Porphyromonas gingivalis FimA monoclonal antibody and application thereof

Abstract

The invention discloses a porphyromonas gingivalis FimA monoclonal antibody and an application thereof, and provides two monoclonal antibodies targeting porphyromonas gingivalis FimA-I type protein, the monoclonal antibodies are named as 1G12 and 4E8, and each monoclonal antibody comprises a heavy chain variable region and a light chain variable region. By exploring the biological functions of the monoclonal antibody, the two antibodies are found to be capable of remarkably inhibiting the adhesion capacity and growth cycle of porphyromonas gingivalis, promoting bacterial agglutination and blocking inflammatory response caused by FimA; meanwhile, the 1G12 and the 4E8 recognize different antigen epitopes of FimA, and an ELISA (enzyme-linked immuno sorbent assay) detection method developed on the basis of the 1G12 and the 4E8 can be used for well detecting the FimA-I type protein and the porphyromonas gingivalis; and a foundation is laid for diagnosis, prevention and treatment of porphyromonas gingivalis and related diseases thereof.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedicine, cell technology and immunology, and relates to a monoclonal antibody and its application. Background Art

[0002] Porphyromonas gingivalis ( Porphyromonas gingivalis , P. gingivalis ) is a Gram-negative, melanin-producing, strictly anaerobic bacterium and is one of the key pathogens causing chronic and aggressive periodontitis. In addition, 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 a variety of virulence factors, such as fimbriae, lipopolysaccharide, gingipains and hemagglutinin. The fimbriae of Porphyromonas gingivalis are proteinaceous filamentous appendages protruding from the bacterial surface and are one of the important virulence factors of Porphyromonas gingivalis. FimA is the main subunit of the fimbriae of Porphyromonas gingivalis and plays a key role in fimbriae assembly and the occurrence and development of periodontal diseases. Studies have shown that FimA can mediate the adhesion of bacteria to host tissues and cells (such as gingival fibroblasts, gingival epithelial cells), participate in bacterial colonization and biofilm formation; promote the invasion of Porphyromonas gingivalis into host cells to escape immune detection; trigger inflammatory responses (IL-1β, IL-6, etc.) through interaction with Toll-like receptors, causing immune dysregulation; and cooperate with proteases such as gingipains to degrade host proteins, thus causing gingival bleeding, alveolar bone resorption and tooth loss; playing a core role in infection establishment, immune evasion and disease progression.

[0003] At present, the FimA genotype has been divided into six types: I-V and Ib, which are different among periodontitis patients in different regions, different populations and different age groups. Some studies have shown that the detection rates of FimA-II and IV types of Porphyromonas gingivalis are relatively high in patients with severe periodontitis, and the FimA-I type ranks second. However, some studies have shown that the FimA-I type of Porphyromonas gingivalis has a relatively high occurrence frequency in patients with chronic periodontitis and puberty gingivitis in China. In some healthy carriers of Porphyromonas gingivalis or patients with early periodontal diseases, the detection rate of the FimA-I type of Porphyromonas gingivalis is even higher. At present, there have been studies on monoclonal antibodies against the FimA-II type of Porphyromonas gingivalis, but there is still no report on monoclonal antibodies against the FimA-I type. At the same time, as the most commonly used model bacterium in scientific research, the lack of monoclonal antibodies against the FimA-I type of Porphyromonas gingivalis also adds difficulties to related scientific research. Based on this, the development of monoclonal antibodies targeting the FimA-I type protein is of great significance for the related scientific research on the FimA-I type of Porphyromonas gingivalis and the diagnosis and treatment of related diseases. Summary of the Invention

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

[0005] To achieve the object of the invention, the present invention provides the following technical solutions: The present application provides a FimA monoclonal antibody, which is selected from one or more of the following antibodies: (1) 1G12 antibody, the heavy chain variable region of the antibody comprises VH-CDR1-3 of the amino acid sequences shown in SEQ ID No.1-3; the light chain variable region of the antibody comprises VL-CDR1-3 of the amino acid sequences shown in SEQ ID No.5-7; 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.

[0006] Preferably, the amino acid sequence of the heavy chain variable region of the antibody (1G12) is as shown in SEQ ID No.4, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No.8.

[0007] (2) 4E8 antibody, characterized in that the heavy chain variable region of the antibody comprises VH-CDR1-3 of the amino acid sequences shown in SEQ ID No.9-11; the light chain variable region of the antibody comprises VL-CDR1-3 of the amino acid sequences shown in SEQ ID No.13-15; Furthermore, the amino acid sequence of the heavy chain variable region of the antibody (4E8) is as shown in SEQ ID No.12, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No.16.

[0008] Furthermore, the monoclonal antibody comprises all or part of the antibody heavy chain constant region and / or the antibody light chain constant region.

[0009] The above FimA monoclonal antibody is named 1G12 antibody or 4E8 antibody.

[0010] The second aspect of the present invention provides nucleic acid sequences encoding antibodies. The nucleic acid sequences include a nucleotide sequence encoding the heavy chain variable region of the 1G12 antibody as shown in SEQ ID No. 17, and 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, and a nucleotide sequence encoding the light chain variable region of the 4E8 antibody as shown in SEQ ID No. 20.

[0011] Furthermore, the DNA sequences encoding the antibody molecules of the present invention can be obtained by methods well-known to those skilled in the art, and the antibodies of the present invention can be expressed in expression systems known to those skilled in the art. For example, the DNA sequences encoding part or all of the antibody heavy chain and light chain can be synthesized based on the corresponding amino acid sequences, and the antibodies can be obtained by expression in a mammalian expression system using a mammalian expression vector.

[0012] The present application also provides a conjugate or fusion product, which is obtained by modifying or fusion-expressing the above-mentioned FimA monoclonal antibody. Regarding the conjugate, the present application takes biotin modification as an example; regarding the fusion product, the present application refers to adding tags or other proteins before and after the antibody when expressing the protein to produce a novel protein containing the antibody sequence.

[0013] The present application also provides application scenarios containing the above-mentioned FimA monoclonal antibody, including: (1) The application of the above-mentioned FimA monoclonal antibody in the preparation of products for diagnosing diseases related to Porphyromonas gingivalis infection; Furthermore, a kit for specifically recognizing Porphyromonas gingivalis is provided, and the kit contains one or two of the FimA monoclonal antibodies of the present invention; (2) The application of the above-mentioned FimA monoclonal antibody in the preparation of a drug combination for treating diseases related to Porphyromonas gingivalis infection; Furthermore, the application of the above-mentioned FimA monoclonal antibody in blocking the adhesion function of Porphyromonas gingivalis; Furthermore, the application of the above-mentioned FimA monoclonal antibody in promoting the aggregation function of Porphyromonas gingivalis; Furthermore, the application of the above-mentioned FimA monoclonal antibody in blocking cytokine inflammation induced by FimA.

[0014] The present invention has the following beneficial effects: 1. The present invention uses type I FimA as a target antigen for immunizing mice, and monoclonal antibodies named 1G12 and 4E8 are prepared by hybridoma technology. Systematic characterization shows that the obtained antibodies have excellent sensitivity and specificity, and can effectively recognize FimA-type I protein and Porphyromonas gingivalis of type I.

[0015] 2. The antibodies obtained in the present invention can effectively inhibit the adhesion ability of Porphyromonas gingivalis; promote the aggregation of Porphyromonas gingivalis and inhibit its growth cycle; block the inflammatory response caused by FimA antigen, providing a solution for the prevention and treatment of Porphyromonas gingivalis and its related diseases.

[0016] 3. The antibodies obtained in the present invention can recognize different antigenic epitopes of FimA-type I protein. Based on this, the developed ELISA detection method can better detect FimA-type I protein and Porphyromonas gingivalis, laying a foundation for rapid clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 For the expression and purification analysis of the antigen.

[0019] Figure 2 For the analysis of the serum titer of immunized mice.

[0020] Figure 3 For the detection of the sensitivity and affinity of the antibody.

[0021] Figure 4 For the specificity analysis of the antibody recognizing Porphyromonas gingivalis and the antigen.

[0022] Figure 5 For the analysis of the differences in the antigenic epitopes recognized by the antibody.

[0023] Figure 6 For the sensitivity analysis of the antibody recognizing Porphyromonas gingivalis.

[0024] Figure 7 For the analysis result that the antibody can aggregate Porphyromonas gingivalis and inhibit its growth.

[0025] Figure 8 For the analysis result that the antibody has the effect of inhibiting the adhesion of Porphyromonas gingivalis.

[0026] Figure 9 Analysis results showing the anti-inflammatory effect of the antibody. Detailed implementation manners

[0027] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0028] Cell lines and experimental animals The myeloma cell line (SP2 / 0) was preserved in the laboratory; primary gingival fibroblasts were obtained from orthodontic patients in the Department of Stomatology and approved by the Medical and Research Ethics Committee of Henan University. BALB / c mice were purchased from Beijing Huafukang Bioscience Co., Ltd.; the animal experiments were approved by the Laboratory Animal Ethics Committee of Henan University.

[0029] The experimental reagents are as follows: Example 1: Expression and purification of FimA recombinant protein Bioinformatics analysis was performed on the FimA gene of Porphyromonas gingivalis fimbrial protein, and the upstream primer FimA-F: CTAGCTAGCATGAAAAAAACAAAG and the downstream primer FimA-R: CCGCTCGAGCCAAGTAGCATTCTG were designed.

[0030] Using the genomic DNA of the reference strain (ATCC-33277) of Porphyromonas gingivalis as a template, the FimA gene fragment was amplified by PCR. Through the NheI and XhoI restriction enzyme sites introduced in the primers, the FimA gene fragment was ligated to the pET28a-His prokaryotic expression vector by restriction enzyme digestion and ligation methods. After digestion and sequencing detection, the correct positive clone vector was named pET28a-FimA / His and stored for later use. The digestion results are shown in Figure 1 A as shown in the figure.

[0031] The pET-28a-FimA / His recombinant plasmid was transformed into the expression strain BL21(DE3). A single colony was picked and cultured until the OD600 reached 0.4 - 0.8, and then 100 mM IPTG (to a final concentration of 0.1 mM) was added for induction expression for 12 - 16 hours, and the cells were collected. After ultrasonic disruption of the cells, the supernatant and precipitate were separated, and the supernatant was filtered through a 0.45 μm filter membrane. The protein expression in the supernatant and precipitate was detected by SDS-PAGE electrophoresis and Coomassie brilliant blue staining method. The results are shown in Figure 1As shown in B, it was found that the FimA recombinant protein was abundantly expressed in the supernatant. The system was scaled up to 2 L, and after ultrasonic disruption, the supernatant was collected. The FimA recombinant protein was purified by nickel column affinity chromatography, and the purified FimA recombinant protein was detected by protein electrophoresis. The results are as Figure 1 shown in C. High-purity FimA recombinant protein could be obtained by elution with 100 - 300 mM imidazole. The elution products were combined and stored for later use.

[0032] Example 2: Mouse Immunization and Antibody Purification The purified FimA recombinant protein in Example 1 was subcutaneously injected at multiple points into four BALB / c mice at a dose of 100 μg / mouse, and another mouse was taken as a negative control. Freund's complete adjuvant (FCA) was used for the first immunization, and Freund's incomplete adjuvant (FIA) was used for the second and third immunizations. A total of 3 immunizations were carried out at 2-week intervals. One week after the third immunization, blood was taken to separate serum, which was diluted according to a ratio and added to a 96-well plate coated with FimA recombinant protein (2 μg / mL). The antibody titer in the mouse serum was detected by ELISA. The results are as Figure 2 shown in A, indicating that the serum titers of the immunized mice all reached above 1 / 320000.

[0033] Two mice with the highest titers were intraperitoneally injected with FimA recombinant protein (50 μg) for boost immunization. Three days later, the spleen cells of the mice were fused with myeloma cells sp2 / 0. When the hybridoma cells grew to 30%, the cell culture supernatant could be collected for ELISA screening. The results are as Figure 2 shown in B. The antibody titers in the supernatants of 1G12 and 4E8 cells were high and their growth states were good. The subtypes of the antibodies were detected by a mouse monoclonal antibody subtype detection kit. The results are as Figure 2 shown in C. The heavy chain subtypes of antibodies 1G12 and 4E8 were IgG1, and the light chain subtypes were Kappa. Subsequently, by the method of collecting antibodies from ascites, the selected monoclonal hybridoma cells were injected into the peritoneal cavity of mice to collect ascites. Protein A / G was selected and the antibodies were purified by an AKTA protein purification system, and stored for later use after detecting the purity.

[0034] Variable region amino acid sequence of 1G12 antibody: VH-CDR 1 (SEQ ID No.1): GYTFTSYV; VH-CDR 2 (SEQ ID No.2): INPYNDDT; VH-CDR 3 (SEQ ID No.3): ARKEFITTVFDY; Amino acid sequence of the heavy chain variable region of the 1G12 antibody (SEQ ID No.4): EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVLHWVKQKPGQGLEWIGYINPYNDDTKYNENFKGKATLTSDKSSSTAYMDLNSLTSEDSAVYYCARKEFITTVFDYWGQGTTLTVSS; VL-CDR 1 (SEQ ID No.5): QDFNTY; VL-CDR 2 (SEQ ID No.6): RAK; VL-CDR 3 (SEQ ID No.7): LQYDEFPYT; Amino acid sequence of the light chain variable region of the 1G12 antibody (SEQ ID No.8): DIKMTQFPSSVYASLGERVTITCKASQDFNTYLTWFQQKPGKSPKTLIYRAKRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK.

[0035] Amino acid sequence of the variable region of the 4E8 antibody: VH-CDR 1 (SEQ ID No.9): GYSITSGYY; VH-CDR 2 (SEQ ID No.10): LNYDGSN; VH-CDR 3 (SEQ ID No.11): ARTYGYETWYFDV; Amino acid sequence of the heavy chain variable region of the 4E8 antibody (SEQ ID No.12): VQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYLNYDGSNYYNPSLKNRISITRDTSKNHFFLKLNSVTTEDTATYYCARTYGYETWYFDVWGAGTAVTVSS; VL-CDR 1 (SEQ ID No.13): QSLLYSNNQKNY; VL-CDR 2 (SEQ ID No.14): WAS; VL-CDR 3 (SEQ ID No.15): QHYYTYPWT; Amino acid sequence of the light chain variable region of the 4E8 antibody (SEQ ID No.16): DIVMTQSPSSLAVSVGEKVTLSCKSSQSLLYSNNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVMAEDLAVYFCQHYYTYPWTFGGGTKLEIK。

[0036] Nucleic acid sequence of the heavy chain variable region of the 1G12 antibody (SEQ ID No.17): GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTAGCTATGTTTTACACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGATACTAAATACAATGAGAACTTCAAAGGCAAGGCCACACTGACCTCGGACAAATCCTCCAGCACAGCCTACATGGATCTCAACAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAAGGAATTTATTACTACGGTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

[0037] Nucleotide sequence of the light chain variable region of the 1G12 antibody (SEQ ID No.18): GACATCAAGATGACCCAGTTTCCATCTTCCGTGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACTTTAATACCTATTTAACCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAAGAGATTGGTAGATGGGGTCCCATCCAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。

[0038] Nucleic acid sequence of the heavy chain variable region of the 4E8 antibody (SEQ ID No. 19): GTTCAGCTGCAGGAGTCTGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACCTAAACTACGACGGTAGCAATTACTACAACCCCTCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCACTTTTTCCTGAAGTTAAATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCAAGGACCTATGGTTACGAAACCTGGTACTTCGATGTCTGGGGCGCAGGGACCGCGGTCACCGTCTCCTCA。

[0039] Nucleic acid sequence of the light chain variable region of the 4E8 antibody (SEQ ID No. 20): GATATTGTGATGACCCAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTCTGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAACAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGATGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCATTATTATACCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

[0040] Example 3: Detection of the sensitivity and affinity of 1G12 and 4E8 The sensitivity of the antibody was detected by enzyme-linked immunosorbent assay (ELISA). The purified recombinant FimA protein was serially diluted with coating buffer and 100 μL of each dilution was coated in a 96-well plate. After sealing, the plate was incubated overnight at 4 °C. The plate was washed 5 times with Wash Buffer working solution, and 200 μL of blocking solution was added to block non-specific binding sites. After blocking at room temperature for 2 hours, the plate was washed with Wash Buffer working solution. Then, 100 μL of FimA monoclonal antibody at a gradient concentration of 2 μg / mL was added to each well and reacted at 37 °C for 2 hours. After washing with Wash Buffer working solution, 100 μL of HRP-labeled anti-mouse secondary antibody diluted 1:10,000 was added and incubated at 37 °C for 1 hour. The plate was washed 3 times with Wash Buffer working solution, TMB chromogenic solution was added, and after incubating at 37 °C for 30 min, the stop solution was added and the OD450 value was measured. The results are as Figure 3 shown in A and B. The IC50 of antibody 1G12 reached 2.3 nM, and the IC50 value of antibody 4E8 reached 3.38 nM, indicating that both antibodies had good recognition and binding sensitivity to FimA.

[0041] Biolayer interferometry (BLI) can measure the association constant (ka or kon), dissociation constant (kd or koff), and initial binding rate by real-time monitoring of the molecular binding process, and analyze the affinity (KD) and concentration information through fitting calculation. Using the BLI detection platform, the HIS1K sensor was immobilized with the recombinant FimA protein, and its affinity for FimA was measured by incubating with serially diluted antibodies. The results are as Figure 3 shown in 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.

[0042] Example 4: Specificity analysis of 1G12 and 4E8 Porphyromonas gingivalis and other common oral bacteria (Streptococcus oralis, Streptococcus mitis, Streptococcus intermedia, Streptococcus gordonii, Streptococcus mutans, Escherichia coli, Staphylococcus aureus) were directly coated in a 96-well ELISA plate at a coating amount of 1×10 8 CFU / well and incubated overnight at 4 °C. After blocking, anti-FimA monoclonal antibody was added for incubation. After washing twice, HRP-labeled anti-mouse IgG secondary antibody was added for binding. After rinsing, TMB was added for color development, and the OD450 value was measured and analyzed. The results ( Figure 4 A and B) showed that 1G12 and 4E8 could specifically recognize Porphyromonas gingivalis.

[0043] Existing studies have shown that FimA has multiple subtypes (I-V and Ib), and FimA-I is the protein subtype of the Porphyromonas gingivalis type strain Porphyromonas gingivalis (ATCC33277) 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) onto 96-well plates overnight at 4°C. After blocking, anti-FimA monoclonal antibody was added for binding. After washing twice, HRP-labeled anti-mouse IgG secondary antibody was added for binding. After rinsing, TMB was added for color development, and the OD450 value was measured and analyzed. The ELISA results are as Figure 4 (C and D) showed that 1G12 and 4E8 could specifically recognize FimA-I protein and had lower sensitivity to other subtypes of FimA (II-V and Ib).

[0044] Example 5: Epitope analysis of 1G12 and 4E8 Antibody pairs against different antigenic epitopes can significantly improve the sensitivity and specificity of detection by specifically binding to multiple sites of the antigen and are the core tools in immunological experiments. To explore the antigenic epitope recognition of 1G12 and 4E8, we analyzed the differences in their recognition sites by BLI. The purified FimA-His recombinant protein was immobilized on the HIS1K sensor, and then the sensor was immersed in the buffer to reach equilibrium. Then the sensor was immersed in the first antibody for the first binding and equilibrated in the buffer; after equilibration, the sensor was immersed in the second antibody to observe the change in the baseline measurement value. As Figure 5 shown, when the two antibodies recognize different epitopes, after the first antibody binds, it does not affect the antigen's continued binding to the second antibody, that is, the measurement value curve will rise again; conversely, if the recognized epitopes are the same or similar, the curve will not have a secondary elevation. The analysis results are as Figure 5 shown that there are differences in the epitopes recognized by antibody 1G12 and 4E8, and they can form a pair of antibodies that recognize FimA-I protein.

[0045] Example 6: Sensitivity analysis of 1G12 and 4E8 for detecting Porphyromonas gingivalis After counting Porphyromonas gingivalis using the standard curve, it was gradient-diluted and coated on a 96-well ELISA plate. 3% BSA buffer was added for blocking at 37°C. After washing three times with Wash Buffer, monoclonal antibody 1G12 or 4E8 was added and incubated at 37°C for 1 hour, followed by washing three times with Wash Buffer. HRP-labeled anti-mouse IgG secondary antibody was added and incubated at 37°C for 30 minutes, then washed three times with Wash Buffer. TMB chromogenic solution was added and incubated at room temperature or 37°C for 15 minutes. The reaction was terminated by adding the stop solution, and the OD450 value was measured using an ELISA reader. A measurement value greater than 2.1 times the negative value was judged as positive. The results are as Figure 6 shown in A of 6 Figure, indicating that both 1G12 and 4E8 can recognize Porphyromonas gingivalis at a concentration of 6.3×10

[0046] CFU / mL or above. To improve the detection sensitivity of Porphyromonas gingivalis, based on the different epitope recognition characteristics of 1G12 and 4E8 antibodies, a double-antibody sandwich ELISA detection system was established in this study. The specific operation process is as follows:

[0047] (1) Preparation of biotin-labeled antibody: Dissolve 2 mg of the antibody 4E8 to be labeled 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 of 20:1, add an appropriate volume of the 10 mM biotin solution to the antibody solution and gently pipette to mix; place the mixed solution in a 37°C incubator in the dark for 30 min or incubate on ice in the dark for 2 hours. After the reaction is completed, add the mixed solution into a dialysis bag for dialysis; and concentrate and collect through an ultrafiltration tube to obtain the biotin-labeled antibody (4E8-Biotin). After detecting the concentration, it is stored for later use. Figure 6 (2) Coat the capture antibody 1G12 on a 96-well ELISA plate. After washing with PBS to remove unbound antibody, add gradient-diluted Porphyromonas gingivalis bacterial solution and incubate at 37°C for 1 hour, then wash 3 times with the washing buffer; subsequently add 3% BSA blocking solution and block at 37°C for 1 hour and then wash again; add the biotin-labeled detection antibody 4E8 and incubate at 37°C for 1 hour and then wash; then add HRP-labeled streptavidin, and after thorough washing, add TMB chromogenic solution and develop color in the dark at room temperature for 15 minutes; finally, add the stop solution to terminate the reaction, and measure the OD450nm absorbance value using an ELISA reader. The judgment criterion is: when the detection value exceeds 2.1 times that of the negative control, it is judged as positive. The experimental results are as 5 shown in B of

[0048] Example 7: Analysis of the Agglutination Effect of 1G12 and 4E8 on Porphyromonas gingivalis Take 1×10 8 CFU of Porphyromonas gingivalis in the logarithmic growth phase and dissolve it in 1 mL of PBS. Stain with FITC at room temperature for 1 h and wash. Take 100 μL of Porphyromonas gingivalis and add 60 μg of 1G12 or 4E8 antibody, and incubate at 37 °C for 1.5 hours. After washing, fix in 4% paraformaldehyde for 15 minutes. After washing 3 times, resuspend the bacteria in PBS. Analyze using a flow cytometer. The control group without monoclonal antibody treatment is used for threshold and gating, and the bacterial agglutination is analyzed by adding FSC and SSC quadrants. The results ( Figure 7 A) show that compared with the isotype antibody control group, significant shifts in FSC and SSC occur in the experimental groups treated with 1G12 and 4E8, indicating that the antibodies 1G12 and 4E8 can significantly induce the agglutination of Porphyromonas gingivalis.

[0049] Take 1×10 8 CFU of Porphyromonas gingivalis in the logarithmic growth phase and add an appropriate amount of GAM medium. Add 1G12 or 4E8 to a concentration of 200 μg / mL, and continuously monitor the absorbance value at OD600 nm (for 48 hours) to analyze its growth. The results ( Figure 7 B) show that compared with the blank control and the isotype control, the antibodies 1G12 or 4E8 significantly inhibit the absorbance value, indicating that 1G12 and 4E8 have a certain inhibitory effect on the growth of Porphyromonas gingivalis.

[0050] Example 8: Analysis of the Adhesion Effect of 1G12 and 4E8 on Porphyromonas gingivalis Due to its high chemical and physical property matching with natural teeth, hydroxyapatite material has become a commonly used material for studying the adhesion effect of Porphyromonas gingivalis. We weigh 2 mg of hydroxyapatite microspheres into a 1.5 mL centrifuge tube, wash twice with ddH2O, and finally add 1 mL of KCl buffer to each tube and incubate at room temperature for 2 hours. Centrifuge and discard the supernatant, add 200 μL of artificial saliva to each tube and incubate overnight at room temperature. Take 1×10 8Porphyromonas gingivalis in the logarithmic growth phase of CFU was dissolved in 100 μL of PBS buffer, and antibodies of 1G12 or 4E8 with a final concentration of 100 µg / mL were added. IgG was used as the control group. It was pre-incubated at 37 °C for 2 hours and mixed every 30 minutes. After the saliva-coated hydroxyapatite microspheres were washed twice with KCl buffer, the hydroxyapatite microspheres were resuspended with 100 uL of KCl buffer, and different treated Porphyromonas gingivalis were added and incubated at room temperature for 2 h to allow binding, and mixed every 30 minutes. Finally, the hydroxyapatite microspheres bound with Porphyromonas gingivalis were washed three times with KCl buffer and then fixed overnight in an appropriate fixing solution (2.5% glutaraldehyde + 4% paraformaldehyde). It was washed three times with 0.1 M PBS, 15 min each time. Gradient dehydration was carried out with ethanol, infiltrated with tert-butanol, then dried in a freeze dryer and images were collected by scanning electron microscopy ( Figure 8 A). To quantify the adhesion number, we extracted the genome of Porphyromonas gingivalis on the samples and analyzed the number of Porphyromonas gingivalis adhering to HAP by fluorescence quantitative PCR ( Figure 8 B); The results of scanning electron microscopy and fluorescence quantitative PCR are as Figure 8 shown. Compared with the blank control and IgG control groups, pretreatment with 1G12 or 4E8 significantly reduced the adhesion ability of Porphyromonas gingivalis.

[0051] Example 9: Analysis of the anti-inflammatory effects of 1G12 and 4E8 Human gingival fibroblasts (HGF) are the most abundant cells in the gingival connective tissue and are crucial for the remodeling and homeostasis of the periodontal tissue. Because of its close relationship with the occurrence and development of periodontal diseases, it has been widely used in the research of periodontal diseases. The primary isolation and culture of gingival fibroblasts were carried out by tissue block isolation method. HGF in passages 3-6 with good growth status was seeded into 6-well plates and cultured in a cell culture incubator; when the cell density reached about 80%, the FimA recombinant protein was co-incubated with 1G12, 4E8 and PBS control for 1 hour and then added to the HGF cells. After co-incubation in the incubator for 4 hours, the total cellular mRNA was extracted; after the total mRNA was reverse transcribed into cDNA, the expression levels of inflammatory cytokines IL-1β, IL-6 and TNF-α were determined by fluorescence quantitative PCR experiments. The experimental results are as Figure 9 shown, indicating that compared with the control group, the mRNA transcription levels of IL-1β, IL-6, and TNF-α in the experimental groups treated with 1G12 and 4E8 monoclonal antibodies were significantly down-regulated, indicating that both 1G12 and 4E8 can inhibit the expression of inflammatory factors caused by FimA protein and have anti-inflammatory effects.

[0052] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

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 heavy chain variable regions as shown in SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11, and light chain variable regions 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 conjugate or fusion, characterized in that: The fusion or conjugate is obtained by conjugating, modifying or fusion expressing the FimA monoclonal antibody protein according to claim 1 or 2.

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

6. A diagnostic reagent or kit, characterized in that: Comprising the FimA monoclonal antibody of claim 1 or 2, or the fusion or conjugate of claim 4.

7. A drug for treating Porphyromonas gingivalis infection-related diseases, characterized in that: Comprising the FimA monoclonal antibody of claim 1 or 2, or the nucleotide fragment of claim 3, or the conjugate or fusion of claim 4.

8. The drug for treating Porphyromonas gingivalis infection-related diseases according to claim 7, characterized in that: The Porphyromonas gingivalis infection-related conditions include cellular inflammation caused by FimA antigen.

9. The drug for treating Porphyromonas gingivalis infection-related diseases according to claim 8, characterized in that: The disease associated with Porphyromonas gingivalis infection is periodontitis.

10. A kind of inhibition P. gingivalis Bacterial adhesion ability, promotion P. gingivalis A biological agent for bacterial agglutination, characterized in that: Comprising the FimA monoclonal antibody of claim 1 or 2, or the biomaterial of claim 3, or the conjugate or fusion of claim 4.

Images