CN1921884B - 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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- CN1921884B CN1921884B CN200480042020XA CN200480042020A CN1921884B CN 1921884 B CN1921884 B CN 1921884B CN 200480042020X A CN200480042020X A CN 200480042020XA CN 200480042020 A CN200480042020 A CN 200480042020A CN 1921884 B CN1921884 B CN 1921884B
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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
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- 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
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- 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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- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (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, and 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.60/532,24,598,2003 applications of 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 accomplishes under the government of the SBIR grant number No.1R43CA101421-01 that National Institute of Health gives supports.Government possibly 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 through 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; Demonstrated through the oral vaccine that changes the Salmonella (Salmonella) to express the antigenic attenuation of Bai Shi plasmodium (Plasmodium berghei) ring spore and can protect mice 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-through I class and II class MHC antigen presentation approach, and its exploitation is used for the antigenic specificity vaccine.Referring to, for example, United States Patent(USP) No. 6,051,237,6,565,852 and 5,830,702.
Listera many years have been studied as stimulating congenital model with 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 through 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 through effectively presenting of I class MHC approach.In the Cytoplasm of antigen-presenting cell (APC), take a sample and degraded through albuminous body with excretory protein by Listera is synthetic.Resulting peptide is shuttled back and forth get into endoplasmic reticulum and be loaded on the I class MHC molecule through TAP protein.MHC I-peptide complexes is delivered to cell surface, its with fully altogether stimulate (signal 2) to 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 through MHC I classpath, expression of heterologous genes is directly related with initiation of CD8+T cell and/or activated usefulness to the excretory efficient that infects (antigen presentation) cell matter from antibacterial with new synthetic protein.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 with 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 in antibacterial, to improve.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, comprise first polynucleotide of 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 (a) coding antibacterial inherent signal peptide, wherein first polynucleotide are optimized and (b) second polynucleotide of coded polypeptide of codon for the expression in this antibacterial; 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.The pharmaceutical composition that comprises Listera also is provided, 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 (like 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, wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide with signal peptide, 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.The pharmaceutical composition that comprises this Listera also is provided, 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.The pharmaceutical composition that comprises this Listera also is provided, immunogenic composition and vaccine.In addition, the invention provides this reorganization Listera of use belong to antibacterial come induce immune response and/or prevention or treatment host 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.The pharmaceutical composition that comprises this Listera also is provided, 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, wherein the promoter of this expression cassette first and second polynucleotide of further comprising 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.The pharmaceutical composition that comprises this Listera also is provided, 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 is (for example; Antigen) second polynucleotide and be operably connected first and the promoter of 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.The pharmaceutical composition that comprises this Listera also is provided, 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, and 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 has comprised 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.The pharmaceutical composition that comprises this Listera also is provided, 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.The pharmaceutical composition that comprises this Listera also is provided, 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, comprise first polynucleotide of (a) coded signal peptide; (b) coding secretory protein or its segmental second polynucleotide, wherein second polynucleotide are arranged in translation frame identical with first polynucleotide and (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, by polypeptide and secretory protein or its segmental protein chimera of the 3rd polynucleotide encoding; 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; Above-mentioned arbitrary aspect in; Or in following detailed Description Of The Invention or embodiment) compositions of described recombinant bacteria gives the host, and 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 is included in this described recombinant bacteria with effective dose gives the host.
The present invention at this (for example further provides; Above-mentioned arbitrary aspect in; 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 said purposes of recombinant bacteria 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 said recombinant bacteria to be used to induce the host to antigenic immunne response, and 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 said recombinant bacteria to be used for prevention or treatment host's disease (like 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, comprises 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 human 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 human EphA2 ectodomain of codon.
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 human 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 human 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 human EphA2 born of the same parents of codon 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 human 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 MCS (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 plain (mesothelium is plain).
Figure 34 has shown the plain aminoacid sequence (SEQ ID NO:35) of people's mesothelium.
Figure 36 has shown the plain coded sequence (SEQ ID NO:36) of Mus mesothelium, 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 EphA2 CO domain but the 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 the directed antitumor efficacy of the hEphA2 of 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 in frame, to be operably connected.5 ' end of hly promoter comprises single Kpn I 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 plain sub-fragment of KpnI-BamHI (SEQ ID NO:43) of plasmid pPL2-hlyP-Np60 CodOp (1-77)-mesothelium.
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 based on chromosomal people's mesothelium plain expression and excretory western blot analysis.The result of the Listera of the mesothelium element that has shown the plain expressed protein engram analysis of mesothelium in the various bacterial cells part and expressed from contrast Listera (the mesothelium element of not encoding) and signal sequence shown in the coding.
Figure 59 A and 59B have shown the diagram of heterologous antigen (AH1-A5) through 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 anti of Listera vaccine mediation as far as 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).
Figure 61 is the diagram that has shown the therapeutic efficiency of the plain Listera of expressing human mesothelium in the vaccinated animal that has a tumor, and is wherein that the tumor cell through engineering approaches is plain with the expressing human mesothelium.
Figure 62 is the diagram that has shown that lung tumor tuberosity level reduces in the mice that has tumor of the Listera of having inoculated expressing human mesothelium element, and is wherein that the tumor cell through engineering approaches is plain with the expressing human mesothelium.
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 plain with the expressing human mesothelium, and the antitumor efficacy that has proved Lm-Meso vaccination is that the mesothelium element is specific.
Figure 64 has shown to have inoculated to express the diagram that the plain Listera of codon optimization people mesothelium 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 wherein the plain nucleic acid of coding human mesothelium has been integrated in the genome of Listera.
Detailed Description Of The Invention
I. introduction
The invention provides multiple polynucleotide, comprise being used for expressing 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) at antibacterial such as Listera.In some embodiments, these polynucleotide can be used for improving antibacterial polypeptide expression and/or secretion.Some expression cassettes comprise the coded sequence of optimized polypeptide of codon and/or signal peptide.In addition, some expression cassettes that are used for antibacterial comprise be derived from other bacterial origins and/or from the signal peptide sequence of multiple different secretory pathways.The antibacterial that comprises expression cassette also is provided, and the compositions that comprises this antibacterial, like vaccine.Also provide and used these polynucleotide, antibacterial and compositions to come the method for induce immune response and/or prevention or treatment host's disease such as disease (for example, cancer).
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 combine) of heterologous polypeptide (like antigen) in recombinant bacteria in the expression cassette; Even signal peptide sequence be antibacterial inherent the time (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 expression and/or the secretion (referring to, for example, following embodiment 19) of heterologous polypeptide in the Listera.Shown that also expression that heterologous protein that the optimization through expression cassette obtains improves and/or secretion cause comprising the immunogenicity (referring to, for example, following embodiment 20) of raising of the antibacterial 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 effective expression and the secretion (referring to, for example, following embodiment 29) of heterologous antigen in the Listera.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, in the one side, 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 second polynucleotide of coded polypeptide (for example, antigen); 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, second polynucleotide also are codon optimized (being often used in expressing in the antibacterial with first polynucleotide same type).In some embodiments; First polynucleotide or first and second polynucleotide are for belong to bacillus (Bacillus), Yersinia pestis (Yersinia pestis) at Listera; Salmonella (Salmonella); Shigella (Shigella), Brucella (Brucella), the expression in mycobacteria or the escherichia coli (E.coli) is that codon is optimized.In some embodiments, polynucleotide are that codon is optimized for belonging to like the expression in the Listeria monocytogenes at Listera.In some embodiments, be (or comprising) antigen by the polypeptide of second polynucleotide encoding, in some cases, the antigen that it can the right and wrong antibacterial.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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, and H-Ras, N-Ras, 12-K-Ras, mesothelium is plain, 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 that mesothelium is plain, or plain antigen fragment or the antigenic variant of mesothelium.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 antibacterial (Listera Listera or non-).In some embodiments, be that antibacterial is inherent by the signal peptide of optimized first polynucleotide encoding of codon.In other embodiments, be external source to antibacterial by the signal peptide of optimized first polynucleotide encoding of codon.In some embodiments; Signal peptide is the secA1 signal peptide; Like LLO signal peptide from Listeria monocytogenes, from the Usp45 signal peptide of lactococcus lactis (Lactococcuslactis), or from the protective antigen 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 Listeria monocytogenes.In addition; Recombinant nucleic acid molecules randomly comprises coding p60 or its segmental polynucleotide sequence; Be arranged in and first and second translation frame that polynucleotide are identical, wherein second polynucleotide are in the 3rd polynucleotide or between first and the 3rd polynucleotide.In the other embodiment, signal peptide is the Tat signal peptide, like bacillus subtilis (B.subtilis) Tat signal peptide (for example, PhoD).The present invention also provides the expression cassette that comprises recombinant nucleic acid molecules and further comprise the promoter of the recombinant nucleic acid molecules that is operably connected (for example, connecting first and second polynucleotide (and the 3rd polynucleotide, if exist)).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 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 disease such as disease.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the second aspect; The invention provides recombinant nucleic acid molecules; First polynucleotide that comprise (a) coding antibacterial inherent signal peptide, wherein first polynucleotide are optimized and (b) second polynucleotide of coded polypeptide of codon for the expression in this antibacterial; 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, second polynucleotide and first polynucleotide are allogenic.In some embodiments, polypeptide to signal peptide be its inherent antibacterial be external source.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, antibacterial is that Listera belongs to antibacterial (for example, Listeria monocytogenes).In some embodiments, second polynucleotide is that codon is optimized for the expression in antibacterial.In some embodiments, the codon optimization of first and/or second polynucleotide has improved the expression of coded fusion rotein in antibacterial and/or the secretion from antibacterial (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 an 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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, 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 Listeria monocytogenes (for example, from).In some embodiments, signal peptide is secA2 signal peptide (for example, the p60 signal peptide of Listeria monocytogenes).The expression cassette of the promoter of first and second polynucleotide that comprise this recombinant nucleic acid molecules and further comprise 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 polynucleotide are that codon is optimized for the expression in recombinant bacteria.In some embodiments, recombinant bacteria is an intracellular bacteria.In some embodiments, recombinant bacteria is selected from Listera and belongs to bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria and escherichia coli.In some embodiments, antibacterial is that the reorganization Listera belongs to antibacterial (for example, the Listeria monocytogenes of reorganization).The immunogenic composition that comprises this recombinant bacteria also is provided, and wherein the polypeptide by second polynucleotide encoding is an antigen.Also provide and induced the method for host to antigenic immunne response, comprised that the compositions that comprises recombinant bacteria with effective dose gives the patient, wherein the polypeptide by second polynucleotide encoding is (or comprising) antigen.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the third aspect; The invention provides the Listera that comprises recombinant nucleic acid molecules and (for example belong to antibacterial; Listeria monocytogenes); Wherein this recombinant nucleic acid molecules comprises first polynucleotide of (a) coded signal peptide, and wherein first polynucleotide are optimized and (b) second polynucleotide of coded polypeptide of codon for belong to expression in the antibacterial at Listera; 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, be allogenic by the polypeptide and the signal peptide of second polynucleotide encoding.In some embodiments, recombinant nucleic acid molecules is a part that further comprises the expression cassette of be operably connected first and the promoter of second polynucleotide.In other words, in some embodiments, this reorganization Listera belongs to antibacterial and comprises the expression cassette that contains this recombinant nucleic acid molecules, wherein the promoter of this expression cassette first and second polynucleotide of further comprising the recombinant nucleic acid molecules that is operably connected.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, the codon optimization of first and/or second polynucleotide has improved coded fusion rotein and has belonged to the expression in the antibacterial and/or belong to the secretion (with respect to the optimized sequence of non-codon) the antibacterial from Listera at Listera.In some embodiments, by the polypeptide of second polynucleotide encoding Listera being belonged to antibacterial is external source (that is it is allogenic, belonging to antibacterial with Listera).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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, or its antigen fragment or antigenic variant.In some embodiments, antigen is that people's mesothelium is plain.In some embodiments, antigen is that people's mesothelium of its signal peptide of disappearance and GPI link field is plain.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 is derived from the antigenic antigen of infectious disease.In some embodiments, signal peptide is non-Listera.In some embodiments, signal peptide is an antibacterial.In some embodiments, it is external source that signal peptide belongs to antibacterial to Listera.In other embodiments, signal peptide is that Listera is inherent.In some embodiments, signal peptide is secA1 signal peptide (for example, from the LLO signal peptide of Listeria monocytogenes, from the Usp45 signal peptide of lactococcus lactis with from the protective antigen signal peptide of Bacillus anthracis).In some embodiments, signal peptide is the secA2 signal peptide p60 signal peptide of 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, it is attenuation that Listera belongs to antibacterial.For example, can the Listera attenuation be used for cell and intercellular diffusion, get into non-phagocytic cell or propagation.In some embodiments, it is to lack ActA that the reorganization Listera belongs to antibacterial, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ actA Δ inlB).In some embodiments, the reorganization Listera belongs to antibacterial and lacks functional ActA, Internalin B, or ActA and Internalin B.In some embodiments, the nucleic acid of recombinant bacteria is through being modified with nucleic acid target compound (for example, psoralen chemical compound) reaction.The present invention also provides and has comprised that the reorganization Listera belongs to the pharmaceutical composition of antibacterial and pharmacological-acceptable carrier, and comprises that the reorganization Listera belongs to the immunogenic composition of antibacterial, and wherein the polypeptide by second polynucleotide encoding is an antigen.The present invention also provides and has comprised that the reorganization Listera belongs to the vaccine of antibacterial.Also provide and induced the method for host to antigenic immunne response, comprised that the compositions 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 disease (for example, disorders such as cancers or infectious disease) also is provided, has comprised that the compositions that this reorganization Listera belongs to antibacterial that comprises with effective dose gives the host.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the fourth aspect; The invention provides recombinant nucleic acid molecules; First polynucleotide and the coded polypeptide that comprise the non-secA1 antibacterial signal peptide of encoding are (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, first polynucleotide and/or second polynucleotide are that codon is optimized for the expression in the particular type antibacterial.In some embodiments, the codon optimization of first and/or second polynucleotide has improved the expression of fusion rotein in antibacterial and/or the secretion from antibacterial (with respect to the optimized sequence of non-codon).In some embodiments, first polynucleotide and/or second polynucleotide are for belong to bacillus at Listera; Yersinia pestis, Salmonella, Shigella; Brucella, the expression in mycobacteria or the escherichia coli are that codon is optimized.In some embodiments, polynucleotide are that codon is optimized for belonging to like the expression in the Listeria monocytogenes at Listera.In some embodiments, be that the optimized antibacterial of codon is inherent 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, 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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, and H-Ras, N-Ras, 12-K-Ras, mesothelium is plain, 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 that mesothelium is plain, or plain antigen fragment or the antigenic variant of mesothelium.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 antibacterial 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 Listeria monocytogenes.In addition; Recombinant nucleic acid molecules randomly comprises coding p60 or its segmental the 3rd polynucleotide; Be arranged in and first and second translation frame that polynucleotide are identical, wherein second polynucleotide are in the 3rd polynucleotide or between first and the 3rd polynucleotide.In the other embodiment, signal peptide is the Tat signal peptide, like bacillus subtilis Tat signal peptide (for example, bacillus subtilis PhoD signal peptide).The present invention also provides the expression cassette of the promoter of first and second polynucleotide that comprise recombinant nucleic acid molecules and further comprise the recombinant nucleic acid molecules that is operably connected.The expression vector and the antibacterial that comprise this expression cassette and/or recombinant nucleic acid molecules also are provided, and the pharmaceutical composition that comprises this antibacterial, immunogenic composition and vaccine.In some embodiments, the recombinant bacteria that comprises this expression cassette or recombinant nucleic acid molecules is an intracellular bacteria.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, antibacterial is that Listera belongs to antibacterial (for example, the member of Listeria monocytogenes kind).In some embodiments, be external source (that is being allogenic) to antibacterial with antibacterial by the polypeptide of second polynucleotide encoding.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).In some embodiments, disease is a cancer.In other embodiments, disease is an infectious disease.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
On the other hand; The invention provides the reorganization Listera that comprises recombinant nucleic acid molecules and belong to antibacterial; Wherein recombinant nucleic acid molecules comprises first polynucleotide and (b) second polynucleotide of coded polypeptide of the non-secA1 antibacterial signal peptide of (a) coding, 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; In some embodiments, be allogenic or be external source antibacterial by the polypeptide of second polynucleotide encoding and signal peptide, or both.In some embodiments, Listera belongs to antibacterial and belongs to the Listeria monocytogenes kind.In some embodiments, recombinant nucleic acid molecules is the part of expression cassette, and this expression cassette further comprises be operably connected first and the promoter of second polynucleotide.In other words, in some embodiments, the reorganization Listera belongs to antibacterial and comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein this expression cassette further comprises the promoter of first and second polynucleotide of the recombinant nucleic acid molecules that is operably connected.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, the codon optimization of first and/or second polynucleotide has improved expression and/or the secretion from this antibacterial (with respect to non-codon optimized sequence) of fusion rotein in this antibacterial.In some embodiments, be allogenic each other with second polynucleotide by first.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, belonging to antibacterial with Listera).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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, or its antigen fragment or antigenic variant.In some embodiments, antigen is that people's mesothelium is plain.In some embodiments, antigen is that people's mesothelium of its signal peptide of disappearance and GPI link field is plain.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 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 polynucleotide are arranged in and first and second translation frame that polynucleotide are identical, and wherein second polynucleotide are in the 3rd polynucleotide of recombinant nucleic acid molecules or between first and the 3rd polynucleotide.In some embodiments, second polynucleotide is positioned at the 3rd polynucleotide.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 an attenuation.For example, can the Listera attenuation be used for cell and intercellular diffusion, get in the non-phagocytic cell or propagation.In some embodiments, the reorganization Listera is to lack ActA, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ act Δ Ain1B).In some embodiments, it is to lack ActA that the reorganization Listera belongs to antibacterial, Internalin B, or ActA and Interfalin B (for example, the two deletion mutants of Δ actA Δ in1B).In some embodiments, the nucleic acid of recombinant bacteria is through being modified with nucleic acid target compound (for example, psoralen chemical compound) reaction.The present invention also provides and has comprised that the reorganization Listera belongs to the pharmaceutical composition that can accept carrier on antibacterial 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 an antigen.The present invention also provides and has comprised that the reorganization Listera belongs to the vaccine of antibacterial.Also provide and induced the method for host to antigenic immunne response, comprised that the compositions 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 disease (for example, disorders such as cancers or infectious disease) also is provided, has comprised that the compositions that the reorganization Listera belongs to antibacterial that contains with effective dose gives the host.Also provide antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
On the other hand; The invention provides and comprise that the coding Listera belongs to the antibacterial allogenic polypeptide (like antigen; Like cancer antigen or non-Listera antigen) the recombinant nucleic acid molecules of polynucleotide, wherein polynucleotide are that codon is optimized for the expression in Listera.In some embodiments, the codon optimization of polynucleotide has improved polypeptide and has belonged to the expression in the antibacterial and/or belong to the secretion (with respect to the optimized sequence of non-codon) the antibacterial from Listera at Listera.In some embodiments, allogenic polypeptide comprises antigen.In some embodiments, allogenic polypeptide is an 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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, and H-Ras, N-Ras, 12-K-Ras, mesothelium is plain, 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 that mesothelium is plain, or plain antigen fragment or the antigenic variant of mesothelium.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 polynucleotide of the signal peptide of the translation frame identical with allogenic polypeptide, makes the recombinant nucleic acid molecules coding comprise the fusion rotein of signal peptide and allogenic polypeptide.In some embodiments, the polynucleotide of coded signal peptide (it is can yes or no natural to Listera) are that codon is optimized for the expression in Listeria monocytogenes.The present invention further provides the expression cassette of the promoter of first and second polynucleotide that comprise recombinant nucleic acid molecules and further comprise 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 reorganization Listera that comprises recombinant nucleic acid molecules and/or expression cassette to belong to antibacterial.In some embodiments, Listera belongs to antibacterial and belongs to the Listeria monocytogenes kind.Also provide and comprised that the reorganization Listera belongs to pharmaceutical composition, immunogenic composition and the vaccine of antibacterial.The present invention further provides and has induced the method for host to antigenic immunne response, comprises that the compositions that the reorganization Listera belongs to antibacterial that contains with effective dose gives the patient, and wherein polypeptide is (or comprising) antigen.In addition, the invention provides use reorganization Listera and belong to the method that antibacterial comes induce immune response and/or prevention or treatment disease (for example, disease).Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein allogenic polypeptide comprises antigen.
On the other hand; The invention provides the reorganization Listera that comprises expression cassette and belong to antibacterial; Wherein expression cassette comprises that the coding Listera belongs to the antibacterial allogenic polypeptide (like antigen; Like cancer antigen or non-Listera antigen) polynucleotide and the promoter of polynucleotide of the encoding exogenous polypeptide that is operably connected, wherein polynucleotide are that codon is optimized for the expression in Listera.In some embodiments, Listera belongs to antibacterial and belongs to the Listeria monocytogenes kind.In some embodiments, the codon optimization of polynucleotide has improved polypeptide and has belonged to the secretion (with respect to the optimized sequence of non-codon) the antibacterial at expression and/or the polypeptide that Listera belongs in the antibacterial from Listera.In some embodiments, allogenic polypeptide comprises antigen.In some embodiments, allogenic polypeptide is an 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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, and H-Ras, N-Ras, 12-K-Ras, mesothelium is plain, 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 that mesothelium is plain, or plain antigen fragment or the antigenic variant of mesothelium.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 promoter that is operably connected and polynucleotide that be arranged in the coded signal peptide of the translation frame identical with allogenic polypeptide, makes the expression cassette coding comprise the fusion rotein of signal peptide and allogenic polypeptide.In some embodiments, the polynucleotide of coded signal peptide (it is can yes or no natural for Listera) are that codon is optimized for the expression in Listeria monocytogenes.Also provide and comprised that the reorganization Listera belongs to pharmaceutical composition, immunogenic composition and the vaccine of antibacterial.The present invention further provides and has induced the method for host to antigenic immunne response, comprises that the compositions that the reorganization Listera belongs to antibacterial that contains with effective dose gives the patient.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 disease (for example, disease).Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein allogenic polypeptide comprises antigen.
In the other aspect, the invention provides the reorganization Listera that comprises recombinant nucleic acid molecules and belong to antibacterial (for example Listeria monocytogenes), 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 coded polypeptide of the translation frame identical with first polynucleotide, wherein the recombinant nucleic acid molecules coding comprises the recombiant protein of non-Listera signal peptide and polypeptide.In some embodiments, recombinant nucleic acid molecules is positioned at the expression cassette that further comprises be operably connected first and the promoter of second polynucleotide.Therefore, in some embodiments, the reorganization Listera belongs to antibacterial and 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.In some embodiments, this expression cassette is polycistronic expression box (for example, a bicistronic mRNA expression cassette).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, the codon optimization of first and/or second polynucleotide has improved expression and/or fusion rotein the secretion (with respect to non-codon optimized sequence) from antibacterial of fusion rotein in antibacterial.In some embodiments, first is allogenic each other with second polynucleotide.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, belonging to antibacterial with Listera).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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, or its antigen fragment or antigenic variant.In some embodiments, antigen is that people's mesothelium is plain.In some embodiments, antigen is that people's mesothelium of its signal peptide of disappearance and GPI link field is plain.In some alternate embodiments, antigen is infectious disease antigen or is derived from infectious disease antigen.In some embodiments, signal peptide is an antibacterial.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 antibacterial 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 antigen 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, it is attenuation that Listera belongs to antibacterial.For example, in some embodiments, the Listera attenuation is used for cell and intercellular diffusion, gets in the non-phagocytic cell or propagation.In some embodiments, it is to lack ActA that the reorganization Listera belongs to antibacterial, Internalin B, or ActA and Internalin B (for example, the two deletion mutants of Δ acta Δ in1B).In some embodiments, the reorganization Listera is to lack functional ActA, Internalin B, or ActA and Internalin B.In some embodiments, the nucleic acid of recombinant bacteria is through being modified with nucleic acid target compound (for example, psoralen chemical compound) reaction.The present invention also provides and has comprised that the reorganization Listera belongs to the pharmaceutical composition that can accept carrier on antibacterial 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 an antigen.The present invention also provides and has comprised that the reorganization Listera belongs to the vaccine of antibacterial.Also provide and induced the method for host to antigenic immunne response, comprised that the compositions that the reorganization Listera belongs to antibacterial that contains with effective dose gives the host, wherein the polypeptide by second polynucleotide encoding is (or comprising) antigen.The method of prevention or treatment host disease (for example disease, like cancer or infectious disease) also is provided, has comprised that the compositions that the reorganization Listera belongs to antibacterial that contains with effective dose gives the host.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
Again in the one side; The invention provides the reorganization 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, is arranged in second polynucleotide and be operably connected first and the promoter of second polynucleotide of the coded polypeptide of the translation frame identical with first polynucleotide.This expression cassette coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.In some embodiments, Listera is belonged to the antibacterial attenuation be used for cell and intercellular diffusion, get in the non-phagocytic cell or propagation.In some embodiments, first polynucleotide, second polynucleotide, or first is that codon is optimized with second polynucleotide for the expression in Listera.In some embodiments, the codon optimization of first and/or second polynucleotide has improved expression and/or the secretion from this antibacterial (with respect to non-codon optimized sequence) of coded fusion rotein in this antibacterial.In some embodiments, first polynucleotide and/or second polynucleotide are that codon is optimized for the expression in 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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, or plain antigen fragment of mesothelium 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 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, like Usp45 signal peptide from lactococcus lactis, or from the protective antigen signal peptide of Bacillus anthracis.In some embodiments, signal peptide is the secA2 signal peptide.In the other embodiment, signal peptide is the Tat signal peptide, like bacillus subtilis Tat signal peptide (for example, PhoD).Also provide and be included in pharmaceutical composition, immunogenic composition and the vaccine that this described reorganization Listera belongs to antibacterial.In addition, the invention provides use reorganization Listera and belong to the method that antibacterial comes induce immune response and/or prevention or treatment disease such as disease.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
The present invention further provides recombinant 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 positioned at 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, first polynucleotide encoding antibacterial autolysin.In some embodiments, the protein chimera has the catalytic activity the same with autolysin.In some embodiments, the antibacterial autolysin is from intracellular bacteria (for example, Listera).In some embodiments, the antibacterial autolysin is the Listera autolysin.In some embodiments; Second polynucleotide of coded polypeptide are positioned at first polynucleotide of coding autolysin or its catalytic activity fragment or catalytic activity variant; And the recombinant nucleic acid molecules polypeptide of encoding wherein is positioned at 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 variant).In some alternate embodiments; Second polynucleotide are positioned at outside first polynucleotide 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 allogenic.In some embodiments, first polynucleotide and second polynucleotide are allogenic each other.In some embodiments; Recombinant nucleic acid molecules comprises that further (c) is arranged in the 3rd polynucleotide of the coded signal peptide of the translation frame identical with first and second polynucleotide; 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 (like 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 a secA2 dependency autolysin.In some embodiments, autolysin is Peptidoglycan hydrolytic enzyme (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 antigenic antigen of infectious disease).In some embodiments, antigen is selected from K-Ras, H-Ras, and N-Ras, 12-K-Ras, mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, or its antigen fragment or antigenic variant.In some embodiments, antigen is that people's mesothelium is plain.In some embodiments, antigen is that people's mesothelium of its signal peptide of disappearance and GPI anchor is plain.The present invention also provides the expression cassette of the promoter of first and second polynucleotide that comprise recombinant nucleic acid molecules and further comprise 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 an intracellular bacteria, belongs to antibacterial (for example, Listeria monocytogenes) like Listera.In some embodiments, be external source to recombinant bacteria by the polypeptide of second polynucleotide encoding.Also provide and comprised the pharmaceutical composition that to accept carrier on (a) recombinant bacteria and (b) materia medica.In addition, the immunogenic composition that comprises recombinant bacteria is provided also, wherein the polypeptide by second polynucleotide encoding is an antigen.The vaccine that comprises recombinant bacteria also is provided, and wherein the polypeptide by second polynucleotide encoding is an antigen.The present invention also provides and has induced the method for host to antigenic immunne response, comprises that the compositions 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 disease also is provided, has comprised that the compositions that contains recombinant bacteria with effective dose gives the host.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in manufacturing, wherein the polypeptide by second polynucleotide encoding comprises antigen.
In the one side, the invention provides the reorganization Listera that comprises the polycistronic expression box and belong to antibacterial again, wherein at least two discrete non-Listera polypeptide of polycistronic expression box coding.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 be operably connected first and the promoter of second polynucleotide.In some embodiments, expression cassette further comprises the intergenic sequence between first and second polynucleotide.In some embodiments, the polycistronic expression box is the bicistronic mRNA expression cassette, two discrete non-Listera polypeptide of its coding.In some embodiments, the reorganization Listera belongs to antibacterial and belongs to the 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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, or its antigen fragment or antigenic variant.In some embodiments, antigen is that people's mesothelium is plain.In some embodiments, antigen is that people's mesothelium of its signal peptide of disappearance and GPI anchor is plain.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 polynucleotide of coded signal peptide, and wherein the polynucleotide of coded signal peptide are that codon is optimized for the expression in Listera.The present invention also provides and has comprised that (a) reorganization Listera belongs to the pharmaceutical composition that can accept carrier on antibacterial and (b) materia medica.Also provide and comprised that the reorganization Listera belongs to the immunogenic composition of antibacterial.Also provide and comprised that the reorganization Listera belongs to the vaccine of antibacterial.Also provide and induced the method for host to antigenic immunne response, comprised that the compositions that the reorganization Listera belongs to antibacterial that contains with effective dose gives the host, wherein at least one non-Listera polypeptide comprises antigen.The method of prevention or treatment host disease also is provided, has comprised that the compositions that the reorganization Listera belongs to antibacterial that contains with effective dose gives the host.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response 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, comprise first Polynucleotide molecule of (a) coded signal peptide, (b) coding secretory protein or its segmental second polynucleotide; Wherein second polynucleotide is positioned at 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 positioned at 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 the 3rd polynucleotide encoding; 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.In some embodiments, secretory protein is natural secretory protein (that is an excretory protein from its n cell).In some embodiments, in recombinant nucleic acid molecules, the 3rd polynucleotide are positioned at second polynucleotide, 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, in recombinant nucleic acid molecules, the 3rd polynucleotide are positioned at outside second polynucleotide, and in the protein chimera, by polypeptide and the secretory protein or the fusion of its fragment 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 polynucleotide.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; Mesothelium is plain, 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, mesothelium is plain, 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 that mesothelium is plain, or its antigen fragment or antigenic variant.For example, in some embodiments, antigen is that people's mesothelium element or the people's mesothelium that has lacked its signal peptide and GPI anchor are plain.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 provide the reorganization Listera to belong to antibacterial (for example, Listeria monocytogenes), and in some embodiments, by the polypeptide of the 3rd nucleotide coding Listera being belonged to antibacterial is external source.The present invention also provides the immunogenic composition that comprises this recombinant bacteria, and wherein the polypeptide by the 3rd polynucleotide encoding is an antigen.Also provide and induced the method for host to antigenic immunne response, comprised that the compositions 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 antibacterial also are provided, and have used this recombinant bacteria or comprise that the compositions of this antibacterial prevents or treat the method for host's disease.Also provide this antibacterial to induce the host to the purposes in the medicine of antigenic immunne response in preparation, wherein the polypeptide by the 3rd polynucleotide encoding comprises antigen.
In the other aspect, the invention provides in host bacteria and to express and the improving one's methods of heterologous protein secretion.The present invention also provides and has improved expression and the excretory method of foreign protein in antibacterial.The present invention further provides this described recombinant nucleic acid molecules, expression cassette, the method for expression vector and recombinant bacteria of being prepared in.
The present invention also provides and has been used for the multiple polynucleotide of optimization heterologous polynucleotide in the expression of antibacterial such as Listera.
Be to be understood that the Ma Kushi group; Ma Kushi claim or through the embodiment described in " or 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, only if stipulate clearly or through context in addition.
Further describing and other embodiments of the present invention and aspect of 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, 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 is provided.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, 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, said recombinant nucleic acid molecules encoding fusion protein 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 said 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 embodiment, 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 optimized and (b) second polynucleotide of coded polypeptide of codon for the expression in antibacterial; 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, and wherein first polynucleotide are optimized and (b) second polynucleotide of coded polypeptide of codon for belong to expression in the antibacterial at Listera; 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; Second polynucleotide of coded polypeptide (for example, heterologous protein and/or antigen) and encode 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, and 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 through 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.Nat1.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 (like Listera albumen, like LLO or p60) of highly expressing, but in another embodiment, can be highly to express to 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, like 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, and wherein the recombinant nucleic acid molecules coding comprises signal peptide, by the polypeptide of 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, the sequence of highly expressing is that wherein to wait to express the antibacterial of this sequence inherent.In other embodiments, the sequence of highly expressing 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 through 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 combines to produce required recombinant nucleic acid molecules relevant polynucleotide.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 said any recombinant nucleic acid molecules to produce the recombinant bacteria method of (for example, the reorganization Listera belongs to antibacterial).In some embodiments, the method for using said recombinant nucleic acid molecules to make recombinant bacteria comprises to be introduced recombinant nucleic acid molecules in the antibacterial.In some embodiments, in the genome with recombinant nucleic acid molecule integrates to 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 through engaging.Can realize getting into the introducing of antibacterial through 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 through 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 in host cell such as antibacterial, expressing 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 transporting of cell membrane that the polypeptide that merges with signal peptide passes cell (for example, bacterial cell), makes polypeptide from cell, secrete.Therefore, in some embodiments, signal peptide is " secreting signal peptide " or " secretion sequence ".In some embodiments, it is terminal that signal peptide is positioned at the N-that treats 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 like 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, like " 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 the 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 said polynucleotide, 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.About the additional information of conserved amino acid change, referring to, for example, the IV part that below details.
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.For example, the ability that is derived from the ultra signal peptide effect of signal peptide of another kind of signal peptide should not receive the influence of the change (disappearance, sudden change etc.) that the primary signal peptide sequence is carried out basically.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 in signal peptide sequence, carrying out is the conserved amino acid replacement.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 to be decided by the type that is positioned at the terminal signal peptide sequence of preceding albumen N-.Most of secretory protein utilizes the Sec approach, and wherein protein is transported through the PS ec hole that is embedded in bacterial membrane with not folding conformation.On the contrary, the protein that utilizes the Tat approach is 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 (Sha rkov 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 through 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 through the relevant signal peptide of the excretory protein of Tat approach with 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, and 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 from antibacterial, to secrete.
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 | |||
| MQKTRKERILEALQEEKKNKKSKKFKTGATIAGVTAIATSITVPGIEVIVSADE(SEQ?IDNO：49) | VSA’DE(SEQ?ID?NO：58) | NamAlmo2691 (autolysin) | Listeria monocytogenes | |
| MKKLKMASCALVAGLMFSGLTPNAFAED(SEQ?ID?NO：50) | AFA’ED(SEQ?ID?NO：59) | *BA_0281 (NLP/P60 family) | Bacillus anthracis | |
| MAKKFNYKLPSMVALTLVGSAVTAHQVQAAE(SEQ?ID?NO：51) | VQA’AE(SEQ?ID?NO：60) | *Atl (autolysin) | Staphylococcus aureus | |
| Tat | MTDKKSENQTEKTETKENKGMTRREMLKLSAVAGTGIAVGATGLGTILNVVDQVDKALT(SEQ?ID?NO：52) | DKA’LT(SEQ?ID?NO：61) | lmo0367 | Listeria monocytogenes |
| MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLSLGLTIAQSVGAFG(SEQ?IDNO：53) | VGA’FG(SEQ?ID?NO：62) | PhoD (alkali phosphatase) | Bacillus subtilis |
* through the excretory antibacterial autolysin of sec approach (not confirming secA1 or secA2).
Therefore, in some embodiments, the sequential coding secA1 signal peptide of coded signal peptide.The instance of SecA1 signal peptide is Listera lysin O (LLO) signal peptide from Listeria monocytogenes.In some embodiments, comprise recombinant nucleic acid molecules or the polynucleotide sequence that expression cassette further comprises coding LLO PEST sequence of the polynucleotide of coding LLO signal peptide.Other instances 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 instance 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, with allogeneic coding sequence and by merging in the heredity of secA2 pathway protein secretion complex identified signal peptide.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 through 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 through 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 through 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 active, this means that heterologous protein secretes as the non-revenue passenger of p60.Can in the frame of any heterologous antigen entering p60 that names a person for a particular job in keeping the proteinic secretion of p60 and the active p60 of autolysin, 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 the 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 heterologous protein effective expression and secretion (referring to, for example, following specific embodiment, embodiment 29).Therefore, in some embodiments, is the p60 signal peptide by the recombinant nucleic acid molecules coding as a part of signal peptide of fusion rotein.In some embodiments, is the NamA signal peptide by the recombinant nucleic acid molecules coding as a part of signal peptide of fused protein.
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 like 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; And in some embodiments, can use the polynucleotide of coding Mol.Microbiology 45:1043-1056 (2002)), 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 illustrative 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 through 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 through 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 the technological secA2 congener that relatively can identify other through 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-deposition and through any proteomic techniques analysis known in the art to confirm that which protein is excretory by wild-type bacterium rather than Δ secA2 mutant.For example, can analyze excretory protein with silver-colored dyeing through SDS-PAGE.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 confirm 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 through the N-of the Edman of automatization degraded.
In the alternate embodiment, in the polynucleotide encoding heredity with the polypeptide (for example, allogeneic polypeptide sequence) that merges by Tat pathway protein secretion complex identified signal peptide.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 ([gi|16802412|ref|NP_463897.1| is with the conservative putative protein matter of bacillus subtilis YwbN protein similar (Listeria monocytogenes EGD (e)]) for Genbank accession number No.NP_463897 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 through 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 through 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 with 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 through the Tat secretory pathway from Listera polynucleotide can be used for said polynucleotide, 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.Instance from the Tat signal peptide of bacillus subtilis like 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 Dij1 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 to have comprised those sequences with following Genbank/Emb1 accession number: CAB15017 [gi|2635523|emb|CAB15017.1| is with the bi-component sensor histidine kinase) (YtsA) (bacillus subtilis) similar] through excretory other protein in bacillus subtilis, identified of Tat approach; CAB12056 [gi|2632548|emb|CAB12056.1| phosphodiesterase/alkali phosphatase D (bacillus subtilis)]; CAB12081 [gi12632573|emb|CAB12081.1, similar] with the protein (bacillus subtilis) of supposition; CAB13278 [gi12633776|emb| CAB13278.1, similar] with the protein (bacillus subtilis) of supposition; CAB14172 [gi|2634674|emb|CAB14172.1| methyl how quinone: cytochrome C oxidoreductase (ferrum-sulfur subunit)) (bacillus subtilis)]; CAB15089 [gi|2635595|emb|CAB15089.11yubF (bacillus subtilis)]; [(29d～similar with the protein (bacillus subtilis) of supposition, wherein all sequences are hereby incorporated by the gene title that gi|2636361|emb|CAB15852.1| is alternative: ipa with CAB15852.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 through its " two-arginine " consensus motif.As stated; And have the structure that be divided into three parts similar through the relevant signal peptide of the excretory protein of Tat approach with 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 through excretory novel protein of SecA2 approach and corresponding SecA2 signal peptide thereof also can be used for identifying through excretory novel protein of Tat approach and signal peptide thereof.List of references Jongbloed etc., J.Biological Chem., 277:44068-44078 (2002) provide and can be used for identifying the instance by the technology of passing through two-identical bacteria types expressed protein of the excretory protein of arginine transport approach.
IV. polypeptide
Said recombinant nucleic acid molecules, and said expression cassette 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 are expressed as fusion rotein (merge with signal peptide and/or other polypeptide, or in other polypeptide) or discrete polypeptide; 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 does not limit for the length or the size of wherein contained aminoacid sequence.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, said any antigen).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.As also be interpreted as the fragment that comprises big antigen protein at this used term " antigen ", 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 combine the algorithm of 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 instances 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 Wiscons in 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.Some algorithms discussed in the listed list of references more than in this paragraph or software kit relate to the prediction of 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 the polynucleotide sequence of this sequence of coding introduced in the expression cassette and introduce in Listera vaccine carrier or other bacterial vaccine carriers.Then through 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 like 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.Below embodiment provide the instance 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 the conserved amino acid replacement.Think that following replacement is the conserved amino acid replacement: 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 replacement 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 basically another antigen that is same as of another antigenic antigen.Have at least about 70% amino acid sequence identity and keep the immunogenicity at least about 70% original antigen if be derived from another antigenic antigen and original antigen, this antigen is first-class basically 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 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 confirm specific derive immunogenicity of antigens and and the immunogenicity of original antigen compare to confirm whether deutero-antigen is equal to original antigen basically, 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 measure those as follows and 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.Below embodiment provide these type algoscopys instance (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 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 plain (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 like 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 situation, 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), tissure specific antigen (for example, PAP; PSA), grow antigen, tumor correlated virus antigen (for example, HPV 16E7), carcinoma of testis antigen (for example, MAGE; BAGE, NY-SEO-1), EA (for example, CEA, α-Jia Taidanbai), oncoprotein antigen is (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), LAGE, LAGE-1, LDLR/FUT, MAGE-1, MAGE-2, MAGE-3, MAGE-6 with codon 12 sudden changes; MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-B5; MAGE-B6, MAGE-C2, MAGE-C3, MAGE-D, MART-1, MART-1/Melan-A, MC1R, MDM-2; Mesothelium is plain, myosin/m, and MUC1, MUC2, MUM-1, MUM-2, MUM-3 is new-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, Pm1/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 plain (Argani etc., Clin Cancer Res.7 (12): 3862-8 (2001)); Sp17 (Lim etc., Blood 97 (5): 1508-10 (2001)), gp100 (Kawakami etc.; Proc.Nat1.Acad.Sci.USA91:6458 (1994)), PAGE-4 (Brinkmann etc., Cancer Res.59 (7): 1445-8 (1999)); TARP (Wolfgang etc., Proc.Nat1.Acad.Sci.USA97 (17): 9437-42 (2000)), EphA2 (Tatsumi etc.; Cancer Res.63 (15): 4481-9 (2003)), PR3 (Muller-Berat etc., Clin.Immunol.Immunopath.70 (1): 51-9 (1994)); Prostate stem cell antigen (PSCA) (Reiter etc., Proc.Nat1.Acad.Sci.USA, 95:1735-40 (1998); Kiessling etc., Int.J.Cancer, 102:390-7 (2002)), or SPAS-1 (the open No.2002/0150588 of U.S. Patent application).
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.Nat1.Cancer Inst., 87:982-90 (1995)), yet; Unfortunately, only obtain limited success.However, researcher has identified by the human T-cell who from the patient of inoculation, produces to be the epitope of the HLA*0201-restriction of identification, CAP-1 (CEA605-613).DC inoculation patient with this epitope pulse fails to induce clinical response (Morse etc., Clin.CancerRes., 5:1331-8 (1999)).Recently, identified the CEA605-613 peptide agonists, had the irregular variation (CAP1-6D) of 610 aspartic acids to agedoite replacement in the position.Although the MHC binding affinity of this this peptide of aminoacid replacement the change, 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 Flt 3-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.Nat1.Acad.Sci.USA., 98:8809-14 (2001)).
In another embodiment, antigen is proteinase-3 or is derived from protease-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: United States Patent(USP) 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 is plain, 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, mesothelium is plain, 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, mesothelium is plain, 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 plain (for example, people's mesothelium is plain).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, mesothelium is plain, 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 that mesothelium is plain.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 that people's mesothelium is plain.
In some embodiments, antigen is that mesothelium is plain, 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 said recombinant nucleic acid molecules, the antigen of the polynucleotide encoding in expression cassette or the expression vector is not Epha 2 (or be derived from Epha2 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 Epha2.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 Epha2.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 Epha2 or is derived from the antigen the Epha2 antigen.
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, mesothelium is plain, 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, mesothelium is plain, 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 to be derived from the plain antigen of mesothelium.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 that mesothelium is plain, 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 that mesothelium is plain, or its antigen fragment or antigenic variant.In some embodiments, be that mesothelium is plain by the polypeptide of polynucleotide encoding, or its antigen fragment or antigenic variant.
In some embodiments, antigen is the mesothelium plain (for example, people's mesothelium is plain) that lacked of the plain signal peptide of mesothelium and/or GPI (glycosyl-phosphatidyl inositol) anchor wherein.Therefore, in some embodiments, comprise that by the polypeptide of polynucleotide encoding the mesothelium that plain signal peptide of mesothelium wherein and/or GPI anchor lacked is plain.In some embodiments, be that wherein the mesothelium that lacked of the plain signal peptide of mesothelium and/or GPI anchor is plain by the polypeptide of polynucleotide encoding.In some embodiments, be that wherein people's mesothelium of having lacked of the plain signal peptide of mesothelium and/or GPI anchor is plain 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 plain (Genbank Acc.No.U40434), people's survivin (Genbank Acc.No.U75285); Human protease 3 (Genbank No.X55668), people K-Ra s (Genbank Acc.Nos.M54969&P01116), people H-Ras (Genbank Acc.No.P01112); People N-Ras (Genbank Acc.No.P01111); With people 12-K-Ras (comprise Gly12Asp sudden change K-Ras) (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 of the amino acid whose tumor associated antigen with at least one conservative replacement.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 instance 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 through Hopp-Woods and Welling antigenicity and to predict these antigens zone.
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 plain (antigenicity) fragment of people's mesotheliums 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, can the polynucleotide of coding idiotypicantigen 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.With comprising that the medicable autoimmune disease of vaccine of the antibacterial that contains expression cassette of the present invention and recombinant nucleic acid molecules includes, but not limited to rheumatoid arthritis, 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 ultra proliferative cell, and antigenic expression that also can targeting.What considered was such will cause the antigen to the immunne response of ultra proliferative cell.
In some embodiments, antigen is the antigen of the unique disease relative protein white matter of targeting structure.One of instance 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 instance of this situation is to introduce to produce the antigen to 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 possibly only be used for the minimizing that speckle forms, it possibly provide medicable vaccine in like the disease condition of 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 be derived from the human immunodeficiency virus (like 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) (like gD, gB, gH; Early protein such as ICP27 immediately), from cytomegalovirus (like gB and gH), from inferior Pneumovirinae (metapneumovirus), from Epstein-Barr virus or from varicella zoster virus (like 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; United States Patent(USP) 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), 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 kinds of 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); FundanentalVirology, 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, like Mycobacterium, and 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 obtain or produce to be applicable to the antigen the present invention that these diseases include, but not limited to diphtheria, pertussis from the known pathogen of disease that causes; 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; Said unconventional pathogen such as Kuru disease, CJ sick (CJD), pruritus is sick; The pathogen of heritability ermine encephalopathy and chronic wasting disease, or from the albumen infectious particles like the Protein virus relevant with bovine spongiform encephalopathy.
Still in other embodiments, antigen is from neurodegenerative disease (like Alzheimer's disease), and related biotic factor obtains or generation in the outbreak of metabolic disease (like type i diabetes) and drug dependence (like 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 said recombinant nucleic acid molecules (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 property 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 the polynucleotide of the coding non-bacterial antigens treating to reach at certain detail strain invading the exterior are that codon is optimized.As another instance; 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 polynucleotide of the coding human cancer antigen 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 (like 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, at least one codon that is replaced in the codon optimization sequence is alternate by target organism be most commonly used to the to encode codon of same amino acid.
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 codon more commonly used than original password of natural coded sequence of wherein having been waited to express coded sequence at least about 20 codons substitutes.
In some embodiments, in the codon optimization polynucleotide at least about 10% codon by target organism (than original password of native sequences) more the codon of (or the most frequently used) commonly used substitute.In some embodiments, in the codon optimization polynucleotide at least about 25% codon by the target organism more the codon of (or the most frequently used) commonly used substitute.In other embodiments, in the codon optimization polynucleotide at least about 50% codon by the target organism more the codon of (or the most frequently used) commonly used substitute.Still in other embodiments, in the codon optimization polynucleotide at least about 75% codon by the target organism more the codon of (or the most frequently used) commonly used substitute.
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 find the codon option table of multiple organism on the internet with www.kazusa.or.jp/codon/ and on the obtainable website of other public.(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 ease.Following table 2B; 2C; 2D also provides Bacillus anthracis, mycobacterium tuberculosis respectively among 2E and the 2F; 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: 3262 CDS ' s (1029006 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
| UUU?29.4(30274)UUC?14.1(14486)UUA?36.8(37821)UUG?12.3(12704) | UCU?13.2(13586)UCC?6.5(6714)UCA?10.4(10751)UCG?6.1(6278) | UAU?22.9(23604)UAC?10.7(11055)UAA?2.2(2307)UAG?0.4(372) | UGU?3.8(3960)UGC?1.9(1972)UGA?0.6(583)UGG?9.3(9580) |
| CUU?21.0(21567)CUC?5.4(5598)CUA?12.9(13279)CUG?5.0(5120) | CCU?8.4(8622)CCC?1.7(1780)CCA?18.5(18996)CCG?7.0(7219) | CAU?12.0(12332)CAC?5.2(5336)CAA?29.9(30719)CAG?5.1(5234) | CGU?12.6(12930)CGC?7.0(7215)CGA?5.6(5732)CGG?2.8(2884) |
| AUU?49.3(50692)AUC?18.4(18894)AUA?9.4(9642)AUG?25.9(26651) | ACU?17.1(17614)ACC?6.9(7089)ACA?26.5(27318)ACG?12.9(13285) | AAU?33.0(33908)AAC?15.3(15790)AAA?61.6(63379)AAG?10.4(10734) | AGU?14.1(14534)AGC?8.8(9031)AGA?6.9(7111)AGG?1.2(1254) |
| GUU?26.4(27202)GUC?8.7(8990)GUA?21.6(22247)GUG?13.1(13518) | GCU?24.3(24978)GCC?8.4(8612)GCA?28.6(29401)GCG?16.6(17077) | GAU?39.8(40953)GAC?14.3(14751)GAA?60.4(62167)GAG?13.1(13507) | GGU?24.2(24871)GGC?14.2(14581)GGA?19.1(19612)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)UUC?10.4(934)UUA?43.7(3931)UUG?11.4(1024) | UCU?17.2(1547)UCC?5.0(453)UCA?14.8(1330)UCG?4.2(375) | UAU?31.9(2876)UAC?9.5(853)UAA?2.2(199)UAG?0.7(66) | UGU?5.1(455)UGC?1.8(164)UGA?0.5(47)UGG?9.3(835) |
| CUU?14.4(1300)CUC?3.7(335)CUA?12.4(1117)CUG?4.4(392) | CCU?10.7(967)CCC?2.7(242)CCA?17.8(1599)CCG?5.9(534) | CAU?15.5(1392)CAC?4.2(379)CAA?32.3(2912)CAG 9.5(859) | CG0?9.8(883)CGC?2.5(223)CGA?6.3(569)CGG?2.0(179) |
| AUU?44.5(4009)AUC?11.9(1072)AUA?22.7(2042)AUG?23.3(2098) | ACU?21.0(1890)ACC?5.0(453)ACA?26.8(2414)ACG?9.4(844) | AAU?44.0(3959)AAC?14.1(1268)AAA?64.3(5786)AAG?22.7(2047) | AGU?17.4(1565)AGC?5.2(467)AGA?13.7(1236)AGG?4.1(368) |
| GUU?20.3(1824)GUC?4.6(414)GUA?26.4(2374)GUG?10.8(973) | GCU?17.8(1598)GCC?4.1(372)GCA?23.5(2117)GCG?7.9(709) | GAU?39.3(3536)GAC?9.0(811)GAA?53.9(4855)GAG?17.9(1614) | GGU?17.9(1611)GGC?5.8(524)GGA?24.5(2203)GGG?12.0(1083) |
The 3rd alphabetical GC25.51% of coding first alphabetical GC44.99% second letter GC33.16% of GC34.55%
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)UUC?25.6(3359)UUA?1.8(231)UUG?14.8(1945) | UCU?2.0(265)UCC?11.4(1499)UCA?4.3(571)UCG?19.2(2522) | UAU?6.0(788)UAC?17.6(2307)UAA?0.4(52)UAG?0.8(103) | UGU?2.5(326)UGC?5.6(738)UGA?1.5(201)UGG?17.9(2352) |
| CUU?5.9(778)CUC?17.7(2329)CUA?4.0(521)CUG?45.9(6032) | CCU?3.9(511)CCC?18.3(2411)CCA?6.4(843)CCG?33.2(4359) | CAU?5.4(711)CAC?14.7(1928)CAA?7.8(1030)CAG?24.2(3176) | CGU?8.0(1048)CGC?26.7(3508)CGA?5.8(764)CGG?21.1(2772) |
| AUU?7.6(993)AUC?32.7(4300)AUA 2.1(282)AUG?19.7(2591) | ACU?4.1(545)ACC?36.0(4735)ACA?4.7(616)ACG?16.4(2158) | AAU?4.8(637)AAC?26.3(3451)AAA?5.8(761)AAG?26.5(3485) | AGU?4.0(531)AGC?15.0(1976)AGA?1.5(192)AGG?3.3(429) |
| GUU?8.3(1095)GUC?32.3(4249)GUA?4.7(622)GUG?35.7(4687) | GCU?11.2(1473)GCC?51.5(6769)GCA?12.4(1625)GCG?41.7(5482) | GAU?15.6(2046)GAC?44.6(5858)GAA?16.8(2211)GAG?35.8(4702) | GGU?18.7(2455)GGC?48.6(6383)GGA?9.0(1183)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 (from www.kazusa.or.jp/codon/) of Salmonella typhimurium
Salmonella typhimurium [gbbct]: 1322CDS ' s (416065 codons)
Zone: [triplet] [frequency: each is thousand years old] ([number])
| UUU?21.7(9041)UUC?15.1(6265)UUA?13.6(5650)UUG?12.1(5025) | UCU?8.5(3518) UCC?10.6(4430) UCA?7.9(3286) UCG?9.4(3924) | UAU?16.5(6853)UAC?11.6(4826)UAA?1.8(731)UAG?0.3(121) | UGU?4.6(1920) UGC?6.1(2524) UGA?1.1(465) UGG?14.1(5851) |
| CUU?12.1(5038)CUC?10.6(4396)CUA?4.7(1958)CUG?49.3(20508) | CCU?7.9(3290) CCC?7.0(2921) CCA?6.5?(2712) CCG?22.7(9463) | CAU?12.1(5047)CAC?9.2(3818)CAA?12.8(5315)CAG?30.8(12803) | CGU?18.1(7542) CGC?20.8(8659) CGA?4.1(1695) CGG?7.2(3004) |
| AUU?28.1(11700)AUC?23.9(9941)AUA?6.7(2771)AUG?26.1(10842) | ACU 8.2(3401) ACC?24.0(9980) ACA?8.0(3316) ACG?18.6(7743) | AAU?19.5(8107)AAC?21.4(8920)AAA?33.0(13740)AAG?12.4(5151) | AGU?8.6(3569) AGC?18.0(7485) AGA?3.2(1348) AGG?2.3(959) |
| GUU?16.4(6831)GUC?17.7(7367)GUA?11.9(4935)GUG?24.3(10092) | GCU?14.4(5985) GCC?27.5(11462) GCA?14.8(6156) GCG?37.0(15387) | GAU?32.9(13700)GAC?21.5(8949)GAA?36.1(15021)GAG?20.9(8715) | GGU?18.1(7541) GGC?33.0(13730) GGA?9.1(3788) 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)UUC?27.4(453)UUA?1.6(26)UUG?14.7(243) | UCU?1.9(31)UCC?11.4(189)UCA?4.5(74)UCG?20.8(343) | UAU?6.6(109)UAC?17.0c281)UAA?0.9(15)UAG?0.8(14) | UGU?2.0(33)UGC?6.7(110)UGA?1.3(22)UGG?14.3(237) |
| CUU?5.6(92)CUC?14.8(244)CUA?5.1(85)CUG?51.5(852) | CCU?2.9(48)CCC?16.3(270)CCA?5.1(84)CCG?31.0(512) | CAU?4.9(81)CAC?17.2(285)CAA?7.3(120)CAG?25.5(421) | CGU?9.4(155)CGC?33.8(559)CGA?7.1(118)CGG?26.7(441) |
| AUU?6.1(100)AUC?39.6(654)AUA?2.2(37)AUG?20.2(334) | ACU?3.1(51)ACC?36.8(609)ACA?4.4(73)ACG?17.4(288) | AAU?4.8(80)AAC?22.3(369)AAA?6.2(102)AAG?24.5(405) | AGU?2.8(46)AGC?14.5(240)AGA?1.1(19)AGG?3.8(62) |
| GUU?7.8(129)GUC?30.1(497)GUA?4.1(67)GUG?37.6(621) | GCU?9.6(158)GCC?54.3(898)GCA?12.5(206)GCG?41.7(689) | GAU?13.4(222)GAC?45.6(754)GAA?16.5(273)GAG?32.7(541) | GGU?16.9(280)GGC?42.6(704)GGA?7.3(120)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)UUC?15.1(2714)UUA?20.8(3756)UUG?13.4(2424) | UCU?16.6(2986)UCC?9.5(1717)UCA?15.6(2821)UCG?6.9(1241) | UAU?21.9(3945)UAC?11.0(1992)UAA?2.0(362)UAG?0.5(91) | UGU?6.9(1252)UGC?5.6(1011)UGA?1.4(254)UGG?13.1(2357) |
| CUU?17.6(3169)CUC?10.4(1878)CUA?7.2(1295)CUG?33.5(6045) | CCU?9.2(1656)CCC?5.9(1072)CCA?9.7(1744)CCG?12.2(2199) | CAU?15.1(2725)CAC?8.2(1472)CAA?15.9(2861)CAG?23.6(4255) | CGU?15.0(2707)CGC?12.6(2269)CGA?5.8(1046)CGG 9.0(1627) |
| AUU?30.0(5417)AUC?16.1(3018)AUA?18.9(3402)AUG?23.3(4198) | ACU?13.8(2480)ACC?13.4(2413)ACA?16.2(2930)ACG?10.0(1809) | AAU?33.5(6044)AAC?18.6(3348)AAA?41.6(7507)AAG?16.4(2961) | AGU?15.3(2764)AGC?12.7(2281)AGA?10.3(1865)AGG 5.7(1029) |
| GUU?19.8(3576)GUC?11.8(2126)GUA?13.1(2370)GUG?16.1(2910) | GCU?19.6(3527)GCC?18.5(3338)GCA?22.2(4009)GCG?15.2(2732) | GAU?34.0(6123)GAC?16.3(2939)GAA?37.5(6763)GAG?21.7(3913) | GGU?19.2(3468)GGC?15.3(2754)GGA?15.1(2727)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, below shown in embodiment in, 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 (like 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 (like 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 is included in this described arbitrary recombinant nucleic acid molecules and further comprises 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; Through site-specific integration or homologous recombination), make therefore to produce this recombinant nucleic acid promoter in the genomic DNA Already in that is operably connected and be integrated into the new expression cassette in the genomic DNA.In some other the embodiment, expression cassette is as the complete unit integration (for example, through site-specific integration or homologous recombination) to genomic DNA that comprises promoter and recombinant nucleic acid molecules.
In some embodiments, the Design Expression box is used at the antibacterial express polypeptide.In some embodiments, the Design Expression box 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 (like 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 the construct in being designed for stable integration to 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) be operably connected first and the promoter of second polynucleotide make 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, and wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) be operably connected first and the promoter of 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, and wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) be operably connected first and the promoter of 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) be operably connected first and the promoter of second polynucleotide make 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, and said 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 is allogenic each other with second polynucleotide.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 those of above description.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) be operably connected first and the promoter of 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 is allogenic each other with second polynucleotide.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, and 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) be operably connected first and the promoter of 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, comprise 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 the translation frame identical with first and second polynucleotide and (d) are operably connected first; The promoter of second and the 3rd polynucleotide; Wherein the recombinant nucleic acid molecules coding comprises signal peptide, by polypeptide and secretory protein or its segmental protein chimera of 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 said expression cassette (or said recombinant nucleic acid molecules) 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 is included in this described recombinant nucleic acid molecules comprises the hly of the polynucleotide of the recombinant nucleic acid molecules that is operably connected, actA or p60 promoter.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; United States Patent(USP) No. 6,566,121).Be applicable to that the promoter in the Bacillus anthracis includes, but not limited to the pagA promoter, AMS promoter (Pamy) and Pntr (referring to, for example, Gat 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 said expression cassette.In other embodiments, used promoter is an inducible promoter in the said expression cassette.With wherein using the endogenous molecule (for example, protein) of the antibacterial of expression cassette can induce inducible promoter.Perhaps, can use the heterologous molecule (for example, micromolecule or protein) that wherein will use 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-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 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.Nat1.Acad.Sci.USA, 97:13853-8 (2000); Garmory etc., JDrug Target, 11:471-9 (2003); Kang etc., FEMS Immunol.Med.Microbiol., 37:99-104 (2003); Garmory etc., Vaccine, 21:3051-7 (2003); Kang etc., Infec t.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, InfectImmun., 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); United States Patent(USP) No. 5,504,005; United States Patent(USP) No. 5,830,702; United States Patent(USP) No. 6,051,237; The open No.2002/0025323 of United States Patent (USP); The open No.2003/0202985 of United States Patent (USP); WO 04/062597; United States Patent(USP) No. 6,566,121; With United States Patent(USP) 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 instance, gene can contain the sequence of coding hydrophobic transmembrane domain, and this domain can suppress the effective secretion from Listera potentially.Therefore, it possibly be ideal coded sequence and the hydrophobic domains of this gene 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 to next start codon subsequently; The 50s ribosomal subunit combines with the 30s ribosomal subunit that has combined 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, express several prfA-dependency virulence factors from polycistronic message 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 through not relying on rho.
Important ground, the secretion of any or whole translated proteins of expressing from polycistronic message if desired connects each coding region on the essential function of signal peptide.In some embodiments, these signal peptides differ from one another.
Therefore, in some embodiments, the said expression cassette that is used for Listera or other antibacterials 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 the sequence that promotes ribosome entering and 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) be operably connected first and the promoter of second polynucleotide, wherein the expression cassette coding is as first and second polypeptide of two discrete polypeptide.In some embodiments, first is the polypeptide that is selected from (for example, in above IV part) said any polypeptide at this with second polypeptide.In some embodiments, at least one comprises antigen in first or second polypeptide.In some embodiments, first comprises (similar and different) fragment of same antigen separately with second polynucleotide.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 signal peptide and first fusion rotein of 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 is the polypeptide that is selected from (for example, in above IV part) described any polypeptide at this with second polypeptide.In some embodiments, at least one comprises antigen in first or second polypeptide.In some embodiments, first comprises the fragment of same antigen separately with second polypeptide.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) be operably connected first and the promoter of 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 signal peptide and first fusion rotein of 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 said arbitrary expression cassette to produce the method for recombinant bacteria (for example, the antibacterial of reorganization Listera genus).In some embodiments, the method for using said expression cassette 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 gets into antibacterial through engaging.The introducing that expression cassette gets in the antibacterial can realize through any standard technique known in the art.For example, through engaging, transduction (transfection) or be converted and carry out the introducing that expression cassette gets into antibacterial.
VII. carrier
The present invention further provides carrier, and like expression vector, it is included in this described arbitrary expression cassette 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 stated, in some embodiments, said expression cassette 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 being used to assemble expression cassette as plasmid construction body skeleton.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 accomplished mixing in the bacterial chromosome that expression cassette (and/or recombinant nucleic acid molecules) gets into Listeria monocytogenes (Listera), and the sequence-specific that said intergrase catalytic carrier gets 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; The open No.20030203472 of United States Patent (USP)).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.Plasmid construction body with required begins, and 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 ORF of comK gene, and pPL2 integrates in the tRNAArg gene, makes the native sequences of gene when successfully integrating, be restored in such a way, therefore keeps the complete of its natural expressive function.PPL1 and pPL2 integration vector comprise the MCS 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, known by one of skill in the art allele switching method is accomplished expression cassette (and/or recombinant nucleic acid molecules) and is got into mixing in the Listera chromosome.Especially, wherein need not 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 under non-permissive temperature and to select the recombinate recombinant clone of the pKSV7 plasmid to the Listera chromosome of representative.PKSV7 allele exchange plasmid vector contains the MCS 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, can pKSV7-expression cassette plasmid be introduced in the required bacterial isolates through 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, possibly hope express polypeptide from stable plasmid episome (comprising the fusion rotein that contains polypeptide).Keeping the plasmid episome through 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., 1986 J.Bacteriol 165:831-836) that selected polypeptide belongs to different gram-positive bacteriums.For further describing of the instance 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 gets into the mixing of bacterial chromosome of Bacillus anthracis, the sequence-specific that said intergrase catalytic carrier gets 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 dissolving the research of source Bacillus strain) I.A bacteriophage specific for Bacillususanthracis (Bacillus anthracis is had specific phage) J.Hyg.49:114-125).
Perhaps, known by one of skill in the art allele switching method can be accomplished the antigen presentation box and get into mixing in the Bacillus anthracis chromosome.Referring to, for example, Gat etc., Infect.Immun, 71; 801-13 (2003).Particularly, wherein need not 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 under non-permissive temperature and to select the recombinate recombinant clone of the pKSV7 plasmid to the bacterial chromosome of representative.PKSV7 allele exchange plasmid vector contains the MCS 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, can pKSV7-expression cassette plasmid be introduced in the required bacterial isolates through electroporation.U.S. Patent Application Serial 10/883,559 provides the limiting examples that in Bacillus anthracis, realizes the allele switching method, 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 in addition, be used for the multiple expression vector of antibacterial adaptable across in gram-positive bacterium, using, and comprises the recombinant bacteria carrier, is that those of ordinary skills are known.Instance 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., 71:29 33-7 (2003); Bao etc., Infec t.Immun., 71:1656-61 (2003); Kawaha ra 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); United States Patent(USP) No. 5,504,005; United States Patent(USP) No. 5,830,702; United States Patent(USP) No. 6,051,237; The open No.2002/0025323 of United States Patent (USP); The open No.2003/0202985 of United States Patent (USP); WO 04/062597; United States Patent(USP) No. 6,566,121; With United States Patent(USP) No. 6,270,776.
The present invention further provides the expression vector that comprises expression cassette, and 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 signal peptide and first fusion rotein of first polypeptide and second fusion rotein that comprises second signal peptide and second polypeptide.
The present invention further provides and has used any said expression vector to produce the method for recombinant bacteria (for example, the antibacterial of reorganization Listera genus).In some embodiments, the method for using said expression vector 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 has been included in this described recombinant nucleic acid molecules, 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, like dendritic cell.In some embodiments, cell is a bacterial cell.In some embodiments, host cell is isolating.
For example, the invention provides and be included in this described recombinant nucleic acid molecules, 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 is included in this described recombinant nucleic acid molecules, 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, has modified the escherichia coli of expressing Listera lysin O (LLO).Therefore, in some embodiments, be included in this described recombinant nucleic acid molecules, 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, be included in this described recombinant nucleic acid molecules, 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; Through being inserted in this described recombinant nucleic acid molecules, expression cassette and/or carrier are modified (for example, 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, like 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, gets into non-phagocytic cell or propagation.Can modify the antibacterial attenuation through sudden change or through 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 get into non-phagocytic cell (for example, Listeria monocytogenes internalin mutant is like the in1B 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 getting into non-phagocytic cell of attenuation.
In some embodiments, the antibacterial attenuation that is included in this described expression cassette 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 is participated in host F-actin belongs to antibacterial at Listera the raising and polymerization an of limit.The polymerization of actin subsequently causes Listera to spread all over cytosolic propelling with dissolving and gets 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 in1B and actA.The Listera bacterial strain of the embodiment of the present invention is an attenuation for getting into 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 cell-to-cells pread (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) carries out the 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 Ar p2/3 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 is that the Listera bacterial strain of selecting carries out cell and iuntercellular diffusion energy force measurement (Glomski, I J.; M.M.Gedde; A.W.Ts ang, J.A.Swanson, and D.A.Portnoy; 2002, J.Cell Biol.156:1029-1038).For example, monolayer infected back 3 days, used that to contain concentration be the culture medium of the gentamycin of 50 μ g/ml when covering, and 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 with 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 through 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 through 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 is included in this described expression cassette 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 through being modified (referring to following) with nucleic acid target compound reaction.Nucleic acid is repaired mutant, like Δ uvrAB Listeria monocytogenes mutant, and the method for preparing this mutant, be described in detail among the open No.2004/0197343 of United States Patent (USP) (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%.
Known by one of ordinary skill in the art several different methods can obtain to exist in the bacterial isolates affirmation of specific sudden change.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 through PCR.Southern blotting technique also can be used for the change in the bacterial detection genome.And, can use standard technique of this area such as western blotting whether to analyze specific protein by this bacterial strain expression.More also can obtain to contain in the required gene confirmation of the bacterial strain of sudden change through 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, 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 like the uvrAB disappearance.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.Nat1.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 get into 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 in1A.In some embodiments, antibacterial comprises the sudden change of in1B.In some embodiments, antibacterial comprises the sudden change of actA and in1B.In some embodiments, antibacterial lacks functional ActA and internalin B.In some embodiments, antibacterial is the two deletion mutants of Δ actA Δ in1B.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 gets into 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 gets into 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 gets into 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 gets into nonphagocytic ability to be reduced at least about 75%.
In some embodiments, the antibacterial of attenuation, like the Listera bacterial strain of sudden change, the non-phagocytic cell that is used to get into more than a type is not an attenuation.For example, it possibly be attenuation that the bacterial strain of attenuation is used to get into hepatocyte, is not attenuation but be used to get into epithelial cell.As another instance, it possibly be attenuation that attenuated strain is used to get into epithelial cell, is not attenuation but be used to get into hepatocyte.It should also be understood that and be used for specific modification antibacterial to get into 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, the Listera Δ in1B mutant strain non-phagocytic cell that is used for get into expressing C-MET HGFr (c-met) comprises that hepatocyte cell line (HepG2) is attenuation with the primary human hepatocyte for example.
In some embodiments, be attenuation even antibacterial (for example, the Listera of sudden change) is used to get into non-phagocytic cell, Listera still can be by phagocyte as dendritic cell and/or macrophage are taken at least.In one embodiment, attenuated bacteria gets into cytophagous ability not to be had because of to the change that bacterial strain carried out, reduce like the sudden change of invasion (that is, after change, keep bacterial strain by about 95% or the ability taken in of more phagocyte that records more).In other embodiments, attenuated bacteria gets into 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 (for example, Listera belongs to antibacterial) gets into the non-phagocytic cell ability is that twice is reduced to higher levels of decay.In some embodiments, antibacterial gets into 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 In1A, In1B, In1C, In1C2, In1D, In1E, In1F, In1G and In1H (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 in1A and in1B has been reported in Gaillard etc., Cell, and 65:1127-1141 (1991) and GenBank accession number are M67471.At Glaser etc., Science has identified 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) other members' of coding internalin GAP-associated protein GAP family gene to comprise lmo0327; Lmo0331, lmo0514, lmo0610, lmo0732; Lmo1136, lmo1289, lmo2396, lmo0171; Lmo0333, lmo0801, lmo1290, lmo2026 and lmo2821.(internalin GAP-associated protein GAP 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.).
In1A (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.
In1B (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, in1B gene regions: nts.97008-98963); With GenBank accession number no.NC_003210 (monocyte Listeria monocytogenes strain EGD; Complete genome; The in1B gene regions: nts.457008-458963), wherein each is incorporated herein by reference with its integral body at this) instruct hepatocyte or epithelial cell like the absorption of the vascular epithelial cell of the brain microvasculature that comprises blood brain barrier to Listera.(describe about the more of internalin B more, 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 get into 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., Cell 65:1127-1141 (1991) have described the algoscopy of the ability of some cell line of screening sudden change monocyte Listeria monocytogenes strain entering.For example; Whether for the non-phagocytic cell that get into 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 gets into the nonphagocytic ability of particular type and belong to antibacterial with the identical Listera that does not change and get into 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 gets into particular type, measure the nonphagocytic ability of sudden change Listera bacterial strain entering particular type and get into nonphagocytic ability and compare with the Listera bacterial strain that does not have sudden change.In addition, also can obtain bacterial strain through 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.Through measuring the LD in mice or other vertebratess
50Can measure the pathogenicity of bacterial isolates.LID
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 instance can find in the open No.2004/0228877 of United States Patent (USP).
As other instance, the degree that can also come the quantitative measurement attenuation through 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 through 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, through 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, through 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, through 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 (CFU that forms; CFU) measure the attenuation of Listera.For example, can comprise intravenous through 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 Listera and through as described competitive assessment of indices method and administration after in a period of time the drug resistance in liver and the spleen (or any other selected organs), 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, be included in this described recombinant nucleic acid molecules, the antibacterial of expression cassette and/or expression vector is the mutant of Listera.In the other embodiment, 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 (application 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 (applications on June 30th, 2004), and open No.2004/0197343 of United States Patent (USP) and US2004/0228877, 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
-In1B
-(for example, DP-L4029in1B was on October 3rd, 2003; According to the regulation of the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose, be preserved in U.S. typical case culture Culture Center (ATCC), 10801 University B1vd.; Manassas (Manassas), Virginia (Virginia) 20110-2209, the U.S. (Unitited States ofAmercian); And preserving number is PTA-5562), actA
-UvrAB
-(for example, DP-L4029 uvrAB was on October 3rd, 2003; Regulation according to the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose is preserved in American type culture collection (ATCC), 10801 University B1vd.; Manassas (Manassas), Virginia (Virginia) 20110-2209, the U.S. (Unitited States ofAmercian); And preserving number is PTA-5563), or actA
-In1B
-UvrAB
-In some embodiments, it is the two deletion mutants of Δ actA Δ in1B that the Listera of attenuation belongs to antibacterial (for example, Listeria monocytogenes).
Can pass through traditional method of mutagenesis, obtain mutant bacteria like mutation chemical drugs or radiation and then select mutant.Those skilled in the art can also obtain mutant bacteria through 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).An exemplary 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 the open No.2004/0197343 of United States Patent (USP) discloses among the No.2004/0228877 with United States Patent (USP), and 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 that (Bacillus anthracis Sterne Δ uvrAB mutant for example is on February 20th, 2004, according to the regulation of the budapest treaty of the internationally recognized microbial preservation that is used for the proprietary program purpose for the Δ uvrAB mutant of Bacillus anthracis; Be preserved in American type culture collection (ATCC); 108011 University B1vd., 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 in Bacillus anthracis, produces sudden change is through 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, the genome area of waiting to lack or suddenly change is carried out pcr amplification with about 1000bp of Bacillus anthracis genome upstream and downstream, 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 carry out through 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 through a plurality of generations is used for getting into 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 discloses No.2004/0197343 with United States Patent (USP), 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 through being modified with the reaction of nucleic acid target compound.In some embodiments, the nucleic acid of the antibacterial of change through 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), like Beta-alanine, and N-(acridine-9-yl), 2-[two (2-chloroethyl) amino] ethyl ester.In some embodiments, activate the nucleic acid target compound through 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-TMP (" S-59 ") and UVA optical processing change antibacterial.In some embodiments, through 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 (application on February 21st, 2003), 60/490; 089 (application on July 24th, 2003); 60/511,869 (application 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, through changing the concentration of S-59, UVA dosage contacted the time of S-59 and changes the time optimization deactivation condition of handling 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 through antibacterial.In addition, those skilled in the art can use
35The S-pulse chase experiment confirms that the expression of heterologous protein and virulence factor such as LLO and p60 of the Listera of S-59/UVA deactivation measures synthesizing of new expressed protein after the S-59/UVA deactivation and secrete.Use
35The LLO of S-metabolic marker and the expression of p60 can conventional determinings.Can
35The S-methionine exists down hatches the Listera 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 through the antibacterial that nucleic acid target compound such as psoralen (combining UVA to handle) 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 through 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 has been that codon is optimized with second polynucleotide for the expression in recombinant bacteria.In some embodiments, first in the recombinant bacteria has been that complete codon is optimized with second polynucleotide 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) be operably connected first and the promoter of 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 optimized and (b) second polynucleotide of coded polypeptide of codon for the expression in antibacterial; 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 be operably connected first and the promoter of 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, and wherein first polynucleotide are optimized and (b) second polynucleotide of coded polypeptide of codon for belong to expression in the antibacterial at Listera; 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 be operably connected first and the promoter of 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, can the Listera attenuation be used for cell and intercellular diffusion, get into 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 in1B) of Internalin B or ActA and Internalin B.In some embodiments, through 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 second polynucleotide of coded polypeptide (for example, antigen); 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 be operably connected first and the promoter of 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) be operably connected first and the promoter of 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 be operably connected first and the promoter of 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, can the Listera attenuation be used for cell and intercellular diffusion, get into 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 in1B).In some embodiments, through 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, the invention provides the antibacterial (for example, Listeria monocytogenes) of the reorganization Listera genus that comprises 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 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, can the Listera attenuation be used for cell and intercellular diffusion, get into 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 in1B).In some embodiments, through 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 is (for example; Non-Listera polypeptide) second polynucleotide and be operably connected first and the promoter of 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 getting into 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 in1B).In some embodiments, through 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, and N-Ras, 12-K-Ras, mesothelium is plain, 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, and H-Ras, N-Ras, 12-K-Ras, mesothelium is plain, 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 that mesothelium is plain, or plain fragment or the variant of mesothelium.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 the USP45 signal peptide 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 embodiment, signal peptide is the Tat signal peptide, like bacillus subtilis Tat signal peptide (for example, PhoD).
The present invention further provides the recombinant bacteria that comprises recombinant nucleic acid molecules, and 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, like 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 be operably connected first and the promoter of 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 signal peptide and first fusion rotein of 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), expression cassette contained in the antibacterial is integrated in the bacterial genomes.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 in which antibacterial, using.In some embodiments, promoter is the antibacterial promoter.In the other embodiment, 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 stated, in some embodiments, the promoter of expression cassette is a constitutive promoter.In other embodiments, also as stated, 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) be operably connected first and the promoter of 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, like actA
-In1B
-Bacterial strain.In some embodiments, expression cassette is 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) be operably connected first and the promoter of 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, like actA
-In1B
-Bacterial strain.In some embodiments, expression cassette is 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, like actA
-In1B
-Bacterial strain.In some embodiments, expression cassette is 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) be operably connected first and the promoter of 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
-In1B
-Mutant.
The present invention further provides antibacterial such as the Listera that comprises more than a said expression cassette.In the specific composition, given proteinic molecular weight can suppress it from the expression of recombinant bacteria like the reorganization Listera.A kind of method that addresses this problem is with " cutting apart " on the gene molecule of coding target protein and with merging 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 of cutting apart on the molecule is introduced the intergenic region that runs through bacterial chromosome, use method well known in the art, for example through the allele exchange.Another instance 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.Said polynucleotide, 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 method for production recombinant bacteria (for example, the antibacterial that the reorganization Listera belongs to), comprises that with said recombinant nucleic acid molecules 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 optimized and (b) second polynucleotide of coded polypeptide of codon for the expression in antibacterial, 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, 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 recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide, with producing recombinant bacteria in this recombinant nucleic acid molecules introducing antibacterial.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 combinations thing such as pharmaceutical composition, immunogenic composition and vaccine.These compositionss be included in this described arbitrary 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).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; (ii) said recombinant bacteria.
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, and wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; With first and the promoter of second polynucleotide of being operably connected, 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 first and the promoter of second polynucleotide of being operably connected, make the expression cassette coding comprise the fusion rotein of signal peptide and polypeptide.
The present invention further provides and has 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) be operably connected first and the promoter of second polynucleotide, wherein the expression cassette coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.
As used at 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.Can compositions of the present invention 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, like subcutaneous injection, carrier comprises water, saline, ethanol, fat, wax or buffer agent.In some embodiments, any of above carrier or solid carrier, like mannitol, lactose, starch, magnesium stearate, saccharin sodium, Talcum, cellulose, glucose, sucrose and magnesium carbonate may be used to oral administration.
Through 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, immunogenic composition is provided also.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, as the part of 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, when compositions being given host (for example mammal such as people), to be enough to induce the level to antigenic immunne response.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 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, and 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) be operably connected first and the promoter of second polynucleotide make 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) be operably connected first and the promoter of second polynucleotide make 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 in Listera, expressing; (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) be operably connected first and the promoter of 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, and 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 for the expression in Listera be codon optimized and (b) coding comprise 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 the 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, and 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 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, and 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, and 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; 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 through 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 through method in external and the 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 through being presenting of delivery cell with recombinant bacteria crowd 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 in MHC I class or II class environment, being through dendritic cell, recombinant bacteria passs T cell.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 through 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.Through the TXi Baoshouti specific recognition by the antigenic stimulus of presenting the T cell; Cause can quantitative measurement (for example; Use the ELISA algoscopy, 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 technology also clear 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 like tilactase.Can come easily to measure the increase that beta galactosidase produces through its activity to substrate such as chlorophenol red-beta galactose glycosides, it causes the change of color.Color change can be used as the next directly measurement of indicator that specific antigen is presented.
Measure other of antigen presentation of recombinant bacteria vaccine of the present invention external with body in method be that those of ordinary skills are known.Also can directly measure the expression of recombinant bacteria to specific heterologous antigen.For example, can radiolabeled aminoacid be added in the cell mass, and measure and mix the radioactivity amount 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 through the whole bag of tricks, like the ELISA algoscopy or through gel electrophoresis and western blot analysis, uses enzyme len antibody or FLA to detect.
Below embodiment 21, how to provide in those of ordinary skills' known technology some are used to measure more immunogenic concrete exemplary 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 are the immunogenic technology that is used to measure well known by persons skilled in the art.Provide antigenic these the technological representativenesses 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 through 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, following examples 20 and the algoscopy described in 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.Can standard technique such as flow cytometry and Western blotting are 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, can tumor cell 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, the vaccine combination that comprises said antibacterial is provided also.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) be operably connected first and the promoter of second polynucleotide make 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) be operably connected first and the promoter of second polynucleotide make 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) be operably connected first and the promoter of 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 is used in this described arbitrary recombinant bacteria infection.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, like antiseptic (for example, thimerosal, 2-phenyl phenol); Stabilizing agent, adjuvant (for example, aluminium hydroxide, aluminum phosphate; Cytokine), antibiotic (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 like 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 the immunne response to vaccine, like adjuvant or costimulatory molecules.Except shown in above those, possible adjuvant comprises chemotactic factor and bacterial nucleic acid sequence, like CpG.In some embodiments, vaccine comprises the antibody of raising to the immunne response of vaccine, like 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 said polynucleotide, 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 said recombinant nucleic acid molecules; 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, 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, 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 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, wherein recombinant nucleic acid molecules coded protein chimera; 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. method for using
Provide and used said recombinant bacteria 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 of treating 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, like mammalian proteins.
As used at 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, the useful or required effect about disease includes, but are 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, comprise about the useful or required effect of disease; But be not limited to following one or more: 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 treating the embodiment of cancer with said compositions, and useful or required effect comprises; But be not limited to following one or more: reduce hypertrophy (or destruction) tumor cell or the cancerous cell of 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; Delay the progress of cancer, and/or prolong cancer patient's survival.
As used at this, term " prevention " disease or " protection host " avoid disease (can be used alternatingly at this) and include, but are 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 comprise; But be not limited to following one or more: stop, postponing; 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 and induce the method for host antigenic immunne response, comprise with the said recombinant bacteria 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) be operably connected first and the promoter of second polynucleotide make 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 giving the host with the compositions that contains the recombinant bacteria of expression cassette comprising of effective dose, 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) be operably connected first and the promoter of second polynucleotide make 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 contains nucleic acid molecules comprising of effective dose is belonged 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) be operably connected first and the promoter of second polynucleotide, wherein the expression cassette coding comprises non-Listera signal peptide and antigenic fusion rotein.
In some embodiments of the method for said induce immune response, 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 through measuring to antigenic antibody titer.In some embodiments, be HI by the inductive immunne response of said compositions.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 said recombinant bacteria 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) second polynucleotide of coded polypeptide (for example, antigen and/or therapeutic mammalian proteins), wherein second polynucleotide is arranged in the translation frame identical with first polynucleotide; (c) be operably connected first and the promoter of second polynucleotide make 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) be operably connected first and the promoter of second polynucleotide make 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 contains nucleic acid molecules comprising of effective dose is belonged 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 contains expression cassette comprising of effective dose is belonged 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) be operably connected first and the promoter of 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 the disease of waiting to treat or prevent 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, give the CD4+T cellular response that the patient has caused the host with the compositions that is included in this described recombinant bacteria.In some other embodiments, give the CD8+T cellular response that the host has caused the host with the compositions that is included in this described recombinant bacteria.In some embodiments, give CD4+T cellular response and the CD8+T cellular response that the host has caused the host with the compositions that is included in this described recombinant bacteria.
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 to estimate at the model of the effect of philtrum 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 the prevention of antagonism specified disease or the ability of therapeutic effect be provided vaccine evaluation.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; Tumor models capable of using; 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 be according to gross tumor volume behind the tumor injection over time or according to the effectiveness (for example, embodiment 31D) of vaccine evaluation in such model over time of survival colony 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 through any suitable method, like Intradermal, subcutaneous; Intraperitoneal, intravenous, intramuscular; In the lymph, oral or intranasal, and through 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 order or separately with the compositions that comprises recombinant bacteria and immunostimulant simultaneously.The instance 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 instances of stimulant are provided among the above part IX.
As used at this, " effective dose " of antibacterial or compositions (like 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 appears in its complication and the disease progression process.For therapeutic use, useful or required effect comprises that clinical effectiveness as suppressing or oppressive disease, reduces the symptom (biochemistry that one or more are caused by disease; Tissue and/or behavior symptom); Comprise the middle pathology phenotype that appears 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.Like what understood in the clinical setting, medicine, the effective dose of chemical compound or pharmaceutical composition can maybe cannot combine 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,, maybe or obtain required result, then can think to have given single kind of medicament with effective dose if combine a kind of and multiple other medicaments.
In some embodiments, for the treatment of treatment for cancer property, effective dose comprises the amount that causes 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 the dosage that causes 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 cancers can start from being diagnosed as cancer maybe can combine other treatment to use as initial therapy.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 cancers 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 said recombinant nucleic acid molecules, the recombinant bacteria of expression cassette and/or expression vector.In some embodiments, recombinant bacteria is an antibacterial that change and/or sudden change, like U.S. Patent Application Serial 10/883; 599, denomination of invention is " microorganism of the free living of change, vaccine combination and a method for using thereof "; Thomas W.Dubensky, Jr etc., application on June 30th, 2004; The open No.2004/0228877 of United States Patent (USP) and United States Patent (USP) disclose those described in the No.2004/0197343, and wherein every piece is incorporated herein by reference with its integral body at this.In some embodiments, the pharmaceutical composition of comprise such change and/or sudden change antibacterial or said any other recombinant bacterias 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, be included in this described reorganization, 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=CFU).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 are released outward.
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 like dendritic cell with being included in this described recombinant nucleic acid molecules.In some embodiments; Antibacterial has been changed and/or has been mutant such as U.S. Patent Application Serial 10/883; 599; Described in open No.2004/0228877 of on June 30th, 2004 application and United States Patent (USP) 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 method for using 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; The open No.2004/0228877 of United States Patent (USP); With the open No.US2004/0197343 of United States Patent (USP), 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 like 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 said any recombinant bacteria (promptly; Be included in this described recombinant nucleic acid molecules; 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 said purposes of recombinant bacteria 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 said recombinant bacteria to be used to induce the host to antigenic immunne response, and 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 said recombinant bacteria to be used for prevention or treatment host's disease (like 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 said antibacterial is contacted with antigen-presenting cell.
The present invention further provides and has induced the method for host to antigenic immunne response, may further comprise the steps: (a) appropriate condition be enough to antigen loaded and be under the time of delivery cell, said recombinant bacteria is contacted with antigen-presenting cell from the host; (b) give the host with antigen-presenting cell.
Recombinant nucleic acid molecules, other of expression cassette and antibacterial possibly purposes be that those of ordinary skills can be cognitive.For example, said recombinant nucleic acid molecules, 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) is with (for example, through transfection, transforming or engage) in said expression cassette 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) is with (for example, through transfection, transforming or engage) in said expression cassette 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 from cell culture, separating excretory or non-secretory proteic method.
Embodiment
Embodiment below providing explains 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 through 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); The open No.2003/0203472 of United States Patent (USP))).ActA
-UvrAB
-The structure of bacterial strain is described in the U.S. Provisional Application 60/446,051, on February 6th, 2003 application, and like L4029/uvrAB (referring to, for example, the embodiment 7 of this application), and the open No.2004/0197343 of United States Patent (USP).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 is 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.Below in application or the publication other descriptions about the sudden change Listera are provided, wherein every piece is incorporated herein by reference with its integral body at this: the open No.2004/0228877 of United States Patent (USP); The open No.US2004/0197343 of United States Patent (USP); 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 instance Listeria monocytogenes Δ actA Δ inIB are on October 3rd, 2003, according to the regulation of 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); The open No.2003/0203472 of United States Patent (USP)) 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 the antigen presentation box (pPL2/LLO-OVA) in the pPL2 integration vector.Through 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, template and primer below using:
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, template and primer below using:
Source: from the colibacillary pDP3616 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 accomplishing building process comprises at first and cuts the LLO amplicon with KpnI and BamHI, and with in the KpnI/BamHI carrier insertion 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 through restriction analysis (KpnI-LLO-XhoI-OVA-NotI) and order-checking.Through 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 like inlB through engaging
-Mutant or inlB
-ActA
-Two deletion mutants).
B. express the structure of the Listera bacterial strain of AH1/OVA or AHI-A5/OV.
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 (AH1 is last):
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 through restriction analysis and/or order-checking.
Through transforming plasmid introduced escherichia coli then, then to introduce and be integrated in the Listera (DPOL4056) through engaging, described according to Lauer etc. fully, or introduce in the another kind of required Listera bacterial strain, like 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), through modify 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, hly promoter and (selecting) signal peptide sequence are inserted between the unique KpnI and BamII site in pPL2 or the pAM401-MCS plasmid vector.Between unique BamHI and SacI site, selected EphA2 gene (is sometimes held the epitope label through modifying to contain N-end and C-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 based on the molecule construction body of pAM401-MCS plasmid vector through electroporation.Verify the plasmid structure of expecting in the Listera transfectant through restriction enzyme analysis through 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, be 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 before described method [Lauer etc., JBacteriol.184,4177-4186 (2002)].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)) through electroporation or through chemical method.Subsequently, will be transferred to the selected Listera bacterial strain from the SM10 that transforms based on the plasmid of pPL2 through engaging.As the described drug selectivity BHI agar plate that contains 7.5 every ml of μ g chloromycetin and the every ml of 200 μ g streptomycins on hatch after, come the selected bacterium colony of purification for 3 times through 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 through 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.Plasmid based on pPL2 mixes the tRNA in the selected Listera strain gene group
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 in the microenvironment of infection cell, is activated.From monocyte Listeria monocytogenes strain DP-L4056 amplification of nucleotide 205586-206000 (414bp), the primer shown in use is following is right through 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 following:
Primer is right:
Forward (KpnI-LLO nts.1257-1276):
5′-CTCT
GGTACCTCCTTTGATTAGTATATTC(SEQ?ID?NO：74)
Oppositely (BamHI-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.
Through from pCR-XL-TOPO-hly promoter plasmid clone, discharging 422bp DNA corresponding to hly promoter and secA1 LLO signal peptide with KpnI and BamHI digestion; And be cloned into pPL2 plasmid vector (Lauer etc.; 2002 J.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 (through 16SrRNA) 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).
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 human 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 human 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.
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. the expression cassette that has optimized secA1 signal peptide of codon and the optimized people EphA2 of codon 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.
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 human 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 human 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.
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.
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 Design Expression box 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 come the Design Expression box 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′ATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCTGACACA?3′(SEQ?ID?NO：86)
For the optimized codon of the expression in Listeria monocytogenes:
5′ATGAAAAAAAAAATTATTAGTGCAATTTTAATGAGTACAGTTATTTTAAGTGCAGCAGCACCATTAAGTGGTGTTTATGCAGATACA?3′(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 combine to express and comprises Usp45 signal peptide and required antigenic fusion rotein with codon optimization or the optimized antigen sequence of non-codon.
Embodiment 13. codings are best with the antigenic codon that secA2 signal peptide (p60) merges
Change expression cassette and carrier
A. the design of codon optimization expression cassette
Use the secA2 secretory pathway to come the Design Expression box 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′ATGAATATGAAAAAAGCAACAATTGCAGCAACAGCAGGTATTGCAGTTACAGCATTTGCAGCACCAACAATTGCAAGTGCAAGTACA3′(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, the primer below using and pPL2-hlyP-OVA (identical with the pPL2/LLO-OVA described in the above embodiment 2A) are as first template:
pPL2-5F：5′-GACGTCAATACGACTCACTATAG(SEQ?ID?NO：92)
p60-hlyP-237R：5′-CTTTTTTCATATTCATGGGTTTCACTCTCCTTCTAC(SHQ?ID?NO：93)
The size of resultant amplicon is 285bp.
Also use Pfx or Vent polymerase to carry out first independent PCR reaction, primer below using and pCR-TOPO-p60 are 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 following:
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:
Kpn1-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 through 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 regional pAM401-p60 plasmid (KpnI/PstI is with the KpnI/BamHI fragment) of p60 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.
It is interior and be arranged in the translation box identical with the p60 sequence to use antigen encoding sequence that technology known to a person of ordinary skill in the art will be required to insert the p60 sequence then.Usually, insertion should make that the terminal signal peptide sequence of p60 N-is complete.Also should make the autolysin sequence of complete that p60 C-is terminal.
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 plain optimized polynucleotide sequence of codon of coding human mesothelium 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 plain optimized polynucleotide sequence of codon of coding Mus mesothelium 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, through 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 through 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) through in not containing the BHI culture medium of chloromycetin, under 30 ℃ permissive temperature, several single bacterium colonies being gone down to posterity.Verify that through 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 EphA2 outside (EX2) and Cytoplasm (CO) domain of EphA2 transbilayer helix both sides, be used for inserting various pPL2-signal peptide expression construct.Use is corresponding to natural mammal sequence or for EphA2 EX2 and the expression codon optimized gene of CO domain 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 through extending eclipsed oligonucleotide.Verify the expected sequence of all synthetic EphA2 constructs through nucleotide sequencing.
The primary amino acid sequence of the EX2 of EphA2 and CO domain, and natural and the optimized nucleotide sequence of codon are shown in (CO domain sequence) among Fig. 4-6 (EX2 sequence) and Figure 15-17.
In addition; In the amino of synthetic EphA2 EX2 and CO gene and carboxyl terminal frame, insert FLAG (Stratagene respectively; La Jolla; CA) and myc epitope label, be used for detecting and express and excretory EphA2 the antibody of use FLAG or protein specific through western blot analysis.Therefore, expressed proteins has following orderly element: NH
2-signal peptide-FLAG-EphA2-myc-CO
2Below shown in be 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 through the western blot analysis of the sedimentary inoculum of trichloroacetic acid (TCA).In brief; With the Listera culture collection that grows in the mid-log phase in the BHI culture medium in 50mL taper centrifuge tube; With bacterial precipitation, and with hatching minimum 90 minutes to [6%] final concentration and on ice in the ice-cold TCA adding bacterial cultures supernatant or spending the night.Through 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 through vortex.If desired, through adding NH
4OH regulates sample pH value, is pink until color.Made all samples in 10 minutes and be used for electrophoresis through adding 100 μ l4 * SDS sample loading buffer 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 through 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).Through 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
With merging in the native sequences of EphA2 CO domain and the heredity of natural secA1 LLO sequence, and as stated between KpnI and SacI site with in the Listera hly promoter control heterologous antigen expression cassette insertion pPL2 plasmid down.As stated through being engaged to Listera bacterial strain DP-L4029 (actA
-) and DP-L4017 (L461T LLO) in introduce pPL2-EphA2 plasmid construction body.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 stated 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) through being engaged to as stated.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 the total length EphA2 EX2 domain protein white matter that optimized signal peptide and heterologous protein cause expecting of expression select to(for) preferred codon in the Listeria monocytogenes.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 as stated between KpnI and SacI site with in the Listera hly promoter control heterologous antigen expression cassette insertion pAM401-MCS plasmid down.Through electroporation pAM401-EphA2 plasmid construction body is introduced among the Listera bacterial strain DP-L4029 (actA) as stated.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 phoDTat of codon signal peptide.This analysis has proved once more and has been used in combination the total length EphA2 CO domain protein that optimized signal peptide and heterologous protein cause expecting of expression select to(for) preferred codon in the Listeria monocytogenes.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.
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 human Eph2A is arranged
The following digital proof that appears 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 combines to have irregular variation for the T-cell receptors that improves) has been given long-term surviving (Figure 41).
EphA2 CO domain has intensive immunogenicity; When with the immunity of the reorganization Listera 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, shown in following table 4.
Table 4. is through 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 multiple external and the 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.Provide test body outer non-limiting example with the interior immunne response method of body among this embodiment.Antigen was pattern antigen described in these tests specifically described, and was not necessarily to use said recombinant nucleic acid molecules, 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 comprises said recombinant nucleic acid molecules, immunne response in the external or body of the antibacterial of expression cassette and/or expression vector.
For example, through 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) through 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, process single-cell suspension liquid and substitute the splenocyte in 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.
Only if point out in addition, 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 use any target antigen to substitute.LLO antigen is specific to Listera.Detect cytokine (for example, IFN-γ) release during through identification specificity antigen and test specific T-cells.With anti-Mus IFN-γ monoclonal antibody (mAb R4; 5 μ g/ml) dull and stereotyped (BD Biosciences, San Jose CA) spends the night based on 96 holes 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 in the presence of the peptide of the various concentration of 0.01 to 10 μ M, hatches 20 to 22 hours at 37 ℃.The peptide that is used for OVA and LLO is SL8, the MHCI class epitope of OVA, LLO
190(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 LLO
91(GYKDGNEYI (SEQ ID NO:102)), the MHCI 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, dull and stereotyped to the biotinylated antibody incubation of the specific secondary of IFN-γ (XMG1.2) of 0.5 μ g/ml with being diluted among the PBS.After 2 hours, washing is dull and stereotyped also with being diluted in the 1nm Kingsoft goat-anti biotin conjugate (GAB-1 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 beam color 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 through the flow cytometry analysis.The splenocyte of inoculation group and control group mice was hatched 5 hours in the presence of brefeldin A (Pharmingen) with SL8 (stimulating OVA specific C D8+ cell) or LLO190 (stimulating LLO specific C D4+ cell).The secretion of the cytokine that the brefeldin A suppressor T cell is produced when stimulating.The splenocyte that the irrelevant MHC I class peptide of usefulness is hatched 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--CD4 mAb (RM4-5) resists with PerCP--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.Through flow cytometer (FACScalibur; Becton Dickinson; MountainView CA) measures the percentage ratio of expressing interior IFN-γ of born of the same parents and/or TNF-α cell and comes analytical data with use CELLQuest software (Becton Dickinson, Immunocymetry System).Because the fluorescent labeling on the various antibody can all be distinguished through FACScalibur, identify suitable cell with any or two kinds of painted those CD8+ among anti-I FN-γ or the anti-TNF-α and CD4+ through 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: come further to estimate OVA specific C D8+T cell at their cellular cytoxicity activity of C57B1/6 mice body build-in test through external or direct.The CD8+T cell is discerned with 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 crowd, 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 the 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 SC according to 100 * (experiment release-spontaneous release)/(maximum release-spontaneous release).Do not have hatching of effect cell through target cell and measure spontaneous release.Through 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 through vortex.The CFSE liquid storage of ten times of dilutions is added in the control cells suspension, and come biased sample through vortex.Cell was hatched 10 minutes at 37 ℃.A large amount of through adding (＞40ml) ice-cold PBS stops dyeing.Cell is used the PBS washed twice in room temperature, then resuspension and counting.Every kind of cell suspending liquid is diluted to 50 * 10
6Every ml, and every crowd 100 μ L are mixed and through 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 through 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 hEphA2 Fc fusion rotein.Use 96 hole speckle readers to measure IFN-γ positive spots and form bacterium colony (SFC).Shown in figure 45, use the splenocyte that is derived from the mice that has inoculated Listera-hEphA2 to observe the IFN-γ SFC of increase.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 through 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) through 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 CD4+ and CD8+ exhaust all fails to be illustrated in 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 is included in this described recombinant nucleic acid molecules 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 in1B gene or both have lacked.A kind of exemplary process that produces the Listera deletion mutant has below been described.
Can exchange through allele and realize the disappearance of internalin B gene (in1B) from Listera DP-L4029 (or from other selected mutants or from the wild type Listera); Like 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 Interialin B gene is with GenBank accession number AL591975 (monocyte Listeria monocytogenes strain EGD, complete genome group, sections 3/12; In1B 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 in1B 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 in1B gene 5 ' and 3 ' end upstream and downstream, template and primer below using:
Template: DP-L4056 or DP-L4029 genomic DNA
Primer (is used for the amplification of in1B 5 ' end upstream region) to 1:
Lm-96031F：5′-GTTAAGTTTCATGTGGACGGCAAAG(SEQ?ID?NO：103)(T
m：72℃)
Lm-(3′in1B-R+)97020R：
5′-
AGGTCTTTTTCAGTTAACTATCCTCTCCTTGATTCTAGTTAT(SEQ?IDNO：104)(T
m：114℃)
(sequence of leukorrhagia line and the downstream area of In1B carboxyl terminal are complementary).
Amplicon size (bp): 1007
Primer (is used for the amplification of in1B 3 ' end downstream area) to 2:
Lm-(5′in1B-F+)98911F：
5′-
CAAGGAGAGGATAGTTAACTGAAAAAGACCTAAAAAAGAAGGC (SEQ?IDNO：105)(T
m：118℃)
(sequence of leukorrhagia line and the aminoterminal upstream region of in1B are complementary).
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 1st pair the forward primer of reverse primer and the 2nd pair through SOE PCR.This has caused the accurate disappearance of in1B 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 accomplishing building process is following:
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 through 1% agarose gel.The reactant gel purification of first PCR is also come eluted dna with GeneClean (BIO101).
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 through 1% agarose gel.Add the adenosine residue with the Taq polymerase at 3 ' end of Listera d1 in1B amplicon.
Then Listera d1 in1B amplicon is inserted in the pCR2.1-TOPO carrier.With XhoI and KpnI digestion pCR2.1-TOPO-d1 in1B DNA and gel-purified 2123bp fragment.The KpnI/XhoI2123bp fragment is inserted through digesting with KpnI and XhoI and handling in the pKSV7 carrier (pKSV7-d1 in1B) that makes with CIAP.Verify the fidelity of d1 in1B sequence among the pKSV7-d1in1B then.Allele exchange through with pKSV7-d1 in1B plasmid lacks the in1B gene from required Listera bacterial strain.
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 | MKKIMLVFTTLILVSLPLAQQTAKDASAFNKENSISSMAPPASPPASPKTPIEKKHAD(SEQ?IDNO：109) 1 | TEA’KD(SEQ?IDNO：54) | ?ATGAAAAAAATAATG?CTAGTTTTTATTACAC?TTATATTAGTTAGTCT?ACCAATTGCGCAACA?AACTGAAGCAAAGGA?TGCATCTGCATTCAAT?AAAGAAAATTCAATT?TCATCCATGGCACCA?CCAGCATCTCCGCCTG?CAAGTCCTAAGACGC?CAATCGAAAAGAAAC?ACGCGGAT(SEQ?ID?NO：110) | ?ATGAAAAAAATTATGTT?AGTTTTTATTACATTAAT?TTTAGTTAGTTTACCAAT?TGCACAACAAACAGAAG?CAAAAGATGCAAGTGCA?TTTAATAAAGAAAATAG?TATTAGTAGTATGGCACC?ACCAGCAAGTCCACCAG?CAAGTCCAAAAACACCA?ATTGAAAAAAAACATGC?AGAT(SEQ?ID?NO：113) | Hly (LLO) [Listeria monocytogenes] |
| MKKKIISAILMSTVILSAAAPLSGVYADT(SEQ?IDNO：46) | VYA’DT(SEQ?IDNO：55) | ?ATGAAAAAAAAGATT?ATCTCAGCTATTTTAA?TGTCTACAGTGATACT?TTCTGCTGCAGCCCCG?TTGTCAGGTGTTTACG?CTGACACA(SEQ?ID?NO：86) | ?ATGAAAAAAAAATTAT?TAGTGCAATTTTAATGAG?TACAGTTATTTTAAGTGC?AGCAGCACCATTAAGTG?GTGTTTATGCAGATACA?(SEQ?ID?NO：87) | Usp45 [lactococcus lactis] | |
| MKKRKVLIPLMALSTILVSSTGNLEVIQAEV(SEQ?IDNO：47) | IQA’EV(SEQ?IDNO：56) | ?ATGAAAAAACGAAAA?GTGTTAATACCATTAA?TGGCATTGTCTACGAT?ATTAGTTTCAAGCAC?AGGTAATTTAGAGGT?GATTCAGGCAGAAGT?T(SEQ?ID?NO：111) | ?ATGAAAAAACGTAAAGT?TTTAATTCCATTAATGGC?ATTAAGTACAATTTTAGT?TAGTAGTACAGGTAATTT?AGAAGTTATTCAAGCAG?AAGTT(SEQ?ID?NO：114) | Pag (protective antigen) [Bacillus anthracis] | |
| secA2 | MNMKKATIAATAGIAVTAFAAPTIASAST(SEQ?IDNO：48) | ASA’ST(SEQ?IDNO：57) | ?ATGAATATGAAAAAA?GCAACTATCGCGGCT?ACAGCTGGGATTGCG?GTAACAGCATTTGCT?GCGCCAACAATCGCA?TCCGCAAGCACT?(SEQ?ID?NO：90) | ?ATGAATATGAAAAAAGC?AACAATTGCAGCAACAG?CAGGTATTGCAGTTCAG?CATTTGCAGcACCAACA?ATTGCAAGTGCAAGTAC?A(SEQ?ID?NO：91) | Iap invades GAP-associated protein GAP p60 [Listeria monocytogenes] |
| Tat | MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLSLGLTIAQSVGAF(SEQ?IDNO：53) | VGA’F(SEQ?IDNO：62) | ?ATGGCATACGACAGT?CGTTTTGATGAATGG?GTACAGAAACTGAAA?GAGGAAAGCTTCAA?AACAATACGTTTGAC?CGCCGCAAATTTATTC?AAGGAGCGGGGAAGA?TTGCAGGACTTTCTCT?TGGATTAACGATTGC?CCAGTCGGTTGGGGC?CTTT(SE?ID?NO：112) | ?ATGGCATATGATAGTCGT?TTTGATGAATGGGTTCAA?AAATTAAAAGAAGAAAG?TTTTCAAATAATACATT?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
Use non-Listera secA1 signal peptide to come the Design Expression box to be 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 combine to be used for to express and comprises Bacillus anthracis PA signal peptide and required antigenic fusion rotein with codon optimization or non-codon optimization antigen sequence.
Embodiment 27. from the reorganization Listera that comprises codon optimization expression cassette express with
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 through 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; From the secretion of reorganization Listera, the codon optimization through conjugated antigen and signal coding sequence has obtained optimization, and described two kinds of antigens are and carcinoma of prostate and ovarian cancer (mesothelium is plain); 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 is with secA1 or comprise that secA2 is relevant with the alternative secretory pathway of two-Arg displacement (Tat), and human tumor antigen-people NY-ESO-1 that frame is interior with selected or people's mesothelium are plain to be merged.(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 through having and has comprised that to express/secrete people's mesothelium plain with the Δ actA Listeria monocytogenes of the construct of the plain LLO signal peptide that merges of people's mesothelium, for LLO and mesothelium element, uses natural codon.Through the western blot analysis of the sedimentary inoculum of TCA, use these constructs not observe the secretion of desired total length mesothelium plain (61kDa), only observe the secretion (Figure 48 A) of several small fragments.
Figure 48 B has shown that this plasmid contains the construct of coding and the plain various signal peptides that merge of people's mesothelium through the plain western blot analysis of Listeria monocytogenes Δ actA expression/secretion people mesothelium that comprises plasmid (pAM401).In each construct, the plain coded sequence of mesothelium is that codon is optimized for the expression in Listeria monocytogenes.Wherein, 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, through preparing this antibody to selected peptide and the IFA of rabbit injection.
Notably, shown in swimming lane 3-5 and the 8-9 among Figure 48 B, have only when signal peptide and the plain coded sequence of 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 plain (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 the plain secretion (62kDa) (Figure 48 B, swimming lane 3) of total length mesothelium, 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 plain secretion (Figure 48 B) of total length mesothelium.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 (Fig. 4 8B, swimming lane 4 and 5) of total length mesothelium plain (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 maybe 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 in1B; Said 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 like the occurrence rate through per 1000 codons in the genomic coded sequence of Listera? Species=Listeria+monocytogenes+ [gbbct]).That belong to other gram-positive bacteriums from Listera 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 needs the optimization (Figure 48 B) of these sequences from effective secretion of the Listera of recombinating with NY-ESO-1.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 through this mechanism.Originally supposed through transporting folding to have the tumor antigen that helps contain remarkable hydrophobic domains before, like 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.
Comparing with parent's Listera Δ actA/ Δ in1B bacterial strain in important ground, utilizes the phenotype of the recombiant vaccine of any tumor antigen secretory pathway not receive appreciable impact.Half fatality rate (the LD of Listera Δ actA/ Δ in1B in the C57BL/6 mice
50) be 1 * 10
8Cfu.The stable single copy locus specificity that uses the pPL2 integration vector to accomplish the harmless site on the tumor antigen expression cassette entering Listera Δ actA/ Δ in1B chromosome mixes.The LD of tumor antigen coding Listera Δ actA/ Δ in1B
50Within 5 times of Listera Δ actA/ Δ in1B.
The structure of embodiment 28. bicistronic mRNA hEphA2 expression vectors
As non-restrictive example, provided the structure of antigen presentation box, wherein from the outside (EX2) of bicistronic mRNA information generating hEphA2 and inner (CO; Kinase dead) expression of domain.Through being connected the secretion of accomplishing 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 through 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 the MluI recognition sequence.
The sub-fragment of synthetic people EphA2 between two MluI restriction endonuclease recognition sequences (membrane spaning domain disappearance, kinases lost efficacy) (through method for synthesizing gene known in the art, for example synthetic through 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 MluI (intergenic region overstriking demonstration) that has shown the codon optimization people EphA2 that contains the actA-plcB intergenic region among Figure 50.In addition, with codon optimization Bs phoD signal peptide be positioned over the actA-plcB intergenic sequence 3 ' end and with EphA2 CO domain coding region, downstream frame endomixis.
Assembling function property people EphA2 bicistronic mRNA box is come in corresponding region through in the people EphA2 sequence that substitutes the kinases inefficacy that membrane spaning domain lacks shown in Figure 49 with the MluI 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 following: 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.
Through 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 EphA2 EX and CO domain.Use said and/or method known to those skilled in the art, the western blot analysis through required antibacterial part detects proteic expression of EphA2 and secretion.
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 heterologous protein functional nucleotide sequence property 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 through 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, mesothelium is plain, between the protein chimera.Through with human tumor antigen; Mesothelium is plain, puts into Listeria monocytogenes albumen p60 at amino acid position 70 exactly and produces 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 in the expression of Listera p60 amino acid/11-70 with the plain coded sequence of complete mesothelium.In addition; With connecting Listeria monocytogenes hly promoter on p60-people's mesothelium fibroin matter chimera function; Mix in the pPL2 carrier, this carrier of use as described herein is subsequently produced and is expressed and the plain reorganization monocyte Listeria monocytogenes strain of secretion people mesothelium.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 protein chimera to secrete through Listeria monocytogenes expressed proteins and the best of the effect each other of 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 said proteinic Peptidoglycan cytohydrolists active.(referring to, Lenz etc., (2003 PNAS 100:12432-12437), for example, are used to describe SecA2-dependency NamA and p60 protein).In some embodiments, hope 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, with connecting prfA-dependency promoter 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 CKIs matter translation efficiency).Synthetic corresponding to the amino acid whose DNA fragment of the terminal p60 of hly promoter-70N-.(this can carry out through method for synthesizing gene known in the art usually, and is for example synthetic through 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 PstI 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 corresponding K pnI and the PstI site of pPL2 carrier, and handles through 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 PstI and BamHI site.Through 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 PstI 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 through 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 that contains total length people mesothelium element from embodiment described in 27 is plain; Or the mesothelium in disappearance signal peptide and GPI connected structure territory plain (the plain Δ SP/ of mesothelium Δ GPI); Contain and be useful on the best codon of in Listeria monocytogenes, expressing, use heat-stabilised poly synthase with proofreading activity and following primer right:
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 (the total length mesothelium is plain) 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); Through using the PstI digestion process, and handle with CIAP.Confirm the N-CO orientation that the plain domain of p60 and mesothelium is consistent through 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 (like the two deletion mutants of Δ actA Δ in1B), as it is described to be applied in this embodiment that comprises.
Shown the plain sub-fragment sequence of KpnI-BamHI of plasmid pPL2-hlyP-Np60 CodOp (1-77)-mesothelium 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.Through come the expression of test protein chimera through the two deletion mutants of reorganization Listeria monocytogenes Δ actA Δ in1B and the secretion to the culture medium through western blot analysis in the method described in this contained embodiment; The tRNA-Arg chromosome that this mutant contains through pPL2-hlyP-Np60 CodOp (the 1-77)-mesothelium element of said method generation 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 terminal aminoacid of the plain N-of h mesothelium (for the plain sequence of people's mesothelium; Referring to, for example, Figure 34 or GenBankAcc.No.BC009272).GPI (Δ GPI) domain of disappearance is corresponding to 42 terminal aminoacid of C-, start from amino acid residue Gly-Ile-Pro with 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.(the Y axle 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 plain expression of total length mesothelium 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 and the nucleotide of V on the coding site 29 and 64 have respectively 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 the 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 merging the plain LLO of total length people mesothelium.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.
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 with 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 in1B 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.Listera promoter hly from the LLO gene is used 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 operationally (on the function) connects the codon optimization sequence (first polypeptide) and the codon optimization sequence (second polypeptide) (swimming lane 1) of bacillus subtilis phoD secretion signal of EphA2 born of the same parents' intracellular domain of being operably connected of encoding of the lactococcus lactis Usp45 secretion sequence of EphA2 ectodomain coded sequence; (2) the coding codon optimization sequence (first polypeptide) and 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 being operably connected that encodes of bacillus anthracis protective antigen secretion sequence of EphA2 ectodomain coded sequence that be 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 in1B 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 the fast band that moves, and wherein moves band soon 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 shows based on the expression of plasmid and secretion conduct and 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 wherein this plasmid-encoded mesothelium is plain.Test is based on the construct of various plasmids, and wherein signal sequence is different.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 that conduct is plain with the Mus mesothelium of the fusant expression of bacillus anthracis protective antigen signal sequence; Swimming lane 3-4 has shown that conduct is plain with the Mus mesothelium of lactococcus lactis Usp45 signal sequence fusant expression; Swimming lane 5-6 has shown that conduct is plain with the Mus mesothelium of the fusant expression of bacillus subtilis phoD signal sequence; Swimming lane 7-8 shown that the Mus mesothelium that the conduct and the fusant of p60 signal sequence express is plain, and swimming lane 9-10 has shown that the plain and swimming lane 11 of Mus mesothelium that the conduct and the fusant of LLO signal peptide express has shown the expressed proteins by contrast host Listera Δ actA Δ in1B.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, expression and secretion that the test person mesothelium is plain.It all is Δ actA Δ in1B Listera and as follows that the Listera of test belongs to antibacterial: Listera Δ actA Δ in1B (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 plain bacillus subtilis protective antigen signal sequence of Δ SS/ Δ GPIh mesothelium; Coding merges the Listera (swimming lane 4-5) of the plain bacillus subtilis phoD signal peptide of Δ SS/ Δ GPI h mesothelium; Coding merges the Listera (swimming lane 6-7) of the plain bacillus anthracis protective antigen signal sequence of total length h mesothelium; Coding merges the Listera (swimming lane 8-9) of the plain bacillus subtilis phoD signal sequence of total length h mesothelium.
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 through integrating with pPL2.
When the plain through engineering approaches of people's mesothelium was lacked its signal sequence with 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 is with 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 through 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 p6 0 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 through 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 axle 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 axle of figure.When use comprises that when inoculating based on the Listera of the construct of LLO, bacterial strain is expressed as " LLO " on the x axle of figure.
Inoculation is expressed based on the full experiment scheme of the Listera of p60 construct following: the Listera (tail vein (i.v.)) of integrating nucleic acid is contained in (1) to mouse inoculation; The nucleic acid coding p60 that wherein integrates, it contains the nucleic acid of the AH1-A5 of nucleotide 199 insertions that are coded in p60.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 (Fig. 5 9A) 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 through 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, the peptide that wherein adds are that the peptide of AH1 (Figure 59 A) or wherein adding is 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, the experiment of promptly carrying out like the identical Listera vaccine with two kinds shown in the B of Figure 59 A.Figure 60 A shown give mouse inoculation through through engineering approaches to contain Listera (" p60 ") or inoculation through the result of through engineering approaches when containing Listera (" LLO ") based on the construct of LLO based on the construct of p60.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 AH1-A5 of encoding 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, combines endogenous p60 and expressed p60 albumen chimera sequence to cause for the construct based on p60.
B. the therapeutic efficiency of the plain Listera of expressing human mesothelium
Result shown in Figure 61 has shown that the Listera of inoculation expressing human mesothelium plain (the hu mesothelium is plain) has prolonged the survival that has mice with tumor, wherein comes the expressing human mesothelium plain tumor cell through engineering approaches in the mice.Tumor cell is the plain CT26 cell of expressing human mesothelium, and mice is the Balb/c mice.(all said CT26 tumor researches 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 plain codon optimization sequence (having lacked its signal sequence and GPI anchor) of coding human mesothelium 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 plain (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 CFU (CFU) Listera (i.v.).
Figure 61 has shown the percentage ratio survival (be shown in y axle on) of mice to the plain CT26 tumor of expressing human mesothelium, and wherein vaccine comprises Hank ' s balanced salt solution (HBSS) (sham vaccine; " HBSS "); Express Listera Δ actA Δ in1B (the positive control vaccine of SF-AH1A5 from the expression cassette of integrating; " SF-AH1A5 "); Or comprise the Listera Δ actA Δ in1B 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 plain (by the sequential coding of codon optimization) merge, wherein hu mesothelium element has lacked signal sequence and the zone (" BaPA-huMeso Δ gpi Δ ss ") that has lacked 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 codon optimization nucleic acid and the coding source of the AH1-A5 that the encodes non-codon optimization nucleic acid from the ovalbumin sequence.
The result has proved with the single immunization of expressing the plain Listera of hu mesothelium and 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 plain (the plain Δ gpi of the BaPA-hu mesothelium Δ ss of Δ signal sequence/Δ gpi hu mesothelium; 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 reduced the level that lung tumor is cut knot through the plain Listera Δ actA Δ in1B of inoculation expressing human mesothelium, wherein come the expressing human mesothelium plain the tumor cell through engineering approaches.Mouse species is Balb/c, and lung tumor cell is the CT26 cell that has the plain carrier of expressing human mesothelium.At the 0th day, with 2 * 10
5The plain CT26 cell intravenous of individual expressing human mesothelium gives Balb/c mice.The sequence of the various signal sequences of coding is operationally connected the plain codon optimization sequence of coding human mesothelium in the frame in expression cassette.The mice that the coding and the expression cassette of the various signal peptides of people mesotehlin fusion is had tumor through 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 in1B 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 in1B 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 plain (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 people's mesothelium element sequence 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 wt CT26 (at the 0th day 2 * 10
5Individual cell (i.v.).The antitumor efficacy that the Listera vaccine of mesothelium plain fusion protein is expressed in the inoculation of research proof is that the mesothelium element is specific.The sequence of the various signal sequences of coding is operationally connected the plain codon optimization sequence of coding human mesothelium in the frame in expression cassette.(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 through 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 the expressing human mesothelium plain.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 HBSS or Listera Δ actA Δ in1B.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 not coming the expressing human mesothelium plain the tumor cell through engineering approaches, 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 plain Listera of inoculation expression codon optimization people's mesothelium has reduced gross tumor volume
Figure 64 has shown that the plain Listera (Δ actA Δ in1B) of people's mesothelium that inoculation is expressed from the expression cassette that comprises the plain codon sequence of codon optimization mesothelium has reduced gross tumor volume.
The sequence of the various signal sequences of coding is operationally connected the plain codon optimization sequence of coding human mesothelium in the frame in expression cassette.The mice that the coding and the expression cassette of the plain various signal peptides that merge of people's mesothelium is had tumor through the Listera vaccine that contains expression cassette.Being used for inoculating the plain Listera vaccine of expressing human mesothelium that has mice with tumor in this research comprises following: the Listera (Δ actA Δ in1B 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 CFU (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 the different time point, measure mean tumour volume.
The result who has shown this research among Figure 64.The result has proved that the plain Listera of people's mesothelium that inoculation is expressed and 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).It is protectiveness (hollow triangle) with the plain Listera of plasmid-encoded people's mesothelium that the LLO signal peptide merges that coding is expressed in inoculation.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 plain (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 in1B-people mesothelium, and wherein Listera contains the chromosomal integration nucleic acid of people's mesothelium plain (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: mice (Balb/c mice or C57BL/6 mice) inoculation (i.v.) Listera is given in (1); 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 plain (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 from spleen, take out 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.Accept medium alone (" ") that does not stimulate, the plain peptide set of mesothelium (" Meso set "), or p60 with the splenocyte of Balb/c mice study
217-225(" p60
217").Accept medium alone (" not stimulating "), the plain peptide set of mesothelium (" Meso set "), or LLO with the splenocyte of C5 7BL/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 plain whole sequence of h mesothelium, 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 expressing the plain Listera vaccine of people's mesothelium that merges with the Bacillus anthracis signal peptide can induce the Balb/c mice to the plain immunne response of mesothelium.Observe the immune height of the immune system of Balb/c mice to the plain IFN γ response ratio C57BL/6 mice of the h mesothelium 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 (66)
1. recombinant nucleic acid molecules comprises:
(a) first polynucleotide sequence of coding antibacterial natural signals peptide, wherein first polynucleotide sequence is that codon is optimized for the expression in Listeria monocytogenes (Listeria monocytogenes); With
(b) second polynucleotide sequence of coded polypeptide, wherein second polynucleotide sequence is arranged in the translation frame identical with first polynucleotide sequence, 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 signal peptide is from the LLO signal peptide of Listeria monocytogenes or from the p60 signal peptide of Listeria monocytogenes.
3. the expression cassette that comprises the recombinant nucleic acid molecules of claim 1 further comprises the promoter of first and second polynucleotide sequences of the recombinant nucleic acid molecules that is operably connected.
4. the reorganization Listeria monocytogenes that comprises the recombinant nucleic acid molecules of claim 1, wherein first polynucleotide sequence is that codon is optimized for the expression in recombinant bacteria.
5. the reorganization Listeria monocytogenes of claim 4 is used to prepare the purposes of inducing the host to resist the compositions of former generation immunne response, and wherein the polypeptide of second polynucleotide encoding comprises antigen.
6. comprise the reorganization Listeria monocytogenes of recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises:
(a) first polynucleotide sequence of coded signal peptide, wherein first polynucleotide sequence is that codon is optimized for the expression in Listeria monocytogenes; With
(b) second polynucleotide sequence of coding and the allogenic polypeptide of signal peptide, wherein second polynucleotide sequence is arranged in the translation frame identical with first polynucleotide sequence,
Wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
7. the reorganization Listeria monocytogenes of claim 6, it comprises the expression cassette that contains recombinant nucleic acid molecules, wherein expression cassette further comprises the promoter of first and second polynucleotide sequences of the recombinant nucleic acid molecules that is operably connected.
8. the reorganization Listeria monocytogenes of claim 6; Wherein the polypeptide of second polynucleotide sequence coding 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.
9. the reorganization Listeria monocytogenes of claim 8, wherein the polypeptide of second polynucleotide sequence coding comprises and is selected from K-Ra s, H-Ra s, N-Ra s, 12-K-Ra s, mesothelium is plain, 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-Ra s, H-Ra s, N-Ra s, 12-K-Ra s; Mesothelium is plain, PSCA, NY-ESO-1, WT-1, survivin, gp100; PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigenic polypeptide of TARP and CEA.
10. the reorganization Listeria monocytogenes of claim 9, wherein the polypeptide of second polynucleotide sequence coding comprises that mesothelium is plain, or its antigen fragment or antigenic variant, or comprises NY-ESO-1, or its antigen fragment or antigenic variant.
11. the reorganization Listeria monocytogenes of claim 10, wherein the polypeptide of second polynucleotide sequence coding comprises that people's mesothelium of its signal peptide and GPI junctional complex domain disappearance is plain.
12. the reorganization Listeria monocytogenes of claim 6, wherein the polypeptide of second polynucleotide sequence coding and signal peptide are allogenic.
13. the reorganization Listeria monocytogenes of claim 6, wherein Listera is belonged to antibacterial is external source to signal peptide.
14. the reorganization Listeria monocytogenes of claim 6, wherein Listera is belonged to antibacterial is natural to signal peptide.
15. the reorganization Listeria monocytogenes of claim 6; 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).
16. the reorganization Listeria monocytogenes of claim 6, it gets into non-phagocytic cell or propagation and weakens for cell and intercellular diffusion.
17. the reorganization Listeria monocytogenes of claim 6, it lacks ActA, Internalin B, or ActA and Internalin B.
18. the reorganization Listeria monocytogenes of claim 6 is wherein through reacting the nucleic acid of having modified recombinant bacteria with the nucleic acid target compound.
19. comprise the immunogenic composition or the vaccine of the reorganization Listeria monocytogenes of claim 6.
20. the reorganization Listeria monocytogenes of claim 6 is used to prepare the purposes of inducing the host to resist the compositions of former generation immunne response, wherein the polypeptide of second polynucleotide encoding comprises antigen.
21. the purposes that the reorganization Listeria monocytogenes of claim 6 is used to prepare prevention or treats the compositions of host's disease.
22. the recombinant nucleic acid molecules of claim 1, wherein antibacterial natural signals peptide right and wrong secA1 antibacterial signal peptide.
23. the recombinant nucleic acid molecules of claim 22, wherein first and second polynucleotide sequences are that codon is optimized for the expression in antibacterial.
24. the recombinant nucleic acid molecules of claim 22, wherein signal peptide is the Listera signal peptide.
25. the recombinant nucleic acid molecules of claim 22, wherein signal peptide is secA2 signal peptide or Tat signal peptide.
26. the recombinant nucleic acid molecules of claim 22, wherein signal peptide is the p60 signal peptide of Listeria monocytogenes or the phoD signal peptide of bacillus subtilis.
27. the recombinant nucleic acid molecules of claim 22, wherein the polypeptide of second polynucleotide sequence coding 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.
28. comprise the expression cassette of the recombinant nucleic acid molecules of claim 22, further comprise the promoter of first and second polynucleotide sequences of the recombinant nucleic acid molecules that is operably connected.
29. comprise the reorganization Listeria monocytogenes of the recombinant nucleic acid molecules of claim 22.
30. the reorganization Listeria monocytogenes of claim 29 is used to prepare the purposes of inducing the host to resist the compositions of former generation immunne response, wherein the polypeptide of second polynucleotide encoding comprises antigen.
31. comprise the reorganization Listeria monocytogenes of recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises:
(a) first polynucleotide sequence of the natural non-secA1 antibacterial signal peptide of the said Listeria monocytogenes of coding, wherein said first polynucleotide sequence is that codon is optimized for the expression in said Listeria monocytogenes; With
(b) coding is allogenic or be the polypeptide of external source or both second polynucleotide sequences to antibacterial with signal peptide, and wherein second polynucleotide sequence is arranged in the translation frame identical with first polynucleotide sequence;
Wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of signal peptide and polypeptide.
32. the reorganization Listeria monocytogenes of claim 31, it comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein expression cassette further comprises the promoter of first and second polynucleotide sequences of the recombinant nucleic acid molecules that is operably connected.
33. the reorganization Listera of claim 31, wherein first and second polynucleotide sequences are that codon is optimized for the expression in Listeria monocytogenes.
34. the reorganization Listeria monocytogenes of claim 31, wherein signal peptide is the secA2 signal peptide.
35. the reorganization Listeria monocytogenes of claim 34, wherein recombinant nucleic acid molecules further comprises:
(c) be arranged in coding sevA2 autolysin or its segmental the 3rd polynucleotide sequence of the translation frame identical with first and second polynucleotide sequence, wherein second polynucleotide sequence is in the 3rd polynucleotide sequence or between first and the 3rd polynucleotide sequence.
36. the reorganization Listeria monocytogenes of claim 31, wherein signal peptide is the Tat signal peptide.
37. the reorganization Listeria monocytogenes of claim 31; Wherein the polypeptide of second polynucleotide sequence coding 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.
38. the reorganization Listeria monocytogenes of claim 37, wherein the polypeptide of second polynucleotide sequence coding comprises and is selected from K-Ra s, H-Ra s, and N-Ra s, 12-K-Ra s, mesothelium is plain, 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-Ra s, H-Ra s, N-Ra s, 12-K-Ra s; Mesothelium is plain, PSCA, NY-ESO-1, WT-1, survivin, gp100; PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigenic polypeptide of TARP and CEA.
39. the reorganization Listeria monocytogenes of claim 38, wherein the polypeptide of second polynucleotide sequence coding comprises that mesothelium is plain, or its antigenicity fragment or antigenicity variant.
40. the reorganization Listeria monocytogenes of claim 39, wherein the polypeptide of second polynucleotide sequence coding comprises that people's mesothelium of its signal peptide and GPI anchor disappearance is plain.
41. the reorganization Listeria monocytogenes of claim 31, it gets into non-phagocytic cell or propagation and weakens for cell and intercellular diffusion.
42. the reorganization Listeria monocytogenes of claim 31, it lacks ActA, Internalin B, or ActA and Internalin B.
43. the reorganization Listeria monocytogenes of claim 31 is wherein through reacting the nucleic acid of having modified recombinant bacteria with the nucleic acid target compound.
44. comprise the immunogenic composition or the vaccine of the reorganization Listeria monocytogenes of claim 31.
45. the reorganization Listeria monocytogenes of claim 31 is used to prepare the purposes of inducing the host to resist the compositions of former generation immunne response, wherein the polypeptide of second polynucleotide encoding comprises antigen.
46. the purposes that the reorganization Listeria monocytogenes of claim 31 is used to prepare prevention or treats the compositions of host's disease.
47. comprise the reorganization Listeria monocytogenes of recombinant nucleic acid molecules, wherein recombinant nucleic acid molecules comprises:
(a) first polynucleotide sequence of the signal peptide of the non-Listera of coding, wherein said first polynucleotide sequence is that codon is optimized for the expression in Listeria monocytogenes; With
(b) be arranged in second polynucleotide sequence of the coded polypeptide of the translation frame identical with first polynucleotide sequence,
Wherein the recombinant nucleic acid molecules coding comprises the fusion rotein of non-Listera signal peptide and polypeptide.
48. the reorganization Listeria monocytogenes of claim 47, it comprises the expression cassette that contains recombinant nucleic acid molecules, and wherein expression cassette further comprises the promoter of first and second polynucleotide sequences of the recombinant nucleic acid molecules that is operably connected.
49. the reorganization Listeria monocytogenes of claim 47, wherein first and second polynucleotide sequences are that codon is optimized for the expression in Listeria monocytogenes.
50. the reorganization Listeria monocytogenes of claim 47, wherein signal peptide is an antibacterial.
51. the reorganization Listeria monocytogenes of claim 50, wherein signal peptide is derived from gram-positive bacterium.
52. the reorganization Listeria monocytogenes of claim 51, wherein signal peptide is derived from and belongs to bacillus (Bacillus), the antibacterial of staphylococcus (Staphylococcus) or Lactococcus (Lactococcus).
53. the reorganization Listeria monocytogenes of claim 52, 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.
54. the reorganization Listeria monocytogenes of claim 47; Wherein the polypeptide of second polynucleotide sequence coding 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.
55. the reorganization Listeria monocytogenes of claim 54, wherein the polypeptide of second polynucleotide sequence coding comprises and is selected from K-Ra s, H-Ra s, and N-Ra s, 12-K-Ra s, mesothelium is plain, 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-Ra s, H-Ra s, N-Ra s, 12-K-Ra s; Mesothelium is plain, PSCA, NY-ESO-1, WT-1, survivin, gp100; PAP, protease 3, SPAS-1, SP-17, PAGE-4, the antigenic polypeptide of TARP and CEA.
56. the reorganization Listeria monocytogenes of claim 55, wherein the polypeptide of second polynucleotide sequence coding comprises that mesothelium is plain, or its antigenicity fragment or antigenicity variant.
57. the reorganization Listeria monocytogenes of claim 56, wherein the polypeptide of second polynucleotide sequence coding comprises that people's mesothelium of its signal peptide and GPI anchor disappearance is plain.
58. the reorganization Listeria monocytogenes of claim 47 belongs to antibacterial, it gets into non-phagocytic cell or propagation and weakens for cell and intercellular diffusion.
59. the reorganization Listeria monocytogenes of claim 47, it lacks ActA, Internalin B, or ActA and Internalin B.
60. the reorganization Listeria monocytogenes of claim 47 is wherein through reacting the nucleic acid of having modified recombinant bacteria with the nucleic acid target compound.
61. comprise the immunogenic composition or the vaccine of the reorganization Listeria monocytogenes of claim 47.
62. the reorganization Listeria monocytogenes of claim 47 is used to prepare the purposes of inducing the host to resist the compositions of former generation immunne response, wherein the polypeptide of second polynucleotide encoding comprises antigen.
63. the purposes that the reorganization Listeria monocytogenes of claim 47 is used to prepare prevention or treats the compositions of host's disease.
64. recombinant nucleic acid molecules comprises:
(a) first polynucleotide sequence of coding antibacterial natural signals peptide, wherein first polynucleotide sequence is that codon is optimized for the expression in Listeria monocytogenes;
(b) coding and the allogenic secretory protein of signal peptide or its segmental second polynucleotide sequence, wherein second polynucleotide sequence is arranged in the translation frame identical with first polynucleotide sequence; With
(c) the 3rd polynucleotide sequence of coding and secretory protein or the allogenic polypeptide of its fragment, wherein the 3rd polynucleotide sequence is arranged in the translation frame identical with first and second polynucleotide sequence,
Wherein the recombinant nucleic acid molecules coding comprises polypeptide and secretory protein or its segmental protein chimera of signal peptide, the 3rd polynucleotide sequence coding; And wherein in the protein chimera; The polypeptide of the 3rd polynucleotide sequence coding and secretory protein or its fragment merge, or are positioned at secretory protein or its fragment.
65. comprise the reorganization Listeria monocytogenes of the recombinant nucleic acid molecules of claim 64.
66. the reorganization Listeria monocytogenes of claim 65 is used to prepare the purposes of inducing the host to resist the compositions of former generation immunne response, wherein the polypeptide of the 3rd polynucleotide encoding comprises antigen.
Applications Claiming Priority (21)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 | ||
| US10/773,618 | 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 |
| US10/773,792 | 2004-02-06 | ||
| US55674404P | 2004-03-26 | 2004-03-26 | |
| US60/556,744 | 2004-03-26 | ||
| US10/883,599 | 2004-06-30 | ||
| US10/883,599 US7695725B2 (en) | 2003-02-06 | 2004-06-30 | Modified free-living microbes, vaccine compositions and methods of use thereof |
| 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 |
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| CN1921884A CN1921884A (en) | 2007-02-28 |
| CN1921884B true CN1921884B (en) | 2012-02-08 |
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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 |
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| JP (2) | JP5532280B2 (en) |
| CN (1) | CN1921884B (en) |
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| US9585953B2 (en) * | 2011-03-22 | 2017-03-07 | Mucosis B.V. | Immunogenic compositions in particulate form and methods for producing the same |
| 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 |
| JP2017507165A (en) * | 2013-12-16 | 2017-03-16 | マサチューセッツ インスティテュート オブ テクノロジー | Micromolded or three-dimensional printed pulsed release vaccine formulation |
| HUE032749T2 (en) * | 2014-12-22 | 2017-11-28 | Sandoz Ag | Sequence variants |
| CN105039381B (en) * | 2015-07-21 | 2018-02-13 | 齐鲁工业大学 | A kind of maltose induction type trehalose synthase synthesis engineering bacteria and preparation method 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 |
| 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 |
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|---|---|---|---|---|
| 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 |
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- 2011-05-23 JP JP2011115257A patent/JP2011155988A/en not_active Withdrawn
Non-Patent Citations (2)
| Title |
|---|
| Carlos A. Guzman et al.Attenuated Listeria monocytogenes carrier strainscandeliveran HIV-1 gp120 T helper epitope to MHCclassII-restrictedhuman CD4+ T cells..Eur. J. Immunol28.1998,281808. * |
| Hao Shen et al.Recombinant Listeria monocytogenes as a livevaccinevehiclefor the induction of protective anti-viralcell-mediatedimmunity..Proc. Natl. Acad. Sci. USA92.1995,923988,图1. * |
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