Broad-spectrum antibody targeting porphyromonas gingivalis FimA protein and application thereof

Abstract

The invention belongs to the fields of cellular immunology and genetic engineering, and relates to detection of porphyromonas gingivalis and an antibody. The broad-spectrum antibody is an antibody 4F12, the antibody 4F12 comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises VH-CDR1 of which the amino acid sequence is as shown in SEQ ID No.2, VH-CDR2 of which the amino acid sequence is as shown in SEQ ID No.3 and VH-CDR3 of which the amino acid sequence is as shown in SEQ ID No.4; the light chain variable region comprises a VL-CDR1 of which the amino acid sequence is as shown in SEQ ID No.6, a VL-CDR2 of which the amino acid sequence is as shown in SEQ ID No.7 and a VL-CDR3 of which the amino acid sequence is as shown in SEQ ID No.8. The invention provides a broad-spectrum antibody targeting porphyromonas gingivalis FimA protein, which can be combined with various FimA subtype porphyromonas gingivalis, and can be used for detecting the porphyromonas gingivalis and treating diseases related to porphyromonas gingivalis infection.

Description

Technical Field

[0001] The present invention belongs to the fields of cellular immunology and genetic engineering, and relates to the detection of Porphyromonas gingivalis and antibodies thereof. Background Art

[0002] Porphyromonas gingivalis ( P. gingivalis ) is a Gram-negative anaerobic bacillus and one of the main pathogenic bacteria of periodontitis. This bacterium can damage periodontal tissues, induce host immune inflammatory responses, and promote the formation of oral biofilms, resulting in alveolar bone resorption and tooth loss. Research shows that P. gingivalis the infection is not limited to the oral cavity and is closely related to systemic diseases. For example, P. gingivalis the infection can promote atherosclerosis; trigger autoimmune reactions in rheumatoid arthritis; promote neuroinflammation and β-amyloid deposition in Alzheimer's disease. Surveys show that severe periodontitis affects approximately 19% of adults globally (more than 1 billion cases, WHO, 2022), and there is an urgent need for new prevention and treatment strategies targeting its key virulence factors. The fimbriae formed by the polymerization of FimA protein are the key virulence factors of Porphyromonas gingivalis, which are responsible for mediating the adhesion of Porphyromonas gingivalis to host tissues and cells (such as gingival epithelial cells and fibroblasts), determining the initiation of infection, and participating in the formation and immune escape of oral bacterial biofilms. In addition, research shows that FimA induces the release of pro-inflammatory factors (such as IL-1β, IL-6) by activating the TLR2 / NF-κB signaling pathway, exacerbating periodontal tissue destruction and periodontitis. Given the important role of FimA in Porphyromonas gingivalis infection and related diseases such as periodontitis, it has become an ideal target for the development of diagnostic reagents and therapeutic drugs.

[0003] Currently, the detection of Porphyromonas gingivalis mainly relies on traditional bacterial culture, 16S rRNA gene sequencing, or real-time quantitative PCR. However, the culture method is time-consuming (5 - 7 days) and has low sensitivity; molecular detection is costly and requires professional equipment, making it difficult to popularize. In terms of treatment, conventional mechanical debridement combined with broad-spectrum antibiotics (such as metronidazole) is prone to drug resistance and cannot specifically block the recolonization of Porphyromonas gingivalis. In addition, there are multiple subtypes (I - V and Ib) of Porphyromonas gingivalis FimA protein, and FimA-II and IV subtypes are highly correlated with aggressive periodontitis, cardiovascular diseases, and rheumatoid arthritis, accounting for more than 70% of clinical isolates. The antigenic epitope differences among different subtypes lead to problems such as low coverage and high false-negative risk in traditional detection methods (such as single-subtype antibodies). Currently, most antibodies against Porphyromonas gingivalis FimA protein are of FimA-I type and FimA-II type, which are difficult to cover other highly pathogenic subtypes (such as type IV), resulting in a risk of missed detection. Therefore, the development of broad-spectrum antibodies targeting FimA protein can not only achieve rapid and highly sensitive pathogen detection but also provide precise treatment by blocking bacterial adhesion or neutralizing virulence. Summary of the Invention

[0004] In view of the above technical problems, the present application discloses a broad-spectrum antibody targeting Porphyromonas gingivalis FimA protein and its application.

[0005] To achieve the object of the invention, the present invention provides the following technical solutions: The present application provides a broad-spectrum antibody targeting Porphyromonas gingivalis FimA protein, named antibody 4F12, and the broad-spectrum antibody comprises a heavy-chain variable region and a light-chain variable region.

[0006] The above heavy-chain variable region comprises VH-CDR1 with the amino acid sequence shown in SEQ ID No.2, VH-CDR2 with the amino acid sequence shown in SEQ ID No.3, and VH-CDR3 with the amino acid sequence shown in SEQ ID No.4; the light-chain variable region comprises VL-CDR1 with the amino acid sequence shown in SEQ IDNo.6, VL-CDR2 with the amino acid sequence shown in SEQ ID No.7, and VL-CDR3 with the amino acid sequence shown in SEQ ID No.8.

[0007] The above heavy-chain variable region has a similarity of more than 85% with the amino acid sequence shown in SEQ ID No.1; the light-chain variable region has a similarity of more than 85% with the amino acid sequence shown in SEQ ID No.5.

[0008] Further, the amino acid sequence of the above heavy-chain variable region is as shown in SEQ ID No.1. The amino acid sequence of the light-chain variable region is as shown in SEQ ID No.5.

[0009] A nucleotide molecule encoding the broad-spectrum antibody as described above; further, the nucleic acid molecule encoding the heavy-chain variable region of the antibody comprises a nucleotide sequence as shown in SEQ ID No.9; further, the nucleic acid molecule encoding the light-chain variable region of the antibody comprises a nucleotide fragment sequence as shown in SEQ ID No.10.

[0010] A fusion or conjugate, which is obtained by conjugating or fusing and expressing the above broad-spectrum antibody protein.

[0011] A biological material, which is an expression fragment, recombinant vector, recombinant bacterium or transgenic cell line containing the above nucleotide fragment.

[0012] A kit, which comprises the above broad-spectrum antibody protein obtained by conjugating or fusing and expressing, or the conjugate or fusion as claimed in the claims.

[0013] A drug for treating diseases related to Porphyromonas gingivalis infection, comprising all or part of the variable region protein of the above-mentioned broad-spectrum antibody, or the above-mentioned nucleotide fragment, or the above-mentioned conjugate or fusion, or the above-mentioned biological material.

[0014] The above-mentioned diseases related to Porphyromonas gingivalis infection include cell inflammation caused by FimA-I, FimA-II, FimA-III, FimA-IV and FimA-Ib protein antigens. The antibody 4F12 of the present application can achieve broad-spectrum specific recognition of FimA-I, FimA-II, FimA-III, FimA-IV and FimA-Ib protein antigens.

[0015] The present invention has the following beneficial effects: The broad-spectrum antibody targeting Porphyromonas gingivalis FimA protein disclosed by the present invention can strongly bind to multiple FimA subtype proteins and Porphyromonas gingivalis, can cover most clinical strains to achieve rapid detection of clinical samples; can block the adhesion of Porphyromonas gingivalis and reduce Porphyromonas gingivalis infection; can reduce the expression of cell inflammatory factors induced by FimA protein and inhibit the pro-inflammatory reaction of Porphyromonas gingivalis. Therefore, the present invention provides a broad-spectrum antibody targeting Porphyromonas gingivalis FimA protein, which can bind to multiple FimA subtype Porphyromonas gingivalis, and can be used for the detection of Porphyromonas gingivalis and the treatment of diseases related to Porphyromonas gingivalis infection. Description of the Drawings

[0016] 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.

[0017] Figure 1 For SDS-PAGE analysis of antigens and antibodies.

[0018] Figure 2 For the analysis of the sensitivity and specificity of antibody 4F12.

[0019] Figure 3 For the analysis of the broad-spectrum binding of antibody 4F12 to FimA.

[0020] Figure 4 For the analysis of the diagnostic detection ability of antibody 4F12.

[0021] Figure 5 For the analysis of the blocking adhesion effect of antibody 4F12.

[0022] Figure 6Analysis of the anti-inflammatory effect of antibody 4F12. Detailed implementation manners

[0023] 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 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.

[0024] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0025] The present application provides a broad-spectrum antibody targeting the FimA protein of Porphyromonas gingivalis, named antibody 4F12, and the broad-spectrum antibody includes a heavy-chain variable region and a light-chain variable region.

[0026] The above heavy-chain variable region includes VH-CDR1 with the amino acid sequence shown in SEQ ID No. 2, VH-CDR2 with the amino acid sequence shown in SEQ ID No. 3, and VH-CDR3 with the amino acid sequence shown in SEQ ID No. 4; the light-chain variable region includes VL-CDR1 with the amino acid sequence shown in SEQ ID No. 6, VL-CDR2 with the amino acid sequence shown in SEQ ID No. 7, and VL-CDR3 with the amino acid sequence shown in SEQ ID No. 8.

[0027] The above heavy-chain variable region has a similarity of more than 85% with the amino acid sequence shown in SEQ ID No. 1; the light-chain variable region has a similarity of more than 85% with the amino acid sequence shown in SEQ ID No. 5.

[0028] The amino acid sequence of the above heavy-chain variable region is shown in SEQ ID No. 1. The amino acid sequence of the light-chain variable region is shown in SEQ ID No. 5.

[0029] A nucleotide molecule encoding the broad-spectrum antibody as described above; further, the nucleic acid molecule encoding the heavy-chain variable region of the antibody includes a nucleotide sequence shown in SEQ ID No. 9; further, the nucleic acid molecule encoding the light-chain variable region of the antibody includes a nucleotide fragment sequence shown in SEQ ID No. 10.

[0030] The specific steps are as follows: Example 1: Preparation of monoclonal antibody targeting Porphyromonas gingivalis FimA Design specific primers according to the gene sequence of Porphyromonas gingivalis FimA-I protein: Upstream primer FimA-F: CTAGCTAGCATGAAAAAAACAAAG; Downstream primer FimA-R: CCGCTCGAGCCAAGTAGCATTCTG; Using the genome as a template for PCR amplification, and cloning and constructing it into the pET-28a-His vector to form the pET28a-FimA / His recombinant plasmid.

[0031] Transform the recombinant plasmid into the Escherichia coli BL21( DE3 ) expression strain, and induce the expression of the target protein for 12 hours by IPTG. After the cells were sonicated, the FimA recombinant protein (46 kDa) was detected in the supernatant by SDS-PAGE electrophoresis. Subsequently, the supernatant was purified by Ni column affinity chromatography to obtain a high-purity FimA recombinant protein ( Figure 1 in A), and stored for later use.

[0032] Using the prepared FimA recombinant protein as an antigen, 6-8-week-old BALB / c mice were selected for immunization experiments (100 μg / mouse), and non-immunized mice were set as negative controls at the same time.

[0033] The immunization protocol used the Freund's adjuvant system: complete Freund's adjuvant (FCA) was used for the first immunization, and incomplete Freund's adjuvant (FIA) was replaced for the second and third booster immunizations. A total of three immunizations were carried out at two-week intervals. One week after the last immunization, serum was collected, diluted in gradients, and ELISA titers were detected with the FimA recombinant protein (coating concentration of 2 μg / mL). The mouse with the highest titer was selected for a terminal boost immunization (intraperitoneal injection of 50 μg FimA recombinant protein). Three days later, splenocytes were prepared and fused with SP2 / 0 myeloma cells. When the hybridoma cells grew to 30% confluence, the culture supernatant was collected for ELISA primary screening. The results showed that the antibody titer secreted by the 4F12 hybridoma cells was significant and the cell proliferation was stable. Subsequently, the 4F12 hybridoma cells expressing the positive antibody were expanded in culture, and the 4F12 monoclonal antibody was prepared using the conventional in vivo ascites induction method. The 4F12 monoclonal antibody was purified from mouse ascites using a Protein A / G affinity chromatography column and an AKTA purification system, and two clear bands were obtained by SDS-PAGE electrophoresis ( Figure 1 B): the molecular weight of the heavy chain was 50 kDa, and the molecular weight of the light chain was 25 kDa. Further identification by a monoclonal antibody subtype detection kit determined that the heavy chain of the 4F12 antibody was of the IgG1 type and the light chain was of the κ type ( Figure 1 C).

[0034] Sequencing identified that the amino acid sequence of the heavy chain variable region of the broad-spectrum antibody targeting Porphyromonas gingivalis FimA protein is as shown in SEQ ID No.1; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.2. The gene sequence encoding the heavy chain variable region of the antibody is as shown in SEQ ID No.9; the gene sequence encoding the light chain variable region of the antibody is as shown in SEQ ID No.10.

[0035] SEQ ID No.1 (heavy chain variable region): QAQLKESGPALVAPSRSLSITCTVSGFSLTSSGVHWVRQFPGKGLEWLGVIWSGGSTTYNSALMSRLSISKDNSKSQAFLRMSSLQTDDTAMYYCARDYNYGSYYVPFAYWGQGTLVTVSA; SEQ ID No.2 (VH-CDR1): GFSLTSSG; SEQ ID No.3 (VH-CDR2): IWSGGST; SEQ ID No.4 (VH-CDR3): ARDYNYGSYYVPFAY; SEQ ID No.5 (light chain variable region): VHDIQMTQTTSSLSASLGDRVTISCRTSQDISNFLNWFQQKPDGTVKLLIYFTSRLHSGVPSRFSGSGSGTDYSLTISNLEHEDIATYFCQQGHTLPWTFGGGTKLEIK; SEQ ID No.6 (VL-CDR1): QDISNF; SEQ ID No.7 (VL-CDR2): FTS; SEQ ID No.8 (VL-CDR3): QQGHTLPWT; SEQ ID No.9 (nucleic acid molecule of heavy chain variable region): CAGGCGCAGCTGAAGGAGTCAGGACCTGCCCTGGTGGCGCCCTCACGAAGCCTGTCCATCACTTGCACTGTCTCCGGGTTTTCATTAACCAGTTCTGGTGTACACTGGGTTCGCCAGTTTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGTCTGGTGGAAGCACAACTTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCGAAGAGCCAAGCTTTCTTAAGAATGAGCAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGATTATAACTACGGAAGTTACTACGTCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA; SEQ ID No. 10 (Nucleic acid molecule of light chain variable region): GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGACAAGTCAGGACATTAGCAATTTTTTAAACTGGTTTCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATTTACTTTACATCAAGATTACATTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAACATGAAGATATTGCCACTTACTTTTGCCAACAAGGTCATACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

[0036] Example 2: Sensitivity and Specificity Analysis of Monoclonal Antibody 4F12 The sensitivity of the antibody was detected by enzyme-linked immunosorbent assay (ELISA). The purified recombinant FimA protein was diluted with the coating solution, and 100 μL per well was coated in a 96-well plate. After sealing, it was placed at 4 °C overnight. The plate was washed 5 times with the Wash Buffer working solution. Then, 200 μL of the blocking solution was added to block the non-specific binding sites, and the blocking was carried out at room temperature for 2 hours. After washing with the Wash Buffer working solution, 100 μL of the FimA monoclonal antibody with a gradient of 1 μg / mL was added per well, and the reaction was carried out at 37 °C for 2 hours. After washing with the Wash Buffer working solution, the HRP-labeled rabbit anti-mouse secondary antibody diluted 1:10,000 was added, and it was incubated at 37 °C for 1 hour. The plate was washed 3 times with the Wash Buffer working solution, then the TMB chromogenic solution was added, and after incubating at 37 °C for 15 minutes, the stop solution was added and the OD450 value was measured.

[0037] The results were as Figure 2 shown in A. The IC50 value of the antibody 4F12 was 0.06 μg / mL, indicating high binding sensitivity to the FimA-I protein.

[0038] Porphyromonas gingivalis and other common oral bacteria (Streptococcus oralis, Streptococcus mitis, Streptococcus intermedius, Streptococcus gordonii, Streptococcus mutans, Escherichia coli, Staphylococcus aureus) were coated onto a 96-well ELISA plate, and the coating amount was about 1×10 8 CFU / well, and it was placed at 4 °C overnight. Then it was washed 3 times with PBST; blocked with 3% BSA at 37 °C for 1 h and washed 3 times; the antibody 4F12 (1 μg / mL) was added and incubated at 37 °C for 1 h, and then washed 3 times; the HRP-labeled rabbit anti-mouse IgG secondary antibody was added and incubated at 37 °C for 1 hour. After washing 3 times, TMB was added for color development in the dark for 15 minutes, the stop solution was added, and the absorbance OD450 value was measured immediately.

[0039] Result analysis ( Figure 2 B) showed that the antibody 4F12 showed high binding ability to Porphyromonas gingivalis, while the binding to other common oral bacteria was very weak and there was no significant change compared with the negative control.

[0040] Furthermore, to visually observe the binding of the antibody 4F12 to Porphyromonas gingivalis, the antibody was labeled with FITC and a fluorescence binding experiment was carried out. First, the purified 4F12 antibody was diluted to an alkaline environment with carbonate buffer (pH 9.0 - 9.5), and the FITC solution (final concentration 1 mg / mL) was slowly added at a ratio of 1:30. It was stirred overnight at 4 °C in the dark. 50 mM glycine (pH 7.4) was added to terminate the reaction, and it was incubated at room temperature in the dark for 30 minutes. Subsequently, the free FITC was removed by ultrafiltration tube binding and PBS replacement. After verifying the successful labeling, it was aliquoted and stored in the dark.

[0041] The Porphyromonas gingivalis was evenly smeared and then air-dried, fixed with 4% paraformaldehyde, blocked with 5% BSA and washed three times, 4F12-FITC was added for binding, and an irrelevant antibody was used as a control. It was incubated overnight at 4°C, washed and mounted, and observed, photographed and analyzed under a fluorescence microscope. The results are as Figure 2 shown in Figure C, the antibody 4F12 labeled with FITC could efficiently bind to Porphyromonas gingivalis.

[0042] Example 3: Broad-spectrum analysis of monoclonal antibody 4F12 against FimA protein of Porphyromonas gingivalis Existing studies have shown that FimA has multiple subtypes (I-V and Ib), among which FimA-II and FimA-IV are common clinical strain subtypes. We respectively 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, incubated overnight at 4°C, after blocking, added anti-FimA monoclonal antibody for binding, after washing twice, added HRP-labeled anti-mouse IgG secondary antibody for binding, rinsed and added TMB for color development, measured the OD450 value and analyzed it.

[0043] The ELISA results ( Figure 3 A) showed that the antibody 4F12 could strongly bind to FimA-I, FimA-II, FimA-III, FimA-IV and FimA-Ib, and weakly bind to the FimA-V protein.

[0044] To further detect the detection sensitivity of 4F12 to different subtype FimA proteins, different FimA genotype antigens at 1 μg / mL, including FimA-I, FimA-II, FimA-III, FimA-IV, FimA-V and FimA-Ib recombinant proteins (1 μg / mL), were coated onto ELISA plates and incubated overnight at 4°C. Blocking was carried out by incubating with 3% BSA at 37°C for 1 h, and then washed three times with PBST buffer; different concentrations of the antibody 4F12 were prepared and added to the ELISA plates and incubated at 37°C for 1 hour, and washed three times; HRP-labeled rabbit anti-mouse IgG secondary antibody was added and incubated at 37°C for 1 hour, and washed three times; TMB was added for color development, incubated in the dark at 37°C for 15 minutes, and the OD450 value was detected after adding the stop solution.

[0045] The results ( Figure 3 B) were consistent with the previous results ( Figure 3 A), the antibody 4F12 could strongly bind to FimA-I, FimA-II, FimA-III, FimA-IV and FimA-Ib, and showed weak binding to the FimA-V protein.

[0046] Example 4: Detection and analysis of monoclonal antibody 4F12 in oral swab samples Saliva and plaque samples were collected by rotating a sterile oral swab more than 5 times at the gingival margin of the volunteers' oral cavity. After immersion in pre-cooled PBS, the swab was vortexed to release the bacteria. The supernatant was discarded by centrifugation, and the bacterial suspension was resuspended in 1 mL of PBS. The suspension was divided into two equal parts, and PBS was added as a negative control. After extracting the genomic DNA from the sample suspension in the first part, positive samples for Porphyromonas gingivalis nucleic acid were screened by quantitative PCR. Subsequently, specific primers for different subtypes of FimA (I-V and Ib) (Pan S, etal. PLoS One . 2017) were used for multiplex PCR amplification, and the FimA protein subtypes of Porphyromonas gingivalis detected were analyzed by 2% agarose gel electrophoresis. The results showed ( Figure 4 A, marked with *) that sample 1 was FimA-II type and trace amount of FimA I type; sample 2 was FimA-IV; sample 3 was FimA-II type mixed with a small amount of FimA-Ib type; sample 4 was FimA-I type mixed with trace amount of FimA-II type; there was no specific band in the PBS negative control. In the second part, the sample suspension was coated on a 96-well plate, fixed overnight at 4°C, blocked with 3% BSA buffer at 37°C for 1 hour, the liquid was discarded, washed 3 times, and patted dry. Antibody 4F12 was added, and mouse IgG was used as a control. Incubate at 37°C for 1 hour, wash 3 times; add HRP-labeled rabbit anti-mouse secondary antibody, incubate at 37°C in the dark for 1 hour, wash 4 times to completely remove the unbound secondary antibody; add 100 μL of TMB chromogenic solution to each well, and react at room temperature in the dark for 15 minutes. Add 50 μL of stop solution, and immediately read the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader.

[0047] The results ( Figure 4 B) analysis showed that the broad-spectrum antibody 4F12 of the present invention could efficiently detect Porphyromonas gingivalis infection with different FimA subtypes.

[0048] Example 5: Analysis of the blocking adhesion effect of monoclonal antibody 4F12 Weighed 2 mg of hydroxyapatite (HAP) into a 1.5 mL EP tube, washed it twice with 1 mL of ddH2O, and finally added 1 mL of KCl buffer to each tube and incubated at room temperature for 2 h; centrifuged to discard the supernatant, added 200 µL of artificial saliva, and incubated overnight at room temperature. Took an appropriate amount of Porphyromonas gingivalis in the logarithmic growth phase, washed it with KCl buffer, and adjusted the bacterial titer to 1×10 9CFU / mL, add antibody 4F12 to a final concentration of 100 μg / mL, use IgG as a control, incubate at 37 °C for 2 hours, and mix evenly at intervals. After washing the saliva-coated hydroxyapatite microspheres twice with KCl buffer, resuspend the hydroxyapatite microspheres in 100 μL of KCl buffer, add the antibody-pretreated Porphyromonas gingivalis, and incubate at room temperature for 2 hours to allow pre-HAP binding, mixing once every 30 minutes. Finally, gently wash the hydroxyapatite microspheres bound with Porphyromonas gingivalis three times with KCl buffer.

[0049] On the one hand, prepare the scanning electron microscope sample by conventional fixation, drying, and gold spraying, and use the scanning electron microscope to take pictures and collect images ( Figure 5 A). On the other hand, extract the genome of Porphyromonas gingivalis adhered to the sample, and analyze the number of Porphyromonas gingivalis adhered to HAP by conventional fluorescence quantitative PCR ( Figure 5 B); the results of scanning electron microscopy and fluorescence quantitative PCR show that compared with the IgG control group, antibody 4F12 significantly reduces the adhesion of Porphyromonas gingivalis.

[0050] Example 6: Analysis of the inhibitory effect of monoclonal antibody 4F12 on pro-inflammatory responses Studies have shown that FimA can stimulate the release of pro-inflammatory factors by epithelial cells such as gingival fibroblasts, trigger inflammation, promote osteoclast differentiation and alveolar bone resorption, and exacerbate the progression of periodontitis. Here, we detected whether antibody 4G12 could inhibit the pro-inflammatory effect induced by FimA. First, seed well-grown HGF in a 6-well plate (2×10 5 cells / well), and place it in a cell culture incubator for culture; when the cell density reaches about 80%, co-incubate FimA-I and FimA-IV proteins with 4F12, use PBS as a control, add it to HGF cells after 1 hour, and co-incubate in the incubator for 4 hours, then extract the total cellular mRNA; reverse transcribe the total mRNA into cDNA by conventional methods; finally, detect the expression levels of inflammatory cytokines IL-1β, IL-6, and TNF-α by fluorescence quantitative PCR experiments.

[0051] The experimental results are as Figure 6 shown, antibody 4F12 can effectively reduce the expression of inflammatory factors induced by FimA protein, thereby inhibiting the pro-inflammatory response caused by Porphyromonas gingivalis.

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

Claims

1. A broad-spectrum antibody targeting FimA protein of Porphyromonas gingivalis, characterized in that: The broad-spectrum antibody is antibody 4F12, which includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes an amino acid sequence as shown in SEQ ID No.2, VH-CDR1 as shown in SEQ ID No.3, and VH-CDR3 as shown in SEQ ID No.4; the light chain variable region includes an amino acid sequence as shown in SEQ ID No.6, VL-CDR2 as shown in SEQ ID No.7, and VL-CDR3 as shown in SEQ ID No.

8.

2. The broad-spectrum antibody targeting FimA protein of Porphyromonas gingivalis according to claim 1, characterized in that: The heavy chain variable region has more than 85% similarity with the amino acid sequence shown in SEQ ID No.1; the light chain variable region has more than 85% similarity with the amino acid sequence shown in SEQ ID No.

5.

3. The nucleotide fragment encoding the broad-spectrum antibody targeting the FimA protein of Porphyromonas gingivalis according to claim 2, characterized in that: The nucleotide fragments include a nucleotide fragment encoding a heavy chain variable region as shown in SEQ ID No.9 and a nucleotide fragment encoding a light chain variable region as shown in SEQ ID No.

10.

4. A fusion or conjugate, characterized in that: The fusion or conjugate is obtained by conjugating, modifying or fusion expressing the broad-spectrum 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 1 or 2.

6. A kit, characterized in that: The kit comprises the broad-spectrum antibody protein according to claim 1 or 2 that is subjected to conjugation modification or fusion expression, or comprises the conjugate or fusion according to claim 4.

7. Use of the kit according to claim 6 in preparing a kit for detecting Porphyromonas gingivalis.

8. A medicine, characterized in that: The drug comprises any one of the broad-spectrum antibody according to claim 1 or 2, the fusion or conjugate according to claim 4, and the biomaterial according to claim 5.

9. Use of the drug according to claim 8 in the preparation of a drug for treating diseases associated with Porphyromonas gingivalis infection.

10. The use according to claim 9, characterized in that: The Porphyromonas gingivalis infection-related diseases include cellular inflammation caused by one or more of the FimA-I, FimA-II, FimA-III, FimA-IV and FimA-Ib protein antigens.

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