Chlamydia trachomatis pgp3 protein monoclonal antibody and application thereof

Abstract

The application provides a monoclonal antibody of a Chlamydia trachomatis Pgp3 protein and application thereof, and the monoclonal antibody comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2 and a heavy chain variable region CDR3 with the amino acid sequences shown in SEQ ID NO. 1, 2 and 3 respectively, and a light chain variable region CDR1, a light chain variable region CDR2 and a light chain variable region CDR3 with the amino acid sequences shown in SEQ ID NO. 4, 5 and 6 respectively. Experimental researches prove that the monoclonal antibody of the Chlamydia trachomatis Pgp3 protein can effectively inhibit infection of Chlamydia trachomatis in a mouse genital tract, and reduce occurrence of oviduct edema.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a monoclonal antibody against Chlamydia trachomatis Pgp3 protein and its application. Background Technology

[0002] Chlamydia trachomatis (Ct) is a Gram-negative, obligate intracellular parasite with a unique developmental cycle. Before entering the cell, it exists as a small, dense, infectious chlamydial. After entering the host cell, it gradually enlarges and multiplies to become an infective protozoan. Once mature, it reverts to an infective chlamydial. It is one of the most common bacteria causing sexually transmitted infections in humans. Trachoma infection is often asymptomatic, chronic, and persistent, and is frequently overlooked and treatment is delayed.

[0003] Statistics show that approximately 100 to 150 million new cases of cytokine (Ct) infection occur worldwide each year. It can cause tubal infertility or ectopic pregnancy in women and promote the development of cervical and ovarian cancer, as well as HIV infection. In men, it primarily causes urethritis, epididymitis, and orchitis. Ct infection spreads globally, causing significant global public health problems and imposing a huge socioeconomic burden on human healthcare. Antibiotics are currently the only available treatment; however, the reinfection rate is high, and the emergence of drug-resistant strains leads to treatment failure. Therefore, the development of novel drugs for treating urogenital Ct infections remains a hot topic in this field.

[0004] We know that Pgp3, encoded by the Ct plasmid, is a crucial virulence factor and the only protein that can be effectively secreted into the cytoplasm. In the later stages of infection, it accumulates in large quantities in the host cell cytoplasm as a trimer. After host cell rupture, Pgp3 is released extracellularly before EB, directly contacting the host's internal environment to exert its effects. Existing cell experiments indicate that Pgp3 effectively inhibits host cell apoptosis in vitro, primarily by inhibiting tumor necrosis factor-α-induced apoptosis. On the other hand, it induces inflammatory responses leading to fallopian tube lesions. Animal experiments show that Pgp3 is a key factor in causing fallopian tube inflammation; similarly, in vitro cell experiments demonstrate that Pgp3 effectively induces the production of MIP-2 and IL-6 by mouse bone marrow-derived macrophages and dendritic cells.

[0005] Currently, the treatment of many diseases has entered the era of biological agents, such as tumors, psoriasis, and atopic dermatitis. Considering the important role of Pgp3 in the pathogenicity of Chlamydia trachomatis, this invention studies the effect of anti-Chlamydia trachomatis Pgp3 protein monoclonal antibody targeting and inhibiting Pgp3 on Chlamydia trachomatis genital tract infection. Summary of the Invention

[0006] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes a monoclonal antibody against Chlamydia trachomatis Pgp3 protein and its application.

[0007] To achieve the above objectives, the technical solution created by this invention is implemented as follows:

[0008] In a first aspect, the present invention provides a monoclonal antibody against Chlamydia trachomatis Pgp3 protein, the monoclonal antibody comprising:

[0009] The heavy chain variable regions CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NO.1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NO.4, 5, and 6, respectively.

[0010] SEQ ID NO.1: GFTFNNYA;

[0011] SEQ ID NO.2: IRSNNNNYAT;

[0012] SEQ ID NO.3: VRGDYRPY;

[0013] SEQ ID NO.4: QNVGTN;

[0014] SEQ ID NO.5: SAS;

[0015] SEQ ID NO.6: QQYNTYPLT;

[0016] Preferably, the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.7.

[0017] Preferably, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.8.

[0018] SEQ ID NO.7: EVQLQESGGGLVQPKGSLKLSCTASGFTFNNYAVNWVRQAPGKGLEWVARIRSNNNNYATFYADSVKDRFTISRDDSQSMLYLQMNNLRTEDTAMYYCVRGDYRPYWGQGTTLTVSS;

[0019] SEQ ID NO.8: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQFPKALIYSASYRNSGVPDRFKGSGSGTDFTLTINNVQSEDLAEYSCQQYNTYPLTFGGGTKLEIKR;

[0020] In a second aspect, the present invention provides a nucleic acid encoding a monoclonal antibody against the aforementioned Chlamydia trachomatis Pgp3 protein.

[0021] Preferably, the nucleic acid sequences encoding the heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ. NO. 9, 10, and 11, respectively, or are complementary sequences thereto, and the nucleic acid sequences encoding the light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ. NO. 12, 13, and 14, respectively, or are complementary sequences thereto.

[0022] SEQ ID NO.9: GGATTCACCTTCAATAATTACGCC;

[0023] SEQ ID NO.10:ATAAGAAGTAACAATAATAATTATGCAACA;

[0024] SEQ ID NO.11: GTCAGAGGGGACTATAGACCCTAC;

[0025] SEQ ID NO.12: CAGAATGTGGGTACTAAT;

[0026] SEQ ID NO.13: TCGGCATCC;

[0027] SEQ ID NO.14: CAACAATATAACACCTATCCTTCCACG;

[0028] Preferably, the nucleic acid sequence encoding the heavy chain is as shown in SEQ.NO.15 or a complementary sequence thereto, and the nucleic acid sequence encoding the light chain is as shown in SEQ.NO.16 or a complementary sequence thereto.

[0029] SEQ ID NO.15: GAGGTGCAGCTGCAGGAGTCAGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTACAGCCTCTGGATTCACCTTCAATAATTACGCCGTGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAACAATAATAATTA TGCAACATTTTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAGAACTGAGGACACAGCCATGTATTACTGTGTCAGAGGGGACTATAGACCCTACTGGGGCCAAGGCACCACCCTCACAGTCTCCTCA;

[0030] SEQ ID NO.16: GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATTTCCTAAAGCACTGATTTACTCGGCATCCTAC CGGAACAGTGGAGTCCCTGATCGCTTCAAAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAATGTGCAGTCTGAAGACTTGGCAGAGTATTCCTGTCAACAATATAACACCTATCCTCTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG;

[0031] In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid encoding a monoclonal antibody against the aforementioned Chlamydia trachomatis Pgp3 protein.

[0032] In a fifth aspect, the present invention provides a host cell that has been transformed or transfected with the above-described expression vector.

[0033] In a sixth aspect, the present invention provides the use of the above-mentioned monoclonal antibody against Chlamydia trachomatis Pgp3 protein, or a functional fragment thereof, as well as its nucleic acid molecule, expression vector, and host cell in the preparation of the following drugs:

[0034] a. Drugs that inhibit Chlamydia trachomatis infection;

[0035] b. Medications that inhibit genital chlamydia trachomatis infection;

[0036] c. Medications to reduce fallopian tube edema.

[0037] Compared with existing technologies, the present invention has the following advantages:

[0038] This invention establishes a mouse reproductive tract infection model of *Chlamydia trachomatis* to investigate the effect of this antibody on the infection of the mouse reproductive tract by *Chlamydia trachomatis*. The exploratory experimental studies of this invention confirm that the monoclonal antibody against the *Chlamydia trachomatis* Pgp3 protein can effectively inhibit the infection of *Chlamydia trachomatis* in the mouse reproductive tract and reduce the occurrence of fallopian tube edema. Attached Figure Description

[0039] Figure 1 This is a 1% agarose gel electrophoresis image of total RNA from Example 1, with lane 1 being lane 2 and lane M being the DL2000 DNA Marker.

[0040] Figure 2 This is a gel image of heavy chain variable region PCR amplification in Example 1. Lanes 1-2 are for H chain PCR, and lane M is for DL2000 DNA Marker.

[0041] Figure 3 This is a gel image of light chain variable region PCR amplification in Example 1. Lanes 1-2 are L-chain PCR, and lane M is DL2000 DNA Marker.

[0042] Figure 4 This is a gel image of light chain colony PCR verification in Example 1. Lanes 1-4 are single colonies 1-4, and lane M is the DL2000 DNA Marker.

[0043] Figure 5 This is a gel image of heavy chain colony PCR verification in Example 1. Lanes 1-4 are single colonies 1-4, and lane M is the DL2000 DNA Marker.

[0044] Figure 6 To compare the reproductive tract infection status of mice in each group at different time points, group C1 was the low-concentration subcutaneous injection group, group C2 was the high-concentration subcutaneous injection group, group C3 was the low-concentration vaginal administration group, group C4 was the high-concentration vaginal administration group, and group C5 was the control group. *P<0.05, compared with the control group.

[0045] Figure 7 Samples of fallopian tube edema were collected from each group of mice. Group C1 was the low-concentration subcutaneous injection group, Group C2 was the high-concentration subcutaneous injection group, Group C3 was the low-concentration vaginal administration group, Group C4 was the high-concentration vaginal administration group, and Group C5 was the control group. Detailed Implementation

[0046] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0047] The "monoclonal antibody" mentioned in the specific implementation refers to an antibody molecule with a single molecular composition, derived from a group of essentially identical antibodies. This monoclonal antibody exhibits single binding specificity and affinity for a specific epitope. Typically, immunoglobulins have heavy and light chains. Each heavy and light chain contains constant and variable regions. The variable regions of the light and heavy chains contain four framework regions, interrupted by three hypervariable regions, also known as "complementarity-determining regions" (CDRs). CDRs are primarily responsible for binding to the epitopes of the antigen. The CDRs of each chain are typically designated CDR1, CDR2, CDR3, and are numbered sequentially starting from the N-terminus, and are usually identified by the chain in which the specific CDR is located.

[0048] In the specific implementation, the term "functional fragment" refers to a portion of the antibody that has specific binding affinity for an antigen, or a polypeptide comprising such a portion. For example, a functional fragment may be a portion of the antibody comprising amino acid residues that confer specificity and / or affinity to the antigen by interacting with an antigen (e.g., an epitope), or a polypeptide comprising such a portion. This functional fragment typically contains one or more "complementarity-determining regions (CDRs)" and one or more "framework (FR)" regions. CDRs are amino acid sequences that contribute to the specificity and affinity of antibody-antigen binding, while frame regions are amino acid sequences that help maintain the proper conformation of these CDRs and facilitate binding between the antigen-binding region and the antigen.

[0049] The invention will be described in detail below with reference to specific embodiments.

[0050] Example 1: Preparation of Chlamydia trachomatis Pgp3 protein

[0051] The purified protein Pgp3 encoded by the Chlamydia trachomatis plasmid pORF5 was prepared using prokaryotic expression technology. The description of the chlamydia plasmid-encoded protein pgp3 is as follows:

[0052] (1) This protein is encoded by the Chlamydia trachomatis plasmid pORF5, and its nucleotide sequence is as follows:

[0053] ATGGGAAATTCTGGTTTTTATTTGTATAACACTGAAAACTGCGTCTTTGCTGATAATATCAAAGTTGGGCAAATGACAGAGCCGCTCAAGGACCAGCAAATAATCCTTGGGACAACATCAACACCTGTCGCAGCCAAAATGACAGCTTCTGATGGAATATCTTTAACAGTCTCCAATAATTCATCAACCAATGCTTCTATTACAATTGGTTTGGATGCGGAAAAAGCTTACCAGCTTATTCTAGAAAAGTTGGGAGATCAAATTCTTGATGGAATTGCTGATACTATTGTTGATAGTACAGTCCAAGATATTTTAGACAAAATCAAAACAGACCCTTCTCTAGGTTTGTTGAAAGCTTTTAACAACTTTCCAATCACTAATAAAATTCAATGCAACGGGTTATTCACTCCCAGTAACATTGAAACTTTATTAGGAGGAACTGAAATAGGAAAATTCACAGTCACACCCAAAAGCTCTGGGAGCATGTTCTTAGTCTCAGCAGATATTATTGCATCAAGAATGGAAGGCGGCGTTGTTCTAGCTTTGGTACGAGAAGGTGATTCTAAGCCCTGCGCGATTAGTTATGGATACTCATCAGGCATTCCTAATTTATGTAGTCTAAGAACCAGTATTACTAATACAGGATTGACTCCGACAACGTATTCATTACGTGTAGGCGGTTTAGAAAGCGGTGTGGTATGGGTTAATGCCCTTTCTAATGGCAATGATATTTTAGGAATAACAAATACTTCTAATGTATCTTTTTTAGAGGTAATACCTCAAACAAACGCTTAA(SEQ ID NO.17).

[0054] (2) Purified chlamydia protein pgp3: with a molecular weight of 29 kDa, and its amino acid sequence is as follows:

[0055] MGNSGFYLYNTENCVFADNIKVGQMTEPLKDQQIILGTTSTPVAAKMTASDGISLTVSNNSSTNASITIGLDAEKAYQLILEKLGDQILDGIADTIVDSTVQDILDKIKTDPSLGLLKAFNNFPITNKIQCNGL FTPSNIETLLGGTEIGKFTVTPKSSGSMFLVSADIIASRMEGGVVLALVREGDSKPCAISYGYSSGIPNLCSLRTSITNTGLTPTTYSLRVGGLESGVVWVNALSNGNDILGITNTSNVSFLEVIPQTNA(SEQ ID NO.18).

[0056] Example 2: Preparation of monoclonal antibody against Chlamydia trachomatis Pgp3 protein

[0057] 2.1 Obtaining mouse monoclonal antibodies against Chlamydia trachomatis Pgp3 protein using hybridoma technology, including the establishment of hybridoma cells and antibody preparation, the specific steps are as follows:

[0058] (1) Purified Chlamydia protein Pgp3 was subcutaneously injected into 6-8 week old Balb / c mice. The mice were purchased from Shandong Hengrong Biotechnology Co., Ltd. The antigen immunization dose was 20 μg / mouse, and the injection was performed three times. Blood from the inner canthus vein of the mice was taken to measure the antibody titer. Three days before cell fusion, mice with high immune titers were selected and injected intraspleurally with Chlamydia protein Pgp3 for booster immunization.

[0059] (2) Myeloma cells SP2 / 0 and spleen cells from immunized mice were cultured in 96-well plates that had been lined with feeder cells.

[0060] (3) Change the culture medium every 2 days. When changing the medium, remove 1 / 2 or 2 / 3 of the culture medium and add an equal amount of fresh culture medium. Use HAT culture medium for 7 days after fusion, use HT culture medium from the 7th to the 14th day, and use ordinary complete culture medium after the 14th day.

[0061] (4) ELISA was used to detect specific antibodies. Cells from positive wells were promptly transferred into 24-well plates and cultured using the limiting dilution method.

[0062] (5) Perform chromosome analysis on hybridoma cells that secrete specific antibodies and identify monoclonal antibody subclasses and specificity in the supernatant of hybridoma cells.

[0063] (6) Hybridoma cells that secrete specific antibodies are cultured in large quantities and the supernatant is collected. Monoclonal antibodies are then purified by gel affinity chromatography.

[0064] 2.2 Primer design: Primers used for amplifying the variable region of the antibody are shown in the table below:

[0065] Table 1 Amplification Primers

[0066]

[0067] 3.3 Total RNA detection

[0068] Results of 1% agarose gel electrophoresis are as follows Figure 1 As shown.

[0069] 3.4 RT-PCR

[0070] RT-PCR includes the following steps:

[0071] (1) 5 μl total RNA, 2.5 μl oligoT(18), 2.5 μl Random Primer, 68℃ for 5-10 min, place on ice for 5 min.

[0072] (2) 3 μl dNTPs (2.5 mM stock), 8 μl Bμffer (5X), 1 μl RNase OUT, 4 μl DTT, mix well, 42℃, 2 min, add 2 μl MMLV, 42℃ for 1-2 h, 70℃ for 15 min.

[0073] 3.5 PCR amplification

[0074] The PCR amplification reaction mixture consisted of: 1 μl cDNA, 1 μl dNTPs (2.5 mM), 2 μl Bμffer (10X), 0.5 μl Primer F (5 μm), 0.5 μl Primer R (5 μm), 0.2 μl (5 Unit / μl) Taq polymerase, and 14.8 μl ddH2O. The reaction program was: 94℃ for 5 min; 94℃ for 30 s; 55℃ for 30 s; 72℃ for 30 s; 72℃ for 10 min; 30 cycles. PCR products were recovered.

[0075] 3.6 Connection and Transformation

[0076] (1) Ligation: 1 μl pMD-19T vector, 2 μl enzyme digestion product, 3 μl ligase mixture (TaKaRa, DNAligation Kit Ver 2.0), mix well, and react at room temperature for more than 30 min.

[0077] (2) Transformation: 1) Remove competent cells (DH5α) stored at -80℃ and thaw slowly on ice. 2) Add competent cells to the ligation product, mix well, and place on ice for 30 min. 3) Heat shock at 42℃ for 90 s. 4) After incubating on ice for 2 min, add 800 μl of antibiotic-free LB medium. 5) Incubate at 37℃ for 45 min. 6) Centrifuge at 5000 rpm for 3 min, discard most of the supernatant, and retain about 100-150 μl. Resuspend the cells and spread them on LB plates with Amp+ resistance. 7) Air dry and incubate upside down in a 37℃ incubator overnight. Use blue-white screening and pick cells with white spots for shaking.

[0078] 3.7 Colony PCR

[0079] The reaction system for colony PCR was as follows: 1 μl bacterial culture, 1 μl dNTPs (2.5 mM), 2 μl Bμffer (10X), 0.5 μl Primer F (5 μm), 0.5 μl Primer R (5 μm), 0.2 μl (5 units / μl) Taq polymerase, and 14.8 μl ddH2O. The reaction program was: 95℃ for 10 min; 95℃ for 45 s; 55℃ for 45 s; 72℃ for 1 min; 72℃ for 10 min; 30 cycles.

[0080] 3.8 Sequencing

[0081] Clones with the correct molecular weight from colony PCR were selected for sequencing. The sequencing results are as follows:

[0082] The nucleic acid sequence of the heavy chain is shown in SEQ. NO. 15, and the amino acid sequence is shown in SEQ. NO. 7.

[0083] The nucleic acid sequence of the light chain is shown in SEQ.NO.16, and the amino acid sequence is shown in SEQ.NO.8.

[0084] Test case

[0085] I. Construction of the experimental mouse model

[0086] 1.1 Preparation of experimental animals

[0087] Twenty-five SPF-grade, 5-6 week old, female BALB / C mice weighing 18-20g were housed.

[0088] 1.2 Experimental grouping and intervention

[0089] Twenty-five mice were randomly divided into five groups of five each: a control group, a subcutaneous injection high-concentration group, a subcutaneous injection low-concentration group, a vaginal administration high-concentration group, and a vaginal administration low-concentration group.

[0090] (1) Establishment of a mouse reproductive tract infection model: Five days before infection, each mouse was subcutaneously injected with 2.5 mg of progesterone injection solution; four days after progesterone injection, vaginal secretions of the mice were cleaned with a medical swab; purified mouse Chlamydia trachomatis EB stored at -80℃ was taken out, diluted with SPG buffer, and placed on ice. The grouped mice were fixed, and 20 μL of strain EB containing 2×10^5 IFU was slowly inoculated into the vagina of the mice through the posterior fornix using a micropipette. After inoculation, the mouse was lifted by the tail and inverted to prevent the inoculated infection fluid from flowing out. After the infection fluid was completely absorbed, the mice were returned to the cages, and the above operation was repeated until all mice in each group were completely infected.

[0091] (2) The purified anti-Chlamydia trachomatis Pgp3 protein monoclonal antibody was diluted with sterile PBS to 0.8 mg / kg and 0.4 mg / kg.

[0092] (3) Low concentration group of anti-Chlamydia trachomatis Pgp3 protein monoclonal antibody was injected subcutaneously once a week for three consecutive weeks, with an injection dose of 0.4 mg / kg.

[0093] (4) High concentration group of anti-Chlamydia trachomatis Pgp3 protein monoclonal antibody was injected subcutaneously once a week for three consecutive weeks, with an injection dose of 0.8 mg / kg.

[0094] (5) Low concentration group of anti-Chlamydia trachomatis Pgp3 protein monoclonal antibody vaginal administration: The dosage was 0.4 mg / kg, and the drug was administered vaginally on days 2, 4, 5, 6 and 7 after infection.

[0095] (6) High concentration group of anti-Chlamydia trachomatis Pgp3 protein monoclonal antibody vaginal administration: The dosage was 0.8 mg / kg, and the drug was administered vaginally on days 2, 4, 5, 6 and 7 after infection.

[0096] II. Detection of Chlamydia muscarinii content in exfoliated epithelial cells of the lower genital tract of mice at different time points using indirect immunofluorescence assay

[0097] (1) On days 3, 10, 14 and 21 after infection with mouse chlamydia, vaginal secretions were scraped from mice with a medical swab, immersed in 500 μL of cold SPG buffer, 5-6 sterile glass beads were added, and the mixture was shaken vigorously on a vortex shaker for 2 minutes to fully rupture the cell membranes of vaginal epithelial cells and the inclusion body membranes of mouse chlamydia, and to fully release EB.

[0098] (2) Centrifuge at 4℃ and 3000rpm for 5min and discard the precipitate.

[0099] (3) Take 50 μL of vaginal secretion supernatant mixed with SPG buffer, dilute it to 500 μL of SPG, and then serially dilute it 10-fold, 20-fold, and 40-fold. Inoculate 200 μL into each well of a 24-well plate monolayer of HeLa cells that have been incubated with DEAE-D solution for 30 min in advance. Incubate at 37°C and 5% CO2 for 30 min. After incubation, centrifuge at 37°C and 1500 rpm / min for 1 h. After centrifugation, incubate at 37°C and 5% CO2 for 30 min, discard the centrifuged liquid, add 1 mL of infection solution containing actinomycin to each well, and incubate at 37°C and 5% CO2 for 18–24 h.

[0100] (4) After 18 hours, discard the infection solution, wash three times with PBS for 5 minutes each time, add 500 μL of methanol to each well, and fix at room temperature for 20 minutes.

[0101] (5) Remove methanol, wash three times with PBS for 5 min each time, add 500 μL of PBS diluted with 1% Tritonx-100, and let stand at room temperature for 8 min.

[0102] (6) Aspirate Tritonx-100, wash three times with PBS for 5 min each time, add 500 μL of cell growth medium, and block at room temperature for 1 h.

[0103] (7) Aspirate the growth medium, wash three times with PBS for 5 min each time, mix and dilute the rabbit anti-Cm inclusion body polyclonal antibody with fresh growth medium at a ratio of 1:2000, add 200 μL of primary antibody dilution solution to each well, and incubate at 37°C for 1 h or at 4°C overnight.

[0104] (8) Aspirate the liquid, wash three times with PBS for 5 minutes each time, mix and dilute the FITC-labeled goat anti-rabbit secondary antibody with fresh growth medium at a ratio of 1:200 in the dark, add 50 μL of secondary antibody dilution to each well, gently shake to cover the cell surface with liquid, and incubate at 37°C in the dark for 1 hour.

[0105] (9) Aspirate the liquid in a dark room, wash three times with PBS for 5 min each time, add 50 μL of DAPI staining solution to cover the cell surface, and incubate at 37°C in the dark for 15 min.

[0106] (10) Aspirate the DAPI staining solution, wash three times with PBS for 5 minutes each time, wash with distilled water for 5 minutes each time, discard the liquid in the well, let the residual water stains on the coverslip dry, take it out with tweezers and fix it on a glass slide with a small amount of sealing agent, and store it in the dark.

[0107] (11) Five fields of view were selected under a fluorescence microscope. The number of inclusion bodies in each field of view was counted under different dilution concentrations and microscope magnification. The number of mouse chlamydia trachomatis IFUs in each swab was calculated. The IFUs values ​​were converted to log10 and the average value of each group at different time points was calculated.

[0108] Experimental results: In a mouse reproductive tract infection model, immunofluorescence assays showed that the number of inclusion bodies ( Figure 6 On day 3 post-vaccination, the subcutaneous injection low-concentration group (p = 0.0321) and the subcutaneous injection high-concentration group (p = 0.0117) were lower than the control group (p < 0.05), while there was no difference between the vaginal administration low-concentration group, the vaginal administration high-concentration group, and the control group (p > 0.05). On day 10 post-vaccination, the vaginal administration high-concentration group was lower than the control group (p = 0.0099), while there was no difference between the other groups and the control group (p > 0.05). On day 14 post-vaccination, there were no statistically significant differences among the groups (p > 0.05). On day 21 post-vaccination, the subcutaneous injection low-concentration group (p = 0.0005), the subcutaneous injection high-concentration group (p = 0.0002), and the vaginal administration low-concentration group (p = 0.008) were lower than the control group (p < 0.05), while there was no difference between the vaginal administration high-concentration group and the control group (p > 0.05).

[0109] III. Assessing the degree of hydrosalpinx in mice after infection with Chlamydia trachomatis pneumonia

[0110] Mice were euthanized 60 days after vaginal infection with *Chlamydia trachomatis*. The abdominal cavity was opened, and the complete reproductive tract tissue was separated. Excess adipose tissue was removed, and the degree of hydrosalpinx was observed and photographed. The severity of hydrosalpinx was scored according to the following criteria: 0 points, no hydrosalpinx; 1 point, hydrosalpinx present but not visible to the naked eye, requiring microscopic observation; 2 points, hydrosalpinx visible to the naked eye, the diameter of the lesion smaller than the diameter of the ipsilateral ovary; 3 points, the diameter of the hydrosalpinx approximately equal to the diameter of the ipsilateral ovary; 4 points, the diameter of the hydrosalpinx larger than the diameter of the ipsilateral ovary. The sum of the bilateral hydrosalpinx scores for each mouse was calculated as the severity score. The scores for each group of mice were statistically analyzed, and the mean and standard deviation of each group were calculated. The statistical results are shown in the table below:

[0111] Table 2. Scoring of the degree of oviduct edema in each group of mice.

[0112]

[0113]

[0114] Note: Gross scores of hydrosalpinx in mice in each group, *P<0.05, compared with the control group.

[0115] Experimental results: Macroscopic assessment of the severity of hydrosalpinx ( Figure 7 The incidence of hydrosalpinx was 0 in the subcutaneous injection low-concentration group and 0 in the subcutaneous injection high-concentration group; the incidence of hydrosalpinx was 3 / 5 in the vaginal administration low-concentration group, with a severity score of 1.4±1.67; the incidence of hydrosalpinx was 2 / 5 in the vaginal administration high-concentration group, with a severity score of 0.4±0.55; and the incidence of hydrosalpinx was 4 / 5 in the control group, with a severity score of 2.4±2.07. There was a statistically significant difference between the subcutaneous injection group and the control group (p<0.05).

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against Chlamydia trachomatis Pgp3 protein, characterized in that: The monoclonal antibody comprises: heavy chain variable regions CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NO. 1, 2, and 3, respectively, and light chain variable regions CDR1 and CDR3 with amino acid sequences as shown in SEQ ID NO. 4 and 6, respectively. The monoclonal antibody also includes a light chain variable region CDR2, the amino acid sequence of which is SAS.

2. The monoclonal antibody against Chlamydia trachomatis Pgp3 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.

7.

3. The monoclonal antibody against Chlamydia trachomatis Pgp3 protein according to claim 1, characterized in that: The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.

8.

4. A nucleic acid encoding a monoclonal antibody against the Chlamydia trachomatis Pgp3 protein as described in any one of claims 1-3.

5. The nucleic acid according to claim 4, characterized in that: The nucleic acid sequences encoding the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ.NO.9, 10, and 11, respectively, or are complementary to them. The nucleic acid sequences encoding the light chain variable regions CDR1 and CDR3 are shown in SEQ.NO.12 and 14, respectively, or are complementary to them. The nucleotide sequence encoding the light chain variable region CDR2 is TCGGCATCC or is complementary to it.

6. The nucleic acid according to claim 4, characterized in that: The nucleic acid sequence encoding the heavy chain is shown in SEQ.NO.15 or is complementary to it, and the nucleic acid sequence encoding the light chain is shown in SEQ.NO.16 or is complementary to it.

7. An expression vector comprising a nucleic acid encoding a monoclonal antibody against the Chlamydia trachomatis Pgp3 protein as described in any one of claims 4-6.

8. A host cell that transforms or transfects the expression vector of claim 7.

9. The use of the nucleic acid molecule, expression vector, and host cell expressing the monoclonal antibody against Chlamydia trachomatis Pgp3 protein as described in any one of claims 1-3 in the preparation of the following drugs: a. Drugs that inhibit Chlamydia trachomatis infection; b. Medications to reduce fallopian tube edema.

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