Monoclonal antibody of fimA of porphyromonas gingivalis and application thereof

Abstract

The application discloses a Porphyromonas gingivalis FimA monoclonal antibody and application thereof, and provides two kinds of monoclonal antibodies targeting the Porphyromonas gingivalis FimA-I type protein, which are named 1G12 and 4E8 and respectively comprise a heavy chain variable region and a light chain variable region. Through exploration of the biological function of the monoclonal antibody, it is found that the two antibodies can significantly inhibit the adhesion ability and growth cycle of the Porphyromonas gingivalis, promote bacterial agglutination and block the inflammatory response caused by FimA; meanwhile, 1G12 and 4E8 recognize different antigen epitopes of FimA, and an ELISA detection method developed based on 1G12 and 4E8 can better detect the FimA-I type protein and the Porphyromonas gingivalis; and the application lays a foundation for diagnosis, prevention and treatment of the Porphyromonas gingivalis and diseases related thereto.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, cell technology and immunology, and relates to a monoclonal antibody and its applications. Background Technology

[0002] Porphyromonas gingivalis ( Porphyromonas gingivalis , P. gingivalis *Porphyromonas gingivalis* is a Gram-negative, melanin-producing, strictly anaerobic bacterium and a key pathogen causing chronic and aggressive periodontitis. Furthermore, studies have shown that *Porphyromonas gingivalis* is also closely related to the occurrence and development of diseases such as atherosclerosis, rheumatoid arthritis, Alzheimer's disease, and tumors. *Porphyromonas gingivalis* can express various virulence factors, such as fimbriae, lipopolysaccharides, gingival protease, and hemagglutinin. The fimbriae are filamentous protein appendages protruding from the bacterial surface and are one of the important virulence factors of *Porphyromonas gingivalis*. FimA is the major subunit of *Porphyromonas gingivalis* fimbriae and plays a crucial role in fimbriae assembly and the development and progression of periodontal disease. Studies have shown that FimA can mediate the adhesion of bacteria to host tissues and cells (such as gingival fibroblasts and gingival epithelial cells), participate in bacterial colonization and biofilm formation; promote *Porphyromonas gingivalis* invasion of host cells to evade immune detection; trigger inflammatory responses (IL-1β, IL-6, etc.) through interaction with Toll-like receptors, causing immune dysregulation; and synergistically degrade host proteins with proteases such as gingival proteases, thereby causing gingival bleeding, alveolar bone resorption, and tooth loss; playing a central role in infection creation, immune evasion, and disease progression.

[0003] Currently, the FimA genotype has been classified into six types, IV and Ib, which vary among periodontitis patients in different regions, populations, and age groups. Studies have shown that FimA-II and IV types of *Porphyromonas gingivalis* have a higher detection rate in patients with severe periodontitis, followed by FimA-I. However, some studies indicate that FimA-I type *Porphyromonas gingivalis* is more frequently observed in patients with chronic periodontitis and adolescent gingivitis in my country. The detection rate of FimA-I type *Porphyromonas gingivalis* is even higher in some healthy *Porphyromonas gingivalis* carriers or patients with early periodontitis. Currently, monoclonal antibodies against FimA-II type *Porphyromonas gingivalis* have been studied, but no monoclonal antibodies against FimA-I have been reported. Furthermore, the lack of monoclonal antibodies against FimA-I type *Porphyromonas gingivalis*, a commonly used model bacterium in scientific research, adds to the difficulty of related scientific research. Based on this, the development of monoclonal antibodies targeting FimA-I proteins is of great significance for the research on FimA-I type Porphyromonas gingivalis and the diagnosis and treatment of related diseases. Summary of the Invention

[0004] To solve the above technical problems, the application provides a Porphyromonas gingivalis FimA monoclonal antibody and application thereof.

[0005] To achieve the object of the application, the application provides the following technical solutions.

[0006] The application provides a FimA monoclonal antibody selected from one or more of the following antibodies:

[0007] (1) 1G12 antibody, wherein the heavy chain variable region of the antibody comprises VH-CDR1-3 of the amino acid sequence shown in SEQ ID No. 1-3; and the light chain variable region of the antibody comprises VL-CDR1-3 of the amino acid sequence shown in SEQ ID No. 5-7.

[0008] The amino acid sequence of the heavy chain variable region of the 1G12 antibody has more than 85% similarity with the amino acid sequence shown in SEQ ID No. 4, and the amino acid sequence of the light chain variable region has more than 85% similarity with the amino acid sequence shown in SEQ ID No. 8; the amino acid sequence of the heavy chain variable region of the 4E8 antibody has more than 85% similarity with the amino acid sequence shown in SEQ ID No. 12, and the amino acid sequence of the light chain variable region has more than 85% similarity with the amino acid sequence shown in SEQ ID No. 16.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the antibody (1G12) is shown in SEQ ID No. 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8.

[0010] (2) 4E8 antibody, wherein the heavy chain variable region of the antibody comprises VH-CDR1-3 of the amino acid sequence shown in SEQ ID No. 9-11; and the light chain variable region of the antibody comprises VL-CDR1-3 of the amino acid sequence shown in SEQ ID No. 13-15.

[0011] Further, the amino acid sequence of the heavy chain variable region of the antibody (4E8) is shown in SEQ ID No. 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 16.

[0012] Further, the monoclonal antibody comprises all or part of the antibody heavy chain constant region and / or the antibody light chain constant region.

[0013] The FimA monoclonal antibody is named as 1G12 antibody or 4E8 antibody.

[0014] The second aspect of the present application provides nucleic acid sequences encoding the antibodies, which include a nucleotide sequence encoding the heavy chain variable region of the 1G12 antibody as shown in SEQ ID No. 17, a nucleotide sequence encoding the light chain variable region of the 1G12 antibody as shown in SEQ ID No. 18; a nucleotide sequence encoding the heavy chain variable region of the 4E8 antibody as shown in SEQ ID No. 19, a nucleotide sequence encoding the light chain variable region of the 4E8 antibody as shown in SEQ ID No. 20.

[0015] Further, the DNA sequences encoding the antibody molecules of the present application can be obtained by methods well known to those skilled in the art, and the antibodies of the present application can be expressed in expression systems known to those skilled in the art. For example, the DNA sequences encoding part or all of the heavy and light chains of the antibodies can be synthesized based on the corresponding amino acid sequences, and the antibodies can be obtained by expression in mammalian expression systems using mammalian expression vectors.

[0016] The present application also provides a conjugate or fusion, which is obtained by modification or fusion expression of the FimA monoclonal antibodies described above. As for the conjugate, the present application takes the modification of adding biotin as an example; as for the fusion, the present application refers to the addition of tags or other proteins before and after the antibody when the protein is expressed to produce a new protein containing the antibody sequence.

[0017] The present application also provides application schemes containing the FimA monoclonal antibodies described above, which include:

[0018] (1) the use of the FimA monoclonal antibodies described above in the preparation of products for diagnosing diseases related to P. gingivalis infection;

[0019] Further, a kit for specifically recognizing P. gingivalis is provided, which contains one or two FimA monoclonal antibodies of the present application;

[0020] (2) the use of the FimA monoclonal antibodies described above in the preparation of pharmaceutical combinations for treating diseases related to P. gingivalis infection;

[0021] Further, the use of the FimA monoclonal antibodies described above in blocking the adhesion function of P. gingivalis;

[0022] Further, the use of the FimA monoclonal antibodies described above in promoting the agglutination function of P. gingivalis;

[0023] Further, the use of the FimA monoclonal antibodies described above in blocking the cytokine inflammation induced by FimA.

[0024] The present application has the following beneficial effects:

[0025] 1、The present application uses type I FimA as a target antigen for mouse immunization, and uses hybridoma technology to prepare monoclonal antibodies, which are named 1G12 and 4E8, and the obtained antibodies have excellent sensitivity and specificity, and can effectively recognize FimA-I type protein and FimA-I type Porphyromonas gingivalis.

[0026] 2、The obtained antibodies can effectively inhibit the adhesion ability of Porphyromonas gingivalis, promote the agglutination of Porphyromonas gingivalis and inhibit its growth cycle, and block the inflammatory response caused by FimA antigen, thereby providing a scheme for the prevention and treatment of Porphyromonas gingivalis and related diseases.

[0027] 3、The obtained antibodies can recognize different antigen epitopes of FimA-I type protein, and the ELISA detection method developed on this basis can better detect FimA-I type protein and Porphyromonas gingivalis, thereby laying a foundation for rapid clinical diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0029] Figure 1 Analysis of expression and purification of antigen.

[0030] Figure 2 Analysis of serum titer of immunized mice.

[0031] Figure 3 Sensitivity and affinity detection of antibody.

[0032] Figure 4 Specificity analysis of antibody recognizing Porphyromonas gingivalis and antigen.

[0033] Figure 5 Analysis of antigen epitope difference recognized by antibody.

[0034] Figure 6 Sensitivity analysis of antibody recognizing Porphyromonas gingivalis.

[0035] Figure 7 Analysis results of antibody agglutinating Porphyromonas gingivalis and inhibiting its growth.

[0036] Figure 8 Analysis results of antibody inhibiting adhesion of Porphyromonas gingivalis.

[0037] Figure 9 Results of analysis for the antibody having an anti-inflammatory effect. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0039] Cell lines and experimental animals

[0040] Myeloma cells (SP2 / 0) were from laboratory preservation; primary gingival fibroblasts were from orthodontic patients in the Department of Stomatology, and were approved by the Medical and Scientific Research Ethics Committee of Henan University. BALB / c mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.; the animal experiments were approved by the Experimental Animal Ethics Committee of Henan University.

[0041] The experimental reagents were as follows:

[0042]

[0043] Example 1: Expression and purification of FimA recombinant protein

[0044] Bioinformatics analysis was performed on the fimbrial protein FimA gene of Porphyromonas gingivalis, and the upstream primer FimA-F: CTAGCTAGCATGAAAAAAACAAAG and the downstream primer FimA-R: CCGCTCGAGCCAAGTAGCATTCTG were designed.

[0045] The genomic DNA of the model strain (ATCC-33277) of Porphyromonas gingivalis was used as a template, and the FimA gene fragment was amplified by PCR. The NheI and XhoI enzyme digestion sites were introduced into the primers, and the FimA gene fragment was connected to the pET28a-His prokaryotic expression vector by enzyme digestion and ligation. After enzyme digestion and sequencing detection, the correct positive clone vector was named pET28a-FimA / His and was preserved for future use. The enzyme digestion results are shown in FIG. 1A. Figure 1

[0046] ​The pET-28a-FimA / His recombinant plasmid was transformed into the expression strain BL21(DE3). Single colonies were picked and cultured to OD600 of 0.4-0.8, and then 100 mM IPTG was added (to a final concentration of 0.1 mM) to induce expression for 12-16 hours, and the bacterial cells were collected. After ultrasonic disruption of the bacterial cells, the supernatant and the precipitate were separated, and the supernatant was filtered through a 0.45 um filter. The protein expression in the supernatant and the precipitate was detected by SDS-PAGE electrophoresis and Coomassie blue staining, and the results are shown in Figure 1 Fig. 1A. It was found that the FimA recombinant protein was abundantly expressed in the supernatant. The system was scaled up to 2L, and after ultrasonic disruption, the supernatant was collected, and the FimA recombinant protein was purified by nickel column affinity chromatography. The purified FimA recombinant protein was detected by protein electrophoresis, and the results are shown in Figure 1 Fig. 1B. A high-purity FimA recombinant protein can be obtained by elution with 100-300 mM imidazole. The elution products were combined and stored for later use.

[0047] Example 2: Mouse immunization and antibody purification

[0048] The purified FimA recombinant protein in Example 1 was injected subcutaneously in multiple sites, and four BALB / c mice were immunized at 100 μg per mouse. One mouse was taken as a negative control. FCA adjuvant was used for the first immunization, and FIA adjuvant was used for the second and third immunizations, for a total of 3 immunizations, with an interval of 2 weeks between each immunization. One week after the third immunization, blood was taken to separate serum, which was diluted according to the proportion and added to a 96-well plate coated with FimA recombinant protein (2 μg / mL). The antibody titer of the mouse serum was detected by ELISA, and the results are shown in Figure 2 Fig. 2A, which shows that the serum titer of the immunized mice reached more than 1 / 320000.

[0049] The two mice with the highest titer were selected for intraperitoneal injection of FimA recombinant protein (50 μg) for boost immunization. Three days later, the mouse spleen cells were fused with myeloma cells sp2 / 0. When the hybridoma cells grew to 30%, the cell culture supernatant was collected for ELISA screening, and the results are shown in Figure 2 Fig. 3B. The antibody titers of the supernatants of 1G12 and 4E8 cells were high, and the growth state was good. The subtype of the antibody was detected by a mouse monoclonal antibody subtype detection kit, and the results are shown in Figure 2 Fig. 3C. The heavy chain subtype of the antibodies 1G12 and 4E8 was IgG1, and the light chain subtype was Kappa. Subsequently, the method of collecting antibodies through ascites was used. The screened monoclonal hybridoma cells were injected into the mouse peritoneal cavity, and the ascites was collected. Protein A / G was selected, and the collected antibodies were purified by the AKTA protein purification system. After detection of the purity, the antibodies were stored for later use.

[0050] The variable region amino acid sequence of the 1G12 antibody:

[0051] VH-CDR 1 (SEQ ID No. 1): GYTFTSYV;

[0052] VH-CDR 2 (SEQ ID No. 2): INPYNDDT;

[0053] VH-CDR 3 (SEQ ID No. 3): ARKEFITTVFDY;

[0054] Heavy chain variable region amino acid sequence of the 1G12 antibody (SEQ ID No. 4): EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVLHWVKQKPGQGLEWIGYINPYNDDTKYNENFKGKATLTSDKSSSTAYMDLNSLTSEDSAVYYCARKEFITTVFDYWGQGTTLTVSS;

[0055] VL-CDR 1 (SEQ ID No. 5): QDFNTY;

[0056] VL-CDR 2 (SEQ ID No. 6): RAK;

[0057] VL-CDR 3 (SEQ ID No. 7): LQYDEFPYT;

[0058] Light chain variable region amino acid sequence of the 1G12 antibody (SEQ ID No. 8): DIKMTQFPSSVYASLGERVTITCKASQDFNTYLTWFQQKPGKSPKTLIYRAKRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK.

[0059] Variable region amino acid sequence of the 4E8 antibody:

[0060] VH-CDR 1 (SEQ ID No. 9): GYSITSGYY;

[0061] VH-CDR 2 (SEQ ID No. 10): LNYDGSN;

[0062] VH-CDR 3 (SEQ ID No. 11): ARTYGYETWYFDV;

[0063] Heavy chain variable region amino acid sequence of the 4E8 antibody (SEQ ID No. 12): VQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYLNYDGSNYYNPSLKNRISITRDTSKNHFFLKLNSVTTEDTATYYCARTYGYETWYFDVWGAGTAVTVSS;

[0064] VL-CDR 1 (SEQ ID No. 13): QSLLYSNNQKNY;

[0065] VL-CDR 2 (SEQ ID No. 14): WAS;

[0066] VL-CDR 3 (SEQ ID No. 15): QHYYTYPWT;

[0067] Light chain variable region amino acid sequence of the 4E8 antibody (SEQ ID No. 16): DIVMTQSPSSLAVSVGEKVTLSCKSSQSLLYSNNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVMAEDLAVYFCQHYYTYPWTFGGGTKLEIK.

[0068] Heavy chain variable region nucleic acid sequence of the 1G12 antibody (SEQ ID No. 17): GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTAGCTATGTTTTACACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGATACTAAATACAATGAGAACTTCAAAGGCAAGGCCACACTGACCTCGGACAAATCCTCCAGCACAGCCTACATGGATCTCAACAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAAGGAATTTATTACTACGGTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0069] 1 The nucleotide sequence of the variable region of the light chain of the 1G12 antibody (SEQ ID No. 18): GACATCAAGATGACCCAGTTTCCATCTTCCGTGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACTTTAATACCTATTTAACCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAAGAGATTGGTAGATGGGGTCCCATCCAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

[0070] 4 The nucleotide sequence of the variable region of the heavy chain of the 4E8 antibody (SEQ ID No. 19): GTTCAGCTGCAGGAGTCTGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACCTAAACTACGACGGTAGCAATTACTACAACCCCTCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCACTTTTTCCTGAAGTTAAATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCAAGGACCTATGGTTACGAAACCTGGTACTTCGATGTCTGGGGCGCAGGGACCGCGGTCACCGTCTCCTCA.

[0071] 4E8 antibody light chain variable region nucleic acid sequence (SEQ ID No. 20): GATATTGTGATGACCCAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTCTGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAACAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGATGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCATTATTATACCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0072] Example 3: Sensitivity and affinity detection of 1G12 and 4E8

[0073] The antibody sensitivity was detected by enzyme-linked immunosorbent assay (ELISA), and the purified FimA recombinant protein was diluted with coating solution, 100 μL of which was coated in a 96-well plate, sealed and placed at 4°C overnight, washed 5 times with Wash Buffer working solution, 200 ml of blocking solution was added to block non-specific binding sites, and incubated at room temperature for 2 hours, then washed with Wash Buffer working solution, 100 μL of gradient 2 μg / mL FimA monoclonal antibody was added per well, and incubated at 37°C for 2 hours, then washed with Wash Buffer working solution, 1:10000 diluted HRP-labeled anti-mouse secondary antibody was added, and incubated at 37°C for 1 hour, then washed with Wash Buffer working solution for 3 times, TMB color developing liquid was added, and incubated at 37°C for 30 min, then stop solution was added and OD450 value was measured. The results are shown in Figure 3 As shown in A and B, the IC50 of antibody 1G12 reached 2.3 nM, and the IC50 of antibody 4E8 reached 3.38 nM, indicating that the two antibodies had good recognition and binding sensitivity to FimA.

[0074] Bio-layer interferometry (BLI) can measure the association constant (ka or kon) and dissociation constant (kd or koff), and the initial binding rate by real-time monitoring of the molecular binding process, and obtain the affinity (KD) and concentration information by fitting calculation analysis. Through the BLI detection platform, HIS1K sensor was used to solidify FimA recombinant protein, and its affinity with FimA was determined by incubation with gradient-diluted antibody. The results are shown in Figure 3As shown in Figures C and D, the KD value of antibody 1G12 reached 33 nM, and the KD value of antibody 4E8 was less than 0.1 nM.

[0075] Example 4: Specificity analysis of 1G12 and 4E8

[0076] P. gingivalis and other common oral bacteria (S. oralis, S. mitis, S. intermedia, S. gordonii, S. mutans, E. coli, S. aureus) were coated directly into 96-well enzyme plates at a coating amount of 1 x 10 8 CFU / well overnight at 4°C, and after blocking, the anti-FimA monoclonal antibody was added for incubation. After two washes, the HRP-labeled anti-mouse IgG secondary antibody was added for binding, and after rinsing, TMB was added for color development. The OD450 value was measured and analyzed. The results (Figures A and B) show that 1G12 and 4E8 can specifically recognize P. gingivalis. Figure 4 A and B) show that 1G12 and 4E8 can specifically recognize P. gingivalis.

[0077] Previous studies have shown that FimA has multiple subtypes (types I-V and Ib), and FimA-I is the protein subtype of P. gingivalis model strain P. gingivalis (ATCC 33277) and common clinical strains. We coated different FimA subtypes, FimA-I, FimA-II, FimA-III, FimA-IV, FimA-V, and FimA-Ib recombinant proteins (1 μg / mL) into 96-well plates overnight at 4°C, and after blocking, the anti-FimA monoclonal antibody was added for binding. After two washes, the HRP-labeled anti-mouse IgG secondary antibody was added for binding, and after rinsing, TMB was added for color development. The OD450 value was measured and analyzed. The ELISA results are shown in Figures C and D. Figure 4 (C and D) show that 1G12 and 4E8 can specifically recognize FimA-I protein, and have lower sensitivity for other subtypes of FimA (II-V and Ib).

[0078] Example 5: Analysis of the recognition epitopes of 1G12 and 4E8

[0079] Antibodies against different antigen epitopes significantly improve the sensitivity and specificity of detection by specifically binding to multiple sites of the antigen, and are a core tool in immunological experiments. In order to explore the antigen epitope recognition of 1G12 and 4E8, we analyzed their recognition site differences by BLI. The purified FimA-His recombinant protein was immobilized on a HIS1K sensor, and then the sensor was immersed in buffer to reach equilibrium. Next, the sensor was immersed in the first antibody for the first binding, and was equilibrated in buffer; after equilibration, the sensor was immersed in the second antibody to observe the change in baseline measurement. As shown in Figures A and B, the KD value of 1G12 reached 33 nM, and the KD value of 4E8 was less than 0.1 nM. Figure 5As shown, when the two antibodies recognize different epitopes, the first antibody binds, and does not affect the antigen to continue to bind with the second antibody, that is, the measurement value curve will appear again rising; conversely, if the same or similar epitopes are recognized, the curve will not have a second lift. The analysis results are shown in Figure 5 The antibody 1G12 and 4E8 recognize different epitopes, and can constitute a group of antibody pairs recognizing FimA-I type protein.

[0080] Example 6: Sensitivity analysis of 1G12 and 4E8 for detecting Porphyromonas gingivalis

[0081] Porphyromonas gingivalis was counted by standard curve, and then gradient diluted and coated on a 96-well enzyme-labeled plate. After blocking with 3% BSA buffer at 37°C, washing three times with Wash Buffer, adding monoclonal antibody 1G12 or 4E8, incubating at 37°C for 1 hour, washing three times with Wash Buffer; adding HRP-labeled anti-mouse IgG secondary antibody, incubating at 37°C for 30 minutes, washing three times with Wash Buffer; adding TMB developing solution, incubating at room temperature or 37°C for 15 minutes, adding stop solution to terminate the reaction, and using an enzyme-labeled instrument to measure the OD450 value. When the measured value is greater than 2.1 times the negative value, it is judged as positive. The results are shown in Figure 6 A of FIG. 6 shows that 1G12 and 4E8 can both recognize 6.3 x 10 6 CFU / mL or more of Porphyromonas gingivalis.

[0082] To improve the detection sensitivity of Porphyromonas gingivalis, based on the characteristics of 1G12 and 4E8 antibody recognizing different epitopes, a double antibody sandwich ELISA detection system was established. The specific operation process is as follows:

[0083] (1) Preparation of biotin-labeled antibody: dissolve 2 mg of labeled antibody 4E8 in 1.0 mL of PBS (pH=9); prepare a 10 mM biotin solution (Biotin NHS); according to the molar ratio of biotin to antibody is 20:1, add appropriate volume of 10 mM biotin solution to the antibody solution, and gently blow and mix; the mixed solution is placed in a 37°C incubator for 30 min or on ice for 2 hours. After the reaction is completed, the mixed solution is added to the dialysis bag for dialysis; and the biotin-labeled antibody (4E8-Biotin) is collected by ultrafiltration tube concentration. After detection of the concentration, it is saved for use.

[0084] (2) Capture antibody 1G12 was coated on 96-well enzyme-labeled plate, after PBS washing to remove unbound antibodies, gradient dilution of P. gingivalis was added, 37°C incubation for 1 hour, then washed 3 times with washing buffer; then 3% BSA blocking solution was added, 37°C blocking for 1 hour, then washed again; biotin-labeled detection antibody 4E8 was added, 37°C incubation for 1 hour, then washed; then HRP-labeled streptavidin was added, after washing, TMB color developing solution was added, color development at room temperature for 15 minutes; finally, stop solution was added to stop the reaction, and the OD450nm absorbance value was measured using an enzyme-labeled instrument. The judgment standard is: when the detection value is more than 2.1 times of the negative control, it is judged as positive. The experimental results are shown in Figure 6 B, which shows that based on the 1G12 / 4E8 antibody, the double-antibody sandwich ELISA detection system constructed can detect P. gingivalis as low as 1.6x10 5 CFU / mL.

[0085] Example 7: Analysis of the agglutination effect of 1G12 and 4E8 on P. gingivalis

[0086] Logarithmic growth phase P. gingivalis 1x10 8 CFU was dissolved in 1 mL PBS, FITC was stained at room temperature for 1 hour and washed. 100 μL of P. gingivalis was added to 60 μg of 1G12 or 4E8 antibody and incubated at 37°C for 1.5 hours. After washing, the bacteria were fixed in 4% paraformaldehyde for 15 minutes and washed 3 times, then resuspended in PBS. Flow cytometry was used for analysis, the control group without monoclonal antibody treatment was used for threshold and gating, and the bacterial agglutination was analyzed by adding FSC and SSC quadrants. The results (Fig. Figure 7 A) show that compared with the isotype antibody control group, the FSC and SSC of the experimental group treated with 1G12 and 4E8 are significantly shifted, indicating that antibodies 1G12 and 4E8 can significantly induce the agglutination of P. gingivalis.

[0087] Logarithmic growth phase P. gingivalis 1x10 8 CFU was added to an appropriate amount of GAM medium. 1G12 or 4E8 was added to a concentration of 200 μg / mL, and the absorbance value at OD600 nm was continuously monitored (48 hours) to analyze the growth. The results (Fig. Figure 7 B) show that compared with the blank control and isotype control, antibodies 1G12 or 4E8 significantly inhibit the absorbance value, indicating that 1G12 and 4E8 have a certain inhibitory effect on the growth of P. gingivalis.

[0088] Example 8: Analysis of the adhesion effect of 1G12 and 4E8 on P. gingivalis

[0089] Hydroxyapatite material is a common material for studying the adhesion of P. gingivalis due to its high match with natural teeth in chemical and physical properties. We took 2 mg of hydroxyapatite microspheres into 1.5 mL centrifuge tubes, washed twice with ddH2O, and finally added 1 mL of KCl buffer to each tube, and incubated at room temperature for 2 hours. Centrifuged to discard the supernatant, and added 200 μL of artificial saliva to each tube, and incubated overnight at room temperature. 1 x 10 8 P. gingivalis in the logarithmic growth phase was dissolved in 100 μL of PBS buffer, and 1G12 or 4E8 antibodies were added at a final concentration of 100 μg / mL, with IgG as a control group, and pre-incubated at 37°C for 2 hours, with mixing every 30 minutes. After washing the saliva-coated hydroxyapatite microspheres twice with KCl buffer, the hydroxyapatite microspheres were resuspended with 100 uL of KCl buffer, and different treatments of P. gingivalis were added for binding at room temperature for 2 hours, with mixing every 30 minutes. Finally, the P. gingivalis-bound hydroxyapatite microspheres were washed 3 times with KCl buffer, and then added to an appropriate amount of fixing solution (2.5% glutaraldehyde + 4% paraformaldehyde) for overnight fixation. Washed 3 times with 0.1M PBS, each for 15 minutes. Gradient dehydration with ethanol, and then immersed in t-butanol and dried in a freeze dryer. Scanning electron microscopy was used to take pictures and collect images (Fig. 6A). To quantify the number of adhesions, we extracted the genome of P. gingivalis on the sample, and analyzed the number of P. gingivalis adhered to HAP by fluorescent quantitative PCR (Fig. 6B). Figure 8 A). To quantify the number of adhesions, we extracted the genome of P. gingivalis on the sample, and analyzed the number of P. gingivalis adhered to HAP by fluorescent quantitative PCR (Fig. 6B). Figure 8 B); as shown in Figure 8 Compared with the blank control and IgG control group, 1G12 or 4E8 pretreatment significantly reduced the adhesion of P. gingivalis.

[0090] Example 9: Analysis of the anti-inflammatory effect of 1G12 and 4E8

[0091] Human gingival fibroblasts (HGF) are the most abundant cells in gingival connective tissue, and are crucial for the remodeling and homeostasis of periodontal tissue. They are widely used in the study of periodontal disease because of their close relationship with the occurrence and development of periodontal disease. Primary separation and culture of gingival fibroblasts were performed using tissue block separation method. HGF cells were passaged to 3-6 generations and had good growth state, and were then plated into 6-well plates and cultured in a cell culture incubator. When the cell density reached about 80%, FimA recombinant protein was added to the HGF cells after 1 hour of incubation with 1G12, 4E8 and PBS control. After 4 hours of co-incubation in the incubator, total mRNA was extracted. After reverse transcription of total mRNA to cDNA, the expression levels of inflammatory cytokines IL-1β, IL-6 and TNF-α were determined by fluorescent quantitative PCR. The experimental results are shown in Figure 9The results show that the mRNA transcription levels of IL-1β, IL-6 and TNF-α in the experimental groups treated by 1G12 and 4E8 monoclonal antibodies are obviously down-regulated compared with the control group, indicating that 1G12 and 4E8 can inhibit the expression of inflammatory factors caused by FimA protein and have anti-inflammatory effect.

[0092] The above merely describes preferred embodiments of the present application but is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A FimA monoclonal antibody, characterized in that: The FimA monoclonal antibody is selected from 1G12 antibody or 4E8 antibody; The 1G12 antibody contains heavy chain variable regions as shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, and light chain variable regions as shown in SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7; The 4E8 antibody contains a heavy chain variable region as shown in SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, and a light chain variable region as shown in SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No.

15.

2. The FimA monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the 1G12 antibody has more than 85% similarity to the amino acid sequence shown in SEQ ID No. 4, and the amino acid sequence of the light chain variable region has more than 85% similarity to the amino acid sequence shown in SEQ ID No. 8; the amino acid sequence of the heavy chain variable region of the 4E8 antibody has more than 85% similarity to the amino acid sequence shown in SEQ ID No. 12, and the amino acid sequence of the light chain variable region has more than 85% similarity to the amino acid sequence shown in SEQ ID No.

16.

3. A nucleotide fragment encoding the FimA monoclonal antibody according to claim 1 or 2, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the 1G12 antibody is shown in SEQ ID No. 17, and the nucleotide sequence encoding the light chain variable region of the 1G12 antibody is shown in SEQ ID No.

18. The nucleotide sequence encoding the heavy chain variable region of the 4E8 antibody is shown in SEQ ID No. 19, and the nucleotide sequence encoding the light chain variable region of the 4E8 antibody is shown in SEQ ID No.

20.

4. A biomaterial, characterized in that: The biological material is an expression fragment containing the nucleotide fragment described in claim 3, a recombinant vector, a recombinant bacterium, or a transgenic cell line.

5. A diagnostic reagent or kit, characterized in that: The FimA monoclonal antibody comprising claim 1 or 2.

6. A drug for treating conditions related to Porphyromonas gingivalis infection, characterized in that: It comprises the FimA monoclonal antibody of claim 1 or 2, or the nucleotide fragment of claim 3.

7. The medicament for treating diseases related to Porphyromonas gingivalis infection according to claim 6, characterized in that: The symptoms associated with Porphyromonas gingivalis infection include cellular inflammation caused by the FimA antigen.

8. The medicament for treating diseases related to Porphyromonas gingivalis infection according to claim 7, characterized in that: The condition associated with Porphyromonas gingivalis infection is periodontitis.

9. A kind of inhibition P. gingivalis Bacterial adhesion ability, promoting P. gingivalis Biological agents that agglutinate bacteria are characterized by: Contains the FimA monoclonal antibody of claim 1 or 2, or the biological material of claim 3.

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