAAAAGCAAC(SEQID?NO：88)
pCR-TOPO-2283R：5′-GTGTGATGGATATCTGCAGAATTC(SEQ?ID?NO：89)
The size of resultant amplicon is 1510bp.Use S6 post (Bio-RadLaboratories, Herculed, California) reactant of purification PCR then.
Carry out second PCR reaction then, the reactant that uses about 5 each first PCR of μ l is as template.Following primer is used in second PCR reaction:
Kpnl-LLO 1257F (used before primer):
5 ' CTCTGGTACCTCCTTTGATTAGTATATTC (SEQ ID NO:74) and
pCR-TOPO-2258R：5′-CCCTTGGGGATCCTTAATTATACG(SEQ?IDNO：83)。The size of resultant amplicon is 1715bp.Verify the amplicon size of expection in all PCR reactions by the agarose gel electrophoresis analysis.The reactant of second PCR of purification, with BamHI digestion, purification once more, and digest with KpnI.Then at the pAM401 of pPL2 and modification (pAM401-MCS; Figure 32) connect hlyP-p60 genetic fragment (KpnI-BamHI) (Figure 30) between BamHI in the plasmid and the KpnI site.
Use then BamHI/KpnI (1697,6024bp) and HindIII (210,424,3460,3634bp) digestion confirms the structure of pPL2-p60 plasmid.Confirmed that also the PstI site among the pPL2-p60 is unique.(and, KpnI/PstI digestion will produce 736 and the fragment of 6985bp).Structure from the pAM401-p60 plasmid (KpnI/PstI is with the KpnI/BamHI fragment) in p60 zone is identical with the structure of pPL2 construct.
Use method known to a person of ordinary skill in the art to make a large amount of preparation separators of each plasmid then.
Can use technology known to a person of ordinary skill in the art to insert required antigen encoding sequence in the p60 sequence then and be arranged in the translation box identical with the p60 sequence.Usually, insertion should make that the signal peptide sequence of p60N-end is complete.Also should make the autolysin sequence of complete of p60C-end.
People's mesothelium element that embodiment 14. is used for expressing at Listeria monocytogenes is compiled
The codon optimization of sign indicating number sequence
Shown the optimized polynucleotide sequence of codon of coding people mesothelium element among Figure 33, the mesothelium element is a kind of cancer antigen.Sequence shown in Figure 33 has been that codon is optimized for the expression in Listeria monocytogenes.Shown peptide sequence among Figure 34 by sequential coding among Figure 33.
The Mus mesothelium element that embodiment 15. is used for expressing at Listeria monocytogenes is compiled
The codon optimization of sign indicating number sequence
Shown the optimized polynucleotide sequence of codon of coding Mus mesothelium element among Figure 35, Mus mesothelium element is a kind of cancer antigen.Sequence shown in Figure 35 has been that codon is optimized for the expression in Listeria monocytogenes.Shown peptide sequence among Figure 36 by sequential coding among Figure 35.
As using integration vector such as pPL2, can use the allele exchange with a kind of possible alternative in the chromosome of allogeneic gene expression box insertion Listera.
In brief, by coating the antibacterial of selecting on the BHI agar culture medium that contains chloromycetin (10 μ g/ml) with pKSV7-heterologous protein expression cassette plasmid electroporation, and under 30 ℃ permissive temperature, hatch.Repeatedly select single cross to change integration by in containing the culture medium of chloromycetin, under 41 ℃ non-permissive temperature, several single bacterium colonies being gone down to posterity to bacterial chromosome.Finally, repeatedly obtain plasmid excision and eliminate (double crossing over) by in not containing the BHI culture medium of chloromycetin, under 30 ℃ permissive temperature, several single bacterium colonies being gone down to posterity.Verify that by PCR the heterologous protein expression cassette is integrated in the bacterial chromosome, use amplification right to the primer that is not contained in the zone that the bacterial chromosome targeting sequence the pKSV7 plamid vector construction body limited in the heterologous protein expression cassette.
Clone in embodiment 17.EphA2 to the pPL2 carrier and insertion are used in selected reorganization
Expression in the monocyte Listeria monocytogenes strain
Separately clone the EphA2 outside (EX2) and Cytoplasm (CO) domain of EphA2 transbilayer helix both sides, be used for inserting various pPL2-signal peptide expression construct.Use corresponding to natural mammal sequence or for EphA2 EX2 and CO domain the optimized gene of expression codon in Listeria monocytogenes.For optimized EphA2 EX2 of codon and EphA2 CO, use in the Listera each best codon (referring to, above table 3) in 20 aminoacid.The technology of using those skilled in the art to use always is synthesized optimized Eph2A EX2 of codon and CO domain by extending eclipsed oligonucleotide.Verify the expected sequence of all synthetic EphA2 constructs by nucleotide sequencing.
The primary amino acid sequence of the EX2 of EphA2 and CO domain, and the natural and optimized nucleotide sequence of codon is shown in (CO domain sequence) among Fig. 4-6 (EX2 sequence) and Figure 15-17.
In addition, in the amino of synthetic EphA2EX2 and CO gene and carboxyl terminal frame, insert FLAG (Stratagene respectively, La Jolla, CA) and myc epitope label, be used for detecting expression and excretory EphA2, use the antibody of FLAG or protein specific by western blot analysis.Therefore, expressed proteins has following orderly element: NH
2-signal peptide-FLAG-EphA2-myc-CO
2Shown below is FLAG and myc epitope label aminoacid and the optimized nucleotide sequence of codon:
FLAG：
5′-GATTATAAAGATGATGATGATAAA(SEQ?ID?NO：96)
NH
2-DYKDDDDK-CO
2(SEQ?ID?NO：97)
Myc：
5′-GAACAAAAATTAATTAGTGAAGAAGATTTA(SEQ?ID?NO：98)
NH
2-EQKLISEEDL-CO
2(SEQ?ID?NO：99)
It is proteic synthetic and from the secretion of various selected reorganization Listera-EphA2 bacterial strains to measure EphA2 by the western blot analysis of the sedimentary inoculum of trichloroacetic acid (TCA).In brief, to grow in the Listera culture collection of the mid-log phase in the BHI culture medium in 50mL taper centrifuge tube, with bacterial precipitation, and will hatch minimum 90 minutes to [6%] final concentration and on ice in the ice-cold TCA adding bacterial cultures supernatant or spend the night.By collecting the sedimentary protein of TCA in centrifugal 20 minutes with 2400 * g at 4 ℃.Then precipitate is resuspended among the TE (pH8.0, it is phenol red to contain 15 μ g/ml) of 300-600 μ l volume.Promote sample dissolution by vortex.If desired, by adding NH
4OH regulates sample pH value, is pink until color.Made all samples in 10 minutes and be used for electrophoresis by adding 100 μ l 4 * SDS sample loading buffers and hatching at 90 ℃.Then with sample in micro centrifuge with 14, centrifugal 5 minutes of 000rpm, and collect supernatant is stored in-20 ℃.For western blot analysis, fraction (1-4 * 10 that 20 μ l are made
9The equivalent of inoculum) loads on the 4-12%SDS-PAGE gel electrophoresis, and protein transduction moved on the PDDF film, the method for using always according to those skilled in the art.Hatched 2 hours by stirring at room in 5% milk powder in PBS, the film that makes transfer is used for hatching with antibody.Use antibody under the dilution factor in following PBST buffer (0.1% polysorbas20 among the PBS): (1) 1: 10,000 rabbit is anti--the myc polyclonal antibody (the ICL laboratory, Newberg, Oregon); (2) 1: 2, and 000 mouse-anti FLAG monoclonal antibody (Stratagene, La Jolla, CA); (3) the anti-EphA2 of rabbit (carboxyl terminal is specific) polyclonal antibody (sc-924, Santa CruzBiotechnology, Inc.Santa Cruz, CA).By with hatching the second time of the goat antirabbit of having puted together horseradish peroxidase or anti-mouse antibody and detect, and be exposed to film and estimate antibody and combine with the specificity of protein target with ECL chemical luminescent detecting test kit (Amersham).
The reorganization Listera of embodiment 19. coding various forms EphA2 is to EphA2 albumen
Secretion
A. Listera: [bacterial strain DP-L4029 (actA) or DP-L4017 (LLOL461T)]
Expression cassette construct: LLOss-PEST-CO-EphA2
To merge in the native sequences of EphA2 CO domain and the heredity of natural secA1 LLO sequence, and between KpnI and SacI site, the heterologous antigen expression cassette under the control of Listera hly promoter is inserted in the pPL2 plasmid as mentioned above.Introduce pPL2-EphA2 plasmid construction body by being engaged among Listera bacterial strain DP-L4029 (actA-) and the DP-L4017 (L461T LLO) as mentioned above.Figure 37 has shown the result of western blot analysis of the sedimentary inoculum of TCA of 4029-EphA2 CO and 4017-EphA2 CO.This analytical proof secreted a plurality of EphA2-specific fragments through through engineering approaches to contain less than 52kDa expection molecular weight by the reorganization Listera of the heterologous protein expression cassette of forming corresponding to secA1 and EphA2 CO fusion rotein native sequences, proved modifying the needs of expression cassette.
B. Listera: [DP-L4029 (actA
-)]
The expression cassette construct:
1. natural LLOss-PEST-FLAG-EX2_EphA2-myc-CodonOp
2.(CodonOp)LLOss-PEST(CodonOp)FLAG-EX2_EphA2-myc
With natural secA1 LLO signal peptide sequence or for being expressed as the optimized secA1 LLO of codon signal peptide sequence and merging in Listera, and between KpnI and SacI site, the heterologous antigen expression cassette under the control of Listera hly promoter is inserted in the pPL2 plasmid as mentioned above for being expressed as in the heredity of the optimized EphA2 EX2 of codon domain sequence in Listera.Introduce pPL2-EphA2 plasmid construction body among the Listera bacterial strain DP-L4029 (actA) by being engaged to as mentioned above.Figure 38 has shown the western blot analysis result of the sedimentary inoculum of TCA of Listera actA, the natural or optimized secA1 LLO of the codon signal peptide that this Listera actA coding and the optimized EphA2 EX2 of codon domain merge.This analytical proof be used in combination for preferred codon in the Listeria monocytogenes and select optimized signal peptide and heterologous protein to cause the expression of the total length EphA2 EX2 domain protein white matter expected.Use the codon optimization of EphA2 coded sequence separately, the expression of total length EphA2 EX2 domain protein white matter is poor.When use was the optimized Listeria monocytogenes LLO of codon secA1 signal peptide for the expression in Listeria monocytogenes, the expression of heterologous protein (fragment or total length) was the highest.
C. Listera: [DP-L4029 (actA)]
The expression cassette construct:
3. natural LLOss-PEST-(CodonOp) FLAG-EphA2_CO-myc
4.CodonOp?LLOss-PEST-(CodonOp)FLAG-EphA2_CO-myc
5.CodonOp?PhoD-(CodonOp)FLAG-EphA2_CO-myc
With natural secA1 LLO signal peptide sequence or for the optimized secA1 LLO of the expression codon signal peptide sequence in Listera, perhaps, Tat signal peptide for the optimized bacillus subtilis phoD of the expression codon gene in Listera, with for merging in the optimized EphA2 CO of the expression codon domain sequence heredity in Listera, and between KpnI and SacI site, the heterologous antigen expression cassette under the control of Listera hly promoter is inserted in the pAM401-MCS plasmid as mentioned above.By electroporation pAM401-EphA2 plasmid construction body is introduced among the Listera bacterial strain DP-L4029 (actA) as mentioned above.Figure 39 has shown the western blot analysis result of the sedimentary inoculum of TCA-of Listera actA, natural or the codon optimization secA1 LLO signal peptide that this Listera actA coding and codon optimization EphA2 CO domain merge, or the optimized bacillus subtilis phoD of codon Tat signal peptide.This analysis has proved to be used in combination for preferred codon in the Listeria monocytogenes once more selects optimized signal peptide and heterologous protein to cause the expression of the total length EphA2 CO domain protein expected.In addition, the expression of the total length EphA2 CO domain protein of expectation and secretion come the reorganization Listera of the codon optimization bacillus subtilis phoD Tat signal peptide of own coding and the fusion of codon optimization EphA2 CO domain.This result has proved new and unforeseeable discovery: can use from the signal peptide of different bacterium kind and carry out the secretion of heterologous protein from the reorganization Listera.Only with the codon optimization of Eph2A sequence, the expression of total length EphA2 CO domain protein is poor.When using for the optimized signal peptide of expression codon in Listeria monocytogenes, the heterologous protein expression levels is the highest.
D. use pCDNA4 plasmid transfection 293 cells of coding total length Eph2A expression cassette construct
6.pCDNA4-Eph2A
With natural total length Eph2A gene clone to based on the expression plasmid pCDNA4 of eukaryote CMV promoter (Invitrogen, Carlsbad, CA) in.Figure 40 has shown the western blot analysis result of the lysate that makes from 293 cells with the pCDNA4-Eph2A plasmid transfection, and has proved the great expression of total length Eph2A albumen in mammalian cell.
The reorganization Listera immunity band of the sub-optimization Eph2A of embodiment 20. usefulness coding passwords
The therapeutic efficiency of Balb/C mice that the CT26 tumor of coding people Eph2A is arranged
The following digital proof that presents among Figure 41-44 following:
The Balb/C mice that has CT26.24 (huEphA2+) lung tumor with the reorganization Listera immunity of coding OVA.AH1 (MMTV gp70 immunodominant antigen decision position) or OVA.AH1-A5 (MMTV gp70 immunodominant antigen decision position, for the T-cell receptors that improves in conjunction with having irregular variation) has been given long-term surviving (Figure 41).
EphA2 CO domain has intensive immunogenicity, when using the reorganization Listera immunity of sub-optimization of coding password or natural EphA2 CO domain sequence, the long term significant of observing the Balb/C mice survival that has CT26.24 (huEphA2+) lung tumor improves (Figure 43).
The immunogenicity of EphA2 EX2 domain is poor, only when the reorganization Listera of the codon optimization secA1 signal peptide that merges with coding and codon optimization EphA2 EX2 domain sequence was immune, the survival of observing the Balb/C mice that has CT26.24 (huEphA2+) lung tumor improved.When the reorganization Listera of the natural secA1 signal peptide that merges with coding and codon optimization EphA2 EX2 domain sequence is immune, in mice, do not observe therapeutic efficiency (Figure 42).The desirability of sub-optimization secA1 signal peptide and the EphA2 EX2 domain sequence of accessing to your password has obtained the support of the remarkable antineoplaston effect of statistics, as shown in following table 4.
Table 4. is by the comparison of the logarithm level test of the survival curve shown in Figure 42
| Experimental group | Half survival (natural law) | Significance (p value) with respect to HBSS group | Significance (p value) with respect to the natural secA1/EphA2 EX2 of actA-group |
| HBSS | 19 | - | - |
| | 20 | NS | NS |
| The natural secA1-EphA2 EX2 of actA-(natural) | 19 ? | NS ? | - ? |
| The natural secA1-EphA2 EX2 of actA-(CodOp) | 24 ? | 0.0035 ? | NS ? |
| actA-CodOp secA1-EphA2?EX2(CodOp) | 37 ? | 0.0035 ? | 0.0162 ? |
| The natural secA1-EphA2 CO of actA-(CodOp) | ＞99 ? | 0.0035 ? | 0.0015 ? |
Significantly, even 293 cells of pCDNA4-EphA2 plasmid transfection have produced very high-caliber protein expression, the Balb/C mice that has CT26.24 (huEphA2+) lung tumor with the immunity of pCDNA4-EphA2 plasmid does not cause the observation (Figure 44) of any therapeutic anti-tumour effect yet.
For the in-vivo tumour therapeutic studies, implant 5 * 10 for female Balb/C mice IV
5The CT26 cell of individual stably express EphA2.After three days, with the reorganization Listera bacterial strain of mice random packet and the various coding of IV inoculation EphA2.(do not mark among the figure) in the certain situation, the intramuscular before tibiofibula is given mouse inoculation 100 μ g pCDNA4 plasmid or pCDNA4-EphA2 plasmids.As positive control, give mice IV inoculation coding OVA.AHI or the chimeric reorganization Listera of OVA.AH1-A5 protein bacterial strain.Gave mouse inoculation in behind the implantation tumour cell the 3rd day and the 14th day.To inject Hnaks balanced salt solution (HBSS) mice buffer or unaltered Listera as negative control.All experiment groups comprise 5 mices.For survival research, when they begin to demonstrate nervous or dyspneic any sign, mice is put to death.
The mensuration of antigen-specific immune response after embodiment 21. vaccinations
Use in the multiple external and body method to test vaccine of the present invention.Some algoscopys comprise the analysis of the T cells with antigenic specificity of vaccinated mouse spleen.The non-limiting example of the outer and interior immunne response method of body of test body is provided among this embodiment.Antigen was pattern antigen described in these tests specifically described, and not necessarily used recombinant nucleic acid molecules described herein, the antigen that expression cassette and/or expression vector produced.This area skill those of ordinary skill will readily appreciate that the test described in this embodiment can easily be applied to test and comprise recombinant nucleic acid molecules described herein, immunne response in the external or body of the antibacterial of expression cassette and/or expression vector.
For example, by intravenous injection 0.1 LD
50The Listera bacterial strain of expression OVA (or other suitable antigens) inoculate C57B1/6 or Balb/c mice.Inoculate back seven days, collect the splenocyte of mice (one group of common 3 mice) by spleen being put into ice-cold RPMI 1640 culture medium and being prepared single-cell suspension liquid thus.As alternative scheme, can similarly collect the lymph node of mice, make the splenocyte in single-cell suspension liquid and the alternative the following stated test.Usually, for the intravenous or the intraperitoneal administration of vaccine, the test splenocyte, and for the intramuscular of vaccine, subcutaneous or intradermal administration, the cell of test splenocyte and lymph node.
Unless otherwise noted, all used among these embodiment antibody can be from Pharmingen, San Diego, and CA obtains.
ELISPOT algoscopy: use to have the antigenic Listera bacterial strain of OVA as an example the quantitative frequency of the T cells with antigenic specificity that produces when using the ELISPOT algoscopy to test immunity in the mouse model.The T cells with antigenic specificity of being estimated is OVA specific C D8+ or LLO specific C D8+ or CD4+T cell.This OVA antigen model measurement to the immunne response of inserting the allos tumor antigen in the vaccine and can be alternative with any target antigen.LLO antigen is specific to Listera.Detect cytokine (for example, IFN-γ) release during by identification specificity antigen and test specific T-cells.With anti-Mus IFN-γ monoclonal antibody (mAb R4; 5 μ g/ml) (BD Biosciences, San Jose CA) spends the night based on the 96 hole flat boards of PVDF for 4 ℃ of coatings.Sealed 2 hours with the complete RPMI of 200 μ L with the flat board washing and in room temperature.With 2 * 10
5The every hole of cell adds the splenocyte of the mice (or control mice of non-inoculation) of inoculation, and hatches 20 to 22 hours at 37 ℃ in the presence of the peptide of the various concentration of 0.01 to 10 μ M.The peptide that is used for OVA and LLO is SL8, the MHC I class epitope of OVA, LLO190 (NEKYAQAYPNVS (SEQ ID NO:100) Invitrogen), the MHC II class epitope of Listera lysin O (Listera antigen), LLO
296(VAYGRQVYL (SEQ ID NO:101), the MHC I class epitope of Listera lysin O, or LLO91 (GYKDGNEYI (SEQ ID NO:102)), the MHC I class epitope of Listera lysin O.LLO
190And LLO
296Be used for the C57B1/6 model, and LLO
91Be used for the Balb/c model.After the washing, with the biotinylated antibody incubation flat board of the specific secondary of IFN-γ (XMG1.2) that is diluted among the PBS to 0.5 μ g/ml.After 2 hours, washing is dull and stereotyped also with the 1nm Kingsoft goat-anti biotin conjugate (GAB-1 that is diluted among the PBS that contains 1%BSA in incubated at room; 1: 200 dilution factor; Ted Pella, Redding CA) was hatched 1 hour at 37 ℃.Thoroughly after the washing, (silver is strengthened test kit with the substrate that produces speckle with flat board; The 30ml/ hole; Ted Pella) incubated at room 2 to 10 minutes.Stop substrate reactions with distilled water rinsing flat board then.After dull and stereotyped air drying, (CTL, Cleveland OH) count the speckle in each hole the ELOSPOT flat bed reader of use automatization.For specific T cell of OVA or the specific T cell of Listera, with cytokine response with per 2 * 10
5IFN-γ speckle forms the quantitaes of cell (SFC) in the individual splenocyte.
Intracellular cytokine dyeing test (ICS): for the quantity of further test antigen specific C D8+ or CD4+T cell and make the result with the ELISPOT test in the result of acquisition associated with each other, carry out ICS and estimate cell by the flow cytometry analysis.With SL8 (stimulating OVA specific C D8+ cell) or LLO
190(stimulating LLO specific C D4+ cell) hatches the splenocyte of inoculation group and control group mice 5 hours in the presence of brefeldin A (Pharmingen).The secretion of the cytokine that the brefeldin A suppressor T cell is produced when stimulating.The splenocyte of hatching with irrelevant MHC I class peptide is with comparing.For IFN-γ and the dyeing of TNF-α intracellular cytokine, (phorbol-12-myristinate-13-acetate, Sigma) splenocyte of 20ng/ml and ionomycin (Sigma) 2 μ g/ml stimulation is as positive control for PMA.In order to detect the Cytoplasm cytokine-expressing, with FITC-anti--CD4mAb (RM4-5) and PerCP-resist-CD8mAb (53-6.7) is cell dyeing, fixing and the infiltrationization with Cytofix/CytoPerm solution (Pharmingen), and the anti-TNF-α mAb (MP6-XT22) that puts together with PE and APC-resisting of puting together-IFN-γ mAb (XMG1.2) dyeed on ice 30 minutes.By flow cytometer (FACScalibur, Becton Dickinson, MountainView CA) measures the percentage ratio and the use CELLQuest software (Becton Dickinson, Immunocymetry System) of expressing interior IFN-γ of born of the same parents and/or TNF-α cell and comes analytical data.Because the fluorescent labeling on the various antibody can all be distinguished by FACScalibur, identify suitable cell with any one or two kinds of painted those CD8+ and CD4+ among anti-IFN-γ or the anti-TNF-α by control.
Be excited the cytokine-expressing of splenocyte: the level of mouse boosting cell secrete cytokines that also can test the C57B1/6 mice of contrast and inoculation.With SL8 or LLO
190Stimulated splenocyte 24 hours.Stimulate with comparing with irrelevant peptide HSV-gB2 (Invitrogen, SSIEFARL, SEQ ID NO:4).Collection be excited cell supernatant and use the ELISA algoscopy (eBiosciences, CO) or Cytometric Bead Array Kit (Pharmingen) measure the level of T auxiliary-1 and auxiliary 2 cytokines of T-.
The test of cytotoxic t cell activity: by external or directly further estimate OVA specific C D8+T cell at their cellular cytoxicity activity of C57B1/6 mice body build-in test.The CD8+T cell is discerned in the antigenic specificity mode and their target cells separately of cracking.Use the chromium release assay method to measure vitro cytotoxicity.In order in the splenocyte group, to expand the OVA specific T-cells, (the EL-4 tumor cell line of OVA was expressed in transfection with the EG7.OVA cell that shines with 10: 1 ratios, ATCC, Mannassas VA) or with 100nM SL8 stimulates originally splenocyte with the mice of Listera OVA (inner) inoculation.Cultivate after 7 days, discharge the cellular cytoxicity activity of measuring the effector lymphocyte in the test at standard 4-hour 51Cr-, use EG7.OVA or SL8 pulse the EL-4 cell (ATCC, Mannassas, VA) as target cell and EL-4 cell separately as negative control.For the activity that will cause owing to the T cell with because the activity difference that the NK cell causes is come, (ATCC, Mannassas is VA) as the target of measuring the NK cytoactive with YAC-1 cell line.Calculate the percentage ratio of specific cytotoxicity according to 100 * (experiment release-spontaneous release)/(maximum release-spontaneous release).Do not have hatching of effect cell by target cell and measure spontaneous release.By measure maximum release with 0.1% triton x-100 cell lysis.If the maximum of spontaneous release＜20% discharges, think that then experiment is effective for analysis.
For the test of the cellular cytoxicity activity of OVA specific C D8+T cell in the body, the splenocyte of C57B1/6 mice originally is divided into two equal five equilibriums.Every group of specific peptide with 0.5 μ g/ml, target (SL8) or contrast (HSV-gB2) were 37 ℃ of pulses 90 minutes.Then cell is washed 3 times washed twice in PBS+0.1%BSA in culture medium.With cell with 1 * 10
7Every ml resuspending is in warm PBS+0.1%BSA (10ml or still less), so that with C-FDA succinyl ester (CFSE, Molecular Probes, Eugene, OR) labelling.In target cell suspension liquid, add the 5mM CFSE liquid storage of 1.25 μ L and come biased sample by vortex.The CFSE liquid storage of ten times of dilutions is added in the control cells suspension, and come biased sample by vortex.Cell was hatched 10 minutes at 37 ℃.A large amount of by adding (＞40ml) ice-cold PBS stops dyeing.In room temperature PBS washed twice, resuspension is also counted then with cell.Every kind of cell suspending liquid is diluted to 50 * 10
6Every ml, and every crowd 100 μ L are mixed and by originally or the tail vein injection of inoculation mice.After 12-24 hour, collect spleen and come 5 * 10 of analyzing total with flow cytometer
6Cell.Count high (target) and low (contrast) fluorescence peak, and the ratio at these two peaks is used to establish the cracked percentage ratio of target cell.The cells in vivo toxotest makes can be measured the lytic activity of T cells with antigenic specificity and does not need externally to stimulate again.In addition, this measurements determination the T cell function in the natural surroundings.
Embodiment 22. is by inducing with the Listera inoculation Balb/c mice of expressing EphA2
People EphA2 specific immunity
Be separated by for two weeks with the Listera L461T that expresses hEphA2 born of the same parents' intracellular domain (the Listera hEphA2-ICD among Figure 45) or express from Δ actA (actA for the Listera of the hEphA2 ectodomain (the Listera hEphA2-ECD among Figure 45) of the expression codon optimization sequence in Listeria monocytogenes
-) the next immune Balb/c mice (n=3) of bacterial strain.(born of the same parents' intracellular domain of hEphA2 alternatively is called hEphA2-ICD at this, hEphA2 ICD, EphA2 CO or CO.The ectodomain of hEphA2 alternatively is called hEphA2-ECD at this, hEphA2 ECD, EphA2 EX2 or EX2).Immunity back 6 angel mice euthanasia are collected spleen and concentrated the last time.For the ELISPOT test, with the P815 cell of expressing total length hEphA2 or external this cell that stimulates again of cell lysate that makes from these cells.Parent P815 cell or cell lysate are as negative control.Also stimulated cell with reorganization hEphA 2Fc fusion rotein.Use 96 hole speckle readers to measure IFN-γ positive spots and form bacterium colony (SFC).As shown in figure 45, observe the IFN-γ SFC of increase with the splenocyte that is derived from the mice that has inoculated Listera-hEphA2.The cell or the cell lysate of expressing hEphA2 stimulate the increase that has caused IFN-γ SFC, and this has shown EphA2 specific C D8+ and CD4+T cellular response.The mouse boosting cell of having inoculated the contrast of parent's Listera does not demonstrate the increase of IFN-γ SFC.
Embodiment 23.EphA2 specificity antineoplastic effect needs CD4+ and CD8+T cellular response
Gave Balb/c mice (n=10) i.v. inoculation 2 * 10 at the 0th day
5Individual CT26-hEphA2.The 1st day and the 3rd day by injection 200 μ g anti--CD4 (ATCC hybridoma GK1.5) or anti--CD8 (ATCC hybridoma 2.4-3) exhaust CD4+ cell and CD8+T cell, confirm (data not shown) by facs analysis.Used 0.1 LD then at the 4th day
50Express Listera L461T i.v. immune mouse and the monitoring survival of hEphA2 ICD.
As shown in Figure 46, the group that exhausts of CD4+ and CD8+ all fails to show the antitumor response of being seen in the animal that non-T cell exhausts.Data are summarized in the following table 5:
Table 5
| Inoculation group | Half survival (natural law) | P with respect to HBSS | # survivor (the 67th day) |
| HBSS | 17 | - | 0 |
| Listera-hEphA2-ICD | ＞67 | ＜0.0001 | 7 |
| The anti-CD4 of Listera-hEphA2-ICD+ | 19 | 0.03 | 2 |
| The anti-CD8 of Listera-hEphA2- | 24 | 0.0002 | 0 |
The data of front show in the optimal inhibition of tumor disappearance needs CD4+ and CD8+T cell.
In some embodiments, the antibacterial that comprises recombinant nucleic acid molecules described herein and expression cassette is the sudden change Listera.For example, in some embodiments, the antibacterial that comprises recombinant nucleic acid molecules and expression cassette is an actA gene wherein, the monocyte Listeria monocytogenes strain that inlB gene or both have lacked.A kind of exemplary process that produces the Listera deletion mutant has below been described.
Can exchange by allele and realize the disappearance of internalin B gene (inlB) from Listera DP-L4029 (or from other selected mutants or from the wild type Listera), as Camilli etc., described in the Mol.Microbiol.8:143-147 (1993).Can use montage overlap extension (SOE) PCR to prepare construct used in the allele switching method.The source of Internalin B gene is with GenBank accession number AL591975 (monocyte Listeria monocytogenes strain EGD, complete genome group, sections 3/12; InlB gene regions: the sequence of nts.97008-98963) listing, be incorporated herein by reference with its integral body at this, and/or with GenBank accession number NC_003210 (monocyte Listeria monocytogenes strain EGD, the complete genome group, the inlB gene regions: the sequence of nts.457008-458963) listing is incorporated herein by reference with its integral body at this.
In first PCR reaction, about 1000bp sequence of amplification Listera inlB gene 5 ' and 3 ' end upstream and downstream, use following template and primer:
Template: DP-L4056 or DP-L4029 genomic DNA
Primer (is used for the amplification of inlB 5 ' end upstream region) to 1:
Lm-96031F：5′-GTTAAGTTTCATGTGGACGGCAAAG(SEQ?ID?NO：103)
(T
m：72℃)
Lm-(3′inlB-R+)97020R：
5′-
AGGTCTTTTTCAGTTAACTATCCTCTCCTTGATTCTAGTTAT(SEQ?IDNO：104)(T
m：114℃)
(sequence of leukorrhagia line and the downstream area complementation of InlB carboxyl terminal).
Amplicon size (bp): 1007
Primer (is used for the amplification of inlB 3 ' end downstream area) to 2:
Lm-(5′inlB-F+)98911F：
5′-
CAAGGAGAGGATAGTTAACTGAAAAAGACCTAAAAAAGAAGGC(SEQ?IDNO：105)(T
m：118)
(sequence and the aminoterminal upstream region complementation of inlB of leukorrhagia line).
Lm-99970R：5′-TCCCCTGTTCCTATAATTGTTAGCTC(SEQ?ID?NO：106)(T
m：74℃)
Amplicon size (bp): 1074
In second PCR reaction, merge the amplicon of first PCR, utilize the complementarity between the forward primer of the 1st pair reverse primer and the 2nd pair by SOE PCR.This has caused the accurate disappearance of inlB coded sequence: nts.97021-98910=1889bp.Template and primer below in second PCR reaction, using:
Template: the reactant of first PCR of purification
Primer is right:
Lm-96043F：5′-GTGGACGGCAAAGAAACAACCAAAG(SEQ?ID?NO：107)(T
m：74℃)
Lm-99964R：5′-GTTCCTATAATTGTTAGCTCATTTTTTTC(SEQ?ID?NO：108)(T
m：74℃)
(amplicon size (bp): 2033)
The experimental program of finishing building process is as follows:
Use Vent archaeal dna polymerase (NEB) and the washed 30 ℃ of Listera DP-L4056 of 10 μ l or DP-L4029 overnight culture to carry out first PCR reaction (3 temperature cycles).Verify the expection size (1007bp and 1074bp) of Listera amplicon by 1% agarose gel.The reactant gel purification of first PCR is also come eluted dna with GeneClean (BIO 101).
Carry out second PCR reaction, every kind of first reactant that uses about equivalent is as template (about 5 μ l).Verify the expection size (2033bp) of the Listera amplicon of PCR reaction for the second time by 1% agarose gel.Add the adenosine residue with the Taq polymerase at 3 ' end of Listera d1 inlB amplicon.
Then Listera d1 inlB amplicon is inserted in the pCR2.1-TOPO carrier.With XhoI and KpnI digestion pCR2.1-TOPO-d1 inlB plasmid DNA and gel-purified 2123bp fragment.KpnI/XhoI 2123bp fragment is inserted by digesting with KpnI and XhoI and handling in the pKSV7 carrier (pKSV7-d1 inlB) that makes with CIAP.Verify the fidelity of d1 inlB sequence among the pKSV7-d1inlB then.Lack the inlB gene by allele exchange from required Listera bacterial strain with pKSV7-d1 inlB plasmid.
The optimized signal peptide of codon in the expression cassette of some representational Listeras that can be used for recombinating is provided in the following table 6
Table 6. is used to make up the representative signal peptide of reorganization Listera
| Secretory pathway | The signal peptide aminoacid sequence | Signal peptide site (') | Native sequences | The optimized sequence of codon during Lm expresses | Gene [belong to/kind] |
| ?secA1 ? ? ? ? ? ? ? ? ? ? ? ? | MKKIMLV FITLILVSL PIAQQTEA KDASAFN KENSISSM APPASPPA SPKTPIEK KHAD (SEQID NO：109)1 ? ? ? | TEA’KD (SEQ?ID NO：54) ? ? ? ? ? ? ? ? ? ? | ATGAAAAAAATAATG CTAGTTTTTATTACAC TTATATTAGTTAGTCT ACCAATTGCGCAACA AACTGAAGCAAAGGA TGCATCTGCATTCAAT AAAGAAAATTCAATT TCATCCATGGCACCA CCAGCATCTCCGCCTG CAAGTCCTAAGACGC CAATCGAAAAGAAAC ACGCGGAT(SEQ?ID NO：110) | ATGAAAAAAATTATGTT AGTTTTTATTACATTAAT TTTAGTTAGTTTACCAAT TGCACAACAAACAGAAG CAAAGATGCAAGTGCA TTTAATAAAGAAAATAG TATTAGTAGTATGGCACC ACCAGCAAGTCCACCAG CAAGTCCAAAAACACCA ATTGAAAAAAAACATGC AGAT(SEQ?ID?NO：113) ? ? | Hly (LLO) [monocyte Listeria monocytogenes] |
| MKKKIISA ILMSTVILS AAAPLSG VYADT (SEQ?ID NO：460 ? | VYA’DT (SEQ?ID NO：55) ? ? ? ? | ATGAAAAAAAAGATT ATCTCAGCTATTTTAA TGTCTACAGTGATACT TTCTGCTGCAGCCCCG TTGTCAGGTGTTTACG CTGACACA(SEQ?ID NO：86) | ATGAAAAAAAAAATTAT TAGTGCAATTTTAATGAG TACAGTTATTTTAAGTGC AGCAGCACCATTAAGTG GTGTTTATGCAGATACA (SEQ?ID?NO：87) ? | Usp45 [lactococcus lactis] | |
| MKKRKVL IPLMALSTI LVSSTGNL EVIQAEV (SEQ?ID NO：47) ? | IQA’EV (SEQ?ID NO：56) ? ? ? ? | ATGAAAAAACGAAAA GTGTTAATACCATTAA TGGCATTGTCTACGAT ATTAGTTTCAAGCAC AGGTAATTTAGAGGT GATTCAGGCAGAAGT T(SEQ?ID?NO：111) | ATGAAAAAACGTAAAGT TTTAATTCCATTAATGGC ATTAAGTACAATTTTAGT TAGTAGTACAGGTAATTT AGAAGTTATTCAAGCAG AAGTT(SEQIDNO：114) ? | Pag (protective antigen) [Bacillus anthracis] | |
| ?secA2 ? ? ? ? ? ? | MNMKKAT IAATAGIA VTAFAAPT IASAST (SEQ?ID NO：48) ? | ASA’ST (SEQ?ID NO：57) ? ? ? ? | ATGAATATGAAAAAA GCAACTATCGCGGCT ACAGCTGGGATTGCG GTAACAGCATTTGCT GCGCCAACAATCGCA TCCGCAAGCACT (SEQ?ID?NO：90) | ATGAATATGAAAAAAGC AACAATTGCAGCAACAG CAGGTATTGCAGTTACAG CATTTGCAGCACCAACA ATTGCAAGTGCAAGTAC A(SEQ?ID?NO：91) ? | Iap invasion associated protein p60[Listeria monocytogenes] |
| Tat ? ? ? ? ? ? ? ? ? ? ? | MAYDSRF DEWVQKL KEESFQNN TFDRRKFI QGAGKIA GLSLGLTI AQSVGAF (SEQ?ID NO：53) ? ? ? | VGA’F (SEQ?ID NO：62) ? ? ? ? ? ? ? ? ? | ATGGCATACGACAGT CGTTTTGATGAATGG GTACAGAAACTGAAA GAGGAAAGCTTTCAA AACAATACGTTTGAC CGCCGCAAATTTATTC AAGGAGCGGGGAAGA TTGCAGGACTTTCTCT TGGATTAACGATTGC CCAGTCGGTTGGGGC CTTT(SEQ?ID NO：112) | ATGGCATATGATAGTCGT TTTGATGAATGGGTTCAA AAATTAAAAGAAGAAAG TTTTCAAAATAATACATT TGATCGTCGTAAATTTAT TCAAGGTGCAGGTAAAA TTGCAGGTTTAAGTTTAG GTTTAACAATTGCACAAA GTGTTGGTGCATTT(SEQ ID?NO：115) ? ? | PhoD alkali phosphatase [bacillus subtilis] |
1Shown sequence comprises the PEST sequence from LLO.
Embodiment 26. comprises the codon of bacillus anthracis protective antigen (PA) signal peptide
The optimization expression cassette
Using non-Listera secA1 signal peptide to design expression cassette is used for expressing heterologous antigen at Listeria monocytogenes.The aminoacid sequence (GenBank accession number NC_007322) that has below shown Bacillus anthracis (Ba) protective antigen (PA) signal peptide, its natural coded sequence and for the optimized coded sequence of the expression in Listeria monocytogenes.
Aminoacid sequence:
MKKRKVLIPLMALSTILVSSTGNLEVIQAEV(SEQ?ID?NO：47)
Signal peptidase recognition site: IQA ' EV (SEQ ID NO:56)
The natural nucleus glycoside acid sequence:
ATGAAAAAACGAAAAGTGTTAATACCATTAATGGCATTGTCTACGATATTAGTTTCAAGCACAGGTAATTTAGAGGTGATTCAGGCAGAAGTT(SEQ?ID?NO：111)
For the optimized codon of the expression in Listeria monocytogenes:
ATGAAAAAACGTAAAGTTTTAATTCCATTAATGGCATTAAGTACAATTTTAGTTAGTAGTACAGGTAATTTAGAAGTTATTCAAGCAGAAGTT(SEQ?ID?NO：114)
The part expression cassette sequence that has shown the Listeria monocytogenes hly promoter that comprises the Ba PA signal peptide codon optimization coded sequence that is operably connected among Figure 47.This sequence can comprise Bacillus anthracis PA signal peptide and required antigenic fusion rotein in conjunction with being used for expressing with codon optimization or non-codon optimization antigen sequence.
Embodiment 27. from the reorganization Listera that comprises codon optimization expression cassette express and
Secretion antigen
The codon optimization of signal peptide and tumor antigen provides from the effective expression and the secretion of reorganization Listera: the codon optimization of the genetic elements of coded signal peptide and heterologous protein provides the human tumor antigen that contains hydrophobic domains from secreting based on the best the vaccine of reorganization Listera.Antigen is needed by effectively presenting of causing of MHC I classpath and CD8+T cell from effective secretion of kytoplasm antibacterial, and is therefore directly related with the effectiveness based on the vaccine of Listera.Two kinds of membrane-bound human tumor antigens of malignant cell, mesothelium element and NY-ESO-1, secretion from the reorganization Listera, the codon optimization by conjugated antigen and signal coding sequence has obtained optimization, described two kinds of antigens are and carcinoma of prostate and ovarian cancer (mesothelium element), and melanoma (NY-ESO-1), together with the relevant together immune target of other solid tumor.
Made up multiple expression cassette, these expression cassettes comprise and hly promoter natural or that codon optimization signal coding sequence is connected, signal peptide and secA1 or to comprise secA2 relevant with the alternative secretory pathway of two-Arg displacement (Tat), in the frame with selected human tumor antigen-people NY-ESO-1 or the plain fusion of people's mesothelium.(for antigen sequence and/or signal sequence, referring to above embodiment 11-14 and 25).The western blot analysis of the Listera culture fluid of growing in the sedimentary BHI meat soup of TCA-is used to measure the synthetic and secretion of heterologous protein from the reorganization Listera.(be similar to those method is used for western blot analysis described in the above embodiment 18).
These result of experiment are shown among Figure 48 A-C.Work as signal coding sequence, comprise when being derived from Listeria monocytogenes, select to observe of effective expression and the secretion of total length tumor antigen when being optimized for the codon in Listeria monocytogenes with the coded sequence of the exogenous antigen that is operably connected from the reorganization Listera.Figure 48 A has shown by having the Δ actA Listeria monocytogenes that comprises with the construct of the plain LLO signal peptide that merges of people's mesothelium and has expressed/secreted people's mesothelium element, for LLO and mesothelium element, uses natural codon.By the western blot analysis of the sedimentary inoculum of TCA, use these constructs not observe the secretion of desired total length mesothelium element (61kDa), only observe the secretion (Figure 48 A) of several small fragments.
Figure 48 B has shown the western blot analysis by the Listeria monocytogenes Δ actA expression/secretion people mesothelium element that comprises plasmid (pAM401), and this plasmid contains the construct of coding and the plain various signal peptides that merge of people's mesothelium.In each construct, the plain coded sequence of mesothelium is that codon is optimized for the expression in Listeria monocytogenes.Shown in it, used signal coding sequence contains native sequences (" natural ") or is codon optimized (" CodOp ") for the expression in Listeria monocytogenes.Use the anti-people/mouse antibodies of polyclone of affinity purification to detect excretory mesothelium element, by preparing this antibody to selected peptide and the IFA of rabbit injection.
Notably, as shown in swimming lane 3-5 and 8-9 among Figure 48 B, have only when the plain coded sequence of signal peptide and mesothelium be codon when optimized for the expression in Listera, just observe the secretion of total length mesothelium element (62kDa).This observation comprises significantly that also it is relevant with the secA2 secretory pathway with secA1 respectively from the signal peptide of antibacterial LLO and the proteinic Listera generation of p60, and these two kinds of signal peptides all comprise the codon that seldom uses.(also comprise the LLO PEST sequence with LLO signal peptide, its coded sequence also is that codon is optimized).When the optimized mesothelium element of the optimized Listera LLO of codon signal peptide and codon is connected, observe effective secretion (Figure 48 B of total length mesothelium element (62kDa), swimming lane 8), but when using the natural coded sequence of Listera LLO signal peptide, do not observe secretion (Figure 48 B, swimming lane 7).In addition, when the optimized mesothelium element of the optimized Listera p60 of codon signal peptide and codon is connected, observe secretion (62kDa) (Figure 48 B of total length mesothelium element, swimming lane 3), but when using the natural coded sequence of Listera p60 signal peptide, do not observe secretion (Figure 48 B, swimming lane 6).Finally, when the optimized signal peptide of codon from the antibacterial kind that is different from Listeria monocytogenes is operably connected the optimized mesothelium of codon when plain, observe the secretion (Figure 48 B) of total length mesothelium element.The signal peptide of bacillus anthracis protective antigen (Ba PA), or the signal peptide of lactococcus lactis Usp45 albumen (L1 Usp45) is carried out the effective secretion (Figure 48 B, swimming lane 4 and 5) of total length mesothelium element (62kDa) from reorganization Listera bacterial strain.Bacillus subtilis phoD signal peptide (Bs phoD) is also carried out the effective secretion (Figure 48 B, swimming lane 9) of total length mesothelium element from Listera.Band with about 62,000 molecular weight corresponding to mesothelium plain and two bands to may be corresponding to the plain polypeptide of the mesothelium that adds cutting of non-cutting (that is, corresponding to the part cutting).
Figure 48 C has shown the expression/secretion of NY-ESO-1 from Listeria monocytogenes Δ actA Δ inlB, described bacterial strain comprises the construct of the LLO signal coding sequence that merges with people NY-ESO-1 coded sequence, and these two sequences all are that codon is optimized for the expression in Listera.Use the NY-ESO-1 monoclonal antibody to detect excretory NY-ESO-1.
Among this embodiment, do composite signal peptide and tumor antigen domain use each amino acid whose most preferably codon, (the http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi that limits as the occurrence rate by per 1000 codons in the genomic coded sequence of Listera? species=Listeria+monocytogenes+[gbbct]).That belong to from Listera and other gram-positive bacteriums and secA1, the relevant signal peptide executor tumor antigen of secA2 or two-Arg displacement (Tat) secretory pathway is from effective secretion of reorganization Listera.Surprisingly, contain codon (per 1000 codon＜10 of occurrence rate) separately from the signal peptide of Listera albumen LLO and p60, and mesothelium is plain and NY-ESO-1 needs the optimization (Figure 48 B) of these sequences from effective secretion of the Listera of recombinating.When the secA1 signal peptide that connects bacillus anthracis protective antigen (pagA) and lactococcus lactis Usp45, and during the Tat signal peptide of the phosphodiesterase of bacillus subtilis/alkali phosphatase D gene (PhoD), observe also that mesothelium is plain to be secreted.
To be used to measure the best secretion whether particular approach helps heterologous protein from the signal peptide of different secretory pathways.For example, the Tat approach is used for albumen folding in the secreting bacteria, and bacillus subtilis phoD albumen is secreted by this mechanism.Originally supposed by transporting folding to have the tumor antigen that helps contain remarkable hydrophobic domains before, as the secretion of NY-ESO-1.Yet these results show that effective secretion of mammalian proteins mainly needs the codon optimization of signal peptide and tumor antigen coded sequence, rather than secretory pathway.
Importantly, compare, utilize the phenotype of the recombiant vaccine of any tumor antigen secretory pathway not to be subjected to appreciable impact with parent's Listera Δ actA/ Δ inlB bacterial strain.Half fatality rate (the LD of Listera Δ actA/ Δ inlB in the C57BL/6 mice
50) be 1 * 10
8Cfu.Using the pPL2 integration vector to finish stable single copy locus specificity that the tumor antigen expression cassette enters the harmless site on the Listera Δ actA/ Δ inlB chromosome mixes.The LD of tumor antigen coding Listera Δ actA/ Δ inlB
50Within 5 times of Listera Δ actA/ Δ inlB.
The structure of embodiment 28. bicistronic mRNA hEphA2 expression vectors
As non-restrictive example, provided the structure of antigen presentation box, wherein produce outside (EX2) and the inner (CO of hEphA2 from bicistronic mRNA information; Kinase dead) expression of domain.By being connected the secretion of finishing EX2 and CO domain with Ba PA and Bs PhoD signal peptide and EX2 and CO domain are functional respectively.
Plasmid with the optimized people EphA2 of codon kinases lost efficacy is called phEphA2KD, is used for the structure of bicistronic mRNA hEphA2 expression vector.(EphA2 is a receptor tyrosine kinase, but eliminates kinase activity by the active site at this enzyme from the sudden change of K to M).The coded sequence that has shown phEphA2KD among Figure 49.PhEphA2KD sequence shown in Figure 49 comprises the codon optimization coded sequence of the hEphA2 that has lacked membrane spaning domain, and contains the structure that 5 ' and 3 ' unique Bam HI and Sac I restriction site are helped the functional antigen expression cassette.What overstriking showed in the sequence shown in Figure 49 is Mlu I recognition sequence.
The sub-fragment of synthetic people EphA2 between two Mlu I restriction endonuclease recognition sequences (membrane spaning domain disappearance, kinases lost efficacy) (by method for synthesizing gene known in the art, for example synthetic by oligonucleotide, PCR, and/or Klenow filling, etc.).In building-up process outside the EphA2 born of the same parents that separate by the hydrophobic transmembrane domain in the native protein and the junction point between born of the same parents' intracellular domain accurately insert the actA-plcB intergenic region.The sub-fragments sequence of Mlu I (intergenic region overstriking demonstration) that has shown the codon optimization people EphA2 that contains the actA-plcB intergenic region among Figure 50.In addition, codon optimization Bs phoD signal peptide is positioned over the actA-plcB intergenic sequence 3 ' end and with EphA2 CO domain coding region, downstream frame endomixis.
Come assembling function people EphA2 bicistronic mRNA box by the corresponding region in the people EphA2 sequence that substitutes the kinases inefficacy that membrane spaning domain lacks shown in Figure 49 with the Mlu I fragment that contains actA-plcB intergenic region and Bs phoD signal peptide.This resulting sequence contains unique Bam HI and Sac I restriction enzyme recognition site at 5 ' and 3 ' end respectively, helps insert and the functional hly of connection promoter and initialize signal peptide, for example Ba PA.
Therefore, seven orderly function element of bicistronic mRNA people EphA2 antigen presentation box are as follows: hly promoter-Ba PA signal peptide-EX domain EphA2-termination codon-actA-plcB intergenic region (having the SD sequence)-Bs PhoD signal peptide-CO domain EphA2-termination codon.All EphA2 and signal coding sequence preferably codon are optimized.
By method illustrated in this application, use pAM401, pKSV7 or pPL1 and pPL2 integration vector are produced the reorganization Listera bacterial strain of expressing and secreting EphA2EX and CO domain.Use described herein and/or method known to those skilled in the art, detect proteic expression of EphA2 and secretion by required antibacterial western blot analysis partly.
Embodiment 29. expresses from comprising the chimeric reorganization Listera of antigen-bacterioprotein
And secretion antigen
In embodiments more of the present invention, the sequence of coded signal peptide and heterologous protein fusion partner thereof all is that codon is optimized.In some embodiments, it is desirable to codon optimization heterologous protein sequence is placed in the proteic localized area, its native form is excretory from Listera.With the heterologous protein functional nucleotide sequence be positioned in the qualification sequence of selected excretory Listera protein sequence, make the protein chimera of synthetic justacrine corresponding to the secretory protein of combination molecule amount.Secrete the secretion that system that needed host's Listera belongs to antibacterial promotes heterologous protein by utilizing from the best of body bacterioprotein.Molecular chaperones promotes the secretion of selected bacterioprotein.
As non-restrictive example, produce Listeria monocytogenes albumen p60 and human tumor antigen, the mesothelium element, between the protein chimera.By with human tumor antigen, the mesothelium element is put into Listeria monocytogenes albumen p60 at amino acid position 70 exactly and is produced protein chimera (although certainly selecting any required heterologous protein coded sequence to produce the protein chimera).The protein chimera contains the optimization codon and is used for expression at Listera p60 amino acid/11-70 and the plain coded sequence of complete mesothelium.In addition, Listeria monocytogenes hly promoter will be connected on p60-people's mesothelium fibroin matter chimera function, mix in the pPL2 carrier, this carrier of use as described herein is subsequently produced the reorganization monocyte Listeria monocytogenes strain of expressing and secreting people's mesothelium element.The experimental technique that is used to make up optimum expression and the chimeric reorganization Listera of secretion p60-people's mesothelium fibroin matter bacterial strain has below been described.
In some embodiments, the chimeric key character of protein between selected Listeria monocytogenes gene and the selected heterologous protein sequence is to put into selected Listeria monocytogenes gene and keep the best secretion of protein chimera by the effect each other of Listeria monocytogenes expressed proteins and antibacterial companion and secretion device selected heterologous protein sequence is functional suitably, and keeps the functional activity of Listeria monocytogenes protein in protein chimera environment.In some embodiments, what the functional placement of the heterologous sequence in Listeria monocytogenes secA2 dependence protein NamA and the p60 was expected is to keep these described proteinic Peptidoglycan cytohydrolist activity.(referring to, Lenz etc., (2003 PNAS 100:12432-12437), for example, are used to describe SecA2-dependency NamA and p60 protein).In some embodiments, wish the heterologous protein coded sequence functionally is positioned between signal sequence (SS) and cell wall-bound domain (LySM) and catalyst structure domain Lyz-2 (NamA) and the p60-dom (p60) (Lenz etc., (2003)).
In some embodiments, prfA-dependency promoter will be connected on the expressive function of antigen or heterologous protein.Like this, inducing heterogenous proteic expression in the microenvironment of the cell that the reorganization Listera infects.
The first step that p60-mesothelium fibroin matter chimera makes up comprises that the DNA of the prfA-dependency hly promoter that connects p60 70 amino acid whose DNA sequence of coding on the function is synthetic, uses best excretory codon in the Listera.(in some embodiments, can further change the zone that codon selects to avoid over-drastic RNA secondary structure, its can Profilin matter translation efficiency).Synthetic corresponding to the amino acid whose DNA fragment of the terminal p60 of hly promoter-70N-.(this can be undertaken by method for synthesizing gene known in the art usually, and is for example synthetic by oligonucleotide, PCR, and/or Klenow filling, etc.).
Monocyte Listeria monocytogenes strain 10403S p60 70 amino acid whose sequences are as follows:
MNMKKATIAATAGIAVTAFAAPTIASASTVVVEAGDTLWGIAQSKGTTVDAIKKANNLTTDKIVPGQKLQ(SEQ?ID?NO：116)
Those skilled in the art will recognize that the laboratory and the on-the-spot separator that there are a plurality of Listeria monocytogenes encoding genes, comprise p60, it can contain the transmutability on nucleotide sequence and the amino acid levels, but substantially the same gene and protein.In addition, those skilled in the art will recognize that to use and make up chimeric protein from any laboratory of Listeria monocytogenes or the gene of on-the-spot separator (comprising the bacterial strain that food is infectious or clinical).
Shown amino acid whose synthetic DNA sequence among Figure 51 corresponding to the terminal p60 of hly promoter-70N-.In addition, the codon of modifying coding p60 amino acid residue 69 (L) and 70 (Q) is helped the functional insertion of heterologous sequence to contain unique Pst I enzyme recognition sequence.In addition, the segmental 5 ' end of synthetic son contains unique KpnI enzyme recognition sequence.
The sub-fragment of 447bp KpnI and PstI digestion is connected in the corresponding K pnI and the PstI site of pPL2 carrier, and handles by digesting with KpnI and PstI enzymic digestion with calf intestine alkaline phosphatase (CIAP).This plasmid is called pPL2-hlyP-Np60 CodOp.Subsequently, the remainder with natural p60 gene is cloned in the pPL2-hlyP-Np60 CodOp plasmid between unique Pst I and BamH I site.By the remainder of PCR clone p60 gene, use the correction that contains the heat-stabilised poly synthase and following primer right:
Forward primer:
5′-CGC?CTGCAGGTAAATAATGAGGTTGCTG(SEQ?ID?NO：117)
Reverse primer:
5′-CGCGGATCCTTAATTATACGCGACCGAAG(SEQ?ID?NO：118)
Digest the 1241bp amplicon with Pst I and BamHI, and the 1235bp of purification is connected in the pPL2-hlyP-Np60 CodOp plasmid, digest with PstI and BamHI, and handle with CIAP.This plasmid contains complete Listeria monocytogenes p60 gene, have corresponding to the best codon of amino acid/11-77 with corresponding to the natural codon of aminoacid 78-478, and function connects Listeria monocytogenes hly promoter.This plasmid is called pPL2-hlyP-Np60 CodOp (1-77), has shown among Figure 52 to contain the sub-fragment sequence of KpnI-BamHI that function connects the hlyP of p60 coded sequence.Confirm the expected sequence of pPL2-hlyP-Np60 CodOp (1-77) plasmid by order-checking.
Next step that makes up is at the unique PstI site functional insertion heterologous protein coded sequence of plasmid pPL2-hlyP-Np60 CodOp (1-77), therefore it keep the proteic normal biological function of Listeria monocytogenes between the terminal signal peptide sequence of N-and first LysM cell wall-bound domain of p60.
As non-restrictive example, will be in unique PstI site of the plain pPL2-hlyP-Np60 CodOp (1-77) of insertion of the optimized people's mesothelium of codon plasmid for Listeria monocytogenes albumen optimum expression.Particularly, the plasmid clone total length mesothelium element that contains total length people mesothelium element from embodiment described in 27, or the mesothelium element in disappearance signal peptide and GPI connected structure territory (the plain Δ SP/ of mesothelium Δ GPI), contain and be useful on the best codon of in Listeria monocytogenes, expressing, it is right that use has the heat-stabilised poly synthase and the following primer of proofreading activity:
1. total length
Forward primer (huMeso 3F):
5′-AAACTGCAGGCATTGCCAACTGCACGTCC(SEQ?ID?NO：119)
Reverse primer (huMeso 1935R):
5′-AAACTGCAGAGCTAATGTACTGGCTAATAATAATGCTAAC(SEQ?ID?NO：120)
2. Δ signal peptide, Δ GPI anchor
Forward primer (huMeso 133F):
5′-CGCCTGCAGCGTACATTAGCAGGTGAAACAGG(SEQ?ID?NO：121)
Reverse primer (huMeso 1770R):
5′-CGCCTGCAGGCCTTGTAAACCTAAACCTAATGTATC(SEQ?ID?NO：122)
Pcr amplification of purification 1932bp (total length mesothelium element) and 1637bp (the plain Δ SP/ of mesothelium Δ GPI), with PstI digestion, purification, and be connected in unique PstI site of plasmid pPL2-hlyP-Np60 CodOp (1-77), by using the PstI digestion process, and handle with CIAP.Confirm the N-CO orientation that the plain domain of p60 and mesothelium is consistent by the restriction endonuclease collection of illustrative plates.These plasmids are called the plain and plain Δ SP/ of pPL2-hlyP-Np60 CodOp (the 1-77)-mesothelium Δ GPI of pPL2-hlyP-Np60 CodOp (1-77)-mesothelium, and introduce selected monocyte Listeria monocytogenes strain (as the two deletion mutants of Δ actA Δ inlB), as it is described to be applied in this embodiment that comprises.
The sub-fragment sequence of KpnI-BamHI that has shown plasmid pPL2-hlyP-Np60CodOp (the 1-77)-mesothelium element that contains the hly promoter that connects p60-people's mesothelium fibroin matter chimera encoding gene on the function among Figure 53.
The sub-fragment sequence of KpnI-BamHI that has shown the plain Δ SP/ of plasmid pPL2-hlyP-Np60 CodOp (the 1-77)-mesothelium Δ GIP that contains the hly promoter that connects the plain Δ SP/ of p60 people's mesothelium Δ GPI protein chimera encoding gene on the function among Figure 54.
Chimeric expression of p60-mesothelium fibroin matter and excretory western blot analysis
As it is described to run through embodiment, and the expression of selected heterologous antigen and secretion cause effective initiation of the CD8+T cell effect of MHC I class restriction.By come the expression of test protein chimera by the two deletion mutants of reorganization Listeria monocytogenes Δ actA Δ inlB and the secretion to the culture medium by western blot analysis in the method described in this contained embodiment, the tRNA-Arg chromosome that this mutant contains by pPL2-hlyP-Np60 CodOp (the 1-77)-mesothelium element of method generation described herein or the plain Δ SP/ of pPL2-hlyP-Np60 CodOp (1-77)-mesothelium Δ GIP plasmid inserts, and uses the plain specific polyclonal antibody of mesothelium to test.
Through engineering approaches disappearance (Δ SP Δ GPI shown in the protein h mesothelium element shown in some swimming lanes, be also referred to as Δ SS Δ GPI at this, Δ SP/ Δ GPI, Δ SS/ Δ GPI, Deng) as follows: the signal peptide of disappearance (Δ SP) corresponding to 34 aminoacid of the N-end of h mesothelium element (for the sequence of people's mesothelium element, referring to, for example, Figure 34 or GenBankAcc.No.BC009272).GPI (Δ GPI) domain of disappearance is corresponding to 42 aminoacid of C-end, start from amino acid residue Gly-Ile-Pro and end at amino acid residue Thr-Leu-Ala (referring to, for example, Figure 34).
The protein chimera that the presentation of results of this analysis is made up of the p60 with the plain Δ SP/ of accurate insertion people's mesothelium element or people's mesothelium Δ GPI (inserting in aminoacid 70 frames at p60 between first of N-terminus signal sequence and two LysM cell wall-bound domains) is from Listeria monocytogenes effective expression and the secretion of recombinating.Referring to Figure 55.(Y-axis of Figure 55 has shown the protein molecular weight (representing with kDa) in the ladder that is run in the Far Left swimming lane.Particularly, the swimming lane 1-4 among Figure 55 has proved the chimeric expression of protein and the secretion that contain people's mesothelium element or the plain Δ SP of people's mesothelium Δ/GPI of expectation.The plain Δ SP/ of people's mesothelium Δ GPI is tangible in swimming lane 2 and 4 with respect to the expression of total length mesothelium element and the efficient of excretory raising.In the protein chimera shown in the swimming lane 3 and 4, used the terminal p60 aminoacid of real N-.In the protein chimera that is run in the swimming lane 1 and 2 among Figure 55, aminoacid T on the coding site 29 and 64 respectively and the nucleotide of V have been lacked.Swimming lane 5 has shown expression and the secretion (wherein the plain coded sequence of signal peptide and mesothelium all is that codon is optimized for the expression in Listeria monocytogenes) with the plain Bacillus anthracis PA signal peptide that merges of people's Δ SP/ Δ GPI-mesothelium, and swimming lane 6 has shown expression and the secretion (wherein the plain coded sequence of signal peptide and mesothelium all is that codon is optimized for the expression in Listeria monocytogenes) of the LLO that merges total length people mesothelium element.Swimming lane 8 has shown J293, the human cell line, and to proteic expression, and swimming lane 7 has shown by J293 (" J293/ the total length ") expression and the excretory albumen that contain coding total length people mesothelium quality grain.Swimming lane 10 has shown from the protein expression of the Listera that has lacked endogenous p60 and secretion.Figure below among Figure 55 has shown the excretory western blot analysis of Listeria monocytogenes p60, uses polyclonal α-p60 antibody.The result proves that the excretory protein of the Lm-of equivalent loads on the gel.
The result has proved that p60 can be as secretion heterogenous protein and the molecular chaperones of presenting that promotes HHC I classpath.
Other embodiment
A. use non-Listera signal peptide to express born of the same parents' intracellular domain (ICD) of Eph2A from the bicistronic mRNA construct
Figure 56 has shown the non-Listera of use, and non-secA1 signal sequence is from the western blot analysis of bicistronic mRNA information representation and secretion Eph2A born of the same parents' intracellular domain (ICD).
The protein that Eph2A is made up of ectodomain (ECD) and born of the same parents' intracellular domain (ICD).To express bicistronic mRNA, wherein the bicistronic mRNA coding is as Eph2A ectodomain and born of the same parents' intracellular domain of discrete polypeptide with Listera Δ actA Δ inlB through engineering approaches.(the bacillus subtilis phoD signal peptide of the sequence of all used coded signal sequences in the construct; bacillus anthracis protective antigen signal peptide and lactococcus lactis Usp45 signal peptide) be that codon is optimized for the expression in Listeria monocytogenes.The sequence of coding ECD and ICD domain also is that codon is optimized for the expression in Listeria monocytogenes.From the Listera promoter hly of LLO gene as the promoter in these constructs.
Use will the encode expression cassette of bicistronic mRNA of integration vector pPL2 to be integrated in the genome of Listera.Use the western blot analysis of the various antibacterial fraction of standard technique to be used to detect and measure EphA2 domain in the cumulative born of the same parents.The result has proved and has used born of the same parents' intracellular domain of being expressed and secreting Epha2 by the non-Listera signal peptide of codon optimization sequential coding from the bicistronic mRNA construct.
Expression construct comprises: (1) coding is the be operably connected codon optimization sequence (second polypeptide) (swimming lane 1) of bacillus subtilis phoD secretion signal of EphA2 born of the same parents' intracellular domain of (on the function) codon optimization sequence (first polypeptide) and encoding of connecting the lactococcus lactis Usp45 secretion sequence of EphA2 ectodomain coded sequence operationally; (2) coding be operably connected codon optimization sequence (second polypeptide) (the swimming lane 2-3 (two different clones) of bacillus subtilis phoD secretion sequence of EphA2 born of the same parents' intracellular domain coded sequence of the codon optimization sequence (first polypeptide) and encode of bacillus anthracis protective antigen secretion sequence of EphA2 ectodomain coded sequence that is operably connected; Description referring to this expression cassette structure among the above embodiment 28).Use the comparative study (swimming lane 4) of parent's Listera Δ actA Δ inlB bacterial strain of attenuation to prove that some contrast the detected cross reactivity of variable in traces.Swimming lane 1-3 has shown mobile slowly band and fast mobile band, and wherein fast mobile band is corresponding to born of the same parents' intracellular domain (ICD).In all three antibacterial fraction, observe EphA2 born of the same parents' intracellular domain (swimming lane 1-3) of expressing from all constructs.Swimming lane 4 (contrast) has only shown mobile slowly band.Owing to do not have antibody to obtain for ectodomain, so do not measure the expression/secretion of ectodomain.
B. plain conduct of Mus mesothelium and of expression and the secretion of the terminal function that merges of various codon optimization signal peptide N-based on plasmid
Figure 57 has shown the Mus mesothelium element of expressing from the plain coded sequence of codon optimization mesothelium expression and the secretion based on plasmid, uses various signal peptides, comprises non-Listera signal sequence and non-secA1 signal sequence.Mus mesothelium element based on the expression of plasmid and secretion as and show by the terminal function that merges of the various signal peptide N-of codon optimization sequential coding.In all situations, the sequence of the signal peptide of coding mesothelium plain fusion protein is that codon is optimized, and the plain coded sequence of Mus mesothelium is that codon is optimized for the expression in Listeria monocytogenes.Measured expression and the secretion of Mus mesothelium element from Listeria monocytogenes, wherein Listera has the pAM401 plasmid, and this plasmid-encoded mesothelium element wherein.Test is based on the construct of various plasmids, wherein the signal sequence difference.Carry out western blot analysis, use from selected protein (A) protein that each fraction of cell wall (B) and cell lysate (C) reclaims.For each fraction; swimming lane 1-2 has shown the Mus mesothelium element of conduct with the fusant expression of bacillus anthracis protective antigen signal sequence; swimming lane 3-4 has shown the Mus mesothelium element of conduct with the expression of lactococcus lactis Usp45 signal sequence fusant; swimming lane 5-6 has shown the Mus mesothelium element of conduct with the fusant expression of bacillus subtilis phoD signal sequence; swimming lane 7-8 has shown the Mus mesothelium element of conduct with the fusant expression of p60 signal sequence; swimming lane 9-10 has shown as the Mus mesothelium element and the swimming lane 11 of expressing with the fusant of LLO signal peptide and has shown the expressed proteins by contrast host Listera Δ actA Δ inlB.The result has proved when signal sequence comprises bacillus anthracis protective antigen signal sequence (swimming lane 1-2) and bacillus subtilis phoD signal peptide sequence (swimming lane 5-6), has found the highest expression and secretion.
C. people's mesothelium element is based on chromosomal expression of Listeria monocytogenes and secretion
Figure 58 has shown that people's mesothelium element is based on chromosomal expression of Listeria monocytogenes and excretory western blot analysis in the various bacterial cell fraction (that is, excretory albumen, cell wall and lysate).When merging with non-Listera secA1 and non-secA1 signal peptide, the expression and the secretion of test person mesothelium element.It all is Δ actA Δ inlB Listera and as follows that the Listera of test belongs to antibacterial: Listera Δ actA Δ inlB (not having through engineering approaches to express the contrast Listera of mesothelium element) (swimming lane 1); Coding merges the Listera (swimming lane 2-3) of the bacillus subtilis protective antigen signal sequence of Δ SS/ Δ GPIh mesothelium element; Coding merges the Listera (swimming lane 4-5) of the bacillus subtilis phoD signal peptide of Δ SS/ Δ GPI h mesothelium element; Coding merges the Listera (swimming lane 6-7) of the bacillus anthracis protective antigen signal sequence of total length h mesothelium element; Coding merges the Listera (swimming lane 8-9) of the bacillus subtilis phoD signal sequence of total length h mesothelium element.
The sequence of the signal peptide of coding fusion mesothelium element all is that codon is optimized for the expression in Listeria monocytogenes in all above-mentioned Listeras.In addition, the plain coded sequence of mesothelium (Δ SS/ Δ GPI and total length) is that codon is optimized for the expression in Listeria monocytogenes in each construct.In the Listera of each above-mentioned expression mesothelium element, the plain expression cassette of mesothelium is inserted in the Listera chromosome by integrating with pPL2.
When the plain through engineering approaches of people's mesothelium was lacked its signal sequence and disappearance hydrophobic region (gpi district), use bacillus subtilis phoD secretion sequence had produced high expressed (swimming lane 4-5).
The immunogenicity of embodiment 31. reorganization Listeria monocytogenes vaccines and anti-swollen
Other embodiment of tumor effect
Following examples disclose the result who inoculates Listera of the present invention, for example, the vaccine dependent stimulation of cytokine-expressing, the vaccine dependency with tumor animal is survived, the vaccine dependency of neoplasm metastasis reduces and the vaccine dependency of gross tumor volume reduces.
A. the immunogenicity that comprises the chimeric Listera vaccine of P-60-pattern antigen
Figure 59 A and B have shown that the Listera by expressing p60 antigen chimera or LLO signal peptide antigen coalescence protein is delivered to heterologous antigen in the MHC I classpath.Used heterologous antigen is AH1-A5 in this experiment.Inoculation is to use through through engineering approaches to comprise the Listera of p60 protein chimera expression cassette, this expression cassette coding inserts the AH1-A5 (merging OVA SL8 peptide) (" based on the construct of p60 ") in the p60 peptide sequence that comprises N-end p60 signal peptide sequence, or connect the coding same antigen with coding through through engineering approaches, be embedded in the Listera of LLO signal peptide of the nucleic acid of the AH1-A5 (" based on the construct of LLO ") in the OVA.These two kinds of constructs use Listera promoter hly.P60 is by the excretory Listera Peptidoglycan of secA2 approach autolysin, and LLO is the Listera lysin.
In order to produce the construct based on p60, to contain the PstI cloning site, wherein the PstI cloning site is represented silent mutation, promptly causes coded aminoacid sequence not change with the engineered nucleic acidization of coding p60.The PstI site is in the p60 sequence between first of N-terminus signal sequence and two LysM cell wall-bound domains.Coding comprises in the polynucleotide frame of heterologous polypeptide of AH1-A5 epitope (SPSYAYHQF (SEQ ID NO:73)) and SL8 epitope (SIINFEKL (SEQ ID NO:123)) in the insertion PstI cloning site.The coded sequence of these epitopes is separated by unique XhoI site and is that codon is optimized for the expression in Listeria monocytogenes.The grade that insertion in the PstI site appears at the nucleotide base numbering 199 of p60 exists together.A 1-70 aminoacid of p60 coded sequence is that codon is optimized for the expression in Listeria monocytogenes.Therefore, from being used for expressing 27 aminoacid corresponding to this signal peptide at the best codon of the expression of Listeria monocytogenes.The antigen presentation box further contains 5 ' and 3 ' unique KpnI and SacI site, inserts separately among the MCS of pPL2 plasmid, is used to be adjacent to the genomic tRNA of Listeria monocytogenes
ArgThe site-specific integration of gene.Comprise the be operably connected sequence (not using any codon optimization) of LLO signal sequence of nucleic acid of the AH1-A5 in the coding OVA of coding based on the construct of LLO.Therefore, in this research, signal peptide is from Listera LLO or from Listera p60.
Construct is put into pPL2, and pPL2 is the carrier of the genomic locus specificity reorganization of mediation Listera, and inserts in the Listera genome.
Figure 59 A and B have shown that inoculation (tail vein) expression is had the immunne response of p60 signal sequence/autolysin as the antigenic Listera of the chimeric AH1-A5 of p60, with the immunne response of inoculation being expressed the antigenic Listera of AH1-A5 that is connected with the LLO signal sequence.In the X-axis of figure, " Unstim " meaning is peptide not to be added (that is, cell is not upset) in the hand-hole, and " AH1 " meaning is that the AH1 nonapeptide is added in the hand-hole, and " AH1-A5 " meaning is that the AH1-A5 nonapeptide is added in the hand-hole.With all bacterial vaccine through engineering approaches with the integration nucleic acid that contains the AH1-A5 that encodes (bacterial vaccine do not encode AH1) (referring to, for example, Slansky etc., (2002) Immunity 13:529-538).When use comprises that when inoculating based on the Listera of p60 construct, bacterial strain is expressed as " p60 " on the x of figure axle.When use comprises that when inoculating based on the Listera of the construct of LLO, bacterial strain is expressed as " LLO " on the x of figure axle.
Inoculation is expressed based on the full experiment scheme of the Listera of p60 construct as follows: (1) contains the Listera (tail vein (i.v.)) of integrating nucleic acid to mouse inoculation, the nucleic acid coding p60 of Zheng Heing wherein, it contains the nucleic acid of the AH1-A5 that the nucleotide 199 that is coded in p60 inserts.In other words, will encode and connect and be operably connected p60 signal sequence and p60 autolysin in the antigenic nucleic acid frame of AH1-A5.The nucleic acid of coding AH1-A5 is that codon is optimized for the expression in Listeria monocytogenes; (2) infected back seven days, take out spleen; (3) separating Morr. cell is put into the hole, and at the peptide (Figure 59 A and 59B) that does not have to add, and adds AH1 (Figure 59 A) or adds under the situation of AH1-A5 (Figure 59 B), hatches splenocyte, as shown on the x axle; (4) behind the adding peptide, cell was hatched five hours, then test the percentage ratio of the CD8+T cell of expressing IFN γ by facs analysis.Similarly experimental program is used to inoculate the Listera of expression based on the construct of LLO.
The result has proved Listera boosting vaccine CD8+T cellular expression IFN γ, wherein the peptide of Jia Ruing is that AH1 (Figure 59 A) or the peptide that wherein adds are AH1-A5 (Figure 59 B).When the AH1-A5 that integrates is operably connected the LLO signal sequence, stimulates slightly height, and when the AH1-A5 that integrates is operably connected the p60 signal sequence, stimulate lower slightly (Figure 59 A and B).
Figure 60 A and B have shown with aforesaid, i.e. the experiment of carrying out as the identical Listera vaccine with two kinds as shown in the B of Figure 59 A.Figure 60 A shown give mouse inoculation through through engineering approaches to contain Listera (" p60 ") based on the construct of p60 or inoculation through the result of through engineering approaches when containing Listera (" LLO ") based on the construct of LLO.As shown on the x axle of Figure 60 A, replenishing peptide based on the test of cell (does not stimulate; " unstim ") or replenished LLO
91-99Peptide (" LLO91 "; Badovinac and Harty (2000) J.Imunol.164:64444-6452).The result has shown similar immunne response (IFN γ expression), and wherein the Listera vaccine contains based on the construct of p60 or based on the construct of LLO.The immunne response of the stimulation among Figure 60 A, as based on cell tests the result reflected, be owing to the endogenous expression of the Listera of natural LLO causes.
Figure 60 B has shown and gives mouse inoculation through the result of through engineering approaches when containing the Listera based on the construct of p60, wherein hly promoter and signal peptide sequence be operably connected the coding AH1-A5 nucleic acid, or inoculation is through the result of through engineering approaches when containing the Listera based on the construct of LLO, wherein hly promoter and the signal peptide nucleic acid of coding AH1-A5 that is operably connected.The peptide that adds does not have peptide (not stimulate; " unstim ") or p60
217-225(" p60-217 "; Sijts etc. (1997) J.Biol.Chem.272:19261-19268), as shown on the x axle.The immunne response of the stimulation among Figure 60 B, as based on cell tests the result reflected, for the construct based on LLO is that expression owing to the Listera of endogenous p60 causes, causes in conjunction with endogenous p60 and expressed p60 albumen chimera sequence for the construct based on p60.
B. the therapeutic efficiency of the Listera of expressing human mesothelium element
Result shown in Figure 61 has shown that the Listera of inoculation expressing human mesothelium element (hu mesothelium element) has prolonged the survival that has mice with tumor, wherein comes expressing human mesothelium element with the tumor cell through engineering approaches in the mice.Tumor cell is the CT26 cell of expressing human mesothelium element, and mice is the Balb/c mice.(all CT26 tumor researches described herein all relate to the Balb/c mice.) in a kind of expression cassette, the sequence of the non-Listera signal sequence of coding is operationally connected the codon optimization sequence (having lacked its signal sequence and GPI anchor) of coding people mesothelium element in the frame.Fusion rotein to the tumor immune response Research on effect in, the expression cassette of the signal peptide that merges with people's mesothelium element (Δ GPI Δ SS) of will encoding has the mice of tumor in the Listera vaccine.The expression cassette of coding mesothelium plain fusion protein has been integrated in the Listera chromosome.At the 0th day, with 2 * 10
5CT26 cell (CT.26huMeso+) intravenous injection of individual expressing human mesothelium element is to the Balb/c mice.Give mouse inoculation at tail vein (i.v.).At the 3rd day inoculation 1e7 colony-forming units (CFU) Listera (i.v.).
Figure 61 has shown the percentage ratio survival (be shown in y axle on) of mice to the CT26 tumor of expressing human mesothelium element, and wherein vaccine comprises Hank ' s balanced salt solution (HBSS) (sham vaccine; " HBSS "); Express Listera Δ actA Δ inlB (the positive control vaccine of SF-AH1A5 from the expression cassette of integrating; " SF-AH1A5 "); Or comprise the Listera Δ actA Δ inlB of the expression cassette of the bacillus anthracis protective antigen signal sequence (by the sequential coding of non-codon optimization) that coding and hu mesothelium element (by the sequential coding of codon optimization) merge, wherein hu mesothelium element has lacked signal sequence and has lacked the zone (" BaPA-huMeso Δ gpi Δ ss ") of the hydrophobic gpi-anchoring peptide of encoding.The Listera that has SF-AH1A5 construct and BaPA-huMeso Δ gpi Δ ss construct contains the construct of these constructs as chromosomal integration.Used will the encode nucleic acid molecules of SF-AH1A5 and the nucleic acid molecules of coding BaPA-huMeso Δ gpi Δ ss construct of pPL2 to be integrated in the Listera genome.SF is the writing a Chinese character in simplified form of 8 amino acid whose peptides that is derived from ovalbumin, is also referred to as SL8 (referring to, for example, Shastri and Ganzalez. (1993) J.Immunol.150:2724-2736).Abbreviation " SF-AH1A5 ", " SF-AH1-A5 " refers to the AH1-A5 that is connected the ovalbumin support with " OVA/AH1-A5 "." SF AH1-A5 " refers to AH1-A5 (SPSYAYHQF (SEQ ID NO:73)) and merges the SF peptide of N-terminal of the amino acid/11 38 to 386 of GenBank accession number No.P01012 (ovalbumin).Among this embodiment, the polynucleotide of coding " SF-AH1A5 " comprise the codon optimization nucleic acid of the AH1-A5 that encodes and the coding source non-codon optimization nucleic acid from the ovalbumin sequence.
The result has proved that the single immunization with the Listera of expressing hu mesothelium element has prolonged the survival that has the mice of expressing the plain tumor of hu mesothelium.Use the bacillus anthracis protective antigen signal sequence of chromosomal integration to merge Δ signal sequence/Δ gpi hu mesothelium element (the plain Δ gpi of BaPA-hu mesothelium Δ ss; Closed square), survival percentage ratio is the highest.When using contrast saline solution " inoculation ", it is minimum to survive.
C. inoculated the mice that has tumor of the Listera of expressing human mesothelium element and saved the knot level because the plain specific antitumor efficacy of mesothelium has reduced lung tumor
Digital proof among Figure 62 the Listera Δ actA Δ inlB by inoculation expressing human mesothelium element reduced the level that lung tumor is cut knot, wherein the tumor cell through engineering approaches is come expressing human mesothelium element.Mouse species is Balb/c, and lung tumor cell is the CT26 cell that has the carrier of expressing human mesothelium element.At the 0th day, with 2 * 10
5The CT26 cell intravenous of individual expressing human mesothelium element gives Balb/c mice.The codon optimization sequence that the sequence of the various signal sequences of coding is operationally connected coding people mesothelium element in expression cassette in the frame.The mice that the coding and the expression cassette of the various signal peptides of people mesotehlin fusion is had tumor by the Listera vaccine that contains expression cassette.At the 3rd day, with 1 * 10
7The Listera vaccine intravenous of CFU/100 μ L has the mice of tumor.HBSS or Listera Δ actA Δ inlB are used in the negative control inoculation.The Listera of expressing the OVR fusion rotein (connecting the OVA sequence in the frame) that comprises AH1A5 is used in the positive control inoculation.(the OVA fusion rotein that comprises AH1A5 is by the optimized expression cassette coding of non-codon.) at the 19th day, mice is put to death, collect lung, and counting lung tumor joint knot.
The Listera vaccine has reduced the number of metastatic tumor in the lung.The control vaccine that only relates to HBBS or Listera Δ actA Δ inlB causes 250 metastatic tumors of each lung and average 135 metastatic tumors of each lung of detected unanimity separately.The Listera that has the plasmid (pAM401) (" pAM-LLO-HuMeso) of coding and the plain LLO signal peptide that merges of people's mesothelium demonstrates about 25 metastatic tumors of each lung.The polynucleotide sequence of the pAM-LLO-HuMeso plasmid of the plain sequence of coding LLO signal peptide and people's mesothelium is that codon is optimized for the expression in Listeria monocytogenes.The Listera that has the integration sequence that coding bacillus anthracis protective antigen signal sequence (BaPA) merges hu mesothelium element (Δ gpi/ Δ signal sequence) (" BaPA-HuMeso Δ gpi Δ ss) also demonstrates on average about 25 metastatic tumors of each lung.The polynucleotide of coding bacillus anthracis protective antigen signal sequence are not that codon is optimized among the BaPA-HuMeso Δ gpi Δ ss, and coding to have lacked the polynucleotide of the plain sequence of people's mesothelium of plain signal peptide of mesothelium and GPI anchor be that codon is optimized for the expression in Listeria monocytogenes.
Figure 63 has shown the result of comparative study, uses the mice that comprises lung tumor joint knot, uses CT.26 parent target cell to produce lung tumor joint knot.Use the Balb/c mice, but substitute injection wtCT26 (at the 0th day 2 * 10
5Individual cell (i.v.).Studies have shown that it is that the mesothelium element is specific that the antitumor efficacy of the Listera vaccine of mesothelium plain fusion protein is expressed in inoculation.The codon optimization sequence that the sequence of the various signal sequences of coding is operationally connected coding people mesothelium element in expression cassette in the frame.(in this experiment in used construct and the above experiment used those of data shown in Figure 62 of producing identical.) expression cassette that will encode with the plain various signal peptides that merge of people's mesothelium has the mice of tumor by comprising the Listera vaccine of expression cassette.At the tail intravenous inoculation (the 3rd day i.v.1 * 10
7CFU/100 μ L).In this particular studies, tumor cell does not have expressing human mesothelium element.Measure survival.When data can obtain, also measured the number of pulmonary metastases.Each inoculation group always has five mices.The negative control inoculation relates to HBS S or Listera Δ actA Δ inlB.The positive control inoculation relates to the Listera of expressing the OVA fusion rotein that comprises AH1A5 (non-codon is optimized).
Shown the result among Figure 63.The fork expression fails to survive, and each inoculation group comprises 5 mices.The inoculation of use positive control, the mice survival, detected metastatic tumor number is about 25 of average each lung in the lung.When the tumor cell through engineering approaches not being come the expressing human mesothelium plain, inoculated the not survival of mice of the Listera that has the plasmid (" pAM-LLO-HuMeso ") of expressing the LLO signal peptide that merges with people's mesothelium element.When have a chromosomal integration to mouse inoculation with the plain bacillus anthracis protective antigen secretion sequence (BaPA that merges (Δ gpi/ Δ signal sequence) of people's mesothelium; nucleotide sequence coded by non-codon optimization) Listera (" BaPA-HuMeso Δ gpi Δ ss) time; some survivals, and other not survivals.
D. the Listera of inoculation expression codon optimization people mesothelium element has reduced gross tumor volume
Figure 64 has shown that the Listera (Δ actA Δ inlB) that inoculation is expressed from people's mesothelium element of the expression cassette that comprises the plain codon sequence of codon optimization mesothelium has reduced gross tumor volume.
The codon optimization sequence that the sequence of the various signal sequences of coding is operationally connected coding people mesothelium element in expression cassette in the frame.The mice that the coding and the expression cassette of the plain various signal peptides that merge of people's mesothelium is had tumor by the Listera vaccine that contains expression cassette.The Listera vaccine that is used for inoculating the expressing human mesothelium element that has mice with tumor in this research comprises following: the Listera (Δ actA Δ inlB Listeria monocytogenes) (" pAMopt.LLO-opt.huMeso ") that has the pAM401 plasmid of the plain LLO signal peptide (by for the optimized sequential coding of expression codon at Listeria monocytogenes) that merges of expression and secretion and people's mesothelium; Have and express and the Listera of the pAM401 plasmid of the plain bacillus anthracis protective antigen signal sequence that merges of hu mesothelium (by non-codon optimization expression cassette coding) (" the non-opt.BaPA-opt.huMeso of pAM); With the Listera of the integration expression box that comprises the bacillus anthracis protective antigen signal peptide (by the sequential coding of non-codon optimization) that coding and hu mesothelium element merge, wherein hu mesothelium element has lacked signal sequence and the zone (" Non-opt.BaPA-opt.huMesodelgpi-ss ") that has lacked the hydrophobic gpi-anchoring peptide of encoding.
In this research, give the subcutaneous implantation 2 * 10 of Balb/c mice
5The individual CT26 Mus colon tumor cell (the 0th day) that comes expressing human mesothelium element through through engineering approaches.Each inoculation group comprises five mices.The non-Listera contrast of inoculation or 1 * 10 in the mouse vein is given in behind injection CT26 cell the 3rd day
7The Listera vaccine of colony-forming units (CFU).The negative control inoculation relates to HBSS.The positive control inoculation relates to (codon the is optimized) Listera of expressing SF-AH1A5.(SF is eight amino acid whose peptides that are derived from ovalbumin, be also referred to as SL8 (referring to, for example, Shastri and Ganzalez (1993) J.Immunol.150:2724-2736.) at different time points, measure mean tumour volume.
The result who has shown this research among Figure 64.The result has proved that inoculation is expressed and the Listera of people's mesothelium element that various signal peptides merge has reduced gross tumor volume.The Listera that the bacillus anthracis protective antigen signal peptide that merges with people's mesothelium element is expressed in inoculation is protectiveness (open circles of dotted line).The Listera that plasmid-encoded people's mesothelium element of coding and the fusion of LLO signal peptide is expressed in inoculation is protectiveness (hollow triangle).Inoculation comprises that the Listera of the chromosomal integration expression cassette of bacillus anthracis protective antigen (the non-codon optimization nucleic acid) signal peptide that coding and people's mesothelium element (Δ gpi/ Δ signal peptide sequence) merge also is (the solid line hollow ellipse) of protectiveness.About positive control, the Listera (hollow square) of expressing the SF-AH1A5 of chromosomal integration also is a protectiveness.The earliest time that maximum gross tumor volume and tumor growth begin appears in the mice of accepting sham vaccine (HBSS).
E. express the immunogenicity of the Listera vaccine of the people's mesothelium element that merges with non-Listera signal sequence
Figure 65 has described the immunogenicity of the plain bacterial strain of Listera Δ actA Δ inlB-people mesothelium, and wherein Listera contains the chromosomal integration nucleic acid of people's mesothelium element (optimized Ba PA hMeso Δ GPI Δ SS) of coding and the fusion of Bacillus anthracis signal peptide.The ELISPOT test is used to test immunne response, and the expression of wherein testing interferon gamma is responsive.
This research may further comprise the steps: (1) gives mice (Balb/c mice or C57BL/6 mice) inoculation (i.v.) Listera, this Listera comprises the integration expression box of the bacillus anthracis protective antigen peptide (by the sequential coding of non-codon optimization) that coding and hu mesothelium element (by the sequential coding of codon optimization, wherein plain signal sequence of mesothelium and hydrophobic gpi-anchor series lack) merge; After (2) 7 days, take out spleen; (3) cell distribution that will take out from spleen is in the hole.About 200,000 splenocytes are accepted in each hole; (4) with a kind of the adding in the hand-hole in three kinds of culture medium, as directed.(" ") that Ci Jis not, the plain peptide of mesothelium is gathered (" Meso set "), or p60 to accept independent culture medium with the splenocyte of Balb/c mice study
217-225(" p60
217").Accept independent culture medium (" not stimulating "), the plain peptide set of mesothelium (" Meso set "), or LLO with the splenocyte of C57BL/6 mice study
296-304(" LLO
296-304"); (5) carry out ELISPOT and test the quantity of measuring the immunocyte of peptide that response adds.The plain peptide set of mesothelium comprises 153 different peptides, and wherein these peptides are crossed over the whole sequence of h mesothelium element, and wherein each peptide is 15 amino acid longs, and adjacent peptide has 11 aminoacid overlapping.
The result who has shown the ELISPOT test among Figure 65.This result shows that the Listera vaccine of expressing the people's mesothelium element that merges with the Bacillus anthracis signal peptide can induce the immunne response of Balb/c mice to the mesothelium element.Observe the immune height of the immune system of Balb/c mice to the IFN γ response ratio C57BL/6 mice of the h mesothelium element of Listera expression.P60 and LLO albumen to the natural generation of the ELISPOT signal response Listera of p60 or LLO.
At these all publications of quoting, patent, patent application, internet sites and accession number/database sequence (comprising polynucleotide and peptide sequence) are incorporated herein by reference with its integral body at this and are used for all purposes, with each independent publication, patent, patent application, internet sites or accession number/database sequence particularly with individually shown in the degree that is incorporated herein by reference identical.
Claims (88)
1. recombinant nucleic acid molecules comprises:
(a) first polynucleotide of coding antibacterial natural signals peptide, wherein first polynucleotide are that codon is optimized for the expression in antibacterial; With
(b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide, and wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
2. the recombinant nucleic acid molecules of claim 1, wherein antibacterial is that Listera belongs to the antibacterial of (Listeria).
3. the recombinant nucleic acid molecules of claim 1, wherein the polypeptide of second polynucleotide encoding comprises antigen, this antigen is selected from tumor associated antigen, is derived from the polypeptide of tumor associated antigen, infectious disease antigen and be derived from the antigenic polypeptide of infectious disease.
4. the recombinant nucleic acid molecules of claim 1, wherein the polypeptide of second polynucleotide encoding and signal peptide are allogenic, are external sources to antibacterial, or both.
5. the recombinant nucleic acid molecules of claim 1, wherein signal peptide is from the LLO signal peptide of Listeria monocytogenes (Listeria monocytogenes) or from the p60 signal peptide of Listeria monocytogenes.
6. the expression cassette that comprises the recombinant nucleic acid molecules of claim 1 further comprises the promoter of first and second polynucleotide of the recombinant nucleic acid molecules that is operably connected.
7. the recombinant bacteria that comprises the recombinant nucleic acid molecules of claim 1, wherein first polynucleotide are that codon is optimized for the expression in recombinant bacteria.
8. induce the method for host to antigenic immunne response, comprise that the compositions with the recombinant bacteria that comprises claim 7 of effective dose gives the host, wherein the polypeptide of second polynucleotide encoding comprises antigen.
9. the reorganization Listera that comprises recombinant nucleic acid molecules belongs to antibacterial, and wherein recombinant nucleic acid molecules comprises:
(a) first polynucleotide of coded signal peptide, wherein first polynucleotide are that codon is optimized for the expression that belongs at Listera in the antibacterial; With
(b) second of coded polypeptide polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide,
Wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
10. the reorganization Listera of claim 9 belongs to antibacterial, and it comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein expression cassette further comprises the promoter of first and second polynucleotide of the recombinant nucleic acid molecules that is operably connected.
11. the reorganization Listera of claim 9 belongs to antibacterial, wherein Listera belongs to antibacterial and belongs to the Listeria monocytogenes kind.
12. the reorganization Listera of claim 9 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises antigen, and this antigen is selected from tumor associated antigen, is derived from the polypeptide of tumor associated antigen, infectious disease antigen and be derived from the antigenic polypeptide of infectious disease.
13. the reorganization Listera of claim 12 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises and is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP and CEA, or comprise being derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigenic polypeptide of TARP and CEA.
14. the reorganization Listera of claim 13 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises the mesothelium element, or its antigen fragment or antigenic variant, or comprises NY-ESO-1, or its antigen fragment or antigenic variant.
15. the reorganization Listera of claim 14 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises people's mesothelium element of its signal peptide and GPI junctional complex domain disappearance.
16. the reorganization Listera of claim 9 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding and signal peptide are allogenic.
17. the reorganization Listera of claim 9 belongs to antibacterial, wherein Listera is belonged to antibacterial is external source to signal peptide.
18. the reorganization Listera of claim 9 belongs to antibacterial, wherein Listera is belonged to antibacterial is natural to signal peptide.
19. the reorganization Listera of claim 9 belongs to antibacterial; wherein signal peptide is the LLO signal peptide that is selected from Listeria monocytogenes; the Usp45 signal peptide of lactococcus lactis (Lactococcus lactis); the protective antigen signal peptide of Bacillus anthracis (Bacillus anthracis); the p60 signal peptide of Listeria monocytogenes, and the signal peptide of the PhoD signal peptide of bacillus subtilis (B.subtilis).
20. the reorganization Listera of claim 9 belongs to antibacterial, it is for cell and intercellular diffusion, enters non-phagocytic cell or propagation weakens.
21. the reorganization Listera of claim 9 belongs to antibacterial, it lacks ActA, InternalinB, or ActA and Internalin B.
22. the reorganization Listera of claim 9 belongs to antibacterial, wherein by reacting the nucleic acid of having modified recombinant bacteria with the nucleic acid target compound.
Belong to immunogenic composition or the vaccine of antibacterial 23. comprise the reorganization Listera of claim 9.
24. induce the method for host to antigenic immunne response, comprise that the compositions that the reorganization Listera that comprises claim 9 with effective dose belongs to antibacterial gives the host, wherein the polypeptide of second polynucleotide encoding comprises antigen.
25. the method for prevention or treatment host disease comprises that the compositions that the reorganization Listera that comprises claim 9 with effective dose belongs to antibacterial gives the host.
26. recombinant nucleic acid molecules comprises:
(a) first polynucleotide of the non-secA1 antibacterial signal peptide of coding; With
(b) coding and second polynucleotide of the allogenic polypeptide of signal peptide, wherein second polynucleotide be arranged in the translation frame identical with first polynucleotide and
Wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
27. the recombinant nucleic acid molecules of claim 26, first polynucleotide wherein, second polynucleotide, or first and second polynucleotide are that codon is optimized for the expression in antibacterial.
28. the recombinant nucleic acid molecules of claim 26, wherein signal peptide is the Listera signal peptide.
29. the recombinant nucleic acid molecules of claim 26, wherein signal peptide is secA2 signal peptide or Tat signal peptide.
30. the recombinant nucleic acid molecules of claim 26, wherein signal peptide is the p60 signal peptide of Listeria monocytogenes or the phoD signal peptide of bacillus subtilis.
31. the recombinant nucleic acid molecules of claim 26, wherein the polypeptide of second polynucleotide encoding comprises antigen, and this antigen is selected from tumor associated antigen, is derived from the polypeptide of tumor associated antigen, infectious disease antigen and be derived from the antigenic polypeptide of infectious disease.
32. comprise the expression cassette of the recombinant nucleic acid molecules of claim 26, further comprise the promoter of first and second polynucleotide of the recombinant nucleic acid molecules that is operably connected.
33. comprise the recombinant bacteria of the recombinant nucleic acid molecules of claim 26.
34. the recombinant bacteria of claim 33, it is the antibacterial that Listera belongs to.
35. induce the method for host to antigenic immunne response, comprise that the compositions with the recombinant bacteria that comprises claim 33 of effective dose gives the host, wherein the polypeptide of second polynucleotide encoding comprises antigen.
Belong to antibacterial 36. comprise the reorganization Listera of recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises:
(a) first polynucleotide of the non-secA1 antibacterial signal peptide of coding; With
(b) coding is allogenic or be the polypeptide of external source or both second polynucleotide to antibacterial with signal peptide, and wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide;
Wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
37. the reorganization Listera of claim 36 belongs to antibacterial, it comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein expression cassette further comprises the promoter of first and second polynucleotide of the recombinant nucleic acid molecules that is operably connected.
38. the reorganization Listera of claim 36 belongs to antibacterial, first polynucleotide wherein, and second polynucleotide, or first and second polynucleotide are that codon is optimized for the expression that belongs at Listera in the antibacterial.
39. the reorganization Listera of claim 36 belongs to antibacterial, wherein antibacterial belongs to the Listeria monocytogenes kind.
40. the reorganization Listera of claim 36 belongs to antibacterial, wherein non-secA1 signal peptide is non-Listera signal peptide.
41. the reorganization Listera of claim 36 belongs to antibacterial, wherein non-secA1 signal peptide is the Listera signal peptide.
42. the reorganization Listera of claim 36 belongs to antibacterial, wherein signal peptide is the secA2 signal peptide.
43. the reorganization Listera of claim 42 belongs to antibacterial, wherein recombinant nucleic acid molecules further comprises:
(c) be arranged in coding sevA2 autolysin or its segmental the 3rd polynucleotide of the translation frame identical with first and second polynucleotide, wherein second polynucleotide are in the 3rd polynucleotide or between first and the 3rd polynucleotide.
44. the reorganization Listera of claim 36 belongs to antibacterial, wherein signal peptide is the Tat signal peptide.
45. the reorganization Listera of claim 36 belongs to antibacterial, wherein signal peptide is the PhoD signal peptide of bacillus subtilis or the p60 signal peptide of Listeria monocytogenes.
46. the reorganization Listera of claim 36 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises antigen, and this antigen is selected from tumor associated antigen, is derived from the polypeptide of tumor associated antigen, infectious disease antigen and be derived from the antigenic polypeptide of infectious disease.
47. the reorganization Listera of claim 46 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises and is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP and CEA, or comprise being derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigenic polypeptide of TARP and CEA.
48. the reorganization Listera of claim 47 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises the mesothelium element, or its antigen fragment or antigenic variant.
49. the reorganization Listera of claim 48 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises people's mesothelium element of its signal peptide and GPI anchor disappearance.
50. the reorganization Listera of claim 36 belongs to antibacterial, it is for cell and intercellular diffusion, enters non-phagocytic cell or propagation weakens.
51. the reorganization Listera of claim 36 belongs to antibacterial, it lacks ActA, Internalin B, or ActA and Internalin B.
52. the reorganization Listera of claim 36 belongs to antibacterial, wherein by reacting the nucleic acid of having modified recombinant bacteria with the nucleic acid target compound.
Belong to immunogenic composition or the vaccine of antibacterial 53. comprise the reorganization Listera of claim 36.
54. induce the method for host to antigenic immunne response, comprise that the compositions that the reorganization Listera that contains claim 36 with effective dose belongs to antibacterial gives the host, wherein the polypeptide of second polynucleotide encoding comprises antigen.
55. the method for prevention or treatment host disease comprises that the compositions that the reorganization Listera that contains claim 36 with effective dose belongs to antibacterial gives the host.
Belong to antibacterial 56. comprise the reorganization Listera of recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises:
(a) first polynucleotide of the signal peptide of the non-Listera of coding; With
(b) be arranged in second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide,
Wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.
57. the reorganization Listera of claim 56 belongs to antibacterial, it comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein expression cassette further comprises the promoter of first and second polynucleotide of the recombinant nucleic acid molecules that is operably connected.
58. the reorganization Listera of claim 56 belongs to antibacterial, first polynucleotide wherein, and second polynucleotide, or first and second polynucleotide are that codon is optimized for the expression that belongs at Listera in the antibacterial.
59. the reorganization Listera of claim 56 belongs to antibacterial, wherein antibacterial is a Listeria monocytogenes.
60. the reorganization Listera of claim 56 belongs to antibacterial, wherein signal peptide is an antibacterial.
61. the reorganization Listera of claim 60 belongs to antibacterial, wherein signal peptide is derived from gram-positive bacterium.
62. the reorganization Listera of claim 61 belongs to antibacterial, wherein signal peptide is derived from and belongs to bacillus (Bacillus), the antibacterial of staphylococcus (Staphylococcus) or Lactococcus (Lactococcus).
63. the reorganization Listera of claim 62 belongs to antibacterial, wherein signal peptide is the Usp45 signal peptide that is selected from lactococcus lactis, the signal peptide of protective antigen signal peptide of Bacillus anthracis and the PhoD signal peptide of bacillus subtilis.
64. the reorganization Listera of claim 56 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises antigen, and this antigen is selected from tumor associated antigen, is derived from the polypeptide of tumor associated antigen, infectious disease antigen and be derived from the antigenic polypeptide of infectious disease.
65. the reorganization Listera of claim 64 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises and is selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigen of TARP and CEA, or comprise being derived from and be selected from K-Ras, H-Ras, N-Ras, 12-K-Ras, the mesothelium element, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigenic polypeptide of TARP and CEA.
66. the reorganization Listera of claim 65 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises the mesothelium element, or its antigen fragment or antigenic variant.
67. the reorganization Listera of claim 66 belongs to antibacterial, wherein the polypeptide of second polynucleotide encoding comprises people's mesothelium element of its signal peptide and GPI anchor disappearance.
68. the reorganization Listera of claim 56 belongs to antibacterial, it is for cell and intercellular diffusion, enters non-phagocytic cell or propagation weakens.
69. the reorganization Listera of claim 56 belongs to antibacterial, it lacks ActA, Internalin B, or ActA and Internalin B.
70. the reorganization Listera of claim 56 belongs to antibacterial, wherein by reacting the nucleic acid of having modified recombinant bacteria with the nucleic acid target compound.
Belong to immunogenic composition or the vaccine of antibacterial 71. comprise the reorganization Listera of claim 56.
72. induce the method for host to antigenic immunne response, comprise that the compositions that the reorganization Listera that contains claim 56 with effective dose belongs to antibacterial gives the host, wherein the polypeptide of second polynucleotide encoding comprises antigen.
73. the method for prevention or treatment host disease comprises that the compositions that the reorganization Listera that contains claim 56 with effective dose belongs to antibacterial gives the host.
74. recombinant nucleic acid molecules comprises:
(a) first polynucleotide of coding antibacterial autolysin or its catalytic activity fragment or catalytic activity variant; With
(b) second polynucleotide of coding and the allogenic polypeptide of antibacterial autolysin, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide,
Wherein the recombinant nucleic acid molecules coding comprises the polypeptide of second polynucleotide encoding and the protein chimera of autolysin or its catalytic activity fragment or catalytic activity variant, wherein in the protein chimera, polypeptide and autolysin or its catalytic activity fragment or catalytic activity variant merge, or are positioned at autolysin or its catalytic activity fragment or catalytic activity variant.
75. comprise the recombinant bacteria of the recombinant nucleic acid molecules of claim 74.
76. induce the method for host to antigenic immunne response, comprise that the compositions with the recombinant bacteria that contains claim 75 of effective dose gives the host, wherein the polypeptide of second polynucleotide encoding comprises antigen.
Belong to antibacterial 77. comprise the reorganization Listera of polycistronic expression box, wherein at least two discrete non-Listera polypeptide of polycistronic expression box coding.
78. induce the method for host to antigenic immunne response, comprise that the compositions that the reorganization Listera that contains claim 77 with effective dose belongs to antibacterial gives the patient, wherein at least one in the non-Listera polypeptide of polycistronic expression box coding comprises antigen.
Belong to antibacterial 79. comprise the reorganization Listera of recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises that coding belongs to the polynucleotide that antibacterial is the polypeptide of external source to Listera, and wherein polynucleotide are that codon is optimized for the expression in Listera.
80. induce the method for host to antigenic immunne response, comprise that the compositions that the reorganization Listera that contains claim 79 with effective dose belongs to antibacterial gives the host, wherein allogenic polypeptide comprises antigen.
81. recombinant nucleic acid molecules comprises:
(a) first polynucleotide of coded signal peptide;
(b) coding secretory protein or its segmental second polynucleotide, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; With
(c) the 3rd polynucleotide of coding and secretory protein or the allogenic polypeptide of its fragment, wherein the 3rd polynucleotide are arranged in and first and second translation frame that polynucleotide are identical,
Wherein the recombinant nucleic acid molecules coding comprises polypeptide and secretory protein or its segmental protein chimera of signal peptide, the 3rd polynucleotide encoding, and wherein in the protein chimera, the polypeptide of the 3rd polynucleotide encoding and secretory protein or its fragment merge, or are positioned at secretory protein or its fragment.
82. comprise the recombinant bacteria of the recombinant nucleic acid molecules of claim 81.
83. induce the method for host to antigenic immunne response, comprise that the compositions with the recombinant bacteria that contains claim 82 of effective dose gives the host, wherein the polypeptide of the 3rd polynucleotide encoding comprises antigen.
84. claim 7,9,33,36,56 or 75 recombinant bacteria induces the host to the purposes in the medicine of antigenic immunne response in manufacturing, and wherein the polypeptide of second polynucleotide encoding comprises antigen.
Induce the host to the purposes in the medicine of antigenic immunne response 85. the reorganization Listera of claim 77 belongs to antibacterial in manufacturing, wherein the polycistronic expression box encode in the non-Listera polypeptide at least one comprise antigen.
Induce the host to the purposes in the medicine of antigenic immunne response 86. the reorganization Listera of claim 79 belongs to antibacterial in manufacturing, wherein allogenic polypeptide comprises antigen.
87. the recombinant bacteria of claim 82 induces the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide of the 3rd polynucleotide encoding comprises antigen.
88. claim 7, the purposes of 9,33,36,56,75,77,79 or 82 recombinant bacteria in the medicine of making prevention or treatment host disease.
Applications Claiming Priority (21)
| Application Number | Priority Date | Filing Date | Title |
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| US53259803P | 2003-12-24 | 2003-12-24 | |
| US60/532,598 | 2003-12-24 | ||
| US54151504P | 2004-02-02 | 2004-02-02 | |
| US60/541,515 | 2004-02-02 | ||
| US10/773,792 | 2004-02-06 | ||
| US10/773,792 US7691393B2 (en) | 2003-02-06 | 2004-02-06 | Listeria attenuated for entry into non-phagocytic cells, vaccines comprising the Listeria, and methods of use thereof |
| US10/773,618 US7833775B2 (en) | 2003-02-06 | 2004-02-06 | Modified free-living microbes, vaccine compositions and methods of use thereof |
| US10/773,618 | 2004-02-06 | ||
| US55674404P | 2004-03-26 | 2004-03-26 | |
| US60/556,744 | 2004-03-26 | ||
| US10/883,599 US7695725B2 (en) | 2003-02-06 | 2004-06-30 | Modified free-living microbes, vaccine compositions and methods of use thereof |
| US10/883,599 | 2004-06-30 | ||
| PCT/US2004/023881 WO2005009463A2 (en) | 2003-07-24 | 2004-07-23 | Antigen-presenting cell vaccines and methods of use thereof |
| USPCT/US2004/023881 | 2004-07-23 | ||
| US59937704P | 2004-08-05 | 2004-08-05 | |
| US60/599,377 | 2004-08-05 | ||
| US61528704P | 2004-10-01 | 2004-10-01 | |
| US60/615,287 | 2004-10-01 | ||
| US61675004P | 2004-10-06 | 2004-10-06 | |
| US60/616,750 | 2004-10-06 | ||
| PCT/US2004/044080 WO2005071088A2 (en) | 2003-12-24 | 2004-12-23 | Recombinant nucleic acid molecules encoding fusion proteins comprising antigens and bacterial secretory signal polypeptides, expression cassettes, and bacteria, and methods of use thereof |
Publications (2)
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| CN1921884A true CN1921884A (en) | 2007-02-28 |
| CN1921884B CN1921884B (en) | 2012-02-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200480042020XA Expired - Fee Related CN1921884B (en) | 2003-12-24 | 2004-12-23 | Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof |
Country Status (4)
| Country | Link |
|---|---|
| JP (2) | JP5532280B2 (en) |
| CN (1) | CN1921884B (en) |
| HK (1) | HK1098673A1 (en) |
| TW (1) | TW200530399A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102766601A (en) * | 2011-11-17 | 2012-11-07 | 中山大学肿瘤防治中心 | Cell line containing inducible cancer gene, establishing method and application thereof |
| CN103547676A (en) * | 2011-03-22 | 2014-01-29 | 缪科西斯私人有限公司 | Immunogenic compositions in particulate form and methods for producing the same |
| CN105039381A (en) * | 2015-07-21 | 2015-11-11 | 齐鲁工业大学 | Maltose inducible trehalose synthase synthesis engineering bacterium, method for preparing same and application |
| CN107229842A (en) * | 2017-06-02 | 2017-10-03 | 肖传乐 | A kind of three generations's sequencing sequence bearing calibration based on Local map |
| CN115894639A (en) * | 2022-11-03 | 2023-04-04 | 大连医诺生物股份有限公司 | Auxiliary infection protein residue mutant protein, recombinant thereof and application thereof in exoenzyme immobilization process |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3763383A1 (en) | 2013-12-16 | 2021-01-13 | Massachusetts Institute Of Technology | Micromolded or 3-d printed pulsatile release vaccine formulations |
| EP3037530B1 (en) * | 2014-12-22 | 2017-02-01 | Sandoz Ag | Sequence variants |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5830702A (en) * | 1990-10-31 | 1998-11-03 | The Trustees Of The University Of Pennsylvania | Live, recombinant listeria monocytogenes and production of cytotoxic T-cell response |
| FR2686896B1 (en) * | 1992-01-31 | 1995-01-06 | Pasteur Institut | MUTANT ATTENUE OF LISTERIA MONOCYTOGENES; RECOMBINANT STRAIN OF LISTERIA MONOCYTOGENES, USE AS HETEROLOGOUS VECTORS OF VACCINE ANTIGENS AND USE AS VACCINE OR DIAGNOSTIC COMPOSITION. |
| DE19949594A1 (en) * | 1999-10-14 | 2001-04-26 | Deutsches Krebsforsch | Recombinant attenuated listeria for immunotherapy |
-
2004
- 2004-12-23 JP JP2006547602A patent/JP5532280B2/en not_active Expired - Fee Related
- 2004-12-23 CN CN200480042020XA patent/CN1921884B/en not_active Expired - Fee Related
- 2004-12-23 TW TW093140281A patent/TW200530399A/en unknown
-
2007
- 2007-04-11 HK HK07103786.5A patent/HK1098673A1/en not_active IP Right Cessation
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2011
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103547676A (en) * | 2011-03-22 | 2014-01-29 | 缪科西斯私人有限公司 | Immunogenic compositions in particulate form and methods for producing the same |
| CN103547676B (en) * | 2011-03-22 | 2016-11-16 | 缪科西斯私人有限公司 | The immunogenic composition of particle form and for the method producing it |
| CN102766601A (en) * | 2011-11-17 | 2012-11-07 | 中山大学肿瘤防治中心 | Cell line containing inducible cancer gene, establishing method and application thereof |
| CN102766601B (en) * | 2011-11-17 | 2015-12-16 | 中山大学肿瘤防治中心 | Containing can the clone of induction type oncogene and establishment method thereof, application |
| CN105039381A (en) * | 2015-07-21 | 2015-11-11 | 齐鲁工业大学 | Maltose inducible trehalose synthase synthesis engineering bacterium, method for preparing same and application |
| CN107229842A (en) * | 2017-06-02 | 2017-10-03 | 肖传乐 | A kind of three generations's sequencing sequence bearing calibration based on Local map |
| CN115894639A (en) * | 2022-11-03 | 2023-04-04 | 大连医诺生物股份有限公司 | Auxiliary infection protein residue mutant protein, recombinant thereof and application thereof in exoenzyme immobilization process |
| CN115894639B (en) * | 2022-11-03 | 2024-05-14 | 大连医诺生物股份有限公司 | Auxiliary invasin residue mutant protein, recombinant thereof and application thereof in exoenzyme immobilization process |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5532280B2 (en) | 2014-06-25 |
| JP2007518405A (en) | 2007-07-12 |
| JP2011155988A (en) | 2011-08-18 |
| TW200530399A (en) | 2005-09-16 |
| HK1098673A1 (en) | 2007-07-27 |
| CN1921884B (en) | 2012-02-08 |
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