CN104558196A - Main outer membrane protein epitope vaccine of chlamydia trachomatis based on HBcAg vector and application of main outer membrane protein epitope vaccine - Google Patents
Main outer membrane protein epitope vaccine of chlamydia trachomatis based on HBcAg vector and application of main outer membrane protein epitope vaccine Download PDFInfo
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Abstract
The invention relates to a chimeric vaccine of a hepatitis B virus and chlamydia trachomatis, and an application of the chimeric vaccine, and particularly relates to a recombinant protein containing one or more epitopes with a main outer membrane protein epitope vaccine of the chlamydia trachomatis. The epitope peptides are inserted into the same or different permissive sites of hepatitis B virus core antigen (HBcAg) protein in a form of a single epitope or a plurality of epitopes which are combined together; the recombinant fusion protein capable of stimulating the organism to generate humoral immunity and cellular immunity with respect to the chlamydia trachomatis is formed; the immunogenicity and the immunoprotecive property of various fusion proteins are primarily evaluated; and a foundation is laid for further research and application of preventing and treating chlamydia trachomatis infection and related diseases employing the epitope vaccine based on an HBcAg vector. The vaccine disclosed by the invention has efficient and safe immunoprophylaxis and treatment effects on the related diseases of the chlamydia trachomatis, and is simple in preparation process and immunologic process, obvious in effect and high in repeatability.
Description
Technical field
The present invention relates to biological medicine technology and field of immunology, in particular to a kind of take hepatitis B core antigen as the Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope vaccine of carrier, its preparation method and the application in control chlamydia trachomatis infection thereof.
Background technology
Chlamydia trachomatis (Ct) is the most commonly encountered diseases substance of sexually transmitted disease (STD) (STDs) in the world, Male urethritis and women's cervicitis can be caused, it is reported that having 50-60% to be infected by Ct in Male urethritis causes (Tiodorovi.J, Randelovi.G, Koci.B et al, Bacteriological finding in the urethra in men with and without non-gonococcal urethritis, 2007, 64 (12): 833-6), and normal and other cause of disease polyinfection, by Sex transmitted pathogen female genitourinary tract, cause Disadvantage pregnancy, as miscarriage, stillborn foetus etc., and there is closely related (Alibek K with cervical cancer, Karatayeva N, Bekniyazov I.The role of infectious agents in urogenital cancers.Infect Agent Cancer.2012, 7 (1): 35, de Abreu AL, Nogara PR, Souza RP, et al.Molecular detection of HPV and Chlamydia trachomatis infections in Brazilian women with abnormal cervical cytology.Am J Trop Med Hyg.2012, 87 (6): 1149-1151).Ct still causes trachoma by contact, major cause (the Lu C of blinding, Holland MJ, Gong S, et al.Genome-wide identification of Chlamydia trachomatis antigens associated with trachomatous trichiasis.Invest Ophthalmol Vis Sci.2012; 53 (6): 2551-9), and it is closely related with some autoimmune disorder, as (Costapinto L such as reactive arthritis, rheumatoid arthritis, SLE and arteriosclerosis, Olavarria VG, Grassi MF, et al.Prevalence of Chlamydia trachomatis endocervical infection in systemic lupus erythematosus patients and evaluation of the risk for HPV-induced lesions.Rheumatol Int.2012; Girschick HJ, Guilherme L, Inman RD et al.Bacterial triggers and autoimmune rheumatic diseases.Clin Exp Rheumatol.2008; 26:S12-17).Ct infects and very easily recurs after treatment, WHO estimates that about there are 9,000 ten thousand Ct patients in the whole world, be used for the treatment of Ct every year and infect cost multi-million dollar (Wang SA, Papp JR, Stamm WE, et al, Evaluation of antimicrobial resistance and treatment failures for Chlamydia trachomatis:a meeting report.J Infect Dis.2005,191 (6): 917-23.).In recent years, the STDs morbidity that China is caused by Ct, in continuing ascendant trend, constitutes high risks to human health, but still lacks effectively preventing method so far, thus seek effective vaccine, is one of hot-point and frontier problem of research at present.
Epitope is the fundamental structural unit or special chemical group that can be combined with φt cell receptor (TCR), B-cell receptor (BCR) or antibodies specific in antigen molecule, determined antigen-specific.Based on the vaccine of epitope design, i.e. epiposition vaccine (Epitope vaccine), that the one design that developed recently gets up is unique, with strong points, the vaccine of safety and stability, and can to T, chimeric (the XuW that B cell antigen epi-position is carried out various combination and made for multiple pathogens, Liu J, GongW, et al.Protective immunity against Chlamydia trachomatis genital infection induced by a vaccine based on the major outer membrane multi-epitope human papillomavirus major capsid protein L1.Vaccine.2011, 29 (15): 2672-2678, Robinson JA.Max Bergmann lecture Protein epitope mimetics in the age of structural vaccinology.J Pept Sci.2013, 19 (3): 127-140).But research prompting, the immunogenicity of epiposition vaccine has to be reinforced.Research proves, hepatitis B virus core antigen (HBcAg) has good immunogenicity, with the virus-like particle (VLPs) of HBcAg formation for the carrier carrying heterologous protein has significant advantage: formation and immunogenicity that the MIR district that the VLPs that HBcAg is formed exists is applicable to carrying exogenous peptide and does not affect its VLPs very much; HBcAg VLPs has the immunogenic adjuvant effect of enhancing exogenous peptide; HBcAg VLPs can be prepared on a large scale at prokaryotic expression system, thus reaches economic and practical object (Yang Xingyu, Bao Hong, Su Yuelong; Hepatitis B virus core antigen is used for the major progress of virus sample particle vaccines research as carrier. viral journal .2012,28 (3): 312-316; Bertrand Bellier, David Klatzmann.Virus-like particle-based vaccines against hepatitis C virus infection.Expert Review of Vaccines, 2013,12 (2): 143-154).
Ct has 19 Serotypes, wherein the serotype such as D-H mainly causes urogenital infections, wherein E and D type is then domestic and international report (Chen Lili, Wu Yimou, Deng Zhongliang etc. southern china urban Medicine against Urogenital Chlamydia Trachomatis gene type is studied. Chinese journal of dermatology, 2006,39:275-277; Suchland RJ, Eckert LO, Hawes SE, et al, Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle, public health clinics:1988-1996.Sex Transm Dis, 2003,30:357-361) in the most common type.Ct major outer membrane protein (MOMP) is principal target (the Klein M of neutralizing antibody, Kotz A, Bernardo K, et al.Kronke M.Detection of Chlamydia pneumoniae-specific antibodies binding to the VD2and VD3regions of the major outer membrane protein.J Clin Microbiol.2003,41 (5): 1957-1962), because of but the target antigen of Ct vaccine research.But serious pathologic immune response (Fletcher MA.Vaccine candidates in STD.Int J STD AIDS, 2002,13.Suppl2:38-41) can be produced with when whole thalline or target antigen immunity human body.Thus, the immunodominant epitope on MOMP albumen is utilized to solve the problem to a certain extent, likely will develop into promising vaccine (Ortiz L, Angevine M, Kim SK, et al.T-cell epitopes in variable segments of Chlamydia trachomatis major outer membrane protein elicit serovar-specific immune responses in infected humans.Infect Immun, 2000,68 (3): 1719-1723).In recent years, a lot of laboratory reports a series of cell epitope successively, mostly be positioned on chlamydia trachomatis MOMP, the polypeptide utilizing these cell epitopes to form can induce body to produce specific immune response (Wang Ledan, Zhang Lifang. the progress of Major Outer Membrane Protein of Chla mydia trachomatis epitope. foreign medical science. prevention. diagnosis. biological products fascicle is used in treatment, 2005,28 (5): 196-199).
At present, the research of Ct vaccine mainly comprises whole cell vaccine, subunit vaccine, recombiant vaccine, improvement on synthesis vaccine, but effect is all undesirable.Due to natural whole cell and MOMP albumen possibility induced pathologies immunne response, therefore select t cell epitope and the B cell epi-position in MOMP albumen with immunodominance, the T-B of composition Ct MOMP combines epi-position, this multi-epitope gene is cloned into prokaryotic expression plasmid and the rear immune mouse of qualification, the specific antibody IgG for CT is detected respectively in mice serum and vaginal secretions, slgA and Specific CTL Cells immunity, and mouse model reproductive tract Ct is attacked there is certain provide protection (Xu W, Liu J, Gong W, et al.Protective immunity against Chlamydia trachomatis genital infection induced by a vaccine based on the major outer membrane multi-epitope human papillomavirus major capsid protein L1.Vaccine.2011, 29 (15): 2672-2678).Therefore, epiposition vaccine is one of direction of chlamydia trachomatis vaccine research, and strengthens the focus that the immunogenicity of epi-position and provide protection are research at present.
In sum; this area a kind ofly strengthens chlamydia trachomatis associating epi-position vaccine immunogenicity and the vaccine of immanoprotection action in the urgent need to developing, and is applied to the autoimmune disorder etc. that disease that chlamydia trachomatis infection causes is as relevant in urogenital infections, trachoma and chlamydia trachomatis infection.
Summary of the invention
The object of the present invention is to provide a kind of vaccine that can be used for chlamydia trachomatis infection and relevant autoimmune disorder (as reactive arthritis, rheumatoid arthritis, SLE and arteriosclerosis etc.) prevention and therapy thereof.
In a first aspect of the present invention, provide a kind of aminoacid sequence, described sequence comprises:
The aminoacid sequence of (a) coding Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope peptide; With
The aminoacid sequence of (b) coding hepatitis B virus core antigen.
In a preference, described Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope peptide is the aminoacid sequence containing multiple CTL epi-position, Th epi-position and B cell epi-position.
In another preference, the aminoacid sequence of described multiple CTL epi-position, Th epi-position and B cell epi-position is series connection or overlapping arrangement.
In another preference, the aminoacid sequence in (a) is through the codon optimized modification of protokaryon.B the C end of the aminoacid sequence in () inserts general Th epi-position, and modify at its N end and C end insertion His-tag.
In another preference, the nucleotide sequence of described coding Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope peptide is connected to carboxyl terminal or the aminoterminal of the nucleotide sequence of coding hepatitis B virus core antigen or inserts its MIR district.
In another preference, (a) is directly connected with the aminoacid sequence in (b) or is connected by catenation sequence.
In an embodiment of the invention, the nucleotide sequence of described Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope peptide is as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
In yet another embodiment of the present invention, the nucleotide sequence of described hepatitis B virus core antigen is as shown in SEQ ID NO:12 (wild-type nucleotide sequences).
In yet another embodiment of the present invention, the nucleotide sequence of described coding Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope peptide is selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 (namely without codon optimized sequence) and SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 (namely through nucleotide sequence that protokaryon is codon optimized).
In yet another embodiment of the present invention, the nucleotide sequence of described coding hepatitis B virus core antigen is selected from: SEQ ID NO:12 (i.e. wild-type nucleotide sequences) and SEQ ID NO:13 (namely adding the nucleotide sequence of general Th epi-position and His-tag).
In a second aspect of the present invention, provide a kind of recombinant vectors, described carrier comprises the above-mentioned chimeric nucleic acid sequence of the present invention.
In a preference, described carrier is selected from: carrier for expression of eukaryon or prokaryotic expression carrier, preferred bacterium plasmid, phage, yeast plasmid, vegetable cell virus or mammalian cell virus, more preferably pET21a (+), pET32a (+), pcDNA3.1 (+), pSIREN-NEO, pQE30, pGEX-4T-1, pPICZA.
In a third aspect of the present invention, provide a kind of genetically engineered host cell, described host cell contains the above-mentioned carrier of the present invention.
In a preference, described host cell is selected from prokaryotic cell prokaryocyte, eukaryotic cell or the higher eucaryotic cells such as low, preferred zooblast, intestinal bacteria or yeast, more preferably E.coli, GS115, COS-7 cell, COS-1 cell.
In a fourth aspect of the present invention, provide the above-mentioned chimeric nucleic acid sequence of the present invention, recombinant vectors or the host cell purposes in the vaccine for the preparation of prevention or treatment chlamydia trachomatis infection and relative disease thereof.
In a preference, described chlamydia trachomatis relative disease is selected from: chlamydia trachomatis infection disease: urethritis, cervicitis, trachoma, infertile etc., and chlamydia trachomatis infection associated autoimmune disease: cervical cancer, reactive arthritis, rheumatoid arthritis, SLE and arteriosclerosis etc.
In a fifth aspect of the present invention, provide a kind of composition, it includes the aforesaid chimeric nucleic acid sequence of the present invention of effective amount, recombinant vectors or host cell and acceptable carrier, vehicle or adjuvant pharmaceutically or in immunology.
In a preference, described composition is immune composition or pharmaceutical composition, preferred vaccine.
In a preference, described carrier, vehicle or adjuvant are selected from: liposome, starch, gelatin, Alum adjuvant, saponin adjuvant or Ribi adjuvant.
In another preference, the recombinant vectors according to claim 4 containing 0.01-99.9wt% in described composition, preferred 0.1-99.0wt%.
In another preference, described composition is intramuscular injection, skin immunization or mucosal immunity.
In a sixth aspect of the present invention, provide a kind of method preparing the above-mentioned recombinant vectors of the present invention, described method comprises:
I () provides the nucleotide sequence of coding Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope peptide and the nucleotide sequence of coding hepatitis B virus core antigen;
(ii) connect described nucleotide sequence, form chimeric nucleic acid sequence;
(iii) step (ii) gained chimeric nucleic acid sequence is cloned in carrier.
In a preference, the sequence of described hepatitis B virus core antigen albumen is as shown in SEQ ID NO:10 (not modified former sequence) and SEQ ID NO:11 (namely through sequence that general Th epi-position and His-tag are modified).
In another preference, the nucleotide sequence of described coding hepatitis B virus core antigen is selected from: SEQ ID NO:12 (i.e. wild-type nucleotide sequences), SEQ ID NO:13 (namely through sequence that general Th epi-position and His-tag modify).
In another preference, the nucleotide sequence of described coding Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope peptide is connected to carboxyl terminal or the aminoterminal Huo Qi MIR district of the nucleotide sequence of coding hepatitis B virus core antigen.
It should be understood that other side of the present invention is due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1: the PCR of the HBcAg gene of modification identifies (A) and sequencing result (B).
In Fig. 1 (A), " M " represents: DL2000DNA marks; " 1-4 " all represents: recombinant plasmid pET21a (+)/HBcAg
The PCR primer of gene.Fig. 1 (B) is sequencing result.
Fig. 2 HBcAg/Ct MOMP epi-position recombinant plasmid sequencing result
(A) HBcAg (MIR)/Ct MOMP epi-position 1 sequencing result.
(B) HBcAg (MIR)/Ct MOMP epi-position 1+2 sequencing result.
(C) HBcAg (MIR)/Ct MOMP epi-position 1+2+3 sequencing result.
(D) Ct MOMP epi-position 1/HBcAg sequencing result.
(E) HBcAg/CtMOMP epi-position 1 sequencing result.
SDS-PAGE (Fig. 3 A), protein purification electrophorogram (Fig. 3 B) and WB that Fig. 3 .HBcAg/CtMOMP epi-position is expressed analyze (Fig. 3 C, Fig. 3 D).
" M " represents: standard protein molecular weight marker; " 1 " is E.coli Rosseta bacterial strain; " 2 " are pET21a (+) empty carrier; " 3 " are pET21a (+)/HBcAg; " 4 " are Ct-MOMP epi-position 1/HBcAg (N end); " 5 " are HBcAg/Ct-MOMP epi-position 1 (C end); " 6 " are HBcAg (MIR)/Ct-MOMP epi-position 1 (MIR district).When wherein WB analyzes, primary antibodie is deactivation Ct whole cell immune serum antibody.B: be each purifying protein SDS-PAGE electrophorogram.
SDS-PAGE (Fig. 4 A), WB (Fig. 4 B) that Fig. 4 .HBcAg/Ct MOMP series connection epi-position is expressed analyze and protein purification electrophorogram (Fig. 4 C)
" M " represents: standard protein molecular weight marker; " 1 " is E.coli Rosseta bacterial strain; " 2 " are pET21a (+) empty carrier; " 3 " are pET21a (+)/HBcAg; " 4 " are HBcAg (MIR)/Ct-MOMP epi-position 1; " 5 " are HBcAg (MIR)/CtMOMP epi-position 1+2; " 6 " are HBcAg (MIR)/Ct MOMP epi-position 1+2+3.When wherein WB analyzes, primary antibodie is deactivation Ct whole cell immune serum antibody.B: be each purifying protein SDS-PAGE electrophorogram.
Fig. 5 take HBcAg as the electron microscopic observation of the MOMP neoepitope Western of carrier
" A " is HBcAg; " B " is HBcAg/MOMP epi-position 1; " C " is MOMP epi-position 1/HBcAg; " D " is HBcAg (MIR)/MOMP epi-position 1; " E " is HBcAg (MIR)/epi-position 1+2; " F " is HBcAg (MIR)/epi-position 1+2+3.All virus-like particle can be formed at electric Microscopic observation.
Fig. 6 respectively organizes the dynamic change of change of serum C t specific antibody IgG after mouse immune.
Fig. 7 respectively organizes the dynamic change of genital secretion Ct specific antibody IgA after mouse immune.
Fig. 8 produces the specific CTL killing activity of CtMOMP after respectively organizing mouse immune.
Fig. 9 produces the specific CTL killing activity of HBcAg after respectively organizing mouse immune.
Figure 10 respectively organizes mouse propagation road Ct and attacks rear clearance rate (IFU counting).
Figure 11 respectively organizes mouse Ct and attacks rear outer reproductive tract inflammatory score.
Embodiment
The present inventor is based on Ct MOMP immunodominant epitope (abbreviation epi-position) gene, and codon optimized through protokaryon, is carrier, has prepared HBcAg/Ct MOMP epiposition vaccine with HBcAg.Confirm that HBcAg/CtMOMP epiposition vaccine has the effect of immunoprophylaxis and treatment mouse propagation road Ct infection concurrently through animal immune experiment; especially the series connection epiposition vaccine that carries of HBcAg MIR district is to the immune protective effect of Ct, is significantly better than Ct MOMP epi-position synthetic peptide vaccine and is added on HBcAg N holding or the chimeric of C end.On this basis, contriver completes the present invention.
Specifically, the present inventor is by predicting and screening the total aminoacid sequence containing multiple CTL epi-position, Th epi-position and B cell epi-position, and the codon optimized modification of protokaryon has been carried out to this gene, obtain Ct MOMP immunodominant epitope gene (abbreviation epi-position), and by its further B cell epi-position attested with this laboratory connect and formed.Meanwhile, the present inventor is also to being carrier with HBcAg and adding the modification of general Th epi-position (PADRE) and His-tag.Then, by Protocols in Molecular Biology, prepared HBcAg/CtMOMP epiposition vaccine (namely carrying CtMOMP epi-position respectively in HBcAg-N end, C Duan He MIR district).
Contriver have detected the immanoprotection action of above-mentioned epiposition vaccine further by immune mouse: by ELISA method detect immunized mice serum IgG and vaginal secretions IgA antibody level, splenic lymphocyte CTL specific killing activity etc. evaluate epiposition vaccine immunological effect and in mouse model to Ct reproductive tract attack immanoprotection action.Result shows; HBcAg/CtMOMP epiposition vaccine has immunoprophylaxis and treatment mouse propagation road Ct infection effect concurrently; and it is better than CtMOMP epi-position synthetic peptide vaccine to the immune protection Be very effective of Ct, especially the protected effect of series connection epiposition vaccine that carries of MIR district and chlamydia trachomatis deactivation whole cell vaccine close.
The present invention studies confirmation by experiment; prepared HBcAg/Ct MOMP epiposition vaccine; body can be stimulated to produce stronger for Ct specific humoral immunity effect; especially the protection antibody of local reproductive road mucous membrane; and for the cellular immunization of Ct; there is the immunoprophylaxis and therapeutic action infected Ct, lay a good foundation based on the epiposition vaccine control chlamydia trachomatis infection of VLPs carrier and the further investigation of relative disease thereof and application for adopting, there is important significance of scientific research and using value.
Meanwhile, the present invention also demonstrates HBcAg carrier by experimentation on animals, to the immune-enhancing effect of the Ct MOMP epi-position of heterology.Result of study confirms, HBcAg/CtMOMP neoepitope Western, body can be induced to produce the specific humoral immunization of Ct and the cellular immunization of enhancing, especially the antibody response of the IgA of mucous membrane local, and to the immanoprotection action that Ct reproductive tract is attacked.These immune effects be better than synthesize CtMOMP epitope peptide vaccine, especially with HBcAgB MIR district carry series connection epi-position vaccine be remarkable, thus enhancing immunoprotective effec.
term explanation
Term " immunocompetence " herein or " immunogenicity " refer to by the specificity humoral in the vaccine-induced mammalian body of natural, restructuring or synthesis and/or the ability of cellullar immunologic response.Term used herein " immunodominant epitope vaccine " " synthetic peptide vaccine " or " chimeric (gene) vaccine " refer to cause the chimeric nucleic acid sequence of the present invention of mammalian immune response and the fusion rotein of expression thereof.
Term " immunne response " herein comprises cellularity and/or humoral immune response, and they are enough to suppress or protect from infection; Or prevent or suppress by chlamydia trachomatis infection associated diseases and relevant autoimmune disorder thereof.
Term " object " herein or " individuality " or " patient " refer to any target needing to carry out diagnosing or treating, and especially mammalian object, particularly people, other object comprises ox, dog, cat, cavy, rabbit, rat, mouse, horse etc.Concerned is especially that those are subject to or are subject to the object of chlamydia trachomatis infection.
Term " nucleic acid " herein and " nucleotide sequence " refer to the Nucleotide (ribonucleotide or deoxyribonucleotide) of the random length of polymerized form.It includes, but is not limited to DNA or RNA of strand, double-strand, genomic dna and cDNA.
Term " significant quantity " herein or " immune significant quantity " refer to that it is effective for giving individual amount to treatment or prevention with single dose or a continuous agent part.This consumption according to treat that the classification (as non-human primates etc.) of individuality is treated by individual healthy state and physiological situation, institute, the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treating physician determine the assessment of medical conditions and other correlative factor.Estimate that this consumption is by relatively wide scope, determines by normal experiment.
As used herein, unless otherwise indicated, described " immunodominant epitope of the present invention ", " epi-position ", " neoepitope Western ", " series connection epi-position " etc. are used interchangeably.
chimeric protein and nucleotide sequence
HBcAg/Ct MOMP epitope sequences of the present invention comprises: (a) encodes the amino acid of Major Outer Membrane Protein of Chla mydia trachomatis immunodominant epitope (Ct MOMP) and nucleotide sequence; (b) amino acid of hepatitis B virus core antigen of encoding and nucleotide sequence.
Term " Major Outer Membrane Protein of Chla mydia trachomatis (Ct MOMP) immunodominant epitope " refers to the aminoacid sequence containing multiple MOMP CTL epi-position, Th epi-position and B cell epi-position.The acquisition of described immunodominant epitope, by prediction and the common epitope screening the other Ct MOMP of various serotype, forms immunodominant epitope by containing the specific CTL epi-position of multiple HLA-A2 and Th and B cell epi-position simultaneously.Such as can obtain the MOMP sequencing and analyzing of Ct each serotype by application network resource database, the restricted t cell epitope of HLA-A*0201 (CTL), Th epi-position and B cell Antigen Epitope Prediction are carried out to the Ct MOMP aminoacid sequence of each Serotypes, select the high Dominant Epitopes of score value to connect into multi-epitope gene, be preferably connected in series.Described Dominant Epitopes includes, but is not limited to: HLA-A2 restricted CTL epitope (379-387aa), Th epi-position (373-387aa, 370-384aa) with B cell epi-position (377-386aa), comprising the CTL epi-position (267-275aa of H-2d type, 371-379aa, 370-378aa).
Term " hepatitis B core antigen (HBcAg) " refers to the hepatitis B virus core antigen one by one of the main component of hepatitis B virus envelope, and its encoding sequence is well known in the art.Those of ordinary skill in the art should be understood that in these known HBcAg encoding sequence chimeric amino acid sequence used in the present invention.
Should be understood that nucleotide sequence of the present invention also can comprise hybridize with above-mentioned encoding sequence under strict conditions and there is the molecule of identical or similar activity.Term " stringent condition " refers to: (1) compared with the hybridization under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, 0.1%SDS, 60 DEG C; Or be added with denaturing agent during (2) hybridization, and as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) homogeny only between two sequences is at least 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%25, more than 85% or more than 90%, just hybridize when being more preferably more than 95%.Such as, described sequence can be complementary sequence.
In a preferred embodiment of the present invention, the codon optimized modification of protokaryon is carried out to Ct MOMP encoding sequence row of the present invention, thus makes the gene of gained can obtain higher expression in prokaryotic expression system.One or both in preferred chimeric nucleic acid sequence is the nucleotide sequence through the codon optimized modification of protokaryon.Described " the codon optimized modification of protokaryon " can adopt ordinary method as known in the art to carry out (such as can refer to Pan W, the Vaccine candidate MSP-1 from Plasmodium falciparum:a redesigned4917bp polynucleotide enables synthesis and isolation of full-length protein from Escherichia coli and mammalian cells.Nucleic Acids Res.1999Feb 5 15 of Ravot E, Tolle R etc.; 27 (4): 1094-103).Preferably, make the GC content in nucleotide sequence improve 5-40%, preferred 10-30%, more preferably 13-23%, but the amino acid keeping it to encode is constant.
Nucleotide sequence of the present invention can directly connect, or is connected by catenation sequence.Term " catenation sequence " refers between the nucleotide sequence and the nucleotide sequence of coding hepatitis B virus core antigen of encode Major Outer Membrane Protein of Chla mydia trachomatis epi-position and series connection epi-position thereof herein, plays the sequence of ligation.The length of catenation sequence is not particularly limited, and is generally 0-100.The length of connection peptides also can be 0, now represents that gene order is directly connected.Usually, catenation sequence do not affect or not remarkably influenced institute catenation sequence expression and produce the immunological effect of antigen.
In a preferred embodiment of the present invention, Ct MOMP encoding sequence is connected to the MIR district of HBcAg encoding sequence or carboxyl terminal or aminoterminal.
carrier and host cell
After obtaining coding chimeric nucleic acid sequence of the present invention, suitable expression vector can be connected into, then be proceeded to suitable host cell.
In the present invention, term " carrier " and " recombinant vectors " are used interchangeably, and refer to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral or other carrier.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.The carrier being selected from lower group can be used: prokaryotic expression carrier or carrier for expression of eukaryon in the present invention, preferred bacterium plasmid, phage, yeast plasmid, vegetable cell virus or mammalian cell virus, more preferably pET21a (+), pET32a (+), pcDNA3.1 (+), pSIREN-NEO, pQE30, pGEX-4T-1 or pPICZA.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell, zooblast.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell is as yeast; Vegetable cell etc.In the present invention, E.coli, GS115, COS-7 cell, COS-1 cell is preferably adopted.
Immunodominant epitope vaccine of the present invention is made up of the gene of target protein of encoding and the expression vector of plasmid, do not integrate with host chromosome after direct importing host cell, but passing through the re-reading system expressing protein antigen of cell, induction body produces special cellular immunization and humoral immunization.
composition
Present invention also offers the various compositions comprising recombinant vectors of the present invention, comprise pharmaceutical composition and vaccine composition.Said composition can be used for prevention or treats chlamydia trachomatis infection and relative disease thereof, and described disease includes, but is not limited to: urethritis, cervicitis, trachoma, infertile, cervical cancer, reactive arthritis, rheumatoid arthritis, SLE and arteriosclerosis etc.
The various compositions comprising recombinant vectors of the present invention can comprise by the buffer reagent selected by the practical use of recombinant vectors; Also can comprise other material being applicable to intended purpose.Those skilled in the art are good at the buffer reagent selected, and known in the art have numerous buffers to be applicable to intended purpose.In some example, said composition can contain pharmaceutically acceptable vehicle, known in the art have multiple and without the need to discussing in detail at this.Pharmaceutically acceptable various vehicle is at the existing detailed description of multiple publication, comprise as " Remington ' s Pharmaceutical Sciences " (" Lei Mingdun pharmaceutical science ", the 19th edition (1995) Mack Publishing Co.).
Pharmaceutical composition or vaccine composition can be prepared into various formulation, as injection, granula, tablet, pill, suppository, capsule, suspension, spraying, suppository, transdermal drug (as paster etc.), ointment, lotion etc.Be applicable to oral or local use pharmaceutical grade other organic or inorganic carrier and/or thinner, can be used for preparing the various compositions comprising therapeutical active compound.Thinner known in the art comprises aqueous medium, vegetalitas and animality oil & fat.Also the salt of useful stabilizers, wetting agent and emulsifying agent, change osmotic pressure or the various buffer reagent of maintenance suitable ph and skin penetration enhancer etc. are as auxiliary agents.
When being used as vaccine, recombinant vectors of the present invention can adopt various method to prepare.Usually, by various method well known in the art, prepare vaccine of the present invention or medicine with suitable pharmaceutical carrier and/or vehicle (vehicle).Suitable carrier is Sterile Saline.Also can use other water-based and non-aqueous isotonic sterile injection liquid and water-based and non-aqueous sterile suspensions (known is all pharmaceutically acceptable carrier well-known to those skilled in the art) for this reason.
In addition, the preparation of vaccine composition of the present invention also can contain other composition, comprises as adjuvant, stablizer, pH adjusting agent, sanitas etc.These compositions are known by vaccines arts technician.Adjuvant class comprises (but being not restricted to) Alum adjuvant; Saponin adjuvant; Ribi adjuvant (Ribi ImmunoChem Research In., Hamilton, MT); Montanide ISA adjuvant (Seppic, Paris, France); Hunter ' s TiterMax adjuvant (CytRx Corp., Norcross, GA); Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, 30 Germany) etc.In addition, other composition (IL-12, CpG oligodeoxynucleotide (CpG-ODN) etc.) of immunity moderation response can also be comprised in the formulation.
route of administration and dosage
When being used as vaccine, recombinant vectors of the present invention is applied to object by available known method.These vaccines are used in the route of administration that usual employing is identical with conventional vaccine and/or simulation pathogenic infection path.When can adopt the form of vaccine composition, also can comprise pharmaceutically acceptable carrier.In addition, this composition also can comprise adjuvant, correctives or stablizer etc.
The routine and the pharmaceutically acceptable approach that give pharmaceutical composition of the present invention or vaccine composition comprise: in nose, intramuscular, tracheal strips, in subcutaneous, intracutaneous, lung, intravenously, intranasal, oral administration or other parenteral route of administration.Can combination medicine-feeding approach if needed, or regulate by antigen peptide or disease event.Vaccine composition can single dose or multiple doses give, and can comprise and give booster dose to cause and/or to maintain immunizing power.
Should give recombiant vaccine with " significant quantity ", namely the amount of recombinant vectors is enough to cause immunne response in selected administration routes, can effectively impel protection host to resist chlamydia trachomatis infection.
The amount of recombinant vectors selected in each vaccine dose part is determined without the amount of obvious side effect by causing protective immune response.Usually, after host cells infected, the vaccine of each dose of part is enough to produce about 1 μ g-10mg, is preferably 5 μ g-2mg, more preferably 10pg-1mg protein.The vaccine effective dose calculated based on recombination epitope vaccine, generally includes and gives about
1 μ g-10mg chimeric nucleic acid/kg body weight, preferably 1 μ g-10mg chimeric nucleic acid/kg body weight, more preferably 1 μ g-10mg chimeric nucleic acid/kg body weight.Can with the IgG titers comprised in the object of observation and other reaction standard research techniques determine the optimum amount of concrete vaccine by.The immunity level that monitoring vaccine provides determines whether to need to strengthen dosage.After have evaluated the IgG titers in serum, may need to select enhancing dose immunizations.Use adjuvant and/or immunostimulant and just can improve immunne response to protein of the present invention.
major advantage of the present invention
Major advantage of the present invention is:
(1) a kind of Ct MOMP epiposition vaccine based on HBcAg virus-like particle carrier is provided, it is effective for the specific humoral immunization of Ct and the cellular immunization for HBcAg, chlamydia trachomatis specific that described epiposition vaccine can induce body to produce, and has immunoprophylaxis and therapeutic action to Ct infection and relevant disease thereof;
(2) vaccine of the present invention is for chlamydia trachomatis whole cell or its MOMP protein vaccine, to the effective immunoprotective effec of chlamydia trachomatis infection tool, can be used for more effective prevention and therapy application of chlamydia trachomatis infection and relative disease thereof;
(3) vaccine preparation technology of the present invention simply, low cost, safety easy to use, and can react by long term activation body general immune, has the advantages such as immunologic process is simple, inoculation safety, successful, repeatability are strong.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition (such as can refer to as people such as Sambrook, " Molecular Cloning: A Laboratory room guide " (New York:Cold Spring Harbor Laboratory Press, 1989) or Immunology Methods Manual, Ivan Lefkovits, CRC, 1998) or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
the acquisition of embodiment 1.HBcAg vector gene and modification
With Wenzhou Area hepatitis B virus carriers (great three positive) serum specimen for template, design and synthesis PCR primer, restriction enzyme site (Ned I and HindIII) is introduced at two ends, amplify HBcAg complete open reading frames, PCR primer is connected with carrier pET21a (+) simultaneously after double digestion, construction recombination plasmid pET21a (+)/HBcAg (wt), through order-checking qualification.
For strengthening the support effect of HBcAg and being easy to molecular cloning protocols, HBcAg Nucleotide (SEQ ID NO:3) is modified and designed, namely hold at its C and insert general Th epi-position PADRE, and hold each introducing His-tag sequence, so that purifying at N and C of HBcAg carrier; Qi MIR district (the 78th and the 82nd amino acids) carries out base mutation simultaneously, forms the restriction enzyme site (BamH I and Sac I) that can insert foreign gene, to carry out the insertion of chlamydia trachomatis MOMP epi-position and series connection epi-position thereof.HBcAg base sequence two ends after modification add Ned I and HindIII restriction enzyme site respectively, be connected on carrier pET21a (+) after double digestion, construction recombination plasmid pET21a (+)/HBcAg (modification), and after PCR and order-checking qualification (Figure 1A, Figure 1B), for the preparation of vaccine.
the preparation of embodiment 2.HBcAg/CtMOMP epiposition vaccine and vivoexpression, formation VLPs are analyzed
By based on the codon optimized Ct MOMP immunodominant epitope gene of protokaryon and series connection epitope gene, design and synthesize complementary primer, restriction enzyme site BamH I and Sac I is introduced at two ends respectively, goal gene is obtained after annealing, pET21a (+)/HBcAg of annealed product after cutting through BamH I and Sac I enzyme is connected, builds HBcAg MIR district containing the recombined pronucleus expression plasmid of Ct MOMP epitope gene with series connection epitope gene; Meanwhile, by PCR method, Ct MOMP immunodominant epitope is connected to N and the C end of the HBcAg after modification, carries out research and compare, by order-checking qualification (Fig. 2).Result shows: HBcAg (MIR)/Ct MOMP epi-position 1, HBcAg (MIR)/Ct MOMP epi-position 1+2, HBcAg (MIR)/CtMOMP epi-position 1+2+3, Ct MOMP epi-position 1/HBcAg and HBcAg/Ct MOMP epi-position 1 are fitted together to construction of recombinant plasmid success.Respectively by the Transfected Recombinant Plasmid intestinal bacteria Rossetta bacterial strain through qualification, by IPTG abduction delivering, and carry out SDS-PAGE analysis, WB qualification and purifying (Fig. 3, Fig. 4).Result shows, and analyze through SDS-PAGE, HBcAg relative molecular mass is about 22kDa; The albumen size of HBcAg/Ct MOMP epi-position 1, Ct MOMP epi-position 1/HBcAg and HBcAg (MIR)/MOMP epi-position 1, HBcAg (MIR)/MOMP epi-position 1+2, HBcAg (MIR)/MOMP epi-position 1+2+3 is all about 22-25kDa, and relative molecular mass size is consistent with expection.Simultaneously with deactivation Ct whole cell immune serum for primary antibodie, analyze confirmation through Western blot and occurred specific band, further through electron microscopic observation, visible virus-like particle forms (Fig. 5).
This result shows: based on the Ct MOMP epi-position of HBcAg carrier and series connection epi-position recombinant plasmid thereof at prokaryotic expression system energy effective expression, and can form Hybrid virus like particles.
the immunogenicity of embodiment 3.HBcAg/CtMOMP epiposition vaccine and immunoprotective effec
Select female BAl BIc in 6 ~ 8 week age/c mouse (purchased from Shanghai Slac Experimental Animal Co., Ltd.), be divided into 8 groups at random, often organize 21, be respectively: PBS control group, HBcAg group, HBcAg (MIR)/MOMP epi-position 1 (Dominant Epitopes 1 is carried in MIR district), HBcAg (MIR)/MOMP epi-position 1+2, HBcAg (MIR)/MOMP epi-position 1+2+3 (, Ct MOMP epi-position 1/HBcAg (N end), HBcAg/Ct MOMP epi-position 1 (C end) and Ct MOMP epi-position synthetic peptide group.Get 100 μ l respectively at 0,2,4 week and test sample (1tg/ μ l), through mouse back intradermal injection immunity.After immunity, 0w, 2w, 4w, 6w, 8w, 10w, 12w, 14w, 18w, 20w gather tail vein and vaginal secretions respectively, detect IgA antibody in Ct specific serum IgG antibody and secretory product by ELISA method; Within 7th week, get spleen and prepare splenocyte suspension, adopt serum lactic dehydrogenase (LDH) method for releasing to detect mouse for the specific CTL killing activity of CtMOMP and HBcAg.Often group residue mouse, all attack with E serotype Ct, after attacking, every day observes the active situation of mouse and the severity of episioitis, score (red 0 ~ 3 point of vulva, swollen 0 ~ 3 point, secretory product 0 ~ 3 point) and count its clearance rate by the separation and Culture of mouse secretory product Ct.
(1) measurement result of immune serum Ct specific antibody IgG:
Adopt indirect ELISA method, by the Ct whole cell bag after purifying by polyethylene micro-reaction plate, after closing, add immune mouse 1:50 dilute serum, 37 DEG C of reaction 2h, wash plate, add the HRP-sheep anti-mouse igg 100 μ l of 1:2000 dilution respectively, 37 DEG C of reaction 2h, after washing plate, O-Phenylene Diamine (OPD) develops the color, and adds 2mol/L H
2sO
4termination reaction, microplate reader surveys A490 value.Every part of serum specimen all repeats 3 holes and detects, and measures each group of immune serum specific antibody IgG level.Result as shown in Figure 6.
Result shows, carry the Ct specific serum IgG antibody reaction that epi-position all can strengthen immune mouse in N, C Duan He MIR district of HBcAg, all have statistical significance (P < 0.05) with PBS group, HBcAg vehicle group and synthetic peptide group comparing difference; Produce peak period at the 6th week antibody, the protein antibodies generation of carrying epi-position with the N of HBcAg carrier end is significantly higher than C Duan He MIR district (P < 0.05) (Fig. 6 A).The MIR district of HBcAg carries the Ct specific IgG antibodies that 1 epi-position (HBcAg (MIR)/MOMP epi-position 1) and series connection 2 (HBcAg (MIR)/MOMP epi-position 1+2) and 3 epi-positions (HBcAg (MIR)/MOMP epi-position 1+2+3) produce and reacts, be high with HBcAg (MIR)/MOMP epi-position 1, tool significant difference (P > 0.05) (Fig. 6 B).
(2) mensuration of immune mouse secretory product specific antibody IgA:
Adopt indirect ELISA method, method is the same, and result as shown in Figure 7.
Latter 6th week of immunity, carry the Ct specificity genital secretion IgA antibody reaction that epi-position all can strengthen immune mouse in N, C Duan He MIR district of HBcAg, all have statistical significance (P < 0.05) with PBS group, HBcAg vehicle group and synthetic peptide group comparing difference; N, C Duan He MIR district of HBcAg carry neoepitope Western IgA antibody produce between compare not statistically significant (P > 0.05) (Fig. 7 A).1 epi-position (HBcAg (MIR)/MOMP epi-position 1) and series connection 2 (HBcAg (MIR)/MOMP epi-position 1+2) and 3 epi-positions (HBcAg (MIR)/MOMP epi-position 1+2+3) are carried, Ct Specific IgA antibody reaction there was no significant difference (P > 0.05) (Fig. 7 B) of generation by the MIR district of HBcAg.
(3) immune mouse specificity CTL killing ability result:
Get spleen and prepare splenocyte suspension adjustment concentration to 2 × 10
6individual/ml, adopts serum lactic dehydrogenase (LDH) method for releasing to detect CTL specific killing activity.Result as shown in Figure 8 and Figure 9.
After immunity, N, C Duan He MIR district of HBcAg carry epitopic immune group and epi-position synthetic peptide group mouse effector cell with target cell ratio (E:T) for 40:1 and 20:1 time, for the specific CTL killing activity of Ct MOMP, comparatively PBS group, HBcAg vehicle group have statistical significance (P < 0.05); The killing activity that the protein immunization group that epi-position is carried in the C end of HBcAg, MIR district carries epitopic immune group and epi-position synthetic peptide group compared with N end has significant difference (P < 0.05); But the protein immunization group that epi-position is carried in the C Duan He MIR district of HBcAg compares not statistically significant (P > 0.05) (Fig. 8 A); The MIR district of HBcAg carries Ct specific killing activity that 2 (HBcAg (MIR)/MOMP epi-position 1+2) and 3 series connection (HBcAg (MIR)/MOMP epi-position 1+2+3) produce and comparatively carries 1 epi-position (HBcAg (MIR)/MOMP epi-position 1) and synthetic peptide group has outstanding sex differernce (P < 0.05) (Fig. 8 B).Also create the specific killing activity of HBcAg, the killing activity that the protein immunization group of holding (Fig. 9 A) and MIR district thereof to carry 2 and 3 epitopes (Fig. 9 B) with the C of HBcAg carrier produces is remarkable (P < 0.05) simultaneously.
(4) HBcAg/Ct MOMP epiposition vaccine immanoprotection action that mouse propagation road Ct is infected:
Each group of mouse final immunization, after 2 weeks, carries out genital tract infection attack with E serotype chlamydia trachomatis type strain (purchased from American typical case thing preservation center (ATCC:VR-348B)).Result shows: transvaginal Ct rises after attacking on the 1st day, and each group mouse all starts the infection sign occurred in various degree, and as vulva has redness, secretory product is abnormal.After mouse propagation road Ct attacks, genital secretion Ct separation and Culture IFU count results, compare with synthetic peptide group, PBS group and HBcAg vehicle Control group, whole cell group clearance rate to 18 day disappears; Carry epi-position at N, C end of HBcAg, can significantly improve Ct clearance rate, be zero (Figure 10 A) respectively at 21 and 24 days clearance rates; The clearance rate that MOMP series connection epi-position significantly can strengthen immune mouse is carried in MIR district, and especially 3 series connection groups (HBcAg (MIR)/MOMP epi-position 1+2+3) namely can be zero (Figure 10 B) 18 days clearance rates.
The outer reproductive tract inflammatory score result of mouse is as Figure 11.Observe and the recall rate interpretation of result of reproductive tract Ct separation and Culture from mouse episioitis, show N, C of HBcAg hold (Figure 11 A) and MIR district carry epi-position and multiple epi-position connect (Figure 11 B) all can strengthen the immanoprotection action of mouse.
Conclusion:
Prepared by application has carried out series of animal experiments based on HBcAg virus-like particle support C t MOMP epiposition vaccine, and result confirms:
1. laboratory animal all creates the specific protection antibody of Ct and Ct specificity CTL killing ability for HBcAg/Ct MOMP epiposition vaccine in serum and vaginal secretions.Show that this vaccine can produce humoral immunization and the cell immunoreceptor of stronger anti-Ct.Show the effect with prevention and therapy Ct infection.
2. laboratory animal creates the specific cellular immunization of HBcAg after the immunity of HBcAg/Ct MOMP epiposition vaccine.Show that HBcAg does not affect the cell immunogenicity of himself (HBcAg specificity) as the carrier of heterologous epitope.
3. laboratory animal creates the serum IgG, secretory product IgA and the specificity CTL killing ability that significantly strengthen for HBcAg/Ct MOMP epiposition vaccine (especially HBcAg (MIR)/Ct MOMP connect epiposition vaccine).Result shows that HBcAg can strengthen the immunological effect of CtMOMP epi-position.
4. mouse all has stronger immunoprophylaxis and therapeutic action through the inoculation of HBcAg/Ct MOMP epiposition vaccine to E type Ct infection.Show that HBcAg/Ct MOMP epiposition vaccine (especially HBcAg (MIR)/Ct MOMP connect epi-position 1+2+3) immunity is attacked Ct reproductive tract and had good provide protection, played the effect of vaccine.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (13)
1. a chimeric amino acid sequence, described sequence by comprising hepatitis B virus core antigen albumen that is wild or that modify through transformation, and by the one or more peptide section sequences that are formed by connecting in following member:
The aminoacid sequence (n >=1) of (a) Major Outer Membrane Protein of Chla mydia trachomatis n immunodominant epitope
The short peptide sequence (i >=0) of (b) i flexible amino acid composition.
2. chimeric amino acid as claimed in claim 1, it is characterized in that, described Major Outer Membrane Protein of Chla mydia trachomatis epitope polypeptide is one of following member and the derivative continuously with the amino acid of n contained by it (n >=5):
(1) immunodominant epitope polypeptide 1 aminoacid sequence is as shown in SEQ ID NO:1
(2) immunodominant epitope polypeptide 2 aminoacid sequence is as shown in SEQ ID NO:2
(3) immunodominant epitope polypeptide 3 aminoacid sequence is as shown in SEQ ID NO:3.
3. immunodominant epitope polypeptide 1 as claimed in claim 2, it is characterized in that, the nucleotide sequence of this polypeptide of encoding is through the codon optimized modification of protokaryon, and sequence is selected from: SEQ ID NO:4 and SEQ ID NO:7.
4. immunodominant epitope polypeptide 2 as claimed in claim 2, it is characterized in that, the nucleotide sequence of this polypeptide of encoding is through the codon optimized modification of protokaryon, and sequence is selected from: SEQ ID NO:5 and SEQ ID NO:8.
5. immunodominant epitope polypeptide 3 as claimed in claim 2, it is characterized in that, the nucleotide sequence of this polypeptide of encoding is through the codon optimized modification of protokaryon, and sequence is selected from: SEQ ID NO:6 and SEQ ID NO:9.
6. chimeric amino acid as claimed in claim 1, is characterized in that, described original or be one of following member through the hepatitis B virus core antigen albumen that transformation is modified:
(1) aminoacid sequence shown in SEQ ID NO:10
(2) aminoacid sequence shown in SEQ ID NO:11.
7. the DNA sequence dna of coding hepatitis B virus core antigen according to claim 1-Major Outer Membrane Protein of Chla mydia trachomatis epi-position chimeric amino acid.
8. prepare the method for hepatitis B virus core antigen-Major Outer Membrane Protein of Chla mydia trachomatis epi-position chimeric DNA, utilize the method for PCR to amplify wild hepatitis B virus core antigen albumen and the gene order corresponding to the hepatitis B virus core antigen albumen through transforming respectively, again by Major Outer Membrane Protein of Chla mydia trachomatis epi-position (as sequence SEQ ID NO:1, SEQ ID NO:2, shown in SEQ ID NO:3) insert in some way, be inserted into the identical or different license site of above-mentioned hepatitis B virus core antigen respectively, as C end, N Duan He MIR district etc.
9. insertion optimal way according to claim 8, it is characterized in that: single epi-position repeats to insert, single epi-position repeats to insert behind the small peptide interval that flexible amino acid forms, different single epi-position combined serial is inserted, different single epi-position combined serial repeats to insert, different single epi-position combined serial behind the small peptide interval that flexible amino acid forms is inserted, and different single epi-position mode that combined serial repeats behind the small peptide interval that flexible amino acid forms is inserted.
10. the identical or different license site of hepatitis B virus core antigen as claimed in claim 8, it is characterized in that, the MIR district of original and through transformation modification HBcAg, or any n the amino acid (1≤n≤6) of replacing between HBcAg 78-83 amino acids, or insert aminoterminal and the carboxyl terminal of modifying HBcAg through transformation, become an artificial gene, and purifying after this gene is expressed in expression vector system, obtain hepatitis B virus core antigen-Major Outer Membrane Protein of Chla mydia trachomatis epi-position chimeric amino acid.
11. 1 kinds of recombinant vectorss, described carrier comprises gomphosis DNA array according to claim 7; A genetically engineered host cell, described host cell contains described carrier.
12. chimeric amino acid sequence as claimed in claim 1, recombinant vectors according to claim 11 and the host cell purposes in the chimeric for the preparation of prevention or treatment chlamydia trachomatis relative disease.
13. 1 kinds of compositions, it includes recombinant vectors according to claim 11 and acceptable carrier, vehicle or the adjuvant pharmaceutically or in immunology of effective amount; And a kind of antibody, it is characterized in that, described antibodies specific ground is combined with epitope polypeptide according to claim 2.
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