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

Abstract

The invention relates to a chimeric vaccine of hepatitis B virus and chlamydia trachomatis and its application. More specifically, the present invention relates to a recombinant fusion protein comprising one or several epitopes of the main outer membrane protein of Chlamydia trachomatis, and the epitope polypeptide is in the form of a single epitope or multiple epitopes combined Inserted into the same or different permission sites of the hepatitis B core antigen (HBcAg) protein to form a recombinant fusion protein that can stimulate the body to produce humoral immunity and cellular immunity against Chlamydia trachomatis, and preliminarily evaluated the immunogenicity of various fusion proteins and immune protection, which laid the foundation for the in-depth research and application of epitope vaccines based on HBcAg vectors in the prevention and treatment of Chlamydia trachomatis infection and related diseases. The vaccine of the invention has high-efficiency and safe immune prevention and treatment effects on diseases related to chlamydia trachomatis, and the preparation process and immune process are simple, the effect is obvious, and the reproducibility is strong.

Description

Chlamydia trachomatis main outer membrane protein epitope vaccine based on HBcAg carrier and application thereof

technical field

The invention relates to the fields of biomedicine technology and immunology, in particular to a Chlamydia trachomatis main outer membrane protein immunodominant epitope vaccine with hepatitis B virus core antigen as a carrier, its preparation method, and its role in preventing and treating Chlamydia trachomatis infection in the application. the

Background technique

Chlamydia trachomatis (Ct) is the most common pathogen of sexually transmitted diseases (STDs) in the world, which can cause urethritis in men and cervicitis in women. According to reports, 50-60% of urethritis in men is caused by Ct infection (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 often mixed infection with other pathogens, sexually transmitted infection of female urinary tract Reproductive tract, leading to adverse pregnancy, such as miscarriage, stillbirth, etc., and is closely related to cervical cancer (Alibek K, 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 can still cause trachoma through contact, which is the main cause of blindness (Lu C, 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 is closely related to some autoimmune diseases, such as reactive arthritis, rheumatoid arthritis, SLE and arteriosclerosis, etc. (Costapinto L, 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 infection is very easy to relapse after treatment. WHO estimates that there are about 90 million Ct patients in the world, and billions of dollars are spent on the treatment of Ct infection every year (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 incidence of STDs caused by Ct in my country has been on the rise, which has caused great harm to human health. However, there is still a lack of effective prevention and treatment methods so far. Therefore, seeking effective vaccines is one of the hotspots and frontier topics of current research. . the

An antigenic epitope is a basic structural unit or a special chemical group that can specifically bind to T cell receptors (TCR), B cell receptors (BCR) or antibodies in an antigen molecule, and determines the specificity of the antigen. The vaccine based on epitope design, that is, epitope vaccine (Epitope vaccine), is a uniquely designed, highly targeted, safe and stable vaccine developed in recent years, and can be produced by different combinations of T and B cell antigen epitopes. A chimeric vaccine against multiple pathogens (XuW, 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 capsid1 proteincine L2.0 V1.0 ; 29(15): 2672-2678; Robinson JA. Max Bergmann lecture Protein epitope mimetics in the age of structural vaccination. J Pept Sci. 2013; 19(3): 127-140). However, studies suggest that the immunogenicity of epitope vaccines needs to be enhanced. Studies have proved that hepatitis B virus core antigen (HBcAg) has good immunogenicity, and the virus-like particles (VLPs) formed by HBcAg have significant advantages as carriers for carrying heterologous proteins: the MIR region of VLPs formed by HBcAg is very suitable for carrying foreign proteins. The source peptide does not affect the formation and immunogenicity of its VLPs; HBcAg VLPs have an adjuvant effect that enhances the immunogenicity of foreign peptides; HBcAg VLPs can be produced in a prokaryotic expression system on a large scale, so as to achieve economical and applicable purposes (Yang Xingyu , Bo Hong, Shu Yuelong; Major progress in the research of hepatitis B virus core antigen as a carrier for virus-like particle vaccines. Acta Virus Sinica. 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). the

Ct has 19 serotypes, of which serotypes such as D-H mainly cause urogenital tract infections, and types E and D are reported at home and abroad (Chen Lili, Wu Yimou, Deng Zhongliang, etc. Urogenital trachoma in some cities in southern China Chlamydia genotyping research. 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. The most common type in Sex Transm Dis, 2003, 30:357-361). Ct major outer membrane protein (MOMP) is the main target of neutralizing antibodies (Klein M, Kotz A, Bernardo K, et al. Kronke M. Detection of Chlamydia pneumoniae-specific antibodies binding to the VD2 and VD3 regions of the major outer membrane protein .J Clin Microbiol.2003, 41 (5): 1957-1962), thus is the target antigen of Ct vaccine research. However, when the whole bacterium or target antigen is used to immunize the human body, a severe pathological immune response can be generated (Fletcher MA. Vaccine candidates in STD. Int J STD AIDS, 2002, 13. Suppl2: 38-41). Therefore, using the immunodominant epitope on the MOMP protein can solve the above problems to a certain extent, and it may be developed into a 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, many laboratories have successively reported a series of cell epitopes, most of which are located on the MOMP of Chlamydia trachomatis, and peptides composed of these cell epitopes can induce specific immune responses in the body (Wang Ledan, Zhang Lifang. The main foreign body of Chlamydia trachomatis Research progress of membrane protein epitope. Foreign medicine. Prevention. Diagnosis. Biological products for treatment, 2005, 28(5): 196-199). the

At present, research on Ct vaccines mainly includes whole bacterial vaccines, subunit vaccines, recombinant vaccines, and synthetic peptide vaccines, but the effects are not satisfactory. Since natural whole bacteria and MOMP protein may induce pathological immune response, the T cell epitope and B cell epitope with immunodominance in MOMP protein are selected to form the T-B joint epitope of Ct MOMP, and the multi-epitope gene After cloning the prokaryotic expression plasmid and identifying it, mice were immunized, and specific antibody IgG, slgA against CT and specific CTL cell immunity were detected in mouse serum and vaginal secretions, and they had the ability to attack Ct in the reproductive tract of the mouse model Certain protective effect (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; 2.1 15): 2672-2678). Therefore, epitope vaccine is one of the research directions of Chlamydia trachomatis vaccine, and enhancing the immunogenicity and protective effect of epitope is the focus of current research. the

In summary, there is an urgent need in this field to develop a vaccine that can enhance the immunogenicity and immunoprotective effect of Chlamydia trachomatis combined epitope vaccine, which can be applied to diseases caused by Chlamydia trachomatis infection such as genitourinary tract infection, trachoma and Chlamydia trachomatis infection related autoimmune diseases. the

Contents of the invention

The purpose of the present invention is to provide a vaccine that can be used for the prevention and treatment of Chlamydia trachomatis infection and related autoimmune diseases (such as reactive arthritis, rheumatoid arthritis, SLE and arteriosclerosis, etc.). the

In a first aspect of the present invention, an amino acid sequence is provided, said sequence comprising:

(a) the amino acid sequence encoding the immunodominant epitope peptide of the major outer membrane protein of Chlamydia trachomatis; and

(b) Amino acid sequence encoding hepatitis B virus core antigen. the

In a preferred example, the immunodominant epitope peptide of the major outer membrane protein of Chlamydia trachomatis is an amino acid sequence containing multiple CTL epitopes, Th epitopes and B cell epitopes. the

In another preferred example, the amino acid sequences of the multiple CTL epitopes, Th epitopes and B cell epitopes are arranged in series or overlap. the

In another preferred example, the amino acid sequence in (a) is modified by prokaryotic codon optimization. The C-terminus of the amino acid sequence in (b) was inserted into a universal Th epitope, and His-tag was inserted into its N-terminus and C-terminus for modification. the

In another preferred example, the nucleotide sequence encoding the immunodominant epitope peptide of the major outer membrane protein of Chlamydia trachomatis is connected to the carboxy-terminal or amino-terminal of the nucleotide sequence encoding the hepatitis B virus core antigen or inserted into its MIR region. the

In another preferred example, the amino acid sequences in (a) and (b) are linked directly or via a linking sequence. the

In one embodiment of the present invention, the nucleotide sequences of the immunodominant epitope peptides of the main outer membrane protein of Chlamydia trachomatis are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. the

In another embodiment of the present invention, the nucleotide sequence of the hepatitis B virus core antigen is shown in SEQ ID NO: 12 (wild type nucleotide sequence). the

In another embodiment of the present invention, the nucleic acid sequence encoding the main outer membrane protein immunodominant epitope peptide of Chlamydia trachomatis is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 (i.e. not codon optimized sequence) and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 (i.e. prokaryotic codon optimized nucleotide sequence). the

In another embodiment of the present invention, the nucleotide sequence encoding the core antigen of hepatitis B virus is selected from the group consisting of: SEQ ID NO: 12 (i.e. wild-type nucleotide sequence) and SEQ ID NO: 13 (i.e. adding universal Th epitope and the nucleotide sequence of His-tag). the

In the second aspect of the present invention, a recombinant vector is provided, said vector comprising the above-mentioned chimeric nucleic acid sequence of the present invention. the

In a preferred example, the vector is selected from: eukaryotic expression vectors or prokaryotic expression vectors, preferably bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or mammalian cell viruses, more preferably pET21a (+), pET32a (+ ), pcDNA3.1(+), pSIREN-NEO, pQE30, pGEX-4T-1, pPICZA. the

In the third aspect of the present invention, a genetically engineered host cell containing the above-mentioned vector of the present invention is provided. the

In a preferred example, the host cell is selected from prokaryotic cells, lower eukaryotic cells or higher eukaryotic cells, preferably animal cells, Escherichia coli, or yeast, more preferably E.coli, GS115, COS-7 cells, COS-1 cells. the

In the fourth aspect of the present invention, the use of the above-mentioned chimeric nucleic acid sequence, recombinant vector or host cell of the present invention in the preparation of vaccines for preventing or treating Chlamydia trachomatis infection and related diseases is provided. the

In a preferred example, the Chlamydia trachomatis-related diseases are selected from: Chlamydia trachomatis infectious diseases: urethritis, cervicitis, trachoma, infertility, etc., and autoimmune diseases related to Chlamydia trachomatis infection: cervical cancer, reactive Arthritis, rheumatoid arthritis, SLE and arteriosclerosis, etc. the

In the fifth aspect of the present invention, a composition is provided, which comprises an effective amount of the aforementioned chimeric nucleic acid sequence, recombinant vector or host cell of the present invention and pharmaceutically or immunologically acceptable carriers and excipients or adjuvants. the

In a preferred example, the composition is an immune composition or a pharmaceutical composition, preferably a vaccine. the

In a preferred example, the carrier, excipient or adjuvant is selected from: liposome, starch, gelatin, aluminum salt adjuvant, saponin adjuvant or Ribi adjuvant. the

In another preferred example, the composition contains 0.01-99.9wt% of the recombinant vector of claim 4, preferably 0.1-99.0wt%. the

In another preferred embodiment, the composition is intramuscular injection, skin immunization or mucosal immunization. the

In the sixth aspect of the present invention, there is provided a method for preparing the above-mentioned recombinant vector of the present invention, the method comprising:

(i) Provide the nucleic acid sequence encoding the immunodominant epitope peptide of the main outer membrane protein of Chlamydia trachomatis and the nucleic acid sequence encoding the core antigen of hepatitis B virus;

(ii) connecting the nucleic acid sequences to form a chimeric nucleic acid sequence;

(iii) Cloning the chimeric nucleic acid sequence obtained in step (ii) into a vector. the

In a preferred example, the sequence of the hepatitis B virus core antigen protein is as SEQ ID NO: 10 (unmodified original sequence) and SEQ ID NO: 11 (that is, the sequence modified by the general Th epitope and His-tag) shown. the

In another preferred example, the nucleic acid sequence encoding the hepatitis B virus core antigen is selected from the group consisting of: SEQ ID NO: 12 (i.e. wild-type nucleotide sequence), SEQ ID NO: 13 (i.e. through the general Th epitope and His- tag modified sequence). the

In another preferred example, the nucleic acid sequence encoding the immunodominant epitope peptide of the major outer membrane protein of Chlamydia trachomatis is connected to the carboxy-terminal or amino-terminal of the nucleic acid sequence encoding the hepatitis B virus core antigen or its MIR region. the

It should be understood that other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. the

Description of drawings

Figure 1: PCR identification (A) and sequencing results (B) of the modified HBcAg gene. the

In Figure 1(A), "M" means: DL2000 DNA marker; "1-4" all mean: recombinant plasmid pET21a(+)/HBcAg

PCR product of the gene. Figure 1(B) is the sequencing result. the

Figure 2 HBcAg/Ct MOMP epitope recombinant plasmid sequencing results

(A) HBcAg(MIR)/Ct MOMP epitope 1 sequencing results. the

(B) HBcAg(MIR)/Ct MOMP epitope 1+2 sequencing results. the

(C) HBcAg(MIR)/Ct MOMP epitope 1+2+3 sequencing results. the

(D) Sequencing results of Ct MOMP epitope 1/HBcAg. the

(E) HBcAg/CtMOMP epitope 1 sequencing results. the

Figure 3. SDS-PAGE of HBcAg/CtMOMP epitope expression (Figure 3A), protein purification electrophoresis (Figure 3B) and WB analysis (Figure 3C, Figure 3D). the

"M" means: standard protein molecular weight marker; "1" is E.coli Rosseta strain; "2" is pET21a(+) empty vector; "3" is pET21a(+)/HBcAg; "4" is Ct-MOMP expression Position 1/HBcAg (N terminal); "5" is HBcAg/Ct-MOMP epitope 1 (C terminal); "6" is HBcAg(MIR)/Ct-MOMP epitope 1 (MIR region). The primary antibody used in WB analysis was the serum antibody of inactivated Ct whole bacterium immunized mice. B: SDS-PAGE electrophoresis diagram of each purified protein. the

Figure 4. SDS-PAGE (Figure 4A), WB (Figure 4B) analysis of HBcAg/Ct MOMP tandem epitope expression and protein purification electrophoresis (Figure 4C)

"M" means: standard protein molecular weight marker; "1" is E.coli Rosseta strain; "2" is pET21a(+) empty vector; "3" is pET21a(+)/HBcAg; "4" is HBcAg(MIR) /Ct-MOMP epitope 1; "5" is HBcAg(MIR)/CtMOMP epitope 1+2; "6" is HBcAg(MIR)/Ct MOMP epitope 1+2+3. The primary antibody used in WB analysis was the serum antibody of inactivated Ct whole bacterium immunized mice. B: SDS-PAGE electrophoresis diagram of each purified protein. the

Figure 5 Electron microscope observation of MOMP epitope protein with HBcAg as carrier

"A" is HBcAg; "B" is HBcAg/MOMP epitope 1; "C" is MOMP epitope 1/HBcAg; "D" is HBcAg(MIR)/MOMP epitope 1; "E" is HBcAg(MIR) /epitope 1+2; "F" is HBcAg(MIR)/epitope 1+2+3. Virus-like particles can be formed when observed under an electron microscope. the

Fig. 6 Dynamic changes of serum Ct-specific antibody IgG after immunization of mice in each group. the

Fig. 7 Dynamic changes of Ct-specific antibody IgA in reproductive tract secretion of mice in each group after immunization. the

Figure 8 CtMOMP-specific CTL killing activity produced by mice in each group after immunization. the

Figure 9 The HBcAg-specific CTL killing activity produced by mice in each group after immunization. the

Fig. 10 Clearance rate (IFU count) of reproductive tract Ct of mice in each group after challenge. the

Fig. 11 Scores of external genital tract inflammation of mice in each group after Ct challenge. the

Detailed ways

The present inventors have prepared the HBcAg/Ct MOMP epitope vaccine based on the Ct MOMP immunodominant epitope (referred to as epitope) gene, optimized prokaryotic codons, and used HBcAg as a carrier. Animal immunization experiments have confirmed that the HBcAg/CtMOMP epitope vaccine can both prevent and treat Ct infection in the reproductive tract of mice, especially the tandem epitope vaccine carried by the HBcAg MIR region has a significantly better immune protection effect on Ct than Ct MOMP epitope vaccine. Synthetic peptide vaccines and chimeric vaccines added at the N- or C-terminus of HBcAg. On this basis, the inventors have completed the present invention. the

Specifically, the inventors obtained Ct MOMP immunodominance by predicting and screening shared amino acid sequences containing multiple CTL epitopes, Th epitopes, and B cell epitopes, and optimized prokaryotic codon modification of the gene Epitope gene (referred to as epitope), and further form it in series with the B cell epitope confirmed by our laboratory. At the same time, the inventors also modified HBcAg as a carrier by adding universal Th epitope (PADRE) and His-tag. Then, the HBcAg/CtMOMP epitope vaccine (that is, the HBcAg-N-terminal, C-terminal and MIR regions carry the CtMOMP epitope respectively) was prepared by molecular biology techniques. the

The inventor further tested the immune protection effect of the above-mentioned epitope vaccine by immunizing mice: ELISA method was used to detect the serum IgG and vaginal secretion IgA antibody levels of mice after immunization, and the specific killing activity of splenic lymphocyte CTL to evaluate the epitope vaccine immune effects and immune protection against Ct reproductive tract challenge in a mouse model. The results show that the HBcAg/CtMOMP epitope vaccine has both immune prevention and treatment effects on mouse reproductive tract Ct infection, and its immune prevention and control effect on Ct is significantly better than the CtMOMP epitope synthetic peptide vaccine, especially the tandem epitope vaccine carried by the MIR region The protective effect of Chlamydia trachomatis inactivated whole bacteria vaccine is close. the

The present invention proves through experimental research that the prepared HBcAg/Ct MOMP epitope vaccine can stimulate the body to produce strong humoral immune effects specific to Ct, especially protective antibodies to the local reproductive tract mucosa, and cellular immunity to Ct, It has immune prevention and treatment effects on Ct infection, and has laid a foundation for the in-depth research and application of epitope vaccines based on VLPs vectors to prevent and treat Chlamydia trachomatis infection and related diseases, and has important scientific research significance and application value. the

At the same time, the present invention also verifies the immunoenhancing effect of the HBcAg carrier on the heterologous Ct MOMP epitope through animal experiments. The research results confirmed that HBcAg/CtMOMP epitope protein can induce the body to produce enhanced Ct-specific humoral immunity and cellular immunity, especially the local IgA antibody response in the mucosa, and immune protection against Ct reproductive tract attack. These immune effects are better than those of synthetic CtMOMP epitope peptide vaccines, especially those with tandem epitopes in the MIR region of HBcAgB, thus enhancing the immune protection effect. the

Glossary

The term "immunological activity" or "immunogenicity" herein refers to the ability of a natural, recombinant or synthetic vaccine to induce a specific humoral and/or cellular immune response in a mammal. The terms "immunodominant epitope vaccine", "synthetic peptide vaccine" or "chimeric (genetic) vaccine" as used herein refer to chimeric nucleic acid sequences of the invention and expressed fusion proteins thereof capable of eliciting an immune response in a mammal. the

The term "immune response" herein includes cellular and/or humoral immune responses sufficient to suppress or prevent infection; or to prevent or suppress disease caused by Chlamydia trachomatis infection and its associated autoimmune diseases. the

As used herein, the term "subject" or "individual" or "patient" refers to any subject in need of diagnosis or treatment, especially a mammalian subject, especially a human, other subjects including cattle, dogs, cats, guinea pigs, rabbits, rats , mice, horses, etc. Of particular concern are those subjects who are susceptible or have been infected with C. trachomatis. the

The terms "nucleic acid" and "nucleic acid sequence" herein refer to nucleotides (ribonucleotides or deoxyribonucleotides) of any length in polymeric form. It includes (but is not limited to) single- and double-stranded DNA or RNA, genomic DNA and cDNA. the

The term "effective amount" or "immunologically effective amount" herein refers to an amount administered to an individual as a single dose or a fraction of consecutive doses that is effective for treatment or prophylaxis. The amount depends on the health and physiological conditions of the individual to be treated, the category of the individual to be treated (such as non-human primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection required, the preparation of the vaccine, and the treatment physician's opinion on medical treatment. Depends on the assessment of the situation, and other relevant factors. This amount is expected to lie within a relatively wide range and can be determined by routine experimentation. the

As used herein, unless otherwise stated, the "immunodominant epitope of the present invention", "epitope", "epitope protein", "tandem epitope" and the like can be used interchangeably. the

Chimeric proteins and nucleotide sequences

The HBcAg/Ct MOMP epitope sequence of the present invention comprises: (a) the aminoacid and nucleic acid sequence of coding Chlamydia trachomatis major outer membrane protein immunodominant epitope (Ct MOMP); and (b) the aminoacid and nucleic acid sequence of coding hepatitis B virus core antigen . the

The term "Chlamydia trachomatis major outer membrane protein (Ct MOMP) immunodominant epitope" refers to an amino acid sequence containing multiple MOMP CTL epitopes, Th epitopes and B cell epitopes. The immunodominant epitope can be obtained by predicting and screening the common epitopes of various serotype Ct MOMPs, and the immunodominant table will be composed of multiple HLA-A2 specific CTL epitopes and Th and B cell epitopes. bit. For example, the network resource database can be used to obtain the MOMP gene and amino acid sequence of each serotype of Ct, and the HLA-A*0201-restricted T cell epitope (CTL), Th epitope and B cell For epitope prediction, the dominant epitopes with high scores are selected and connected into multi-epitope genes, preferably in series. The dominant epitopes include (but are not limited to): HLA-A2 restricted CTL epitopes (379-387aa), Th epitopes (373-387aa, 370-384aa) and B cell epitopes (377-386aa), These include CTL epitopes of H-2d type (267-275aa, 371-379aa, 370-378aa). the

The term "hepatitis B virus core antigen (HBcAg)" refers to one of the main components of the hepatitis B virus capsid - hepatitis B core antigen, and its coding sequence is known in the art. Those of ordinary skill in the art should understand that these known HBcAg coding sequences can be used in the chimeric amino acid sequences of the present invention. the

It should be understood that the nucleic acid sequence of the present invention may also include molecules that hybridize to the above-mentioned coding sequence under stringent conditions and have the same or similar activity. The term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2) denaturing agent added during hybridization, Such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 50%, preferably more than 55%, 60% More than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90%, more preferably more than 95%, hybridization occurs. For example, the sequence may be a complementary sequence. the

In a preferred embodiment of the present invention, the prokaryotic codon optimization modification is performed on the Ct MOMP coding sequence of the present invention, so that the resulting gene can obtain higher expression in the prokaryotic expression system. Preferably, one or both of the chimeric nucleic acid sequences are nucleic acid sequences modified by prokaryotic codon optimization. The "prokaryotic codon optimization modification" can be carried out using conventional methods known in the art (for example, 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. 1999 Feb 5 15; 27(4): 1094-103). Preferably, the GC content in the nucleic acid sequence is increased by 5-40%, preferably 10-30%, more preferably 13-23%, but the encoded amino acid remains unchanged. the

The nucleic acid sequences of the present invention may be linked directly, or via a linker sequence. The term "connecting sequence" herein refers to a sequence that is located between the nucleic acid sequence encoding the main outer membrane protein epitope of Chlamydia trachomatis and its tandem epitope and the nucleic acid sequence encoding the hepatitis B virus core antigen. The length of the linker sequence is not particularly limited, usually 0-100. The length of the connecting peptide can also be 0, which means that the gene sequences are directly connected. Typically, the linking sequence has no or no significant effect on the expression of the linked sequence and the immune effect of the antigen produced. the

In a preferred embodiment of the present invention, the Ct MOMP coding sequence is connected to the MIR region or the carboxy-terminal or amino-terminal of the HBcAg coding sequence. the

Vectors and host cells

After obtaining the chimeric nucleic acid sequence encoding the present invention, it can be connected into a suitable expression vector, and then transformed into a suitable host cell. the

In the present invention, the term "vector" and "recombinant vector" are used interchangeably, and refer to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. Carriers selected from the following group can be used in the present invention: prokaryotic expression vectors or eukaryotic expression vectors, preferably bacterial plasmids, phages, yeast plasmids, plant cell viruses, or mammalian cell viruses, more preferably pET21a (+), pET32a (+ ), pcDNA3.1(+), pSIREN-NEO, pQE30, pGEX-4T-1 or pPICZA. the

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell or an animal cell. Representative examples include: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like. In the present invention, E.coli, GS115, COS-7 cells, and COS-1 cells are preferably used. the

The immunodominant epitope vaccine of the present invention is composed of the gene encoding the target protein and the expression vector of the plasmid. After being directly introduced into the host cell, it does not integrate with the host chromosome, but expresses the protein antigen through the transcription system of the cell, and induces the body to produce specific cell Immunity and humoral immunity. the

combination

The present invention also provides various compositions comprising the recombinant vector of the present invention, including pharmaceutical compositions and vaccine compositions. The composition can be used to prevent or treat Chlamydia trachomatis infection and related diseases, including (but not limited to): urethritis, cervicitis, trachoma, infertility, cervical cancer, reactive arthritis, rheumatoid Arthritis, SLE and arteriosclerosis, etc. the

Various compositions comprising the recombinant vector of the present invention may contain a buffer selected according to the actual use of the recombinant vector; and may also contain other substances suitable for the intended use. The choice of buffering agent is well within the skill of the art, and a variety of buffering agents are known in the art to be suitable for the intended use. In some instances, the composition may contain a pharmaceutically acceptable excipient, many of which are known in the art and need not be discussed in detail here. Various pharmaceutically acceptable excipients are described in various publications, including, for example, "Remington's Pharmaceutical Sciences" ("Remington's Pharmaceutical Sciences", 19th Edition (1995) Mack Publishing Co.). the

The pharmaceutical composition or vaccine composition can be prepared into various dosage forms, such as injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, suppositories, transdermal drugs (such as patches, etc.), ointments, Lotion etc. Pharmaceutical grade organic or inorganic carriers and/or diluents, suitable for oral or topical use, may be used in formulating various compositions containing the therapeutically active compounds. Diluents known in the art include aqueous media, vegetable and animal oils and fats. Stabilizers, wetting agents and emulsifiers, salts to change the osmotic pressure or various buffers and skin penetration enhancers to maintain a suitable pH value can also be used as auxiliary materials. the

When used as a vaccine, the recombinant vector of the present invention can be formulated in various ways. Generally, the vaccine or medicine of the present invention is formulated with suitable pharmaceutical carriers and/or vehicles according to various methods well known in the art. A suitable carrier is sterile saline. Other aqueous and nonaqueous isotonic sterile injection solutions and aqueous and nonaqueous sterile suspensions (all known pharmaceutically acceptable carriers well known to those skilled in the art) may also be used for this purpose. the

In addition, the preparation of the vaccine composition of the present invention may also contain other components, including, for example, adjuvants, stabilizers, pH regulators, preservatives and the like. These components are well known to those skilled in the vaccine art. Adjuvants include (but are not limited to) aluminum salt adjuvants; saponin adjuvants; Ribi adjuvants (Ribi ImmunoChem Research In., Hamilton, MT); Montanide ISA adjuvants (Seppic, Paris, France); Hunter's TiterMax Adjuvant (CytRx Corp., Norcross, GA); Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, 30 Germany) and the like. In addition, other components that modulate the immune response (IL-12, CpG oligodeoxynucleotides (CpG-ODN), etc.) may also be included in the formulation. the

Route of Administration and Dosage

When used as a vaccine, the recombinant vector of the present invention can be administered to a subject by a known method. These vaccines are usually administered by the same route of administration as conventional vaccines and/or by simulating pathogen infection routes. When in the form of a vaccine composition, a pharmaceutically acceptable carrier may also be included. In addition, this composition may also include adjuvants, flavoring agents or stabilizers and the like. the

Conventional and pharmaceutically acceptable routes of administration of the pharmaceutical or vaccine compositions of the present invention include: intranasal, intramuscular, intratracheal, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral Route of administration. The routes of administration can be combined if desired, or adjusted according to the antigenic peptide or disease state. Vaccine compositions may be administered in single or multiple doses, and may include administration of booster doses to elicit and/or maintain immunity. the

The recombinant vaccine should be administered in an "effective amount", that is, the amount of the recombinant vector is sufficient to trigger an immune response in the selected route of administration, and can effectively promote the protection of the host against Chlamydia trachomatis infection. the

The amount of recombinant vector used in each vaccine dose is determined according to the amount that can elicit an immune protective response without obvious side effects. Typically, each dose of vaccine is sufficient to produce about 1 μg-10 mg, preferably 5 μg-2 mg, more preferably 10 pg-1 mg of protein after infection of host cells. The effective dose of the vaccine calculated based on the recombinant epitope vaccine usually includes administration of about

1 μg-10 mg chimeric nucleic acid/kg body weight, preferably 1 μg-10 mg chimeric nucleic acid/kg body weight, more preferably 1 μg-10 mg chimeric nucleic acid/kg body weight. The optimal amount for a particular vaccine can be determined by standard research methods, including observation of antibody titers and other responses in subjects. Monitor the level of immunity provided by the vaccine to determine if a booster dose is needed. After assessment of antibody titers in sera, a booster dose of immunization may be required. Administration of adjuvants and/or immunostimulants can enhance the immune response to the proteins of the invention. the

Main advantages of the invention

The main advantages of the present invention are:

(1) provide a kind of Ct MOMP epitope vaccine based on HBcAg virus-like particle carrier, described epitope vaccine can induce body to produce effective humoral immunity against Ct specificity and specific cellular immunity against HBcAg, Chlamydia trachomatis, It has immune prevention and treatment effects on Ct infection and related diseases;

(2) Compared with the whole bacterium of Chlamydia trachomatis or its MOMP protein vaccine, the vaccine of the present invention has an effective immune protection effect on Chlamydia trachomatis infection, and can be used for more effective prevention and treatment of Chlamydia trachomatis infection and related diseases;

(3) The preparation process of the vaccine of the present invention is simple, low in cost, convenient and safe to use, and can activate the overall immune response of the body for a long time. It has the advantages of simple immunization process, safe inoculation, obvious effect, and strong repeatability. the

Example

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions (for example, with reference to Sambrook et al., "Molecular Cloning Laboratory Guide" (New York: Cold Spring Harbor Laboratory Press, 1989) or Immunology Methods Manual, Ivan Lefkovits, CRC, 1998) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. the

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only. the

Embodiment 1. Obtaining and modification of HBcAg carrier gene

Using the serum samples of hepatitis B virus carriers (Dasanyang) in Wenzhou as a template, PCR primers were designed and synthesized, and enzyme cutting sites (Ned I and Hind III) were introduced at both ends to amplify the complete open reading frame of HBcAg. The PCR product and The vector pET21a(+) was ligated after double digestion at the same time to construct the recombinant plasmid pET21a(+)/HBcAg(wt), which was identified by sequencing. the

In order to enhance the carrier effect of HBcAg and facilitate the molecular cloning operation, the HBcAg nucleotide (SEQ ID NO: 3) was modified and designed, that is, the universal Th epitope PADRE was inserted at its C-terminus, and the N- and C-terminals of the HBcAg carrier Each introduces a His-tag sequence to facilitate purification; at the same time, base mutations are carried out in its MIR region (78th and 82nd amino acids) to form restriction sites (BamH I and Sac I) that can be inserted into foreign genes, In order to carry out the insertion of the Chlamydia trachomatis MOMP epitope and its tandem epitope. Ned I and HindIII restriction sites were added to both ends of the modified HBcAg base sequence respectively, and after double digestion, it was connected to the vector pET21a(+) to construct the recombinant plasmid pET21a(+)/HBcAg(modified), and carried out PCR And sequence identification (Figure 1A, Figure 1B), for the preparation of vaccines. the

Example 2. Preparation and in vitro expression of HBcAg/CtMOMP epitope vaccine, analysis of VLPs formation

Based on the prokaryotic codon-optimized Ct MOMP immunodominant epitope gene and tandem epitope gene, complementary primers were designed and synthesized, and the two ends were respectively introduced with enzyme cutting sites BamH I and Sac I, and the target gene was obtained after annealing. The annealed product was ligated with the pET21a(+)/HBcAg digested by BamH I and Sac I to construct a recombinant prokaryotic expression plasmid containing the Ct MOMP epitope gene and the tandem epitope gene in the HBcAg MIR region; at the same time, the Ct MOMP Immunodominant epitopes were linked to the N and C terminals of the modified HBcAg, compared for research and identified by sequencing (Fig. 2). The results showed that: HBcAg(MIR)/Ct MOMP epitope 1, HBcAg(MIR)/Ct MOMP epitope 1+2, HBcAg(MIR)/CtMOMP epitope 1+2+3, Ct MOMP epitope 1/HBcAg and HBcAg /Ct MOMP epitope 1 chimeric recombinant plasmid was constructed successfully. The identified recombinant plasmids were transfected into Escherichia coli Rossetta strain, induced by IPTG, and subjected to SDS-PAGE analysis, WB identification and purification (Figure 3, Figure 4). The results showed that by SDS-PAGE analysis, the relative molecular mass of HBcAg was about 22kDa; The protein sizes of bit 1+2 and HBcAg(MIR)/MOMP epitope 1+2+3 are all about 22-25kDa, and the relative molecular mass is consistent with the expectation. At the same time, the serum of mice immunized with inactivated Ct whole bacteria was used as the primary antibody, and Western blot analysis confirmed the appearance of specific bands. Further electron microscope observation showed the formation of virus-like particles (Figure 5). the

The results indicated that the Ct MOMP epitope and its tandem epitope recombinant plasmid based on the HBcAg vector could be efficiently expressed in the prokaryotic expression system, and chimeric virus-like particles could be formed. the

Example 3. Immunogenicity and immune protection effect of HBcAg/CtMOMP epitope vaccine

Female BALB/c mice aged 6-8 weeks (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were randomly divided into 8 groups, 21 mice in each group, respectively: PBS control group, HBcAg group, HBcAg(MIR) /MOMP epitope 1 (MIR region carries dominant epitope 1), HBcAg(MIR)/MOMP epitope 1+2, HBcAg(MIR)/MOMP epitope 1+2+3(, Ct MOMP epitope 1/HBcAg( N-terminal), HBcAg/Ct MOMP epitope 1 (C-terminal) and Ct MOMP epitope synthetic peptide group. Take 100μl test samples (1tg/μl) at 0, 2, and 4 weeks respectively, and inject them into the back of the mouse for immunization Collect tail vein blood and vaginal secretions at 0w, 2w, 4w, 6w, 8w, 10w, 12w, 14w, 18w, and 20w after immunization, and detect Ct-specific IgG antibodies in serum and IgA antibodies in secretions by ELISA; In the 7th week, the spleen was taken to prepare splenocyte suspension, and the specific CTL killing activity of mice against CtMOMP and HBcAg was detected by lactate dehydrogenase (LDH) release method. The remaining mice in each group were challenged with E serotype Ct, Observe the activity of the mice and the severity of vulva inflammation every day after the challenge, score (0-3 points for vulva redness, 0-3 points for swelling, 0-3 points for secretions) and count through the separation and culture of mouse secretion Ct its clearance rate.

(1) Determination results of Ct-specific antibody IgG in serum of immunized mice:

Using the indirect ELISA method, the purified Ct whole cells were coated with a polyethylene micro-reaction plate, and after sealing, 1:50 diluted serum of immunized mice was added, reacted at 37°C for 2 hours, washed the plate, and added 1:2000 diluted HRP respectively - Goat anti-mouse IgG 100 μl, react at 37°C for 2 hours, after washing the plate, o-phenylenediamine (OPD) develops color, add 2 mol/L H ₂ SO ₄ to stop the reaction, measure the A490 value with a microplate reader. Each serum sample was tested repeatedly in 3 wells, and the serum specific antibody IgG level of immunized mice in each group was determined. The result is shown in Figure 6.

The results showed that carrying epitopes in the N, C and MIR regions of HBcAg could enhance the Ct-specific serum IgG antibody response of immunized mice, and the differences were statistically significant compared with the PBS group, HBcAg carrier group and synthetic peptide group ( P<0.05); at the peak period of antibody production at week 6, the antibody production of the protein carrying the epitope at the N-terminal of the HBcAg carrier was significantly higher than that at the C-terminal and MIR region (P<0.05) ( FIG. 6A ). The MIR region of HBcAg carries 1 epitope (HBcAg(MIR)/MOMP epitope 1) and tandem 2 (HBcAg(MIR)/MOMP epitope 1+2) and 3 epitopes (HBcAg(MIR)/MOMP epitope The Ct-specific IgG antibody response generated by the epitope 1+2+3) was higher in HBcAg(MIR)/MOMP epitope 1, with significant difference (P>0.05) (Fig. 6B). the

(2) Determination of specific antibody IgA in the secretion of immunized mice:

The indirect ELISA method was adopted, and the method was the same as above, and the results were shown in FIG. 7 . the

At the sixth week after immunization, the epitopes carried in the N, C and MIR regions of HBcAg can all enhance the IgA antibody response of Ct-specific reproductive tract secretions of immunized mice, and the difference is compared with that of PBS group, HBcAg carrier group and synthetic peptide group All had statistical significance (P<0.05); there was no statistical significance in the production of IgA antibodies carrying epitope proteins at the N, C and MIR regions of HBcAg (P>0.05) (Fig. 7A). The MIR region of HBcAg carries 1 epitope (HBcAg(MIR)/MOMP epitope 1) and tandem 2 (HBcAg(MIR)/MOMP epitope 1+2) and 3 epitopes (HBcAg(MIR)/MOMP epitope 1+2+3), there was no significant difference in the Ct-specific IgA antibody response (P>0.05) (Fig. 7B). the

(3) Results of specific CTL killing activity of immunized mice:

Spleen was taken to prepare splenocyte suspension to adjust the concentration to 2×10 ⁶ cells/ml, and the specific killing activity of CTL was detected by lactate dehydrogenase (LDH) release method. The results are shown in Figure 8 and Figure 9.

After immunization, when the effector cell to target cell ratio (E:T) of mice carrying epitopes in the N, C and MIR regions of HBcAg immunization group and epitope synthesis peptide group was 40:1 and 20:1, the Ct MOMP The specific CTL killing activity was statistically significant compared with the PBS group and the HBcAg carrier group (P<0.05); the C-terminus and MIR region of HBcAg carrying epitope protein immunization group were compared with the N-terminal epitope-carrying immunization group and epitope synthetic peptide There was a statistical difference in the killing activity of the two groups (P<0.05); however, there was no statistical significance in the comparison between the C-terminus of HBcAg and the protein carrying the epitope in the MIR region (P>0.05) (Figure 8A); the MIR region of HBcAg carried 2 One (HBcAg(MIR)/MOMP epitope 1+2) and three tandem (HBcAg(MIR)/MOMP epitope 1+2+3) produced more Ct-specific killing activity than carrying one epitope (HBcAg(MIR) )/MOMP epitope 1) and the synthetic peptide group had a significant difference (P<0.05) ( FIG. 8B ). At the same time, HBcAg-specific killing activity was also produced, and the killing activity produced by the C-terminal of the HBcAg carrier (Figure 9A) and its MIR region carrying 2 and 3 antigenic epitopes (Figure 9B) was the most significant (P<0.05). the

(4) Immunoprotective effect of HBcAg/Ct MOMP epitope vaccine on mouse genital tract Ct infection:

Two weeks after the last immunization of the mice in each group, the challenge of reproductive tract infection was carried out with the standard strain of Chlamydia trachomatis E serotype (purchased from the American Type Collection Center (ATCC: VR-348B)). The results showed that from the first day after transvaginal Ct challenge, the mice in each group began to show signs of infection in different degrees, such as redness and swelling of the vulva and abnormal secretions. After the mouse reproductive tract was challenged with Ct, the reproductive tract secretion Ct was isolated and cultured for IFU counting results. Compared with the synthetic peptide group, PBS group and HBcAg carrier control group, the clearance rate of the whole bacteria group disappeared after 18 days; in the HBcAg N, Carrying epitopes at the C-terminus can significantly increase the clearance rate of Ct, and the clearance rates are zero at 21 and 24 days respectively (Fig. 10A); carrying MOMP tandem epitopes in the MIR region can significantly enhance the clearance rate of immunized mice, especially three tandem groups (HBcAg(MIR)/MOMP epitope 1+2+3) clearance rate can be zero in 18 days ( FIG. 10B ). the

The results of the inflammation score of the external genital tract of mice are shown in Fig. 11 . From the observation of mouse vulva inflammation and the detection rate analysis of reproductive tract Ct isolation and culture, it was shown that the N and C terminals of HBcAg (Fig. 11A) and the epitopes carried in the MIR region and the series of multiple epitopes (Fig. 11B) can enhance the expression of small immune protection in mice. the

in conclusion:

A series of animal experiments were carried out using the prepared HBcAg virus-like particle carrier Ct MOMP epitope vaccine, and the results confirmed that:

1. The experimental animals produced Ct-specific protective antibodies and Ct-specific CTL killing activity against HBcAg/Ct MOMP epitope vaccine in serum and vaginal secretions. It shows that the vaccine can produce strong anti-Ct humoral and cellular immune effects. It shows that it has the effect of preventing and treating Ct infection. the

2. The experimental animals produced HBcAg-specific cellular immunity after being immunized with HBcAg/Ct MOMP epitope vaccine. It was shown that HBcAg as a carrier of heterologous epitope does not affect its own (HBcAg-specific) cellular immunogenicity. the

3. The experimental animals produced significantly enhanced serum IgG, secreted IgA and specific CTL killing activity against the HBcAg/Ct MOMP epitope vaccine (especially the HBcAg(MIR)/Ct MOMP tandem epitope vaccine). The results showed that HBcAg could enhance the immune effect of CtMOMP epitope. the

4. Mice vaccinated with HBcAg/Ct MOMP epitope have strong immunopreventive and therapeutic effects on E-type Ct infection. It shows that HBcAg/Ct MOMP epitope vaccine (especially HBcAg(MIR)/Ct MOMP tandem epitope 1+2+3) immunization has a good protective effect on Ct reproductive tract challenge, and the effect of the vaccine has been exerted. the

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. the

Claims (13)

1. A chimeric amino acid sequence, which consists of wild or modified hepatitis B core antigen protein, and a peptide sequence that is connected by one or more of the following members: (a) Amino acid sequences of n immunodominant epitopes of the main outer membrane protein of Chlamydia trachomatis (n≥1) (b) A short peptide sequence consisting of i flexible amino acids (i≥0). the 2. The chimeric amino acid as claimed in claim 1, characterized in that, the main outer membrane protein epitope polypeptide of Chlamydia trachomatis is one of the following members and derivatives that continuously have n amino acids (n≥5) contained therein one: (1) The amino acid sequence of immunodominant epitope polypeptide 1 is shown in SEQ ID NO: 1 (2) The amino acid sequence of immunodominant epitope polypeptide 2 is shown in SEQ ID NO: 2 (3) The amino acid sequence of the immunodominant epitope polypeptide 3 is shown in SEQ ID NO:3. the 3. The immunodominant epitope polypeptide 1 as claimed in claim 2, wherein the nucleotide sequence encoding the polypeptide is optimized and modified by prokaryotic codons, and the sequence is selected from: SEQ ID NO: 4 and SEQ ID NO :7. the 4. The immunodominant epitope polypeptide 2 as claimed in claim 2, wherein the nucleotide sequence encoding the polypeptide is optimized and modified by prokaryotic codons, and the sequence is selected from: SEQ ID NO: 5 and SEQ ID NO :8. the 5. The immunodominant epitope polypeptide 3 as claimed in claim 2, wherein the nucleotide sequence encoding the polypeptide is optimized and modified by prokaryotic codons, and the sequence is selected from: SEQ ID NO: 6 and SEQ ID NO :9. the 6. The chimeric amino acid as claimed in claim 1, wherein said original or modified hepatitis B core antigen protein is one of the following members: (1) The amino acid sequence shown in SEQ ID NO: 10 (2) The amino acid sequence shown in SEQ ID NO: 11. the 7. The DNA sequence encoding the hepatitis B core antigen-Chlamydia trachomatis major outer membrane protein epitope chimeric amino acid described in claim 1. the 8. The method for preparing hepatitis B core antigen-Chlamydia trachomatis main outer membrane protein epitope chimeric DNA is to amplify the gene sequences corresponding to the wild hepatitis B core antigen protein and the transformed hepatitis B core antigen protein respectively by using the PCR method, and then The main outer membrane protein epitope of Chlamydia trachomatis (as shown in the sequence SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) is inserted in a certain way, respectively inserted into the same or different epitopes of the above-mentioned hepatitis B core antigen Licensing sites, such as C-terminal, N-terminal and MIR regions, etc. the 9. The insertion optimization method according to claim 8, characterized in that: repeated insertion of single epitopes, repeated insertion of single epitopes separated by short peptides composed of flexible amino acids, tandem insertion of different single epitope combinations, different single epitopes Combined tandem repeat insertion, different single epitopes are inserted in combination and tandem after being spaced by short peptides composed of flexible amino acids, and different single epitopes are inserted in the form of combined tandem repeats after being spaced by short peptides composed of flexible amino acids. the 10. The same or different permission sites of hepatitis B core antigen as claimed in claim 8, characterized in that, the MIR region of the original and modified HBcAg, or replace any of the 78th-83rd amino acids of HBcAg n amino acids (1≤n≤6), or inserted into the N-terminal and carboxyl-terminal of modified HBcAg to become an artificial gene, and express the gene in an expression vector system and then purify it to obtain hepatitis B core antigen-Chlamydia trachomatis main Outer membrane protein epitope chimeric amino acids. the 11. A recombinant vector, said vector comprising the chimeric DNA sequence of claim 7; a genetically engineered host cell, said host cell comprising said vector. the 12. Use of the chimeric amino acid sequence as claimed in claim 1, the recombinant vector as claimed in claim 11 and the host cell in preparing a chimeric vaccine for preventing or treating diseases related to Chlamydia trachomatis. the 13. A composition comprising an effective amount of the recombinant carrier of claim 11 and a pharmaceutically or immunologically acceptable carrier, excipient or adjuvant; and an antibody, wherein the The antibody specifically binds to the epitope polypeptide according to claim 2. the