WO2004020471A1 - Recombinant protein for preventing human chlamydia trachomatis and the uses thereof - Google Patents

Abstract

Recombinant protein vaccine is a fusion protein formed from connection of BCG vaccine heat shock protein 65 with the major outer membrane protein (MOMP) of Chlamydia trachomatis being able to activate cytotoxic T -lymphocytes. The present invention also provide the nucleotide sequence for coding the recombinant protein, the expression vector containing above nucleotide sequence, host cell containing the expressing vector, and the method for preparing recombinant protein vaccine. After the fusion protein is applied in the human body, it can effectively prevent Chlamydia infections.

Description

 Recombinant protein for preventing human Chlamydia trachomatis infection and use thereof

Field of invention

 The invention relates to the field of genetic engineering, in particular to genetically engineered recombinant protein vaccines (hereinafter sometimes referred to as "genetically engineered recombinant proteins"), in particular to a method for preventing infection of human Chlamydia trachomatis, especially for preventing urogenital infection A recombinant protein vaccine; a nucleotide sequence encoding the recombinant protein vaccine (sometimes referred to as a "gene" below); an expression vector containing the nucleotide sequence; a host cell containing the expression vector; and a recombinant protein vaccine Preparation method: The present invention also relates to the use of the genetically engineered recombinant protein in preparing a vaccine product for preventing human Chlamydia trachomatis infection and a vaccine product containing the genetically engineered recombinant protein. Background of the invention

 Chlamydia is a non-moving, specialized intracellular parasitic microorganism. It has a unique life cycle: it has an elementary body (hereinafter sometimes referred to as "EB") and an initial body (reticulate body). Sometimes called simply "RB") two developmental stages. EB has the ability to infect, is adsorbed to the host cell, and is swallowed into the cytoplasm by the host cell membrane. About 10 hours after infection, EB began to differentiate, and became RB. RB is a proliferative form of chlamydia and has the ability to proliferate. RBs are proliferated by a two-division method. After repeated divisions, inclusion bodies are formed, which are filled with EB, RB, and intermediates. About 2-4 days, the RB stopped dividing, and the inclusion body was filled with small EB-dominated chlamydia colonies. The inclusion body membrane and the host cell membrane were successively ruptured. EB was released outside the parasitic host, and then infected other cells to start a new life cycle .

 Chlamydia Trachomatis (hereinafter sometimes referred to as "CT") can be divided into mouse biotypes and human biotypes according to the infected host. Mouse biotypes can cause mouse pneumonia and have nothing to do with humans, while human biotypes have 15 A serotype A, B, Ba, C, D, E, F, G, H, I, J, K, Ll, L2, L3, all of which are pathogens that take humans as the sole host.

Infection of A, B, Ba, and C can cause trachoma. Humans can repeatedly infect Chlamydia trachomatis and develop severe corneal vascular crests and scars. This damage can cause inverted eyelids and trichiasis. Corneal turbidity can lead to blindness. Trachoma is the main cause of human blindness in the world.

 D ~ K can cause inclusion body conjunctivitis and genitourinary diseases. Its genitourinary infection is a sexually transmitted disease. Epidemiological investigations show that 3 to 4 million people in the United States are infected each year. Because chlamydia itself is not easy to infect squamous epithelial cells and easily invade columnar epithelial cells, the cervical epidermis is columnar epithelial cells, so the cervix is a common site for CT infection, and then CT travels along columnar epithelium, leading to endometritis and fallopian tube inflammation. , Pelvic inflammatory disease, and can cause tubal infertility and ectopic pregnancy, which seriously threatens women's health.

 The increased susceptibility to CT during pregnancy leads to an infection rate of 20-30% in pregnant women, and vertical transmission of CT can cause fetal infection. Some scholars have reported that about 50-70% of fetuses who become infected with CT become CT-infected neonates through the infected cervix and suffer from CT-containing conjunctivitis and pneumonia.

 STD lymphogranuloma (LVG) is also transmitted mainly through sexual contact. Inguinal diaphragms are common in men, and anorectal lymph nodes are more common in women. Lesions gradually spread to other lymph nodes, and some lymph nodes swell and pus, perforate to form fistulas, and late lesions are external genital rubber swollen and rectal stenosis. Therefore, the harmfulness of CT infection is quite large. Because of this, the development of a Chlamydia trachomatis vaccine is particularly important and urgent.

 After decades of research, no ideal vaccine has been available, in part because the immune mechanisms that mediate the genital tract mucosa against Chlamydia infection are not fully understood. However, some experiments based on gene knockout mice and T cell clones provide evidence that CD4 + T cells play a major role in the protection of Chlamydia genital tract infections, while CD8 + T cells and humoral immunity play a lesser role. The above points have reached consensus in this research area.

 The current research hotspots of C. trachomatis vaccine focus on the following aspects:

 DNA vaccine (Chlamydia trachomatis) There are still many problems with DNA vaccines such as: only partial protection can be induced, and the Thl immune response and antibody response are weak.

Live attenuated chlamydia vaccine, Hua Su et al. (Hua Su, Qonald Messer, William Whitmire et al., Subclinical chlamydia infection of the female mouse reproductive tract produces a strong protective immune response: conclusions from the development of live attenuated vaccine strains (Subclinical Chlamydial Infection of the Female Mouse Genital Tract Generates a Potent Protective Immune Response: Implications for Development of Live Attenuated Chlamydial Vaccation Strains), Infection and Immunity, January 2000, January, P.192-196, vol.68.) Infection with Chlamydia trachomatis After intervention treatment with a small amount of oxytetracycline, the mice were placed in a subclinical state with mild infection to achieve an effect similar to a live attenuated vaccine. The experimental results show that compared with natural infection, the body produced Good protective immunity, it is concluded that live attenuated vaccines may have very good prospects. However, in fact, the genetic system of C. trachomatis is not fully understood, and attenuating mutations cannot be achieved. Therefore, the current vaccine may cause severe autoimmune or immunopathological reactions.

 DC (Dendritic Cells) cells (chlamydia-pulsed DC), Hua Su et al. Used heat-killed chlamydia-pulse DC (auto-infusion) to find a very good immune effect (Hua su, Ronald Messer, William, et al., 1998. Vaccination against Chlamydial Tract Infection after Immunization with Dendritic Cells Pulsed Ex Vivo with Dendritic Cells Immunized with Dendritic Cells Loaded In Vitro with Inactive Chlamydia Nonviable Chlamydiae). J. Exp. Med. Volume 188, Number 5, September 7, 1998 809-818). At present, it is unrealistic to use this method to achieve immune protection, but it suggests that immunity to non-live bacterial infections can also achieve immune protection.

 Epitopes vaccine, the major outer membrane protein (MOMP) is the most important outer membrane protein of Chlamydia trachomatis. Many experiments have shown that MOMP of Chlamydia trachomatis can produce a certain protective immunity. More than 20 immunogenic epitopes have been found in MOMP (Linette Ortiz, Karen RDemick, Jean W. Petersen, et al., 1996. Activation of type II HLA-restricted T cells from infected humans is mainly caused by C. trachomatis Membrane protein (Chlamydia trachomatis Major Outer Membrane Protein (MOMP) Epitopes That Activate HLA Class II-Restricted T Cells from infected Humans), The Journal of Immunology, 1996, 157: 4554-4567. The design of the vaccine has laid a certain foundation.

 The core of the current work is how to improve the immunogenicity and protective ability of the vaccine and prolong the duration of immune protection. Linking heat shock protein 65 to some of the epitopes on MOMPs has been found to provide a possibility to achieve this.

Heat shock proteins (hereinafter sometimes referred to as "HSP") are a family of molecular chaperone proteins that exist in many organisms. In the process of immune response, heat shock proteins can perform the following three basic functions: 1. Assist the entry of antigenic substances into the tree Antigen-presenting cells including protruding cells; 2. Interact with processed antigen materials in antigen-presenting cells to enter the MHC class I antigen-presenting pathway, thereby activating antigen-specific CTL; 3. Stimulus tree Synaptic cells express co-stimulatory molecules (such as B7, etc.) and secrete cytokines (Suzue K, Zhou X, Eisen HN, Young RA. Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway. Proc Natl. Acad. Sci. USA 1997 Nov 25; 94 (24): 13146-13151). Due to the above characteristics, the heat shock protein 65 is linked to some epitopes on the MOMP that have been discovered to form a fusion protein to achieve the purpose of improving immunogenicity and protective ability, and prolonging the time of immune protection. Summary of the invention

 One of the present invention is to provide a genetically engineered recombinant protein vaccine formed by fusion of a BCG heat shock protein 65 and a polypeptide capable of activating the epitope of the main outer membrane protein of Chlamydia trachomatis, which can prevent human Chlamydia trachomatis infection.

 The second aspect of the present invention provides a nucleotide sequence encoding the recombinant protein.

 The third aspect of the present invention is to provide an expression vector containing the nucleotide sequence.

 The fourth aspect of the present invention is to provide a host cell containing the expression vector.

 The fifth aspect of the present invention is to provide a method for preparing the recombinant protein vaccine.

 The sixth aspect of the present invention relates to the use of the genetically engineered recombinant protein in the preparation of a vaccine product for preventing human Chlamydia trachomatis infection.

 The seventh aspect of the present invention relates to a vaccine product containing the genetically engineered recombinant protein.

 Therefore, it is the inventor who solved the existing technical problems after a long period of extensive research, and for the first time pioneered the BCG heat shock protein 65 and some epitopes on C. trachomatis MOMP to form a new genetically engineered recombinant protein. Therefore, the immunogenicity and protective ability of the vaccine against Chlamydia infection are improved, and the duration of the immune protection is extended.

In addition, it should be pointed out that, on the basis of the disclosure content in the context of the present application, other aspects and inventive beneficial effects of the present invention are obvious to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of HSP65- Chlamy built on PET28 (a +).

 Figure 2 shows the results of three rounds of agarose gel electrophoresis, Lane 1: DL2000 DNA marker; 2: First round of PCR; 3: Second round of PCR; 4: Third round of PCR.

 Figure 3 is an agarose gel electrophoresis picture of pMD18T-chlaray recombinant plasmid digested with EcolRI and Hindlll, Lanel: DL2000 DNA marker; 2: Restriction digestion of recombinant plasmid.

 Figure 4 is an agarose gel electrophoresis picture of the recombinant plasmid cloned with the HSP65 coding gene into pET28 (a +) and digested with NcoLI and EcoRI: Lane 1: Restriction identification of the recombinant plasmid; 2: DL2000 DNA marker.

 Figure 5 shows the agarose gel electrophoresis pictures of pET28 (a +)-HSP65- chlamy recombinant plasmid digested with EcolRI and Hindlll: Lane 1: DL2000 DNA marker; 2: Recombinant plasmid digested identification.

 Figure 6 is a photograph of a large number of inclusion body growth (20x) seen 48-72 hours after CT-D inoculation on Hela monolayer cells. The white arrow indicates C. trachomatis inclusion bodies.

 Figure 7 shows the SDS photo of recombinant protein vaccine expression and purification: 1 is the protein marker, 2 is the expression of the target protein, and 3, 4, and 5 are the purification of the target protein.

 Figure 8 is a photo of mild inflammation of vaginal tissue in mice immunized with C. trachomatis infection vaccine: white coils show lamina propria dilated and congested, scattered lymphocytes infiltrate, and fibrous tissue hyperplasia.

 Figure 9 is a photograph of moderate inflammation of vaginal tissue in mice after Chlamydia trachomatis infection: The white coils show that there are more lymphocyte infiltration in the lamina propria.

 Figure 10 shows third degree inflammation of vaginal tissue in mice after Chlamydia trachomatis infection: the white coil shows a large number of lymphocytes infiltrating the solid layer of blood vessels.

 Figure 11 is a histogram showing that the antibody titer of the vaccine immunization group is significantly higher than that of the C57 mouse antibody titer detected by ELISA method in the PBS immunization group: 1: blank control; 2: PBS immunization; 3: vaccine immunization; specimen 1 -10. detailed description

 In the context of the present invention, the terms used have the meanings generally understood by a person of ordinary skill in the art unless otherwise stated. In particular, the following terms have the following meanings:

"Recombinant protein vaccine" means that the vaccine is derived from a prokaryotic or Protein expressed in nuclear cells.

 "BCG vaccine heat shock protein 65" refers to a heat shock protein with a molecular weight of 65 kDal derived from BCG vaccine, and its amino acid sequence is shown in SEQ ID NO: 4.

 The "epitope polypeptide capable of activating the main outer membrane protein of Chlamydia trachomatis human" is a polypeptide formed by connecting 9 epitopes derived from the main outer membrane protein of Chlamydia trachomatis, and its amino acid sequence is shown in SEQ ID NO: 2 .

 "Chlamydia trachomatis infection" refers to the state in which Chlamydia trachomatis enters the human body through a certain route and infects various organs of the human (such as the urogenital system, respiratory system, eyelid conjunctiva, etc.), including acute and chronic infections and asymptomatic state .

 • "Strong hybridization conditions" refer to hybridization conditions used to avoid non-homologous or partially homologous nucleic acid sequences in the reaction system from forming hybrid complexes, such as higher reaction temperatures and low ionic strength.

 "Treatment" or "prevention" includes:

 (1) preventing disease, that is, preventing the clinical symptoms of the disease from developing in mammals that may be in contact with the pathogen of the disease or susceptible to the disease but have not experienced or developed the symptoms of the disease,

 (2) Inhibiting a disease, that is, preventing or reducing the development of the disease or its clinical symptoms, or

(3) Relieve the disease, that is, cause the deterioration of the disease or its clinical symptoms.

 The present invention provides a recombinant protein vaccine, which is a fusion protein formed by linking BCG heat shock protein 65 and an epitope of a major outer membrane protein of Chlamydia trachomatis capable of activating human T cells, wherein the human T can activate human T The polypeptide of the main outer membrane protein epitope of C. trachomatis cells can be any amino acid sequence capable of activating the main outer membrane protein epitope of human T cells, and preferably has the following amino acid sequence shown in SEQ ID NO: 2: or its function equivalent.

 Glu Phe | Pro Ala Tyr Gly Arg His Met Gin Asp Ala

Glu Met Phe Thr Asn Ala Ala Cys Met Ala Leu Asn lie Trp Asp Glu Leu Asn Val Leu Cys Asn Ala Ala

Glu Phe Thr lie Asn Lys Pro Lys Gly Tyr Val Gly

Lys Glu Phe Pro Leu Ala Leu Asp Ala Ala Thr Gly Thr

Lys Asp Ala Ser lie Asp Tyr His Glu Trp Gin

Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr Pro Tyr lie Gly Val Lys Tip Ser Arg Ala Ser Phe Asp Ala Asp Thr Tyr | Lys Leu | (SEQ ID N0: 2)

 In the recombinant protein vaccine of the present invention, the BCG heat shock protein 65 may be a heat shock protein with a molecular weight of 65 kDal derived from BCG, and its amino acid sequence is preferably as shown in SEQ ID NO: 4 or a functional equivalent thereof. It can be located at the amino terminus of the fusion protein, and the epitope of the major outer membrane protein of C. trachomatis capable of activating human T cells is located at the carboxyl terminus of the fusion protein.

 The recombinant protein vaccine of the present invention has any amino acid sequence selected from the following:

 1) the amino acid sequence shown in SEQ ID NO: 4; and

 2) An amino acid sequence encoded by a nucleotide sequence that hybridizes with a nucleotide sequence encoding the amino acid sequence of 1) under stringent hybridization conditions.

 The present invention also provides a nucleotide sequence encoding the recombinant protein vaccine of the present invention, and the nucleotide sequence may have any sequence selected from the following:

 1) the nucleotide sequence shown in SEQ ID NO: 3; and

 2) A nucleotide sequence that hybridizes to the nucleotide sequence of 1) under stringent hybridization conditions. The nucleotide sequence of the epitope polypeptide of the major outer membrane protein of Chlamydia trachomatis capable of activating human T cells is shown in SEQ ID NO: 2 or a functional equivalent thereof.

 BCG heat shock protein 65 is a BCG-derived protein that is fused to an epitope polypeptide that is a major outer membrane protein of C. trachomatis that can activate human T cells. The sequence is shown in SEQ ID N0: 4. After entering the human body, it can stimulate human DC cells to up-regulate the levels of MHC (class I and II) and co-stimulatory molecules (B7.2), secrete cytokines, and BCG heat shock protein 65 has a unique feature that can assist with it The fusion protein epitope polypeptide is formed, enters the antigen-presenting cells including dendritic cells, and enters the MHC class I pathway for processing and presentation, and then activates antigen-specific cytotoxic T lymphocytes (CTLs) for pathogenic bacteria. Specific attack and killing, and the induction of this CTL does not require the involvement of exogenous adjuvants or CD4 + cells.

 The recombinant protein vaccine of the present invention can be produced by a method known in the art, for example, as described in Molecular Cloning-Book (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989). The following examples are exemplified in detail. A method for producing the recombinant protein vaccine of the present invention is provided.

Therefore, the present invention also provides a nucleoside containing the above-mentioned recombinant protein vaccine encoding the present invention. An expression sequence of an acid sequence; a host cell containing the expression vector, which can be various prokaryotic cells, eukaryotic cells or mammalian cells conventional in the art; and a genetic engineering preparation method of the recombinant protein vaccine.

 In addition, the present invention also relates to the use of the genetically engineered recombinant protein in the preparation of a vaccine product for preventing human Chlamydia trachomatis infection, and a vaccine product containing the genetically engineered recombinant protein. Those skilled in the art will understand that these vaccine preparations can be prepared by various conventional methods known in the art.

The recombinant protein vaccine of the present invention can be inoculated to humans by subcutaneous injection, and the inoculation dose is 100-500 μ _§ . In order to enhance the effect, booster can be carried out 2-3 times. The time interval can be from 2 weeks to February.

 The present invention will be further described in detail below with reference to specific preparation examples and biological effect examples, and with reference to the drawings. It should be understood that these examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Examples

 In the following examples, various processes and methods that are not described in detail are conventional methods well known in the art, such as the methods described in the Molecular Cloning book (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).

 In the following examples, the source of the reagents used, the trade name and / or the need to list their components are indicated only once. Unless otherwise specified, the same reagents used thereafter will not repeat the above.

Example 1 Artificial construction of epitope genes

 The selected epitope genes on the major outer membrane proteins were stringed together, and the EcoRI digestion site was constructed at the amino terminus, and the Hindlll digestion site was constructed at the carboxyl terminus, to obtain human T cell activated Chlamydia trachomatis Gene encoding a major outer membrane protein epitope (hereinafter referred to as "chlamy").

 The specific method is: artificially construct the selected epitope gene through 3 rounds of PCR cycle. Primer 3 and primer 4 serve as templates and primers for each other, and perform the first round of PCR; then use the first round of PCR products as templates, and use primers 2 and 5 as primers to perform the second round of PCR; and then use the second round of PCR products as For the template, primers 1 and 6 were used as primers for the third round of PCR.

The primer sequences are as follows: primer 1 (chlamy- 1)

5 '(

 TGGCT3 '(SEQIDNO: 5)

primer2 (chlamy-2)

 GAGTTT 3 '(SEQ ID NO: 6)

primer3 (chlamy-3)

5'TGCAACGCTGCT (

 CTGGCTCTG 3, (SEQ ID NO: 7)

primer4 (an-chlamy-3)

5 'CTGCCATTCGTGGXA

GGAAATTC3 '(SEQ ID NO: 8)

primer5 (an-chlamy-2)

5 'GATGTACGGGGTAAi

GTAGTC3 '(SEQIDNO: 9)

primer6 (an-chlamy- 1)

5 'AAGCTTGTAGGTGT (

 AAACAT3 '(SEQ ID NO: 10) Primer 3 and primer 4 are used as templates and primers to perform the first round of PCR reaction as follows: Add the following reagents to a 500 ul microcentrifuge tube:

 Primer 3 2.5ul (10umol / L)

 Primer 4 2.5ul (10umol / L)

 10x PCR buffer (TakaRa, see product description for ingredients) 5 μ 1

 dNTPs (10ramol / L) lul

 TaqDNA polymerase (TakaRa) (5 μm / μ1) 0.5 μ1

 Add deionized water to a final volume of 50 μΐ

 Add 3 drops of mineral oil after mixing

Reaction conditions: 94 ° C, lmin; 55 ° C, 1 min; 72 ° C, 1 min, after 30 cycles, 72 ° C extended for 10 min. The first round of PCR products (product 1) is formed as follows: (the complement is not written (SEQ ID NO: 11) The second round of PCR cycle uses product 1 as a template, and primers 2 and 5 are 5'-end primers and 3'-end primers, respectively. The reaction system is as follows:

 Product 1 lul

 10 (PCR buffer (with magnesium chloride) 5 μ 1

 dNTPs (10mmol / L) lul

 Primer 2 and Primer 5 each 2.5 μΐ

 TaqDNA polymerase (5ιι / μ1) 0.3μ1

 Add deionized water to a final volume of 50 μΐ

 Add 3 drops of mineral oil after mixing

Reaction conditions: 94V, Γ; 55. C, Γ; 72V, 1 ', after 30 cycles, 72 ° C extension for 10min. The second round of PCR products (product 2) is formed as follows: (its complementary strand is not written)

ATCGTCTGAACATGTTTACCCCGTACATC (SEQ ID NO: 12)

 The third PCR cycle uses product 2 as the template, and primers 1 and 6 are the 5 'and 3' primers respectively. The reaction system is as follows-product 2 lul

 lOxPCR buffer (with magnesium chloride) 5 μ 1

 dNTPs (10mmol / L) lul

 Primer 1 and Primer 6 each 2.5 μΐ

 Taq DNA polymerase (5u / μ 1) 0.3 μ 1

 Add deionized water to a final volume of 50 μΐ

 Add 3 drops of mineral oil after mixing

Reaction conditions: 94 ° C, 1 min; 55 ° C, 1 min; 72 ° C, 1 min, after 30 cycles, Extend at 72 ° C for 10 min. Formation of the third PCR product (final product) SEQ ID No: 1:

 AGCTT (SEQ ID NO: 1).

 The agarose gel electrophoresis results of the three rounds of PCR products are shown in Figure 2.

Example 2 TA cloning of epitope genes

 The TA cloning method (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989) was used to clone the PCR products. The TA cloning vector was PMD18-T Vector

(TakaRa). The specific method is as follows:

A DNA recovery:

 1. Run the third round of PCR products on a 2% agarose gel (lxTAE, 150-200mA, 0.5 hours);

 2. Cut the gel containing the DNA fragments from the agarose gel and put it into a centrifuge tube;

 3. Add 3 times the volume of sol (Beijing Dingguo Company), 45-55Ό water bath for 5-10min to completely melt the glue;

 4. Add lOul glass milk (Beijing Dingguo Company), flick the bottom of the tube and mix well, then mix in a water bath at 45-55 ° C for 5-1 Omin, mix every 2-3min;

 5. Centrifuge at 5000g for 60sec and discard the supernatant.

 6. Add 400ul of rinsing solution, flick the bottom of the tube to mix the glass milk, then centrifuge as above, discard the supernatant;

 7. Add 400ul of rinsing solution, flick the bottom of the tube to mix the glass milk, then discard the supernatant by centrifugation as above, and use a sampler to remove the rinsing solution as much as possible; then dry the glass milk at room temperature;

 8. Force mouth 10-30ul of sterile double distilled water to suspend the glass milk, 45-55 ° C water bath for 5-10 min;

 9. Centrifuge at 10,000g for 2min and recover the supernatant for later use.

Two connection reactions:

 1. Take 3ul of the supernatant for electrophoresis, and observe the electrophoresis band under a UV lamp. Compare with the marker to estimate the recovered DNA concentration;

2. Connection reaction system: Insert DNA (see SEQ ID NO: 1 for sequence) 0.05-0.3 pmol

 pMD18-T Vect (TakaRa) 0.5-lul Solution I (TakaRa) 5 ul

DH ₂ 0 up to 10 ul

 16 ° C reaction lh

Three transformations: The method for preparing competent cells is: Streak E. coli JM109 (Novagen) on LB agar medium, culture at 37 ° C for 12-16h; take a single colony from agar plate the next day and culture in 2ml LB Medium, shake culture at 37 ° C for 12-16h at 225r / min; take 1ml of the above culture and inoculate in 100ml LB medium, shake culture at 37 ° C at 225r / min until the OD value is about 0.5 (about 3h) ); The bacterial solution was ice-baked for 2h, then 2,500 g, and centrifuged at 4 ° C for 20min to collect the bacterial cells; 100ml of ice-cold Trituration buffer (100mmol / L CaC12, 70mmol / L MgC12, 40mmol / L sodium acetate, pH5.5) Mix well, place on ice for 45min; centrifuge at 1,800g, 4 ° C for 10min, discard the supernatant, add 10ml ice-cold Trituration buffer to suspend cells; aliquot 200ul each, and store at 4 ° C for 1-2 weeks. For long-term storage, add glycerol to a final concentration of 15% and store at -70 ° C until use.

 The transformation method is: 200 ul competent cells are thawed on ice, then 3 ul DMSO or β-mercaptoethanol is added, and after mixing, 10 μ 反应 ligation reaction solution (containing recombinant plasmid) is added, mixed gently, and placed on ice for 30 min; 42 ° C for 45sec, then quickly put back on ice for 1-2min; add 2ml of LB culture medium, shake and culture for 1h at 225r / min at 37 ° C; centrifuge at 4,000g for 10sec, discard the supernatant, and resuspend in 200ul of LB medium Bacterium; spread the bacterial solution on an LB agar culture plate containing appropriate antibiotics, spread evenly, leave it at room temperature for 20-30 minutes, and invert in a 37 ° C incubator for 12-16h.

 Four identification: preliminary identification of recombinant clones by restriction enzyme digestion. DNA sequencing was performed by Beijing Dingguo Biotechnology Development Center. The sequencing results were correct (see SEQ ID No. 1 for specific sequences). The recombinant pMD-18-chlamy plasmid was digested with EcoRI and Hindlll to release a chlamy (300bp) fragment (see Figure 3). Five strains and preservation: Recombinant plasmids were stored frozen at -20 ° C. Recombinant plasmid-containing strains were stored at -20 ° C or -70 ° C in 20% glycerol-containing culture broth. Example 3

Obtain and encode the gene encoding Mycobacterium tuberculosis 65 D heat shock protein (BCG HSP65) Cloned into pET28 (+) expression vector

 The B. tuberculosis mycobacterium tuberculosis originated from the Changchun Institute of Biological Products. Mycobacterium tuberculosis BCG vaccine was cultured at 37-39 ° C using Starch Potato Code NO C250-1 Beijing Dingguo Biological. Mycobacterium tuberculosis grew dry and light yellow. Bacterial film. Collect the bacterial membrane and extract the B. tuberculosis genome from M. tuberculosis. The method for extracting the genomic DNA of M. tuberculosis from J. Sambrook, Isolation of high-molecular-weight DNA from mammalian cells ), 9.16-9.22, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).

 The heat shock protein 65 (HSP65) structural gene was isolated from Mycobacterium tuberculosis by PCR. The 5 'end primer sequence is 5'CCATG GCC AAG ACA ATT GCG3' (SEQ ID NO: 13), and the 3 'end primer sequence is 5' GAA TTC GCT AGC CAT ATG GAA ATC CAT GCC ACC CAT 3 '(SEQ ID NO: 14).

 The PCR procedure is as follows: Add the following reagents to a 500 μ1 microcentrifuge tube: Template cDNA 5 μl (mmol / L)

 10x PCR buffer (with magnesium chloride) 5 μ 1

 dNTPs (10 sides ol / L) 1 μ 1

 5 'and 3' primers (0.01 mmol / L) each 0.5 μ 1

 Taq DNA polymerase (5u / μ 1) 0.25 μ 1

 Add deionized water to a final volume of 50 μΐ

 Add 3 drops of mineral oil after mixing

 Reaction conditions: 94 ° C, 30sec; 55 ° C, lmin; 72 ° C, 2 min, after 30 cycles, 72 ° C extended for 10 min.

 The TA cloning method was used to clone the PCR product. The methods of DNA extraction, ligation and transformation are described in Example 2.

 Recombinant pMD18-T vector containing HSP65 and pET28a (+) (Novagen) were digested with restriction enzymes Ncol (TakaRa) and EcoRI (TakaRa). The reaction system was as follows:

7 μ 1 plasmid DNA lul IOχ buffer (TakaRa)

 1 ul 0.1% BSA (TakaRa)

 0.5 μ 1 restriction enzyme Ncol (10 units / μ 1)

 0.5 μ 1 restriction enzyme EcoRI (10 units / μ ΐ)

 37 ° C 4h

 Extract the HSP65 DNA insert and the digested pET28a (+) linear vector (for the method, see Example 2. Estimate the concentration of these two types of DNA under UV light after electrophoresis. The ligation reaction system is established as follows:

 pET28a (+) linear plasmid DNA 5ul

 HSP65DNA insert 3 ul

 10x T4 DNA ligase buffer 1 ul

 T4DNA ligase 1 ul 16 V I2h

See Example 2 for the transformation method after ligation. See specific sequences SEQ ID No: 3 (1- 1638bp ) Q digestion identification results see Figure 4. Example 4 Construction of a BCG heat shock protein 65-activating human T cell C. trachomatis major outer membrane protein epitope polypeptide fusion protein gene

 The expression vector pET28a (+) plasmid into which the HSP65 gene was inserted was extracted (see Example 3), and the recombinant plasmids were digested with restriction enzymes EcoRI and Hindin (TakaRa). The membrane protein epitope gene (chlamy) of pMD18-T (see Example 2), the reaction system is as follows:

 8 n g plasmid (recombinant pSP28a of HSP65 or recombinant pMD18-T of chlamy)

1 μ 1 10 X buffer (TakaRa)

 0.5 U l restriction enzyme EcoRI (10 units / μ ΐ)

 0.5 μ 1 restriction enzyme Hindlll (10 units / μ 1)

After mixing, incubate at 37 ° C for 30-120min.

 As described above, the digested linear plasmid pET28a (+) and chlamy inserts were extracted by the same method. And connect the two:

 Connection reaction:

^ DNA (0.5 g / l) 3 l DNA insert (for sequence see SEQ ID N0.2) (300ng / μ 1) 5 U 1

 10xT4 DNase ligation buffer 1 μ 1

 Τ4 DNA ligase Ι μ ΐ

 After mixing, place in a water bath at 14-16 ° C for 6-12h.

 This recombinant pET-28a (+) plasmid was transformed into JM109 E. coli. The method is the same as before.

 BCG heat shock protein 65-activating human T cells Chlamydia trachomatis major outer membrane protein epitope peptide fusion protein gene sequencing was undertaken by Beijing Dingguo Biotechnology Development Center. The gene and design of human T cell Chlamydia trachomatis main outer membrane protein epitope polypeptide fusion protein are exactly the same. Recombinant pET-28a (+)-HSP65-chlamy plasmid was digested with EcoRI and Hindlll to release chlamy

(300bp) fragment is shown in Figure 5. Example 5

BCG heat shock protein 65 can activate human T cells Chlamydia trachomatis major outer membrane protein epitope polypeptide fusion protein gene expression

 The recombinant plasmid finally obtained in Example 4 was transformed into an expression strain BL21 (DE3) (Novagen), and a single colony of bacteria was inoculated into 50 ml of LB medium in a 250 ml Erlenmeyer flask with 37.0 water bath shaking Cultivate to an OD600 of 0.6. Add IPTG to a final concentration of 01 mM, 37. C. Cultivate with water bath for 2-3h. Place the Erlenmeyer flask on ice for 5 min and centrifuge at 4 ° C for 5 min (5000 g). Aspirate and discard the supernatant and collect bacteria for immediate use or frozen storage. Example

BCG heat shock protein 65 can activate human T cell C. trachomatis major outer membrane protein epitope polypeptide fusion protein purification. Preparation of samples

 1. Resuspend the bacteria in the cell lysate;

 Cell lysate: 20mM Tris, 0.5 M NaCl

 5mM imidazole 0.1 mM PMSF adjust pH to 7.9

2. Add 10mM MgSO ₄ and 20 ug / ml Dnase I (GIBCO) to the cell lysate. Incubate on ice for 30 min to remove DNA;

 3. Centrifuge at 12000r / min for 15min to collect the precipitate, and then wash it once with cell lysate;

4. Dissolve the pellet in binding buffer and incubate on ice for 1-2 hours.

 Binding buffer: 20mM Tris, 0.5 M Nacl

 5mM imidazole 6M urea to adjust pH to 7.9

 5. Centrifuge at 12000r / min for 15min to remove floc. Preparation of columns

 1. Align the chromatography column to the desired capacity and mark it with a waterproof pen;

 2. Pack the column with phosphate buffer solution and seal the outlet;

 Phosphate buffer: 20 mM phosphate, pH 7.2 lM NaCl

Preparation method: solution A Na ₂ HP0 ₄ .12H ₂ 0 35.8g double distilled water is added to 100ml

 Solution B NalLPOOLO 13.8g double distilled water was added to 100ml solution C. Solution B was gradually added to solution A until the pH was 7.2.

3. Start adding chromatography medium (Ni ²⁺ -Saphrose-4-B)

 4. Allow the column to begin to flow and add more medium until the medium level reaches the marked column level;

5. Open the outlet, add phosphate buffer, and hit the side of the column to sink the medium, and continue to bounce until the height of the column bed is constant

 6. Add more media and repeat steps 1-6. When the bomb is not changing the volume of the column bed, close the outlet of the column.

 7. Add phosphate buffer to fill the column, cover the top of the column, and open the outlet;

 8. Allow the column to flow at a minimum of 100 bed volumes / h, with a minimum flow of 30 minutes;

 9. If the volume of medium in the column needs to be adjusted, close the outlet of the column, open the lid of the column, add more medium, and then rinse the column at the maximum flow rate for at least 30min. Charge the medium with metal ions

 Before the chelating medium can be used in the purification process, it must be charged with the selected metal ion.

 1. Wash the column with at least 5 bed volumes of double distilled water

2. Add 3-5 column bed volumes of 20mM metal ion solution to the column; Nickel ion solution prepared 50mM NiS0 ₄ .6H ₂ 0 13. lg double distilled water was added to 1000ml.

3. Wash the column with 5 bed volumes of elution buffer

 Elution buffer: 20mM Tris, H 7.9

 0. 5M Nacl

 1 M imidazole

 6M urea

 4. Wash the column with 10 bed volumes of binding buffer.

 Binding buffer: 20mM Tris, pH7.9

 0.5M NaCl

 5mM imidazole

 6M urea

Adsorption

 1. Add the sample to a metal-chelated column and flow it through the bed at a predetermined optimal flow rate;

 2.Wash the column with binding buffer containing 16 mM imidazole until the absorbance of the effluent returns to the baseline level.

Elution When eluting with the eluent, a peak appears. Continue to elute until the absorption value does not drop. Take the effluent from the container for different periods of time and perform SDS electrophoresis to determine that the purity of the purification effect can reach more than 98%. See Figure 7 for purified SDS-PAGE.

 Imidazole eluent: 20mM Tris, pH 7.9, 0.5 M NaCl,

 1 M imidazole, 6M urea. Example 7 Culture and Purification of Chlamydia

 A training

1. Add Hda cells (Department of Immunology, Basic Medical College, Xinmin Campus, Jilin University) in a 24-well plate with 1-2X10 ⁵ cells / well, and l-2d fusion into a dense monolayer.

 The culture medium was IMDM (Iscove's modified Dulbecco's medium, GIBCO) + 10% fetal bovine serum (Tianjin TBD Biotechnology Development Center) 100 iig gentamicin / ml.

2. Discard the culture solution, inoculate the strain (Peking Union Medical College Hospital, Chlamydia trachomatis D) on the monolayer of the cell at 3000 r / min / h at room temperature, and remove the sucrose-phosphate transport solution ( Short name SPG: Sucrose 75g, KH ₂ P0 ₄ 0.52g, Na ₂ HP0 ₄ 1.22g, glutamic acid 0.72g, H ₂ 0 to 1 liter, pH7.4-7.6).

 3. Add Chlamydia growth solution lml IMDM + 10% fetal bovine serum 100 ug gentamicin / ml lug / ml actinomycin. After 48-72 h, more than 90% of the cells can be seen under the inverted microscope. Remove the growth solution, add a little cold SPG, and repeatedly blow the cells with a pipette to remove them. The cell suspension was stored in a -70 ° C refrigerator.

 Purification (density shaving centrifugation)

 1. Ultrasonication of cell suspension (20 seconds)

 2. Centrifuge the suspension at 500g for 15min at 4 ° C to remove the supernatant.

 3. The supernatant was tiled on 8ml 35% (vol / vol) renal contrast agent (76 ° /., Shanghai Huaihai Pharmaceutical Factory).

 35% renal contrast agent 65% 0.01M HEPES with 0.1M NaCl

 4. 4 centrifugation 43000g for 1 h

 5. The precipitate is suspended again by SPG and concentrated together and mixed.

 6. The mix is spread on a contrast agent of discontinuous density.

 13ml 40%, 8ml 44%, 5ml 52%, 43000g lh at 4 ° C, EB is located at 44/52% contrast medium interface.

 7. Collect 3 volumes of SPG dilution and centrifuge at 30,000g for 30 min.

 8.Resuspend the pellet with SPG and store at -70 ° C.

 Hela monolayer cells were inoculated with Chlamydia trachomatis 48-72 h after growth of inclusion bodies (see Figure 6). Example 8 Protective experiment of mouse genital chlamydia infection after vaccine immunization

A material

 1. C57 female mice (Chinese Academy of Military Medical Sciences) 6-8 weeks old

 2. D-chlamydia

 3. Fusion protein of antigen HSP65 and major outer membrane protein epitope of C. trachomatis which can activate human T cells (hereinafter referred to as "HSP65chlamy"), PBS

Two methods

1. Twenty mice were randomly divided into two groups, one was a vaccine immunization group, and the other was a PBS immunization group. The vaccine group was injected at four axillary sites with a total of 50ug at 0, 14, 4, and 2 days. In the PBS group, PBS was injected by the same method.

 2.On the 21st day, each mouse was injected with 5 mg of progesterone

3. vaginal vaccination with Chlamydia 1.5xl0 ⁴ IFU on the 28th day

 4. On the 35th day, the vagina, uterus, and fallopian tubes of 20 rats were removed for histopathological examination. The pathological conditions of vaccine immunized group and PBS immunized group were compared.

Three pathological grading standards:

 0 Normal

 1+ minimal response (a small number of inflammatory cells)

 2+ mild response (increase inflammatory cells with mild interstitial thickening and spread to surrounding adipose tissue)

 3+ Moderate response (significant presence of inflammatory cells, with clearance of adjacent adipose tissue) 4+ Serious response (moderate to severe response, presence of affected tissue, with multiple focal necrosis of adjacent adipose tissue).

Experimental results and conclusions

 PBS immunization group: 10 cases of reproductive system specimens with normal ovaries and endometrium;

 Vaginal inflammation: 3+ in 7 cases and 2+ in 3 cases

 Vaccination group: 10 cases of reproductive system specimens with normal ovaries and endometrium;

 Vaginal inflammation: 2+ in 2 cases and 1+ in 8 cases

 Conclusion: After immunizing with the vaccine, inflammation in the reproductive system of the mice is reduced, and the vaccine has a certain protective effect.

 See sections 8, 9, and 10 for the pathological sections of inflammatory reactions at all levels.

Example 9 Plant foot experiments after vaccine immunization

A material

 1. C57 male mice (Chinese Academy of Military Medical Sciences), 6-8 weeks of age

 2. D-chlamydia

 3. Antigen HSP65 and fusion protein (hereinafter referred to as "HSP65chlamy"), PBS, which can activate the main outer membrane protein epitope of Chlamydia trachomatis in human T cells.

Two methods

1. Twenty mice were randomly divided into two groups, one was a vaccine immunization group, the other was a PBS immunization group, and the vaccine group was injected at four axillary sites at a total of 5 days. 0 ug. In the PBS group, PBS was injected by the same method.

2. On the 28th day of each mouse, 25ul PBS was injected subcutaneously in the plantar of the left hind foot and 25ul 2x10 ⁴ IFU heat-inactivated CT (56 ° C 30min) was injected subcutaneously in the right hind foot.

 3. Measure the thickness of the sole at 72h.

Experimental results and conclusions

 1. PBS group: average thickness of left foot 2.8 ± 0.2cm, average thickness of right foot 3.1 ± 0.4

 2. Vaccine group: left foot average thickness 2.95 ± 0.3, right foot average thickness 6.7 ± 0.2

 The experimental results show that: after injection of CT-D in the right foot of the vaccine immunized group, the swelling degree of the mouse was significantly higher than that of the left foot injected with PBS, and also significantly higher than that of the PBS immunized group. Example 10 CTL killing experiment after vaccine immunization

A material

 1. C57 male mice, 6-8 weeks of age

 2. D-chlamydia

 3. Fusion protein of antigen HSP65 and the major outer membrane protein epitope of C. trachomatis that can activate human T cells (hereinafter referred to as "HSP65chlamy"), PBS

Two methods

 1. 20 mice were randomly divided into two groups, one group was a vaccine immunization group, the other was a PBS immunization group, and the vaccine group was injected at four axillary positions at a total of 5 days. 0 ug. In the P B S group, the same method was used to inject P B S in the same volume.

 2. Preparation of "condition medium": Take a normal mouse, sacrifice the neck, sterilize the abdomen with alcohol, remove the spleen with a sterilized device, place it in a sterile dish, and transfer it to ultra-clean Ground, ground with frosted glass, suspend spleen cells in complete culture, filter with gauze, 2500r / min, 5min, remove the supernatant, resuspend with 0.83% ammonium chloride 1ml, 2min on ice, 2500 r / min, 5min, remove Supernatant, spleen cells were suspended in complete culture solution, transferred to a culture flask, and 25ug concanavalin A (ConA) was added, and the complete culture solution was added to a total volume of 5ml. Culture at 37 ° C for 24h, 2500 r / min, 5min, and collect the supernatant as the conditioned medium.

3. Target cell antigen presentation and labeling of Na ₂ ⁵¹ Cr0 ₄

1) When the cells have grown into a sparse monolayer of about 2 × ^{10 6} cells in a 100 ml culture flask, pour out the culture medium, add serum-free IMDM + 10% conditioned medium, and culture for 16-24 h. 2) Add 5ul of lipofectin to 1ml of 5xl0 ⁶ IFU heat-inactivated CT EBs, mix for 5min, and then add to 1) at 37 ° C for 4h.

3) Pour out the liquid in the bottle, digest with 0.25% trypsin (Huanhua Biotechnology), add 5ml complete culture medium, count, 1200 r / min, 10min, resuspend in 100ul complete culture medium, and transfer to sterile plastic In a tube, add 100ul N ⁵¹ CrO ₄ (including 200uCi) (PerkinElmer), and shake culture for 1h.

 4) Wash the complete culture solution 3-4 times.

5) Adjust the cell concentration to 1 × ^{10 5} cells / ml with complete culture medium.

4. The spleen and lymph nodes and PBS immunized mice vaccinated extraction, grinding process, splenocytes as described above, and the spleen and lymph node cells were counted, transferred into 2xl0 ⁷ cells / ml. 100ul of spleen and lymphocytes were added to a 96-well plate, and 3 concentration gradients were set to 2 × 10 ⁷ ′ 1 × ^{10 7} ′ 5 × ^{10 6} and 3 replicates were set.

 5. Add 100ul of labeled B16 cells to the wells containing effector cells, and set three additional wells for spontaneous release (100ul target cells + 100ul complete culture solution) and maximum release (100ul target cells + 100ul 2mol hydrochloric acid) .

6. 37 5% C0 ₂ , 12-16h.

 7. Centrifuge the 96-well culture plate at 150g for 5min.

 8. Take the lOOul supernatant and move it to a plastic tube (for Y counting)

 9. Use a Y counter to determine the cpm value of each tube.

10. Calculation of killing rate:

Kill rate = (experimental hole value-spontaneous release hole value / maximum release hole value-spontaneous release hole value) * 100% III. Experimental results and conclusions

 The experimental results show that the spleen cells and lymphocytes of the vaccine-immunized group have a certain killing effect on the target cells. With an effective target ratio of 100: 1, the killing rate is 50-60% on average. The average lethality of the control group was 5-10%. Example 11 ELISA to detect antibody titers of C57 mice after immunization with vaccine

A material

C57 mice (male 6-8 weeks old), Chlamydia CT-D, PBS, coating buffer 1% BSA, goat anti-mouse IgG-HRP (Beijing Dingguo Company), OPD-substrate buffer (0.2M Na ₂ HP0 ₄ 25.7ml; 0.1M citric acid 24.3ml)

Two methods

I. Place the purified chlamydia in a 56 ° C water bath for 30 min. After pre-experiment, 1:80 was diluted with coating buffer (Na ₂ C0 ₃ 1.59g, NaHC0 ₃ 2.93g with water to 1000ml).

2. Coating: Add 96-well plate and hide 1 / well. 4. C, overnight.

 3. Washing: Wash the plate 3 times with washing solution, 5min / time.

 4. Sealing: 1% BSA closed, 200ul / hole. 40 ° C, lh.

 5. Washing: Wash the plate 3 times with washing solution, 5min / time.

 6. Adding the sample: The PBS immunized mice and the vaccine immunized mice (the same immunization method as above) were used to make 100-fold dilutions of the serum of each sample with 100 μl / well. Set the compound hole. 40 ° C, 30min.

 7. Add enzyme-labeled antibody: Dilute sheep anti-mouse IgG-HRP (Beijing Dingguo Biological, 0.1ml, 1: 1000) with washing solution 500 times, 100ul / well. 40 ° C, 30min.

 8. Washing: Wash the plate three times with washing solution, 5min / time.

9. Color development: Freshly prepared OPD-substrate buffer, lOOul / well. Avoid reaction at room temperature for 5-10min. 10. Termination: 2mol / LH ₂ S0 ₄ , 50ul / well.

I I. Test A ₄₉₀ .

Blank control PBS group vaccine immunization group

 1 0.004 0 0.005 0.003 ± 0.003 0.166 0.082 0.123 0.124 + 0.042 0.293 0.467 0.358 0.382 ± 0.087

2 0.077 0.143 0.201 0.140 ± 0.062 0.327 0.542 0.410 0.426 ± 0.11

3 0.131 0.122 0.178 0.144 + 0.03 0.410. 0.412 0.378 0.4 ± 0.019

4 0.83 0.120 0.141 0.115 ± 0.029 0.418 0.322 0.438 0.393 ± 0.062

5 0.119 0.142 0.09 0.117 ± 0.026 0.342 0.198 0.201. 0.247 ± 0.082

6 0.100 0.123 0.175 0.133 ± 0.038 0.211 0.105 0.156 0.157 + 0.053

7 0.092 0.108 0.087 0.096 ± 0.01 0.478 0.412 0.388 0.426 ± 0.047

8 0.115 0.141 0.176 0.144 ± 0.031 0.378 0.422 0.398 .0.399 ± 0.022

9 0.092 0.126 ■ 0.242 0.153 ± 0.079 0.412 0.402 0.423 0.412 ± 0.011

10 0.087 0.125 0.092 0.101 ± 0.0 7 0.510 0.378 0.469 0.452 ± 0.068 Experimental results and conclusions

 1 blank control: 0.003 ± 0.0008;

 2 PBS immunization group: 0.1038 ± 0.0095;

 3 Vaccination group: 0.435 ± 0.012.

 The experimental results show that the antibody titer of the vaccine immunization group is significantly higher than that of the PBS immunization group.

Claims

Claim

1. A recombinant protein vaccine comprising a BCG heat shock protein 65 and an epitope of a major outer membrane protein of C. trachomatis that can activate human T cells.

 2. The recombinant protein vaccine according to claim 1, which has any amino acid sequence selected from the group consisting of:

 1) the amino acid sequence shown in SEQ ID No: 4; and

 2) An amino acid sequence encoded by a nucleotide sequence that hybridizes with a nucleotide sequence encoding the amino acid sequence of 1) under stringent hybridization conditions.

 3. The recombinant protein vaccine according to claim 1, wherein the polypeptide capable of activating an epitope of a major outer membrane protein of Chlamydia trachomatis in human T cells has an amino acid sequence shown in SEQ ID NO.2.

 The recombinant protein vaccine according to claim 1, wherein the BCG heat shock protein 65 is located at the amino terminus of the fusion protein, and the epitope of the major outer membrane protein of C. trachomatis capable of activating human T cells is located at the carboxyl terminus of the fusion protein .

 The recombinant protein vaccine according to claim 1, which has an amino acid sequence represented by SEQ ID N (κ4).

 A nucleotide sequence encoding the recombinant protein vaccine according to any one of claims 1 to 5.

 7. The nucleotide sequence according to claim 6, which has any sequence selected from the group consisting of:

1) the nucleotide sequence shown in SEQ ID NO: 3; and

 2) A nucleotide sequence that hybridizes to the nucleotide sequence of 1) under stringent hybridization conditions.

The gene according to claim 6, which has a nucleotide sequence shown in SEQ ID NO: 3.

 9. An expression vector comprising the nucleotide sequence of any one of claims 5-7.

 10. A host cell containing the expression vector of claim 10.

 The host cell according to claim 9, which is a prokaryotic cell, a eukaryotic cell, or a mammalian cell.

 A method for preparing a recombinant protein vaccine according to any one of claims 1 to 5, comprising culturing a host cell according to claim 9 or 10.

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13. Use of the recombinant protein according to any one of claims 1-5 in the preparation of a vaccine product for the prevention of human Chlamydia trachomatis infection.

 14. The use according to claim 12, wherein the human Chlamydia trachomatis infection is a urogenital infection.

 15. A vaccine product for the prevention of human Chlamydia trachomatis infection, comprising the recombinant protein according to any one of claims 1 to 5 and a carrier or excipient.

 16. The vaccine product according to claim 12, wherein the human Chlamydia trachomatis infection is a urogenital infection.

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