A humanized Chlamydia trachomatis Pgp3 antibody and its application

Abstract

The present invention discloses a humanized Chlamydia trachomatis Pgp3 antibody and its application. The humanized Chlamydia trachomatis Pgp3 antibody comprises: heavy chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; and light chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO.4, SEQ ID NO.21, and SEQ ID NO.5, respectively; and a constant region, wherein the constant region is of human origin. The humanized Pgp3 antibody 2B2A2 of the present invention has high affinity, broad-spectrum anti-infective ability, and significant in vitro and in vivo protective effects. It can effectively reduce Chlamydia load and inhibit pathological damage, providing an innovative solution for the prevention and treatment of Chlamydia-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a humanized Chlamydia trachomatis Pgp3 antibody and an application thereof. Background Art

[0002] CDR grafting is a commonly used method for antibody humanization. It involves transplanting the CDRs of a non-human monoclonal antibody (donor) into the FRs of a human monoclonal antibody (recipient). When selecting the recipient FR region, high FR homology is an important criterion. Human antibody germline genes are also an important source of FR regions for humanization. Compared to selecting FRs from mature IgG, antibody germline genes do not introduce somatic mutations, and their FR regions are considered to have lower immunogenicity.

[0003] Simple CDR grafting can result in a significant loss of affinity. Backmutating key amino acids to their donor counterparts can effectively reverse this affinity loss. These key amino acids typically play a significant role in the proper conformation of the CDRs and are typically located within the β-sheet FRs.

[0004] Alterations in antibody structure can lead to reduced antibody binding affinity. Some mouse residues in the framework regions, known as Vernier region residues, have been shown to influence CDR loop conformation and antibody affinity. These residues are located in the β-sheet framework region adjacent to the CDRs. Therefore, after selecting the desired human framework, these residues are retained in the human antibody.

[0005] Current humanization technology for Chlamydia trachomatis Pgp3 antibodies is mainly limited by conformational stability, immunogenicity, affinity and specificity, as well as insufficient functional validation models. Summary of the Invention

[0006] In view of this, the present invention aims to provide a humanized Chlamydia trachomatis Pgp3 antibody and its application to solve at least one technical problem in the background technology.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] A humanized Chlamydia trachomatis Pgp3 antibody, comprising: heavy chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; and light chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO.4, SEQ ID NO.21, and SEQ ID NO.5, respectively.

[0009] SEQ ID NO.1: GFTFNNYA; SEQ ID NO.2: IRSNNNNYAT; SEQ ID NO.3: VRGDYRPY; SEQ ID NO.4: QNVGTN; SEQ ID NO.21: SAS; SEQ ID NO.5: QQYNTYPLT.

[0010] The heavy chain variable region amino acid sequence is shown in SEQ ID NO. 6, and the light chain variable region amino acid sequence is shown in SEQ ID NO. 7.

[0011] SEQ ID NO.6:

[0012] QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAVNWVRQAPGKGLEWVARIRSNNNNYAFYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRPYWGQGTLVTVSS;

[0013] SEQ ID NO.7: DIQMTQSQSTLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASYRNSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYPLTFGGGTKVEIK;

[0014] Furthermore, the constant region includes a human immunoglobulin IgG1 constant region sequence and a human immunoglobulin Kappa constant region.

[0015] A nucleic acid encoding the above-mentioned humanized Chlamydia trachomatis Pgp3 antibody.

[0016] A vector comprising the nucleic acid encoding the above-mentioned gene.

[0017] A cell comprising the above nucleic acid or the above vector.

[0018] The use of the above-mentioned humanized Chlamydia trachomatis Pgp3 antibody, the above-mentioned vector or the above-mentioned cell in the preparation of the following drugs: a. a drug for inhibiting Chlamydia trachomatis infection; b. a drug for reducing fallopian tube edema; c. a drug for inhibiting salpingitis; d. a drug for inhibiting cervicitis.

[0019] Compared with the prior art, the humanized Chlamydia trachomatis Pgp3 antibody and its application described in the present invention have the following advantages:

[0020] 1. High-affinity antibody properties: The humanized Pgp3 antibody 2B2A2 of the present invention has been verified by ELISA, Western Blotting and surface plasmon resonance (SPR), showing excellent antigen-binding affinity, ensuring its efficient recognition and targeting effects.

[0021] 2. Broad specific recognition capability: 2B2A2 can specifically recognize endogenous Pgp3 protein, providing a precise target for the detection and treatment of chlamydia infection.

[0022] 3. Highly effective in vitro anti-infection effect: 2B2A2 can significantly inhibit the in vitro infection of Chlamydia muridarum (Cm) and Chlamydia trachomatis serotype D (Ct-D), indicating that it has broad-spectrum anti-chlamydial activity.

[0023] 4. Significantly Reduces Pathogen Load and Pathological Damage in Acute Infection Models: In a mouse model of acute Cm infection, 2B2A2 significantly reduced the chlamydial load in the lower genital tract, effectively controlling infection; inhibited the formation of hydrosalpinx, reducing genital tract structural damage; alleviated fallopian tube dilation and inflammatory infiltration, and reduced histopathological damage. Effectively intervened in persistent infection and prevented chronic pathological damage. In a mouse model of persistent Cm infection, 2B2A2 consistently reduced the chlamydial load in the lower genital tract, reducing the risk of chronic infection; inhibited the development of hydrosalpinx, preventing long-term complications; alleviated fallopian tube dilation and inflammatory infiltration, and improved the genital tract microenvironment.

[0024] 5. Potential clinical application value: The 2B2A2 antibody can be used for: prevention and treatment of chlamydial infection, reducing the risk of pelvic inflammatory disease (PID), infertility, and ectopic pregnancy; intervention of chronic infection, blocking chlamydial immune escape and persistent infection; and development of new antibody drugs or vaccines, providing new strategies for anti-chlamydial immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 Schematic diagram of the amino acid sequence analysis results described in the embodiments of the present invention (A is the heavy chain amino acid sequence analysis result of mhPgp3Ab, B is the light chain amino acid sequence analysis result of umhPgp3Ab);

[0027] Figure 2 Schematic diagram of the SDS-PAGE results of the chimeric antibody described in the examples of the present invention (M is a marker, B is a BSA standard (66 kDa), 1 is a reduced mhPgp3Ab, and 2 is a non-reduced mhPgp3Ab);

[0028] Figure 3 Alignment analysis of the heavy and light chain amino acid sequences of the humanized Pgp3 antibodies described in the Examples of the present invention (A is a multiple comparison of the heavy chain amino acid sequences of the humanized Pgp3 antibodies; B is a multiple comparison of the light chain amino acid sequences of the humanized Pgp3 antibodies. * indicates that the residues at the same position in all sequences are exactly the same; : indicates a highly conserved substitution; . indicates a less conserved substitution; blank indicates an irrelevant substitution or deletion);

[0029] Figure 4 The SDS-PAGE results of the 11 hPgp3Ab strains described in the Examples of the present invention are shown (A: 1, 3, 5, 7, 9, and 11 are reduced hPgp3Ab, and 2, 4, 6, 8, 10, and 12 are non-reduced hPgp3Ab, corresponding to 1A1A3, 1B1B2, 3B2B3, 3B2A2, 1B2A2, and 2B2A2, respectively; B: 1, 3, 5, 7, and 9 are reduced hPgp3Ab, and 2, 4, 6, 8, and 10 are non-reduced hPgp3Ab, corresponding to 1B1A2, 1B2B2-6-2, 3B2B2, 1B2B2-9-2, and 1A2B3, respectively. M is a marker, and B is a BSA standard (66 kDa));

[0030] Figure 5 The affinity ranking results of the ELISA assay described in the Examples of the present invention (Pgp3mAb was used as a positive control, PBS was used as a negative control, 11 hPgp3Abs and 1 mhPgp3Ab were constructed as primary antibodies, and the affinity of the above antibodies to the His-Pgp3 protein was detected by ELISA. The six concentration gradients on the X-axis were 10 μg / mL to 0.3125 μg / mL (doubling dilutions));

[0031] Figure 6 The Western-blot affinity ranking results described in the examples of the present invention;

[0032] Figure 7 Affinity determination curves for the candidate antibodies described in the Examples of the present invention and His-Pgp3 (a is 2B2A2, b is 3B2B2, c is 1A2B3, d is 1B1A2, e is 1B2B2-6-2, f is 1B2A2, g is hPgp3Ab, h is 1B1B2, i is 1B2B2-9-2, j is mhPgp3Ab, the abscissas of a, b, c, d, e, f, g, h, i, and j are all antibody concentrations, and the ordinates are the binding signals);

[0033] Figure 82B2A2 described in the examples of the present invention can specifically bind to endogenous Pgp3 (A shows Hela cells infected with Cm and fixed and permeabilized 23 hours after infection; B shows Hela cells infected with Ct-D and fixed and permeabilized 46 hours after infection; Pgp3 was labeled with 2B2A2 and a secondary antibody conjugated with Alexa Fluor 594 (red); inclusion bodies were labeled with a rabbit anti-Chlamydia antibody and a secondary antibody conjugated with Alexa Fluor 488 (green); and cell nuclei were labeled with DAPI (blue);

[0034] Figure 9 2B2A2 described in the examples of the present invention inhibits infection of Cm-infected cell lysates in vitro (supernatant of Hela cell lysates continuously infected with Cm for 24 hours was incubated with different concentrations of 2B2A2 for 2 hours. Each mixture was inoculated onto Hela cells grown on coverslips in 24-well plates. Immunofluorescence assay was performed 24 hours later. A shows Cm EB infection in each group observed under a fluorescence microscope (20×). Inclusion bodies were labeled with rabbit anti-Chlamydia antibodies and Alexa Fluor 488-conjugated (green) secondary antibodies, and cell nuclei were labeled with DAPI (blue). Merge is the overlay of layers at the same position. B shows the count of inclusion bodies and host cells under a fluorescence microscope. Each coverslip was randomly observed five times. The results are expressed as the infection rate (number of inclusion bodies per 100 cells). The mean ± SD of each group was calculated from three independent experiments. ns, P>0.05; **, P<0.01; ***, P<0.001; One-way ANOVA test was used).

[0035] Figure 10 2B2A2 described in the examples of the present invention can inhibit the infectivity of Ct-D in vitro (the supernatant of Hela cell lysate continuously infected with Ct-D for 48 hours was incubated with different concentrations of 2B2A2 for 2 hours. Each mixture was inoculated into Hela cells grown on coverslips in 24-well plates. Immunofluorescence assay was performed 24 hours later. A shows the infection status of Cm EB in each group observed under a fluorescence microscope. Inclusion bodies were labeled with rabbit anti-Chlamydia antibodies and Alexa Fluor 488-conjugated (green) secondary antibodies, and cell nuclei were labeled with DAPI (blue). Merge is the overlay of layers at the same location. B shows the count of inclusion bodies and host cells under a fluorescence microscope. Each coverslip was randomly observed five times. The results are expressed as the infection rate (number of inclusion bodies per 100 cells). ns, P>0.05; **, P<0.01; ****, P<0.0001; One-way ANOVA test was used).

[0036] Figure 11The effect of 2B2A2 described in the examples of the present invention on the chlamydial load in the lower genital tract of mice with acute Cm infection (24 BALB / c mice were randomly divided into 4 groups: Cm acute infection group; Cm acute infection + subcutaneous injection of 0.8 mg / kg Pgp3mAb group; Cm acute infection + subcutaneous injection of 2A2B2 0.1 mg / kg group; Cm acute infection + subcutaneous injection of 2A2B2 0.8 mg / kg group. After acute infection with Cm, the mice were dosed according to the group, and vaginal swabs were collected at designated time points after infection to monitor the shedding of live C. muris in the lower genital tract. A shows the change in the chlamydial load in the lower genital tract over time in each group, with the X-axis representing the fixed sampling interval and the Y-axis representing the chlamydial load in the lower genital tract (IFUs measured in each swab). B shows the chlamydial load in the lower genital tract in each group at different time points, with the X-axis representing the fixed sampling interval and the Y-axis representing the chlamydial load in the lower genital tract (Log10 IFUs), ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; One-way ANOVA test);

[0037] Figure 12 2B2A2 described in the examples of the present invention can inhibit the pathogenicity of Cm in the upper reproductive tract of mice (A shows the mouse reproductive tract was harvested and the gross pathology of the upper reproductive tract was assessed 60 days after infection. B shows the visual score of the severity of hydrosalpinx, ns, P>0.05; ***, P<0.001; ****, P<0.0001; tested by One-way ANOVA);

[0038] Figure 13 The 2B2A2 described in the embodiment of the present invention can reduce the pathogenicity of acute Cm infection to the upper reproductive tract of mice (A is Figure 12 Histopathological examination of the reproductive tract tissues of mice in the middle stage. Microscopic images of H&E-stained sections of oviduct tissue were obtained using a 10x objective lens. D is a microscopic image of H&E-stained sections of oviduct tissue obtained using a 40x objective lens. IDscore is the dilation score, IN score is the inflammation score, B is the score for chronic inflammatory infiltration, and C is the score for lumen dilation (**, P < 0.01; ANOVA test).

[0039] Figure 14The effect of 2B2A2 described in the examples of the present invention on the chlamydial load in the lower genital tract of mice with persistent Cm infection (24 BALB / c mice were randomly divided into 4 groups: a Cm persistent infection group; a Cm persistent infection + subcutaneous injection of 0.8 mg / kg Pgp3mAb group; a Cm persistent infection + subcutaneous injection of 2A2B2 0.1 mg / kg group; and a Cm persistent infection + subcutaneous injection of 2A2B2 0.8 mg / kg group. After persistent Cm infection, mice were dosed according to group, and vaginal swabs were collected at designated time points after infection to monitor the shedding of live C. muris in the lower genital tract. A shows the change in the chlamydial load in the lower genital tract over time in each group, with the X-axis representing the fixed sampling interval and the Y-axis representing the chlamydial load in the lower genital tract (IFUs measured in each swab). B shows the chlamydial load in the lower genital tract in each group at the same time point, with the X-axis representing the group and the Y-axis representing the chlamydial load in the lower genital tract (Log10 IFUs). ns, P>0.05; ***, P<0.001; ****, P<0.0001; One-way ANOVA test);

[0040] Figure 15 All groups of mice described in the examples of the present invention were sacrificed after inhalation anesthesia 60 days after infection, and the reproductive tract tissues 60 days after infection were gross (A is the mice in the Pgp3mAb group and the mice in the high-dose subcutaneous injection group of 2B2A2, and B is the visual scoring of hydrosalpinx in all mice);

[0041] Figure 16 The 2B2A2 described in the embodiment of the present invention can inhibit the pathogenicity of Cm persistent infection to the upper reproductive tract of mice (A is Figure 14 Histopathological examination of the reproductive tract tissues of mice was performed. Microscopic images of H&E-stained sections of oviduct tissue were obtained using a 10x objective lens. (D) Microscopic images of H&E-stained sections of oviduct tissue were obtained using a 40x objective lens. (B) Chronic inflammatory infiltration was scored, and (C) Lumen dilation was scored (*, P < 0.05; **, P < 0.01; ANOVA test). DETAILED DESCRIPTION

[0042] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0043] The "monoclonal antibody" mentioned in the specific embodiments refers to an antibody molecule with a single molecular composition, obtained from a group of substantially identical antibodies. The monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. Typically, immunoglobulins have heavy chains and light chains. Each heavy chain and light chain contains a constant region and a variable region. The light chain and heavy chain variable regions contain four framework regions, interrupted by three hypervariable regions, also known as "complementarity determining regions," or CDRs. CDRs are primarily responsible for binding to the epitope of the antigen. The CDRs of each chain are typically CDR1, CDR2, and CDR3, numbered consecutively starting from the N-terminus, and are usually also identified by the chain in which the specific CDR is located.

[0044] Example 1. Construction and expression of human-mouse chimeric Pgp3 antibody;

[0045] 1. Construction of human-mouse chimeric Pgp3 antibody;

[0046] (1) The amino acid sequence of the variable region of the Pgp3 monoclonal antibody prepared by hybridoma cell technology is as follows: Heavy chain SEQ ID NO.8:

[0047] EVQLQESGGGLVQPKGSLKLSCTASGFTFNNYAVNWVRQAPGKGLEWVARIRSNNNNYATFYADSVKDRFTISRDDSQSMLYLQMNNLRTEDTAMYYCVRGDYRPYWGQGTTLTV.

[0048] Heavy chain variable region CDR1: GFTFNNYA; CDR2: IRSNNNYAT; CDR3: VRGDYRPY;

[0049] Light chain SEQ ID NO.9:

[0050] DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQFPKALIYSASYRNSGVPDRFKGSGSGTDFTLTINNVQSEDLAEYSCQQYNTYPLTFGGGTKLEIKR;

[0051] Light chain variable region CDR1: QNVGTN; CDR2: SAS; CDR3: QQYNTYPLT.

[0052] (2) The sequencing results were entered into the IMGT website (https: / / www.imgt.org / IMGT_vquest / analysis) and the framework region (FR) and complementarity decision region (CDR) of the amino acid sequences of the heavy and light chains of Pgp3 mAb were annotated using the IMGT numbering scheme.

[0053] (3) The monoclonal antibody subtype of Pgp3mAb has been identified, and the results show that the heavy chain subtype of Pgp3mAb is IgG2b, and the light chain subtype is kappa.

[0054] (4) The heavy chain and light chain variable region sequences of Pgp3mAb were linked to the human immunoglobulin IgG1 constant region sequence and the human immunoglobulin Kappa constant region sequence, respectively, and the corresponding signal peptide and enzyme cleavage sites were added, and synthesized into the pCDNA3.4 expression vector to construct the recombinant plasmid pCDNA3.4-mhPgp3Ab.

[0055] 2. Construction of humanized Pgp3 antibody;

[0056] (1) Antibody model construction: The heavy chain and light chain sequences of the Pgp3 monoclonal antibody were input into AlphaFold to construct the antibody structure in the multimer mode.

[0057] (2) Energy minimization: The above antibody model is input into GROMACS for energy minimization and the new antibody structure is output.

[0058] (3) Sequence renumbering: The amino acid sequence in the antibody structure model is renumbered using the Kabat naming scheme.

[0059] (4) Select human antibody germline genes: The mouse heavy chain and light chain sequences are distinguished into FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 according to the Kabat naming scheme, and the antibody germline templates are obtained using the complete variable region, FR1+FR2+FR3+FR4, and CDR1+CDR2+CDR3, respectively.

[0060] (5) CDR transplantation: Using the same Kabat nomenclature, the mouse CDRs were transplanted to the corresponding CDR positions of the template germline. This version is named version B.

[0061] (6) Generating back-mutated sequences: Based on the key amino acid positions of the prior art (Foote J, Winter G. Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 1992 Mar 20; 224(2): 487-99. doi: 10.1016 / 0022-2836(92)91010-m. PMID: 1560463.), the key amino acid positions of version B were back-mutated into the corresponding mouse amino acids to form version A.

[0062] (7) Energy calculation and sequence selection: Each heavy and light chain combination formed in steps (5) and (6) above is compared with the wild-type antibody structure. First, the energy change is evaluated based on each differential amino acid. Amino acids that meet the energy change requirements will be selected and combined for optimization, and finally the sequence corresponding to the most stable structure will be selected.

[0063] (8) Generate the final sequence: Weigh the energy and degree of humanization and finally select the heavy chain and light chain variable region sequences of the candidate humanized Pgp3 antibody.

[0064] (9) The final candidate heavy chain and light chain variable region sequences were inserted into the pCDNA3.4 expression vector containing the antibody constant region sequence (IgG1 / Kappa) to construct the complete heavy / light chain humanized plasmid pCDNA3.4-hPgp3Ab-H / L.

[0065] 3. Eukaryotic expression of human-mouse chimeric Pgp3 antibodies and humanized Pgp3 antibodies;

[0066] 1) Transfection-grade plasmid transformation, amplification, extraction, and plasmid sequencing verification;

[0067] (1) Add the recombinant plasmid to the prepared E. coli Transformation was performed in JM108 competent cells by heat shock.

[0068] (2) Add the transformed competent cells to LB medium and culture in a shaking incubator at 37°C and 200 rpm for about 30 minutes.

[0069] (3) Take out the competent cells after culture, aspirate part of the suspension and smear it on the LB plate containing Amp+, place it in the incubator, and culture it at 37℃ overnight.

[0070] (4) Pick a single clone from a fresh culture plate and place it in 2-5 mL of LB medium. Set the shaker at 37°C, 200 rpm, and culture for 8 hours.

[0071] (5) Inoculate into 200 mL of LB medium at a ratio of 1:500 and culture in a shaker at 37°C and 200 rpm for 16 h.

[0072] (6) Collect the cultured bacterial solution and centrifuge it, remove the supernatant, and keep the bacterial pellet.

[0073] (7) Extract the plasmid according to the instructions of the Qiagen transfection-grade plasmid extraction kit. (8) Verify the correctness of the recombinant plasmid sequence using Sanger sequencing technology.

[0074] 2) HEK293 cell recovery and passaging;

[0075] (1) Open the constant temperature water bath in advance and set its temperature to 37℃. Preheat the culture medium at 37℃. Prepare a 15mL sterile centrifuge tube containing 10mL of culture medium in the clean bench.

[0076] (2) Remove the frozen HEK293 cells from the liquid nitrogen tank.

[0077] (3) Place the well-sealed cryopreservation tube in a 37°C water bath and shake it gently to melt it quickly. The cell freezing and thawing time should be controlled within 1 minute.

[0078] (4) Disinfect the outer wall of the cryopreservation tube with 75% ethanol, place it in a clean bench, and quickly transfer it to the above-mentioned 15 mL centrifuge tube. Centrifuge at 800 rpm for 5 minutes.

[0079] (5) Discard the cell supernatant after centrifugation, take 1 ml of culture medium to resuspend the cells, make the cells disperse evenly, take the cell suspension for cell counting and viability test, and adjust the cell density to 3.0-5.0×10 5 cells / mL, with viability >95%, transfer the cell suspension with adjusted density into culture flask.

[0080] (6) Place in an incubator at 110 rpm, 37°C, and 8% CO2.

[0081] (7) After 2-3 days of cell culture, the cell density reaches 4.0-6.0×10 6 Cells were passaged when the concentration was around cells / mL.

[0082] (8) Aspirate part of the culture medium from the culture flask and add fresh culture medium to the culture flask to dilute the remaining cell culture (the remaining cell culture depends on the cell density, culture volume, density after dilution, etc.).

[0083] (9) Place in an incubator at 110 rpm, 37°C, 8% CO2 and continue culturing.

[0084] (10) Cell density and viability should be tested every day. When the cell density reaches 2.0-3.0×10 6 When the cell count is around 500 cells / mL, the cells should be passaged.

[0085] 3) Plasmid transfection into HEK293 (human embryonic kidney 293) cells;

[0086] (1) One day before transfection, cells were suspended and cultured at a seeding density of 2.0×10 6 cells / mL and cultured in an incubator at 110 rpm, 37°C, and 8% CO2.

[0087] (2) On the day of transfection, the cells grew to 3.0-5.0×10 6 cells / mL, and diluted with fresh culture medium to a density of 3.0×10 6 cells / mL for transfection.

[0088] (3) DNA-transfection reagent mixture: Add DNA and transfection reagent to transfection buffer, mix well, and incubate at 37°C.

[0089] (4) Add the DNA-transfection reagent mixture to the cells to be transfected and culture in an incubator at 110 rpm, 37°C, and 8% CO2.

[0090] (5) 24 hours after transfection, add 5% feed and continue culturing.

[0091] (6) Collection: About 4-6 days after transfection, remove the cell culture, centrifuge, and collect the supernatant or cells.

[0092] 4) Protein purification: Centrifuge the cell culture medium after 5 days of transfection and filter the cell supernatant through a 0.22 μm filter. Dialyze the supernatant into 1× PBS (pH 7.4) at 4°C. Purify the protein using a Protein A column after dialysis.

[0093] 5) Protein purity and molecular weight determination:

[0094] (1) The purified 11 humanized Pgp3 antibodies and 1 human-mouse chimeric Pgp3 antibody were used as target proteins and mixed with 5×SDS reducing and non-reducing loading buffer at a volume ratio of 4:1. The sample mixed with the reducing loading buffer was placed in a 100℃ water bath for 10 min.

[0095] (2) Load the target protein and protein marker onto a 12% SDS-PAGE precast gel as needed.

[0096] (3) Perform electrophoresis at a constant voltage of 180 V until the protein marker and target protein bands are separated to the appropriate positions.

[0097] (4) Remove the gel, cut off the excess part, immerse it in sufficient Coomassie Brilliant Blue staining solution and stain it slowly at room temperature for 2 hours.

[0098] (5) Pour out the staining solution, add decolorizing solution and shake slowly to decolorize. Change the decolorizing solution every hour. After 4 times, shake slowly overnight.

[0099] (6) The next day, when the color is decolorized to the ideal state, take photos and record the results for analysis.

[0100] 6) Protein stability test (freeze-thaw experiment): Take a piece of protein frozen at -80℃ and place it in an ice water bath for 5-10 minutes to slowly thaw. After thawing, place it in a 4℃ refrigerator for 0.5 hours. No abnormal phenomenon indicates that the protein freeze-thaw experiment is normal.

[0101] 7) Protein concentration determination: The concentration of the constructed humanized Pgp3 antibody was determined using the A280 UV absorption method.

[0102] Results: 1. Construction and expression of human-mouse chimeric Pgp3 antibody;

[0103] The recombinant plasmid pCDNA3.4-mhPgp3Ab was successfully constructed, and the sequence of the recombinant plasmid was verified to be correct by Sanger sequencing. The human-mouse chimeric Pgp3 antibody (mhPgp3Ab) was successfully constructed and expressed. The amino acid sequence analysis results of the heavy and light chains of mhPgp3Ab are shown in Figure 1 .

[0104] 2. Purity and concentration determination of human-mouse chimeric Pgp3 antibody;

[0105] The recombinant plasmid pCDNA3.4-mhPgp3Ab was transfected into eukaryotic cells and expressed in large quantities. The protein purity was detected by SDS-PAGE gel electrophoresis after purification of mhPgp3Ab by Protein A affinity chromatography. Two clear bands at 50KDa and 25KDa were visible, which were the heavy chain and light chain of mhPgp3Ab, respectively. Figure 2 As shown in the figure, the 150 kDa band is the non-reduced mhPgp3Ab. The concentration of mhPgp3Ab was determined by A280 UV absorption method and was 0.14 mg / mL.

[0106] 3. Construction and expression of humanized Pgp3 antibody;

[0107] The humanized antibody germline gene template was obtained using the nucleotide sequences of the FR1+FR2+FR3+FR4 and CDR1+CDR2+CDR3 regions of the heavy and light chains of Pgp3mAb, and the template combination was finally determined (Table 1). The CDRs of Pgp3mAb were transplanted into the FRs of the corresponding germline templates. Based on the key amino acid sequences reported in the literature (Foote J, Winter G. Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 1992 Mar 20;224(2):487-99. doi:10.1016 / 0022-2836(92)91010-m. PMID:1560463.), the key amino acid positions were backmutated to the corresponding amino acids of mouse Pgp3mAb to increase the affinity of the constructed humanized Pgp3 antibody (hPgp3Ab). The amino acid sequences of the heavy and light chains of the humanized Pgp3 antibody were compared and analyzed in Clustal Omega. Figure 3 As shown in Table 2, 11 humanized antibodies were constructed by combining the heavy and light chain amino acid sequences of the humanized Pgp3 antibody. The combinations are shown in Table 2. Eleven recombinant plasmids, pCDNA3.4-hPgp3Ab-H / L, were successfully constructed. Sanger sequencing verified the correctness of the recombinant plasmid sequences. Eleven mhPgp3Ab strains were successfully constructed and expressed.

[0108] Table 1: Human antibody germline gene templates;

[0109] ;

[0110] Table 2: Sequence combination results of humanized Pgp3 antibodies;

[0111] ;

[0112] Heavy_1 is SEQ ID NO.10, light_1 is SEQ ID NO.11;

[0113] Heavy_2 is SEQ ID NO. 12, light_2 is SEQ ID NO. 13;

[0114] Heavy_3 is SEQ ID NO. 14, light_3 is SEQ ID NO. 15;

[0115] Heavy_4 is SEQ ID NO. 16, light_4 is SEQ ID NO. 17;

[0116] light_5 is SEQ ID NO.18; the heavy chain of Pgp3mAb is SEQ ID NO.19, and the light chain of Pgp3mAb is SEQ ID NO.20.

[0117] 4. Purity and concentration determination of humanized Pgp3 antibody;

[0118] The recombinant plasmid pCDNA3.4-hPgp3Ab-H / L was transfected into eukaryotic cells and expressed in large quantities. Eleven humanized Pgp3 antibodies (hPgp3Ab) were successfully expressed, namely 1A1A3, 1A2B3, 1B1A2, 1B1B2, 1B2A2, 1B2B2-6-2, 1B2B2-9-2, 2B2A2, 3B2A2, 3B2B2, and 3B2B3. The 11 hPgp3Abs were purified by protein A affinity chromatography and then assayed for protein purity by SDS-PAGE gel electrophoresis. Two clear bands at 50 kDa and 25 kDa were observed, representing the heavy and light chains of the 11 hPgp3Abs, respectively. Figure 4 As shown, the 150 kDa band represents the non-reduced mhPgp3Ab of 11 strains. The concentrations of hPgp3Ab of 11 strains were determined by A280 UV absorption method: 1A1A3 (0.51 mg / mL), 1A2B3 (1.60 mg / mL), 1B1A2 (0.35 mg / mL), 1B1B2 (0.67 mg / mL), 1B2A2 (0.35 mg / mL), 1B2B2-6-2 (1.28 mg / mL), 1B2B2-9-2 (0.27 mg / mL), 2B2A2 (0.49 mg / mL), 3B2A2 (0.39 mg / mL), 3B2B2 (2.11 mg / mL), and 3B2B3 (0.63 mg / mL).

[0119] Example 2: In vitro evaluation of affinity and function of humanized Pgp3 antibodies, affinity ranking of humanized Pgp3 antibodies;

[0120] 1. Enzyme-linked immunosorbent assay (ELISA) affinity assay;

[0121] (1) ELISA coating solution: Dilute the His-Pgp3 protein to 10 μg / mL, add 100 μL to each well of a 96-well ELISA plate, and coat overnight at 4°C.

[0122] (2) Wash the coated ELISA plate three times with a plate washer and dry it, add 100uL of 5% BSA solution to each well for blocking, incubate in a 37℃ incubator for 1 hour, and then wash the blocked ELISA plate five times with a plate washer and dry it.

[0123] (3) Pgp3 mAb was used as a positive control, PBS was used as a negative control, and the wells not coated with His-Pgp3 protein were used as blank controls. Humanized Pgp3 antibody was used as the primary antibody and diluted to 6 concentrations (10 μg / mL to 0.3125 μg / mL) using a 0.1% BSA solution by serial dilution. 100 μL was added to each well and incubated at 37°C for 90 min. The plate was removed and washed five times with a plate washer and then blotted dry.

[0124] (4) Use HRP-labeled goat anti-mouse IgG / goat anti-human IgG as the secondary antibody and dilute it with 0.1% BSA solution at a ratio of 1:5000. Add 100 μL to each well and incubate at 37°C for 60 minutes. After incubation, wash the plate five times in a plate washer and aspirate dry.

[0125] (5) Add 100 μL of single-component TMB colorimetric solution to each well, protect from light, and incubate at 37°C for 30 minutes.

[0126] (6) Add 100 μL of ELISA stop solution to each well to terminate the reaction.

[0127] (7) Place the plate in a microplate reader and read the results. Set the detection wavelength to 450 nm and read the results. If the OD value is 3 times greater than the negative control, the sample in this well is considered positive.

[0128] 2. Western blotting to determine affinity;

[0129] (1) Prepare clean glass plates and a glue rack. Ensure that the lower edges of the two glass plates are intact, align them, and then place them in the glue rack.

[0130] (2) According to the molecular weight of His-Pgp3 protein, prepare 12% separation gel and 5% stacking gel according to the reagent ratio described in the table below. After mixing, add the separation gel to the appropriate position of the glass plate and slowly add pure water to flatten the separation gel liquid surface. Let it stand for 30 minutes until the separation gel solidifies, then pour out the upper layer of pure water and dry it. Fill the remaining space of the glass plate with stacking gel and insert a comb of appropriate size. Be careful to operate quickly and horizontally to avoid bubbles. Let it stand for 30 minutes to wait for the stacking gel to solidify.

[0131] Table 3: Reagent ratios for 12% separation gel and 5% stacking gel;

[0132] ;

[0133] (3) Remove the glass plate, wrap it with absorbent paper soaked in electrophoresis buffer, and use it immediately or temporarily store it in a 4°C refrigerator (for use within 3 days).

[0134] (4) Mix 4× protein loading buffer and His-Pgp3 protein, seal well and place in a 100℃ water bath for 15 minutes. It can be stored in a -20℃ refrigerator.

[0135] (5) After loading the gel plate into the electrophoresis tank, add sufficient electrophoresis buffer and slowly pull out the comb horizontally to remove residual gel and avoid band deformity. Load 3ul of protein marker and 3ul of target protein as needed. Set the electrophoresis conditions to 80V for 30min for the stacking gel and 110V for 90min for the separation gel. Maintain constant pressure until the protein marker and target protein are separated to the appropriate position. If necessary, place the electrophoresis tank in an ice box to keep it low.

[0136] (6) Prepare the transfer buffer in advance and store it in a refrigerator at 4°C. Soak the transfer clip, sponge, and filter paper with transfer buffer in advance. Gently pry open the glass plate and cut off the concentrated gel and some unnecessary separation gel. Activate the PVDF (0.45um) membrane with methanol for 30 seconds, rinse the membrane surface with pure water, and place it in transfer buffer for 1 minute. Clamp the transfer clip from bottom to top according to the position of sponge, filter paper, gel, PVDF membrane, filter paper, and sponge, ensuring that there are no bubbles between each layer. Place it in the transfer tank, confirm the correct position, and add enough transfer buffer. Set the transfer current to 300mA and transfer in a constant current ice bath for 25 minutes.

[0137] (7) After the transfer is complete, remove the PVDF membrane, rinse once with TBST, add 5% skim milk to block, and shake slowly at 4°C for 60 minutes. After blocking, wash quickly with TBST three times, each time for 5 minutes.

[0138] (8) Dilute humanized Pgp3 antibody as the primary antibody with 3% skim milk powder (1:1000) and add it to the incubation box and shake slowly at 4℃ overnight. The primary antibody was recovered the next day and stored in a refrigerator at -20℃. Wash quickly with TBST three times, 5 minutes each time.

[0139] (9) Horseradish enzyme-conjugated goat anti-mouse IgG and horseradish enzyme-conjugated goat anti-human IgG were used as secondary antibodies. 3% skim milk powder was used to dilute the secondary antibody at a dilution of 1:5000 and added to the incubation box to cover the PVDF membrane. The membrane was shaken slowly on ice for 90 minutes. The membrane was washed quickly with TBST three times, each time for 5 minutes.

[0140] (10) In the dark, mix ECL chemiluminescent reagent solution A and solution B in a ratio of 1:1. Use a pipette to drop the ECL luminescent solution onto the PVDF membrane, ensuring that the working solution is evenly covered. Place the membrane on the stage of the chemiluminescent imager and collect images.

[0141] 3. Surface plasmon resonance affinity determination;

[0142] (1) Fix the CM5 chip and activate the sensor surface by injecting a mixture of 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for 7 minutes.

[0143] (2) The antigen (His-Pgp3 protein) was diluted to 10 μg / mL with 10 mM Acetate (pH 4.5), and the flow rate was 10 μL / min. The target coupling amount was set to 300 RU and fixed on the surface of the CM5 chip. (3) The surface was blocked with 1 M ethanolamine (pH 8.5). The final coupling amount was 289.6 RU. (4) The binding characteristics of the antigen and each antibody were preliminarily determined and evaluated in manual mode, and 200 nM was determined as the analytical concentration of the antibody. (5) The settings for sample analysis were: antibody flow rate of 30 μL / min, association time of 120 s, dissociation time of 600 s, and regeneration of Gly-HCl (pH 1.5) for 15 s. (6) The experiment was run in multiple cycles, with the response signal plotted with analysis time as the horizontal axis and response value as the vertical axis. Biacore T200 analysis software was used for fitting. The fitting model used was a 1:1 Langmuir binding model. Kinetic constants such as the association rate constant, dissociation rate constant, and association dissociation constant were determined, and the antibodies were sorted according to their association dissociation constants.

[0144] 4. Verify affinity by indirect immunofluorescence;

[0145] (1) Place a 14mm cell slide in a 24-well plate in the clean bench in advance, select Hela cells in good condition, digest them with trypsin and centrifuge them, adjust the cell density, add them to the 24-well plate, keep the plate stable and place it in the cell incubator to culture until the cell density in the well reaches 80%. (2) Preheat sterile DADE-D solution, centrifugation solution (DMEM culture medium), and infection solution at 37℃. Aspirate the growth solution in the 6-well plate in the clean bench, add 500uL DADE-D solution to each well, and place it in the incubator for 25 minutes to increase the permeability of the cell membrane. (3) Take out the EP tubes (pre-loaded with 3-5 sterile glass beads) containing Cm and Ct-D from the -80 refrigerator. Vortex and shake for 2 minutes to lyse the cell membrane and release the chlamydia from the cells. Set the centrifuge to 3000rpm and centrifuge at 4℃ for 5 minutes. Store on ice for later use. (4) Take out the 24-well plate, aspirate the DADE-D solution in the well, and add 1ml centrifugation solution to each well. Pipette 30-50uL of chlamydia supernatant and add it to the wells. Shake well using the figure-eight method. Place in the incubator for 30 minutes. (5) Preheat the centrifuge at 37℃. Place the 24-well plate from step 6 into the centrifuge and centrifuge at 3000rpm for 1 hour. After centrifugation, continue to place it in the incubator and let it stand for 1 hour. (6) Take out the 6-well plate and aspirate the centrifuge liquid in the wells in the clean bench. Add 2ml of infection liquid to each well and place it in an incubator with a CO2 concentration of 5% and 37℃. Hela cells can be infected with Cm2 for 2-23 hours and Ct-D for 44-46 hours before starting the immunofluorescence step. Add ice-cold PBS phosphate buffer to the wells and rinse them 4 times for 3 minutes each time. Discard the ice-cold PBS phosphate buffer and add 1mL of ice-cold 4% paraformaldehyde per well to fix for 15 minutes. Then rinse them 3 times with ice-cold PBS phosphate buffer for 3 minutes each time. (7) Add 1 mL / well of 0.1% Tritonx-100 solution (diluted with PBS) and permeabilize at room temperature for 10 minutes. Then, wash three times with ice-cold PBS phosphate buffer, each for 3 minutes. Add 500 μL of cell growth medium to each well for 1 hour to reduce nonspecific background staining, and then wash three times with ice-cold PBS phosphate buffer, each for 3 minutes. (8) Screen the humanized Pgp3 antibody with the best affinity as the primary antibody, dilute it with cell growth medium at a ratio of 1:1000, add 200 μL / well to the 24-well plate, seal it and refrigerate it at 4°C overnight. (9) Take out the 24-well plate the next day, let it stand at room temperature for 30 minutes to rewarm, and wash it four times with ice-cold PBS phosphate buffer, each for 3 minutes. (10) Rabbit anti-chlamydia antibody is used as the second primary antibody, diluted with cell growth medium (1:1500), add 200 μL to each well, seal it and refrigerate it at 4°C overnight. (11) Take out the well plate and rinse it with ice-cold PBS phosphate buffer 4 times, 3 minutes each time.Under dark conditions, dilute Alexa Fluor 594-labeled goat anti-mouse IgG (1:400) in cell growth medium, add 50µL to each well, incubate in a CO2 incubator at 37°C for 1 hour, and rinse four times with ice-cold PBS phosphate buffer, each for 3 minutes. (12) Protect from light, dilute Alexa Fluor 488-labeled goat anti-rabbit IgG (1:400) in cell growth medium as the second secondary antibody, add 50µL to each well, incubate in a CO2 incubator at 37°C for 1 hour, and rinse four times with ice-cold PBS phosphate buffer, each for 3 minutes. (13) Add 50µL DAPI to each well, let it stand at room temperature for 3 minutes, and then rinse four times with ice-cold PBS phosphate buffer, each for 3 minutes. (14) Label the slide with the strain, fluorescent type, date, and name in advance. Use clean tweezers to remove the cell slide, place it upside down on a slide with anti-fluorescence attenuation mounting medium, and gently press the slide to remove bubbles. (15) Store the prepared slides in a light-proof box at 4°C and observe the distribution of different fluorescent dyes under a fluorescence microscope.

[0146] 5. Humanized Pgp3 antibodies can inhibit Cm infection in vitro;

[0147] (1) Evenly plate the Hela cell suspension in good condition into a 24-well plate with a cell slide in advance, and incubate for 24 hours until the cell density reaches 80%. (2) After incubating the Hela cells infected with Cm for 24 hours, collect them into EP tubes, shake them on a vortex shaker for 2 minutes, and centrifuge them at 4℃ pre-cooled, 3000rpm, and centrifuge for 5 minutes. Store them on ice for later use. (3) Place the Cm-infected cell lysate prepared above into 3 EP tubes, 50uL / tube. Place appropriate volumes of humanized Pgp3 antibody 2B2A2 into the tubes so that the final concentrations of the antibodies are 5μg / mL, 25μg / mL, and 125μg / mL, respectively. Incubate at 37℃ for 2 hours. Use the Cm-infected cell lysate without antibody as the control group. (4) Use the mixed solution after incubation to infect Hela cells grown in a 24-well plate with a cell slide, inoculate 1uL into each well, and mix thoroughly using the figure-eight method. Centrifuge at 1500 rpm and 32°C for 1 hour. Place in a 37°C incubator and let it rest for 1 hour before replacing the infection solution. (5) 24 hours after infection, stain and count the inclusion bodies and host cells using a fluorescence microscope. Five fields of view were randomly observed on each coverslip. The results were expressed as infection rate (number of inclusion bodies per 100 cells) and the data were presented as mean ± standard deviation.

[0148] 6. Humanized Pgp3 antibodies can inhibit Ct-D infection in vitro;

[0149] IFA assay was performed 48 h after Ct-D infection, and the operating procedures were the same as those for humanized Pgp3 antibody to inhibit Cm infection in vitro, except that Ct-D was used for infection.

[0150] result:

[0151] 1. ELISA affinity test results;

[0152] The results of ELISA showed that 11 humanized hPgp3Abs and 1 human-mouse chimeric mhPgp3Ab and Pgp3mAb all had high affinity to His-Pgp3 protein, among which 3B2B2 had the best affinity, followed by 2B2A2 and 1A2B3, and the values ​​of the three were not much different. Figure 5 The specific values ​​and affinity ranking results are shown in Table 4.

[0153] Table 4: ELISA affinity ranking

[0154] ;

[0155] 2. Western Blotting to determine affinity results;

[0156] His-Pgp3 was used as the target protein, and the top three antibodies with the best affinity in the ELISA test results, 3B2B2, 2B2A2, and 1A2B3, were selected as primary antibodies, and Pgp3 mAb was used as a positive control. Western Blotting analysis showed that humanized Pgp3 antibodies 3B2B2, 2B2A2, and 1A2B3 could all recognize the His-Pgp3 protein at 28 kDa. Compared with Pgp3 mAb, the affinity of the candidate antibodies was good, and there was no significant difference between 3B2B2 and 2B2A2, such as Figure 6 shown.

[0157] 3. Surface plasmon resonance affinity determination results;

[0158] Through ELISA screening, 8 humanized antibodies were selected as candidates. The His-Pgp3 protein was immobilized on a CM-5 chip, and the affinity of these selected candidate humanized hPgp3Abs, human-mouse chimeric mhPgp3Abs, and Pgp3mAbs to the His-Pgp3 protein was determined using surface plasmon resonance. The binding curves are shown in Figure 7 The results of the determination of kinetic constants such as the association rate constant (Ka), dissociation rate constant (Kd), and association dissociation constant (KD = Kd / Ka) are shown in Table 5. The association dissociation constant, also known as the affinity constant, reflects the magnitude of the binding force of the interaction. The smaller the KD value, the stronger the affinity. The results showed that the association rate constant (Ka) of 2B2A2 with His-Pgp3 protein was 2.00×10 4 The dissociation rate constant (Kd) is 3.302×10 -5The corresponding calculated binding dissociation constant (Kd / Ka=KD) is 1.651×10 -9 , and its KD value was the smallest, indicating that 2B2A2 had the strongest affinity to His-Pgp3 protein, which was higher than that of Pgp3mAb.

[0159] Table 5: Affinity determination results of candidate antibodies and His-Pgp3 by SPR;

[0160] ;

[0161] 4. 2B2A2 can recognize endogenous Pgp3;

[0162] The above affinity screening experiment results showed that 2B2A2 had the best affinity for His-Pgp3 protein. To study whether 2B2A2 could recognize endogenous Pgp3 secreted by Hela cells after Chlamydia infection, Hela cells infected with Cm for 23 hours and Ct-D for 46 hours were washed, fixed, and permeabilized. 2B2A was used as the primary antibody and Alexa Fluor 594-labeled secondary antibody was used to label Pgp3. Indirect immunofluorescence detection was performed. The results showed that 2B2A2 could specifically recognize the Pgp3 protein secreted by Cm and Ct-D, see Figure 8 .

[0163] 5. Humanized Pgp3 antibody 2B2A2 can inhibit Cm infection in vitro;

[0164] Taking into account the affinity ranking results of the above-mentioned humanized Pgp3 humanized antibodies, 2B2A2 was selected for large-scale expression. The above experimental results show that 2B2A2 has the best affinity with His-Pgp3 protein, and the SPR affinity test results are better than Pgp3mAb. Previous studies have shown that Pgp3mAb can effectively inhibit Cm infection in vitro, and Pgp3mAb mainly binds to secreted Pgp3. To study whether 2B2A2 can effectively inhibit the infectivity of Cm in vitro, we incubated 5μg / mL, 25μg / mL, and 125μg / mL of 2B2A2 with equal amounts of Cm-infected cell lysates and evaluated their infectivity. The results showed that as the concentration of 2B2A2 increased, its inhibitory effect on Cm infectivity increased ( Figure 9 A).

[0165] Compared with the Cm group, the IFUs formed by Hela cells infected with 25 μg / mL 2B2A2 after co-incubation with Cm-infected cell lysate were reduced (P < 0.01); the IFUs formed by Hela cells infected with 125 μg / mL 2B2A2 after co-incubation with Cm-infected cell lysate were significantly reduced (P < 0.001); while the IFUs formed by Hela cells infected with 5 μg / mL 2B2A2 after co-incubation with Cm-infected cell lysate had no significant difference (P > 0.05) ( Figure 9 B).

[0166] 6. Humanized Pgp3 antibody 2B2A2 can inhibit Ct-D infection in vitro;

[0167] To investigate whether the constructed humanized Pgp3 antibody 2B2A2 could inhibit the infectivity of Ct-D in vitro, we mixed different concentrations of 2B2A2 with equal amounts of Ct-D infected cell lysate for pretreatment, inoculated them onto Hela cell monolayers and cultured them for 44-46 hours before counting the number of inclusion bodies formed ( Figure 10 A). The results showed that 2B2A2 inhibited the infectivity of Ct-D in a concentration-dependent manner. After 5μg / mL 2B2A2 was co-incubated with Ct-D infected cell lysate, the IFUs formed in Hela cells were reduced compared with Ct-D (P < 0.01); after 25μg / mL 2B2A2 was co-incubated with Ct-D infected cell lysate, the IFUs formed in Hela cells were significantly reduced (P < 0.0001); after 125μg / mL 2B2A2 was co-incubated with Ct-D infected cell lysate, the IFUs formed in Hela cells were significantly reduced (P < 0.0001) ( Figure 10 B).

[0168] Example 3: In vivo experiments to evaluate the function of humanized Pgp3 antibodies;

[0169] 1. Construction of a mouse model of acute Cm infection;

[0170] (1) Five-week-old female SPF BALB / c mice (n=24) were housed in an animal room for one week and their feeding status was observed daily. This animal experiment was approved by the Ethics Committee of Tianjin Medical University General Hospital. (2) The mice were randomly divided into four groups (n=6): acute Cm infection group; acute Cm infection + subcutaneous injection of 0.8 mg / kg Pgp3 mAb group; acute Cm infection + subcutaneous injection of 0.1 mg / kg 2A2B2 group; and acute Cm infection + subcutaneous injection of 0.8 mg / kg 2A2B2 group. After infection, the drugs were administered twice a week for three weeks. (3) Five days before infection, mice were subcutaneously injected with 2.5 mg of progesterone to synchronize the estrous cycle and increase their susceptibility to Cm. Disposable sterile swabs were used to clean the lower genital tract secretions of the mice one day before infection to reduce their impact on Cm infection in the lower genital tract. (4) Dilute the Cm EB stored in -80℃ refrigerator with ice SPG in a clean bench to 2×10 5 IFU / mL, store on ice. (5) Pipette 20uL of purified Cm EB (2×10 5 IFU / mL), fix the mouse with one hand and turn it upside down. Hold the pipette with the other hand and gently place the pipette tip against the mouse's posterior fornix and slowly inject it into the mouse's vagina. (6) After removing the pipette tip, observe whether there are bubbles at the vaginal opening. Place the mouse upside down for 2 minutes to ensure that all Cm EBs are absorbed and then return the mouse to its cage.

[0171] 2. Construction of a mouse model of persistent Cm infection;

[0172] (1) Five-week-old female SPF BALB / c mice (n=24) were acclimated in the animal house for one week, and the feeding status of the mice was observed daily. (2) The mice were randomly divided into four groups (n=6) after one week of feeding and ear-tagged: Cm persistent infection group; Cm persistent infection + subcutaneous injection of 0.8mg / kg Pgp3mAb group; Cm persistent infection + subcutaneous injection of 2A2B2 0.1mg / kg group; Cm persistent infection + subcutaneous injection of 2A2B2 0.8mg / kg group. After infection, the drug was administered twice a week for 3 weeks. (3) The procedures for infection of the lower genital tract of mice and enhancement of susceptibility before infection can refer to steps (3)-(6) of 3.1.4.3. The infection dose was 20uL Cm EB (2×10 6 IFU / mL). (4) The gavage needle was sterilized with high-pressure steam in advance. The day of Cm infection in the reproductive tract of mice was set as Day 0. From Day 5, 2 mg / Kg Amox was gavaged twice a day until Day 11.

[0173] Example 4: Effect of humanized Pgp3 antibody 2A2B2 on Cm infection in vivo;

[0174] 1. Chlamydia load in the lower genital tract;

[0175] (1) Example 3 was used to construct a Cm acute infection mouse model and a Cm persistent infection mouse model. (2) After infection, the Cm acute infection mice and Cm persistent infection mice were subcutaneously injected with Pgp3mAb or 2A2B2. The administration frequency was twice a week for 3 consecutive weeks, and the administration time was fixed. The weight of each mouse was weighed before each administration, and the drug was administered strictly according to the weight and the set drug concentration. (3) A sterile sampling tube containing a disposable swab was placed in the clean bench in advance, and 500ul of ice sampling special SPG and 3 to 4 glass beads were added to the sampling tube. In order to monitor the shedding of chlamydia organisms in the lower genital tract of mice, the sampling period was set at 3, 6, 9, 12, 15, 18, 21, and 28 days after the mice were infected with chlamydia. (4) After manually securing the mouse, place it head down and slowly insert a disposable sterile swab into the vagina. Gently scrape the vaginal wall clockwise and counterclockwise, then place it in a sampling tube containing SPG. Store on ice and vortex for 2 minutes to fully release the chlamydia. (5) Set up three dilution gradients and dilute the liquid in the sampling tube with ice-cold DMEM medium. Infect Hela cells grown in a 24-well plate containing a cell slide. (6) Count the number of inclusion bodies in five random fields of view at different dilutions using a fluorescence microscope. Calculate the IFUs of chlamydia in each swab using the formula and convert the calculated IFUs to a logarithm with a base of 10.

[0176] 2. Visual scoring of hydrosalpinx in mice;

[0177] (1) The day of vaginal infection with Chlamydia was designated as Day 0. To evaluate the histopathology of the mouse reproductive tract, the mice were sacrificed 60 days after vaginal infection under isoflurane inhalation anesthesia, and the complete reproductive tract from the vagina to the ovary of each mouse was aseptically isolated. (2) The bilateral oviducts of each mouse were visually scored according to their dilation size using a stereo magnifying glass. The specific scoring criteria were as follows: no hydrosalpinx, score 0; hydrosalpinx visible after magnification, score 1; hydrosalpinx visible to the naked eye but smaller than the ovary, score 2; hydrosalpinx similar in size to the ovary, score 3; hydrosalpinx larger than the ovary, score 4. The scores from both sides of the oviduct of the same mouse were combined into the total score of the mouse, and the score of each mouse was recorded in detail. The mouse reproductive tract was placed on a blue background and photographed as soon as possible. After recording, it was immersed in 4% tissue cell fixative for 48 hours.

[0178] 3. Scoring of oviduct dilatation and inflammatory cell infiltration in H&E-stained sections of the mouse reproductive tract;

[0179] (1) Fix the mouse reproductive tract tissue thoroughly to ensure that the fixative penetrates into the tissue block, and then rinse thoroughly with distilled water to remove the residual fixative. (2) Place the mouse reproductive tract tissue in a gradient of alcohol for step-by-step dehydration, in the order of 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol (I)-100% alcohol (II), with each dehydration time of 1-2 hours. (3) Soak the mouse oviduct tissue in two different xylenes in turn, in tank I for 20 minutes and in tank II for 30 minutes, until the tissue becomes transparent. (4) Immediately place the transparent tissue in pre-melted paraffin wax, immerse the paraffin wax in the tissue, and replace the xylene (2-3 hours). (5) Place the dissolved paraffin wax in an embedding tank, take out the wax-soaked tissue, and place it in the embedding tank containing the wax wax in turn, wait for the wax wax to solidify into a block, and then shape it into a rectangular wax block. (6) The trimmed wax block is pre-cooled and fixed on a paraffin slicer, with the section parallel to the blade. The slice thickness is adjusted to 5-7 μm, and slices of uniform thickness are made, including the cervix, uterine horns, and fallopian tubes as much as possible. (7) The slide machine is adjusted to the appropriate temperature. Use tweezers to gently place the tissue slice on the 40°C warm water of the slide machine to flatten the tissue grain. Pick up the tissue slice with a slide, place it on a 60°C slide machine and bake it for 1 hour, then bake it in an oven for 1 hour. After taking it out, place it in a 37°C constant temperature box for staining. (8) Dewaxing and hydration: Place the paraffin slice in xylene I for 10 minutes, xylene II for 10 minutes, 100% alcohol I for 2 minutes, 100% alcohol II for 2 minutes, 95% alcohol for 2 minutes, 90% alcohol for 2 minutes, 80% for 2 minutes, and 70% for 2 minutes. Finally, rinse with distilled water and dry. (9) Staining: Hematoxylin stains cell nuclei and eosin stains cytoplasm. Distilled water 5s - hematoxylin 5min - water 1min - hydrochloric acid alcohol 5s - running water rinse 1min - eosin 5min (10) Sealing: 70% alcohol 1s - 80% alcohol 1s - 90% alcohol 10s - 95% alcohol 1min - 100% alcohol I 2min - 100% alcohol II 2min - xylene I 5min - xylene II 5min - neutral gum - glass slide (11) Observe the prepared stained tissue sections under an inverted microscope and score the pathology and inflammation severity of the hydrosalpinx. The scoring rules are as follows: Fallopian tube dilatation score: no obvious dilatation, scored as 0 points; single cross-section dilatation, scored as 1 point; 1-3 cross-sections dilatation, scored as 2 points; 3 cross-sections dilatation, scored as 3 points; obvious fusion dilatation, scored as 4 points. Inflammatory cell infiltration score: no obvious inflammatory cell infiltration, 0 points; inflammatory infiltration in a single lesion, 1 point; inflammatory infiltration in 2 to 4 lesions, 2 points; inflammatory infiltration in more than 4 lesions, 3 points; inflammatory infiltration in the entire oviduct wall, 4 points. (12) The scores of both sides of each mouse were added together to obtain the total score of the mouse.

[0180] Results: 1. 2B2A2 can reduce the chlamydial load in the lower genital tract of mice acutely infected with Cm;

[0181] After in vitro experiments confirmed that the humanized Pgp3 antibody 2B2A2 can reduce the infection rate of Cm and Ct-D, we established a Cm acute infection mouse model and subcutaneously injected different doses of 2B2A2 to intervene. At given time intervals, swabs of the lower genital tract of mice were collected and the shedding content was detected to evaluate the effect of 2B2A2 on mice with acute Cm infection. Figure 11 The data showed that the chlamydial loads in the lower genital tract of mice in the high-dose subcutaneous injection 2B2A2 and Pgp3mAb groups were significantly lower than those in the acute Cm infection group on days 3, 6, 9, 12, 15, 18, 21, and 28 after infection (P < 0.05). However, the chlamydial shedding levels in the lower genital tract of mice in the low-dose subcutaneous injection 2B2A2 group were similar to those in the Cm group on days 3, 6, 9, 12, 15, 18, 21, and 28 after infection, with no significant difference (P > 0.05). There was no significant statistical difference in the chlamydial loads in the lower genital tract of mice in the high-dose subcutaneous injection 2B2A2 group and the Pgp3mAb group on days 3, 6, 9, 12, 15, 18, 21, and 28 after infection (P > 0.05). On day 28 after infection, mice in the acute Cm infection group and the low-dose 2B2A2 subcutaneous injection group still shed live Chlamydia trachomatis in the lower genital tract, whereas the Chlamydia load in the high-dose 2B2A2 subcutaneous injection and Pgp3mAb groups was zero. Compared with the acute Cm infection group and the low-dose 2B2A2 subcutaneous injection group, the high-dose 2B2A2 subcutaneous injection and Pgp3mAb groups showed a shortened Cm infection cycle in the lower genital tract.

[0182] 2. 2B2A2 can inhibit hydrosalpinx in mice with acute Cm infection;

[0183] All mice in the Cm acute infection group were euthanized 60 days after infection, and the complete reproductive tract from the vagina to the oviduct was isolated. The reproductive tracts of mice in the Cm acute infection group and the low-dose subcutaneous injection group showed severe hydrosalpinx ( Figure 12 A). The severity of tubal dilatation was visually scored using the previously described scoring criteria ( Figure 12B) The scores of each group were statistically analyzed. Compared with the acute Cm infection group, the severity of hydrosalpinx in the high-dose subcutaneous injection 2B2A2 group and the Pgp3mAb group was significantly reduced (0.50±0.55 vs 4.00±1.55; 0.83±0.75 vs 4.00±1.55; P<0.001). There was no statistically significant difference in the hydrosalpinx scores between the low-dose subcutaneous injection 2B2A2 group and the acute Cm infection group (3.67±1.03 vs 4.00±1.55, P>0.05). The hydrosalpinx in the high-dose subcutaneous injection 2B2A2 group was significantly alleviated compared with the low-dose subcutaneous injection 2B2A2 group. The hydrosalpinx scores of the high-dose subcutaneous injection 2B2A2 group and the Pgp3mAb group were 0.50±0.55 and 0.83±0.75, respectively, and there was no statistically significant difference between the two groups.

[0184] 3. 2B2A2 can reduce the dilation and inflammatory infiltration of the oviduct in mice with acute Cm infection;

[0185] The reproductive tract tissues of all mice were fixed, stained with H&E, and then observed under a microscope for oviduct dilatation and inflammatory infiltration ( Figure 13 A and Figure 13 D). The results showed that the oviducts of mice in the acute Cm infection group and the low-dose subcutaneous injection group of 2B2A2 showed obvious inflammatory cell infiltration, tubal dilation, thinning, myometrial disappearance, and tissue fibrosis. However, the oviducts of mice in the high-dose subcutaneous injection group of 2B2A2 and the Pgp3mAb group showed no obvious inflammatory cell infiltration or tubal dilation. Figure 13 B and Figure 13 C). The inflammatory cell infiltration and fallopian tube dilation scores in the high-dose subcutaneous 2B2A2 group (inflammatory cell infiltration score: 1.17±0.75; dilation score: 0.83±0.98) were significantly lower than those in the acute Cm infection group (inflammatory cell infiltration score: 3.67±1.51; dilation score: 3.17±1.47) and the low-dose subcutaneous 2B2A2 group (inflammatory cell infiltration score: 3.33±1.03; dilation score: 3.17±1.17). There was no significant difference in the inflammatory cell infiltration and fallopian tube dilation scores between the high-dose subcutaneous 2B2A2 group and the Pgp3 mAb group (inflammatory cell infiltration score: 1.00±0.89; dilation score: 0.67±0.82).

[0186] 4. 2B2A2 can reduce the chlamydial load in the lower genital tract of mice persistently infected with Cm;

[0187] The above animal experiments found that 2B2A2 can effectively reduce the chlamydia load in the lower genital tract of mice with acute Cm infection, inhibit the severity of hydrosalpinx in mice with acute Cm infection, and reduce the dilation of the fallopian tubes and the degree of inflammatory cell infiltration in mice with acute Cm infection. A Cm persistent infection mouse model was established through antibiotic intervention to evaluate the effect of 2B2A2 on persistent Cm infection. Figure 14 As shown, the genital tract chlamydial load of all mice decreased by more than 90% on day 6 post-infection, reaching zero on days 9 and 12 post-infection. On day 15 post-infection, genital chlamydial shedding resumed in mice in the persistent Cm infection group and the low-dose subcutaneous 2B2A2 injection group, and genital chlamydial load continued to increase on days 18, 21, and 28 post-infection. In contrast, no live murine chlamydial shedding was detected in the Pgp3mAb and high-dose subcutaneous 2B2A2 injection groups on days 15, 18, 21, and 28 post-infection, with chlamydial loads reaching zero, indicating no secondary peak of chlamydial infection. These data indicate that Pgp3mAb and high-dose 2B2A2 can reduce genital chlamydial load in mice with persistent Cm infection.

[0188] 5. 2B2A2 can inhibit hydrosalpinx in mice with persistent Cm infection;

[0189] All mice in the groups were killed after inhalation anesthesia 60 days after infection, and gross specimens of reproductive tract tissues were collected 60 days after infection, as shown in Figure 2. Figure 15 As shown in A, mice in the Pgp3mAb group and the high-dose subcutaneous injection 2B2A2 group had no obvious hydrosalpinx. Visual scoring of hydrosalpinx in all mice was performed, as shown in Figure 15 As shown in Figure 2, mice in the Cm persistent infection group and the low-dose subcutaneous injection group had varying degrees of hydrosalpinx, and there was no significant difference in the visual score between the two groups (4.33±1.03 vs. 4.00±1.10, P>0.05). The degree of hydrosalpinx in the high-dose subcutaneous injection group and the Pgp3mAb group was significantly alleviated compared with the Cm persistent infection group (1.00±0.63 vs. 4.33±1.03; 0.83±0.75 vs. 4.33±1.03; P<0.0001). Compared with the low-dose subcutaneous injection group, the degree of hydrosalpinx in the high-dose subcutaneous injection group was significantly alleviated. There was no statistically significant difference in the hydrosalpinx score between the high-dose subcutaneous injection group (1.00±0.63) and the Pgp3mAb group (0.83±0.75).

[0190] 6. 2B2A2 can reduce the dilation and inflammatory infiltration of the oviduct in mice with persistent Cm infection;

[0191] Cm infection of mice induces oviduct dilation and inflammatory cell infiltration. The reproductive tract tissues of the mice in the above groups were stained with H&E and the gross pathological changes were verified under a microscope, such as Figure 16 As shown, it was found that the oviducts of mice in the high-dose subcutaneous injection 2B2A2 group and the Pgp3mAb group had no obvious dilation and inflammatory cell infiltration, while the oviducts of mice in the Cm persistent infection group and the low-dose subcutaneous injection 2B2A2 group showed varying degrees of dilation and inflammatory cell infiltration. The inflammatory cell infiltration and fallopian tube dilation scores in the high-dose subcutaneous injection 2B2A2 group (inflammatory cell infiltration score: 1.00±0.89; dilation score: 0.50±0.55) were significantly lower than those in the persistent Cm infection group (inflammatory cell infiltration score: 3.50±0.84; dilation score: 3.17±1.47) and the low-dose subcutaneous injection 2B2A2 group (inflammatory cell infiltration score: 3.33±1.37; dilation score: 3.00±1.41). There was no significant difference in the inflammatory cell infiltration and fallopian tube dilation scores between the high-dose subcutaneous injection 2B2A2 group and the Pgp3mAb group (inflammatory cell infiltration score: 1.33±1.21; dilation score: 1.00±0.89).

[0192] The above description is only 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 in the scope of protection of the present invention.

Claims

1. A humanized Chlamydia trachomatis Pgp3 antibody, characterized in that: The humanized Chlamydia trachomatis Pgp3 antibody includes the heavy chain variable region amino acid sequence shown in SEQ ID NO.16; The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 15; It also includes a constant region, and the species origin of the constant region is human.

2. The humanized Chlamydia trachomatis Pgp3 antibody according to claim 1, characterized in that: The constant region includes the human immunoglobulin IgG1 constant region sequence and the human immunoglobulin Kappa constant region.

3. A nucleic acid, characterized in that It encodes the humanized Chlamydia trachomatis Pgp3 antibody according to claim 1 or 2.

4. A carrier, characterized in that It contains the nucleic acid encoding the protein according to claim 3.

5. A cell, characterized in that It contains the nucleic acid according to claim 3 or the vector according to claim 4.

6. Use of the humanized Chlamydia trachomatis Pgp3 antibody according to claim 1 or 2, the vector according to claim 4, or the cell according to claim 5 in the preparation of the following medicament, characterized in that: a. Drugs that inhibit Chlamydia trachomatis infection.

7. Use of the humanized Chlamydia trachomatis Pgp3 antibody according to claim 1 or 2, the vector according to claim 4, or the cell according to claim 5 in the preparation of the following medicament, characterized in that: b. Drugs that reduce fallopian tube edema caused by Chlamydia trachomatis infection; c. Drugs that inhibit fallopian tube inflammation caused by Chlamydia trachomatis infection.

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