Single-chain antibody for specifically recognizing porphyromonas gingivalis

Abstract

The invention belongs to the technical field of biology, and discloses porphyromonas gingivalis resistant single-chain antibodies MB004, MB005, MB022, MB023, MB028 and MB032, the six single-chain antibodies are all combined with porphyromonas gingivalis rHA2, the binding force between the MB004 antibody and HA2 protein is the highest, and gt is recognized; the porphyromonas gingivalis of 5 * 10 < 6 > CFU / ml has no cross reaction with F.nucleatum, S.mutans, A.viscosus, P.intermedia and A.actinocula, has good specificity and sensitivity, and can be applied to preparation of drugs for preventing and treating diseases caused by the porphyromonas gingivalis, preparation of detection tools for the porphyromonas gingivalis and preparation of diagnosis tools for the diseases caused by the porphyromonas gingivalis.

Description

Technical Field

[0001] The present invention belongs to the fields of gene and antibody engineering, relates to the biomedical field, and new technologies for the diagnosis and treatment of immune-related diseases. The present invention relates to antibodies that specifically recognize Porphyromonas gingivalis and their applications. Background Art

[0002] Periodontal disease, as a common and frequently-occurring oral disease, is a chronic inflammatory disease caused by bacterial infection. As the disease progresses, periodontal disease can lead to the destruction of periodontal supporting tissues and ultimately cause tooth loss. Epidemiological surveys show that more than half of the adults worldwide suffer from periodontitis, and about 10% suffer from severe periodontitis. Periodontal disease is associated with systemic diseases such as chronic obstructive pulmonary disease, cardiovascular and cerebrovascular diseases, diabetes, preterm birth, Alzheimer's disease, rheumatoid arthritis, oral cancer, and colorectal cancer.

[0003] P.g is an obligately anaerobic Gram-negative bacterium. As an important virulence factor, HA2 binds to the porphyrin ring of heme in hemoglobin to provide the iron and porphyrin necessary for the survival of P.g. P.g adheres to and colonizes the periodontal tissue and is the main pathogenic bacterium of periodontal disease. The number of P.g bacteria at the infection site of periodontal disease is closely related to the prognosis of patients. Inhibiting the growth of P.g can prevent and treat periodontitis. The oral level of P.g can predict the progression of periodontal disease in the next 3 months. P.g is also associated with systemic diseases such as chronic obstructive pulmonary disease, cardiovascular and cerebrovascular diseases, diabetes, preterm birth, Alzheimer's disease, rheumatoid arthritis, oral cancer, and colorectal cancer.

[0004] Clinically, antibiotics and antibacterial agents such as metronidazole and chlorhexidine are commonly used for the treatment of P.g infections. However, the treatment with antibiotics and antibacterial agents is likely to cause side effects and the emergence of drug-resistant strains. There is a need for P.g antibacterial drugs with strong specificity, low side effects, and no drug resistance. Single-chain antibodies have the advantages of strong specificity, low side effects, and no drug resistance.

[0005] P.g detection methods such as bacterial culture, nucleic acid detection, enzymatic detection, and antigen-antibody detection can specifically detect Porphyromonas gingivalis, but they are time-consuming and laborious, require special equipment and professional technical personnel, and do not meet the requirements of simple operation, rapidity, low cost, and easy readability of results. It is difficult to be promoted clinically and in the market. There is a need for a P.g detection tool that is fast, simple, low-cost, and easy to interpret. Colloidal gold test strips have the advantages of being fast, simple, low-cost, and readable with the naked eye, and can be used for auxiliary screening, diagnosis, and monitoring of diseases caused by P.g. The preparation of colloidal gold test strips requires anti-P.g monoclonal antibodies. Using genetic engineering technology, single-chain antibodies can be used to prepare anti-P.g antibodies. Summary of the Invention

[0006] In view of this, the present invention provides a single-chain antibody that specifically recognizes P.g, including the DNA sequence and amino acid sequence of the P.g single-chain antibody.

[0007] The DNA sequence of the single-chain antibody (scFv) against Porphyromonas gingivalis is shown in SEQ ID No. 1.

[0008]

[0009]

[0010] The amino acid sequence of the single-chain antibody (scFv) against Porphyromonas gingivalis is shown in SEQ ID No. 2.

[0011]

[0012]

[0013]

[0014] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0015] The present invention provides an antigen for preparing an anti-P.g single-chain antibody and an animal immunization method.

[0016] The present invention provides a method for constructing an anti-P.g hemagglutinin 2 single-chain antibody library by phage display technology, including primer sequences for PCR amplification of antibody heavy chain and light chain variable region genes, synthesis of single-chain antibodies, phagemid construction methods, phage library screening methods, antibody expression vector construction methods, and antibody expression methods.

[0017] The present invention provides that the P.g single-chain antibody can be modified into antibodies such as bispecific variable region antibodies, trispecific variable region antibodies, multispecific variable region antibodies, bivalent tandem antibodies, trivalent tandem antibodies, multivalent tandem antibodies, and intact antibodies containing Fc fragments.

[0018] The present invention also provides the application of the anti-P.g single-chain antibody (scFv) in the preparation of prevention and treatment tools, diagnostic detection tools, and scientific research fields for P.g-related diseases. The P.g-related diseases include, but are not limited to, infectious diseases caused by P. gingivalis, gingivitis, periodontitis, chronic obstructive pulmonary disease, cardiovascular and cerebrovascular diseases, diabetes, premature birth, Alzheimer's disease, rheumatoid arthritis, oral cancer, intestinal cancer, and other tumors.

[0019] Drawings and their brief descriptions

[0020] Figure 1 Showing total cellular RNA. Note: M: Maker, 1: RNA

[0021] Figure 2Show the PCR amplification products of VL and VH genes. Note: A: VL gene, M: Maker, 1: VL (Kappa chain), 2: VL (Lamda chain); B: VH gene, M: Maker, 3: VH

[0022] Figure 3 Show the PCR amplification products of scFv gene. Note: M: Maker, 1, 2: VL (Lamda chain)-linker-VH, 3: VL (Kappa chain)-linker-VH

[0023] Figure 4 Show the library capacity of the phage antibody library after 3 rounds and 4 rounds of panning.

[0024] Figure 5 Show the binding ability of the monoclonal antibody to HA2.

[0025] Figure 6 Show the PCR amplification products of the target genes. Note: M: Maker, 1: MB004-VH, 2: MB004-VL, 3: MB005-VH, 4: MB005-VL, 5: MB022-VH, 6: MB022-VL, 7: MB023-VH, 8: MB023-VL, 9: MB028-VH, 10: MB028-VL, 11: MB032-VH, 12: MB032-VL

[0026] Figure 7 Show the detection of the target protein by SDS-PAGE

[0027] Figure 8 Show the binding activity of the single-chain antibody to P.g rHA2

[0028] Figure 9 Show the titer of the single-chain antibody against P.g

[0029] Figure 10 Show the specificity of the single-chain antibody in recognizing P.g

[0030] Figure 11 Show the sensitivity of the single-chain antibody in recognizing P.g Detailed implementation manners

[0031] The present invention discloses a single-chain antibody against Porphyromonas gingivalis. Those skilled in the art can refer to the content herein, appropriately modify the process parameters to obtain the antibody, and realize its application. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The preparation method and application of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the preparation method and application herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] The reagents and raw materials used in the anti-Porphyromonas gingivalis single-chain antibody provided by the present invention can all be purchased from the market.

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further elaborated below in conjunction with embodiments:

[0034] Term Explanation:

[0035]

[0036] Example 1: Mouse Immunization and Serum Titer Detection

[0037] 1 Mouse Immunization

[0038] Using rHA2 as the immunogen, 3 healthy female Balb / c mice were selected. For the first immunization, after emulsifying 50 μg / mouse of antigen rHA2 with an equal volume of Freund's complete adjuvant, the mice were immunized by multi-point subcutaneous injection in the abdomen; two weeks later, after emulsifying 50 μg / mouse of antigen HA2 with an equal volume of Freund's incomplete adjuvant, the mice were immunized by multi-point subcutaneous injection in the abdomen. Immunization was carried out once every 2 weeks, and the protocol was the same as the second immunization. Starting from the 3rd immunization, blood was collected 7 days after immunization and the antibody titer in the mouse blood was measured by indirect ELISA. If the titer did not reach above 1:104, a booster immunization was carried out again after an interval of two weeks.

[0039] 2 ELISA Titer Detection

[0040] (1) Protein Coating: In the experimental group, the immunizing antigen rHA2 was diluted to 5 μg / mL with the coating solution, and the control group was added with the coating solution, 100 μL / well, coated overnight at 4°C, washed 2 times with PBST, and the plate was patted dry.

[0041] (2) Blocking: 3% skim milk powder, 380 μL / well, incubated at room temperature for 1 h, washed 2 times with PBST, and the plate was patted dry.

[0042] (3) Sample Addition: The serum was diluted to the specified concentration, 100 μL / well, incubated at room temperature for 1 h, washed 2 times with PBST, and the plate was patted dry.

[0043] (4) Addition of Secondary Antibody: Rabbit anti-mouse IgG-HRP 1:4000, 100 μL / well, incubated at room temperature for 1 h, washed 3 times with PBST, and the plate was patted dry.

[0044] (5) Color Development: TMB Color Development Solution A:B = 1:1, 100 μL / well, reacted at room temperature for 20 min.

[0045] (6) Termination: 2M sulfuric acid, 50 μL / well, immediately measure the OD value at 450 nm in each well.

[0046] (7) Result determination: Taking the ratio of the OD value (T) of the serum group to the OD value (C) of the control group being greater than 2 (T / C > 2) as the limit, it is determined as the titer critical value.

[0047] Example 2 Total RNA Extraction and Reverse Transcription

[0048] 1 RNA Extraction

[0049] After grinding the tissues of immunized mice, add Trizol. After adding chloroform, shake the test tube vigorously for 1 min and let it stand for 5 min. Centrifuge at 4°C, 11000 rpm for 10 min, and take the supernatant. Repeat step 2. Add an equal volume of isopropanol to the supernatant, mix well, and let it stand at room temperature for 30 min. Centrifuge at 4°C, 11000 rpm for 10 min, and discard the supernatant. Add 1 mL of 75% ethanol to the precipitate and wash the precipitate twice. Centrifuge at 4°C, 7000 rpm for 5 min, and discard the supernatant. Air-dry at room temperature and dissolve with an appropriate amount of DEPC water.

[0050] 2 cDNA Synthesis

[0051] Synthesize cDNA by reverse transcription according to the requirements of the reverse transcription kit instructions. The input amount of RNA template is 20 μg, and the reverse transcription volume is 100 μL.

[0052] The reverse transcription system is as follows:

[0053]

[0054] Heat at 65°C for 5 min, quickly place it on ice for rapid cooling, and let it stand on ice for 2 min.

[0055]

[0056] Gently pipette and mix well. Incubate at 42°C for 2 min.

[0057]

[0058] Incubate at 50°C for 50 min, 85°C for 5 s.

[0059] Example 3 Construction of Phagemid

[0060] 1 PCR Amplification of VL and VH Genes

[0061] PCR amplify the antibody heavy chain and light chain variable region gene sequences. The amplification system (20 μL) for each pair of primers is shown in Table 20, and the amplification program is shown in Table 21. The primer sequences for amplifying the antibody light chain and heavy chain variable region genes are shown in Table 22.

[0062] Table 20 PCR Amplification System

[0063]

[0064] Table 21 PCR Amplification Program

[0065]

[0066] Table 22 Primer Sequences of PCR Amplifying Antibody Light Chain and Heavy Chain Variable Region Genes

[0067]

[0068]

[0069] 2 Synthesis of scFv Single-chain Antibody

[0070] The PCR products of the light chain and heavy chain variable region genes were mixed in equal amounts, and the single-chain antibody gene was obtained by SOE-PCR technology. The expected fragment size was 800 bp. SOE-PCR primer sequences (5′→3 ′ ): F-ACTGCTGCTTGGCCCAAGCGGCC; R-GAGGAGGAGGGCCGACGGGGCCTG. The amplification system (taking 50 μL as an example) is shown in Table 23, and the amplification program is shown in Table 24.

[0071] Table 23 Overlap PCR Amplification System

[0072]

[0073] Table 24 Overlap PCR Amplification Program

[0074]

[0075] 3 Phagemid Construction

[0076] The scFv fragment was recovered by gel using a gel extraction kit according to the instructions, and the recovered scFv fragment and the pcomb3X vector were digested with sfiI enzyme respectively (the digestion system is shown in Table 25). After digestion, the scFv fragment was directly purified using a gel extraction purification column, and the pcomb3X vector was recovered as a linear vector using a gel extraction kit. The scFv fragment and pcomb3X were mixed and ligated overnight at 4°C using T4 ligase (the ligation system is shown in Table 26).

[0077] Table 25 Digestion System (taking 50 μL of digestion system as an example)

[0078]

[0079] After mixing the above digestion systems, they were placed in a 50°C water bath for 60 min, and then gel extraction was performed.

[0080] Ligate the recovered vector with the target fragment, and incubate overnight at 4°C. The reaction system is 600 μL.

[0081] Table 26 Ligation reaction system

[0082]

[0083] Example 3: Electroporation and library quality analysis

[0084] 1 Mix the purified ligation product with XL1-Blue competent cells on ice, aliquot 80 μL per cup into electroporation cuvettes, and perform electroporation at 1800 V. After electroporation, transfer the mixture into 2YT-ATG medium containing ampicillin and tetracycline, and make up to 200 mL with 2×YT-ATG. Incubate the library at 37°C and 250 rpm for 1 hour to recover, and this is the antibody bacterial liquid library. Take 100 μL of the bacterial liquid from the library and dilute it 1000 times by the 10-fold dilution method (100 μL of bacterial liquid + 900 μL of 2×YT) using 2×YT medium. Then, pipette 100 μL of the diluted bacterial liquid and spread it on a 2×YT-ATG semi-solid medium plate, and incubate overnight at 37°C. The next day, count the monoclonal colonies and calculate the number of transformants.

[0085] 2 Library quality analysis

[0086] Randomly pick 15 single colonies with good growth and obvious characteristics in the medium, inoculate them into 10 mL of 2×YT (containing ampicillin) liquid medium, and culture them overnight at 37°C with shaking at 225 rpm. Take 10 mL of the bacterial solution cultured in a shaker at 37°C for 15 h, and collect the bacteria at 5000 rcf for 10 min at room temperature. Discard the medium, add 500 μL of SalutionI / RNaseA mixture, and shake to completely suspend the cells. Transfer the solution to a new 2 mL centrifuge tube, add 500 μL of solutionII, and gently invert to mix evenly. Let the mixed solution stand at room temperature for 2 - 3 min. Add 250 μL of pre-cooled N3 Buffer, gently invert the centrifuge tube up and down several times until a white flocculent precipitate forms. Centrifuge at a maximum speed of ≥13000 rcf for 10 min at room temperature, and gently transfer the supernatant to a new 1.5 mL centrifuge tube. Add 0.1 volume of ETR Solution to the supernatant, invert 10 times to mix evenly, and incubate on ice for 10 min. Incubate at 42°C for 5 min, and the solution becomes turbid again. Centrifuge at 12000 rcf for 3 min at 25°C, and transfer the upper aqueous phase (containing DNA) to a new 1.5 mL centrifuge tube. Add 0.5 volume of absolute ethanol, invert up and down 6 - 7 times to mix evenly, and let it stand at room temperature for 1 - 2 min. Transfer the mixed solution (transfer up to 700 μL at a time) to a HiBind DNA binding column sleeved with a 2 mL collection tube, centrifuge at 10000 rcf for 1 min at room temperature, and discard the filtrate. Repeat the steps until all the mixed solution is transferred through the column. Reinstall the column into the collection tube, add 500 μL of HBC Buffer, centrifuge at maximum speed for 1 min, and discard the filtrate. Reinstall the column into the collection tube, add 700 μL of DNA Wash Buffer, centrifuge at maximum speed for 1 min, and discard the filtrate. Reinstall the column into the collection tube, centrifuge the empty column at ≥13000 rcf for 2 min to dry the column matrix. Install the column in a clean 1.5 mL centrifuge tube, add 80 - 100 μL of Elution Buffer to the column matrix, let it stand for 1 min, and centrifuge at maximum speed for 1 min to elute the DNA. Discard the column, and store the DNA product at -20°C. Sequence the DNA product.

[0087] Expression amplification of phage library

[0088] The remaining library bacterial liquid from the previous step was continuously shaken and cultured at 37°C and 250 rpm. After 1 hour of culture, helper phage was added and allowed to stand and infect for half an hour. Then, it was further cultured at 37°C and 200 rpm for 1 hour. Finally, the bacterial cell pellet was collected by centrifugation and resuspended in 100 mL of 2×YT-ATK medium (containing ampicillin, tetracycline, and kanamycin), and amplified by overnight expression at 30°C and 225 rpm. The supernatant was collected the next day, and 4% PEG800 and 3% NaCl were used for ice bath and centrifugation to obtain the phage pellet. The obtained phage pellet was resuspended and dissolved in PBS (pH 7.4) and filtered through a 0.2 μm needle filter to remove bacteria. 10 μL of the filtered library was taken and diluted 10 8 times by the 100-fold dilution method of 2×YT medium (10 μL + 990 μL). Then, 10 μL of the diluted solution was taken to infect 100 μL of XL1-blue. After 30 minutes, it was spread on a 2×YT-ATG semi-solid medium plate. The next day, the number of monoclonal colonies was counted to calculate the concentration of the amplified library phage. The filtered library was added with 7% DMSO by volume and stored at -80°C.

[0089] Example 4: Screening of phage library

[0090] 1 Panning of amplified library

[0091] The antigen (antigen: HA2) was coated on a high-affinity ELISA plate overnight. After washing the plate with 0.1% PBST, it was blocked with 3% skim milk powder at room temperature for 1 h. The blocking solution was discarded and the plate was washed and ready for use. Approximately 5×10 12 PFU was added to the above-mentioned blocked antigen plate and incubated with the antigen at 37°C for 2 h. The plate was washed with 0.1% PBST. After elution with Glycine-HCl elution buffer, the pH was adjusted to 7.4 with Tris-HCl. The above eluate was mixed with Escherichia coli XL1-Blue, incubated at 37°C for 30 min and then shaken and cultured. After rescuing the phage, it was shaken and cultured overnight at 30°C to obtain the amplified antibody 1st library. The above process was repeated successively to obtain the 2nd, 3rd, and 4th libraries.

[0092] 2 ELISA detection of the 3rd and 4th amplified libraries

[0093] Coating antigen rHA2: Coating sample concentration 5 μg / mL, overnight at 4°C. Blocking: 3% milk, whole well, room temperature, 1 h. Adding sample: Supernatant of phage expression library, diluted 2-fold, 100 μL per well, room temperature, 1 h.

[0094] Adding secondary antibody: M13-HRP (0.2 μg / mL), 100 μL per well, room temperature, 1 h. TMB chromogenic solution 200 μL per well, chromogenic for 20 min; chromogenic termination solution 50 μL per well. Reading value: Immediately read the OD450 at 450 nm.

[0095] 3 Monoclonal Screening

[0096] Forty-eight monoclonal antibodies were selected from the rHA2-3rd library and 144 monoclonal antibodies were selected from the HA2-4th library. The supernatant after phage expression was used for ELISA detection. Coating antigen rHA2: Coating sample concentration 0.5 μg / mL, overnight at 4°C. Blocking: 3% milk, whole well, room temperature, 1 h. Adding sample: Phage expression supernatant, diluted 10-fold, 100 μL per well, room temperature, 1 h. Adding secondary antibody: M13-HRP (0.2 μg / mL), 100 μL per well, room temperature, 1 h. TMB chromogenic solution 200 μL per well, chromogenic reaction for 20 min; chromogenic termination solution 50 μL per well. Absorbance was detected at 450 nm. Monoclonal antibodies were selected for sequencing according to the detection results, and the sequencing results were analyzed using NCBI BLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ).

[0097] Example 5: Construction of Expression Vector

[0098] 1 Amplification of Target Sequence

[0099] The target gene was synthesized and the target sequence was amplified by PCR. The amplification system (50 μL) is shown in Table 27, and the amplification program is shown in Table 28.

[0100] Table 27 PCR Amplification System

[0101]

[0102] Table 28 PCR Amplification Program

[0103]

[0104] 2 Plasmid Digestion

[0105] The pABm-mIgG2a vector was double digested with EcoRI / EcoRV. The double digestion system is shown in Table 29.

[0106] Table 29 Double Digestion System

[0107]

[0108] The pABm-mKappa vector was double digested with EcoRI / EcoRV. The double digestion system is shown in Table 30.

[0109] Table 30 Double Digestion System

[0110]

[0111]

[0112] After mixing the above digestion system, place it in a water bath at 37°C for 15 minutes, and then perform gel extraction and recovery.

[0113] 3 Homologous recombination

[0114] Recombinase ligation was performed on the gel-extracted and recovered pABm-mIgG2a vector and the PCR-amplified VH fragment, and recombinase ligation was performed on the gel-extracted and recovered pABm-mKappa vector and the PCR-amplified VL fragment. React at 50°C for 15 - 20 minutes, and the reaction system (20 μL) is shown in Table 31.

[0115] Table 31 Recombinase ligation reaction system

[0116]

[0117] Example 6: Transformation

[0118] Transform the ligation system in Table 30 into DH5α competent cells. Take 1 tube of competent cells from the -80°C refrigerator and immediately place it on ice. Add the ligation system (volume not exceeding 10 μL), gently mix, and place on ice for 30 minutes. Heat shock accurately in a 42°C water bath for 90 seconds, and quickly place it on ice to cool for 3 - 5 minutes after heat shock.

[0119] Add 1 mL of LB liquid medium (without antibiotics) to the tube, mix well, and culture it on a shaker at 37°C and 200 rpm for 1 hour to allow the bacteria to resume normal growth. Centrifuge the above bacterial solution, remove 800 μL of the supernatant, pipette and mix the remaining medium, and spread it on a screening plate containing the corresponding antibiotic. Invert the culture dish and culture it at 37°C for 16 - 24 hours. Pick colonies from the transformed plate and sequence the cloned bacterial solution.

[0120] Example 7:: Antibody expression

[0121] 1 Cell culture

[0122] Subculture HEK293 cells with 293 serum-free CD medium. Mix the VH and VL plasmids to be expressed with the transfection reagent TF2 and co-transfect them into the cells. Add 293 serum-free feeding solution on the 1st, 3rd, and 5th days after transfection. Shaking flask culture conditions: 5% CO2, temperature 37°C, shaker speed 175 rpm.

[0123] 2 Protein purification

[0124] Centrifuge the cell supernatant and filter (0.22 μm). Take the filtered sample and perform protein purification through a Ni affinity chromatography column. Collect the eluted sample and dialyze it overnight at 4°C. Dialysis buffer: PBS. Take the dialyzed sample and concentrate it by ultrafiltration (ultrafiltration tube)

[0125] Example 8: Identification of Single-chain Antibody

[0126] 1 Identification of Antibody Purity by SDS-PAGE Electrophoresis

[0127] Install the electrophoresis tank and electrophoresis gel, and add the corresponding volume of electrophoresis solution into the electrophoresis slot and gel tank respectively. Sample treatment: After determining the concentration of monoclonal antibody by BCA method, take an appropriate amount of monoclonal antibody, add an appropriate amount of SDS-PAGE protein loading buffer, denature at 99 °C for 5 minutes, and then place it on ice to cool to room temperature. Loading: Add 15 μL of sample to each well, and add 5 μL of Marker to both sides. Electrophoresis: Connect the power supply and stop electrophoresis when the bottom line is 1 cm away. Development: Take out the gel, place it in a petri dish, add an appropriate amount of SolarFast SDS-PAGE Coomassie Brilliant Blue staining solution, and stain overnight at 4 °C. Result analysis: Analyze the electrophoresis results using ImageJ software.

[0128] 2 Antibody Binding Detection

[0129] Dilute HA2 to 2 μg / mL with coating buffer, 100 μL / well, coat overnight at 4 °C, wash twice with PBST, and pat dry. Prepare 3% skim milk powder, 340 μL / well, incubate at room temperature for 1 h, wash twice with PBST, and pat dry.

[0130] Gradient-dilute the above antibody starting from 1 μg / mL, and at the same time add an irrelevant monoclonal antibody (D1) to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, incubate at room temperature for 1 h, wash twice with PBST, and pat dry. Dilute the secondary antibody Goat anti-Mouse IgG-HRP to the working concentration of 0.1 μg / mL, 100 μL / well, incubate at room temperature for 1 h, wash three times with PBST, and pat dry. Color with TMB chromogenic solution, 100 μL per well, develop color in the dark for 15 min. Determine the time to add the termination solution according to the color development degree of the highest concentration microplate well. Terminate with ELISA termination solution, 100 μL per well. Immediately read the OD450 at 450 nm.

[0131] 3 Antibody Specificity Detection

[0132] Coat antigens P. gingivalis, P. intermedia, F. nucleatum, A. viscosus, A. actinomycetemcomitans and S. mutans (5×10 8 CFU / mL), after blocking and washing, add 1 μg / mL single-chain antibody, and use the negative antibody (D1) as a control. The remaining steps are the same as above.

[0133] 4 Antibody Sensitivity Detection

[0134] Coat P. g bacteria solution 5×10 7 、107 , 5×10 6 , 10 6 , 5×10 5 , 10 5 CFU / mL. After blocking and elution, add 1 μg / mL single-chain antibody, with negative antibody (D1) as the control, and the remaining steps are the same as above.

[0135] 5 Antibody titer detection

[0136] Coat the antigen P.gingivalis (5×108 CFU / mL). After blocking and elution, add antibodies diluted at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, with negative antibody (D1) as the control, and the remaining steps are the same as above. Result determination: The OD450nm value of the single-chain antibody is (P), the OD450nm of D1×2 is the negative control (N), and P / N > 2 is positive.

[0137] Effect example 1: Serum titer

[0138] The serum titer of mice immunized with rHA2 was determined by indirect ELISA. When the antiserum titer reached 1:104 or higher, the immunization was effective, as shown in Table 1. Select mice with relatively high antiserum titers and sacrifice them 3 days after booster immunization.

[0139] Table 1 Serum titers of mice after the third immunization

[0140] Serum dilution factor Mouse No. 549 Mouse No. 550 Mouse No. 551 Negative 1：1000 4.393 2.8016 2.7247 0.0894 1：3000 2.7316 3.0332 2.8738 0.0613 1：9000 3.4687 2.5549 2.8657 0.0496 1：27000 2.5037 1.5788 2.5084 0.0465 1：81000 1.9509 0.802 1.7489 0.0553 1：243000 1.0782 0.4156 0.832 0.05 1：729000 0.6041 0.2944 0.4821 0.0453

[0141] P / N > 2 (i.e., the OD value ratio of the positive group to the negative group is greater than 2), and the titer is > 10 4 , and the immunization is effective.

[0142] Effect example 2: Amplification of VL and VH genes

[0143] Extract the total RNA of spleen cells from immunized mice, as shown in Figure 1 , and the RNA meets the requirements. PCR amplifies the antibody VL and VH gene sequences. Specific fragments of 350 bp and 400 bp can be seen by agarose gel electrophoresis respectively, and their sizes are consistent with the target fragments, as shown in Figure 2 .

[0144] Effect example 3: Amplification of single-chain antibody gene (scFv)

[0145] VH and VL are connected by linker, and VH-linker-VH is synthesized in vitro. The constructed target band is 800 bp, and its size is consistent with the target fragment, as shown in Figure 3 .

[0146] Effect Example 4: Construction, Expression and Amplification of Phage Single-chain Antibody Library

[0147] Calculate the number of transformants in the transformant library:

[0148] HA2 = Number of monoclonal colonies counted × Dilution factor per unit volume × Total library volume = 729 × 10000 × 200 = 7.29×10 8 PFU Library quality analysis: Randomly pick 15 monoclonal colonies from the library-coated petri dish for sequencing, 1 original vector, 1 special structure, 13 correct sequences, sequence correct rate 86.6%

[0149] Calculate the library titer from this:

[0150] HA2 = Number of monoclonal colonies counted × Dilution factor per unit volume × Total library volume = 729 × 10000 × 200 = 7.29×10 8 ×86.6% = 6.31×10 8 pFU

[0151] Amplified library quality analysis: Calculate the amplified phage concentration of the library according to the number of monoclonal colonies counted as follows:

[0152] Library amplified phage concentration = Number of monoclonal colonies counted × Dilution factor per unit volume = 152×(10 8 ×100) = 1.52×10 12 PFU Effect Example 5: Screening of Phage Single-chain Antibody Library

[0153] The phage library was subjected to 4 rounds of "adsorption - elution - enrichment", and the titers of the input and output phages in each round were measured. The harvest rate of phage single-chain antibodies increased. After calculation, the enrichment degree of phage single-chain antibodies after 4 rounds of panning was about 3623 times that of the first round, as shown in Table 2, effectively enriching phages with better specificity. ELISA detected the antigen-binding ability of the amplified libraries of the 3rd and 4th rounds, see Figure 4 . Select monoclonal colonies from the amplified libraries of the 3rd and 4th rounds for ELISA detection, and sequence the phage single-chain antibodies that meet the positive standard (OD450 value of the test group / OD450 value of the control group > 2.5 is positive). For phage single-chain antibodies with relatively high binding ability, the OD450 values of HA2 - MB001, HA2 - MB004, HA2 - MB005, HA2 - MB022, HA2 - MB023, HA2 - MB028 and HA2 - MB032 were 1.5761, 1.466, 1.4472, 1.1343, 1.0776, 0.8169 and 1.252 respectively. The OD450 value of the control group was 0.128, see Figure 5Sequencing showed that the six single-chain antibody genes were identical to the phage single-chain antibody structure constructed. The sequencing results are shown in Table 3, and the HA2-MB001 sequencing had double peaks.

[0154] Table 2 Library capacity of the phage antibody library after 4 rounds of panning

[0155]

[0156] Sequencing results of 3 single-chain antibodies

[0157]

[0158]

[0159] Effect Example 6: Construction of single-chain antibody expression vector

[0160] The target gene with correct sequencing was synthesized, and the target gene was amplified by PCR, see Figure 6 . The pABm-mIgG2a vector and pABm-mKappa vector were double digested with EcoRI / EcoRV. The gel-extracted pABm-mIgG2a vector was ligated with the PCR-amplified VH fragment by recombinase, and the gel-extracted pABm-mKappa vector was ligated with the PCR-amplified VL fragment by recombinase. The expression vector was designed using Snapgene software.

[0161] Effect Example 7: Identification of single-chain antibody

[0162] SDS-PAGE identified that the purity of 4 single-chain antibodies was relatively high, see Figure 7 . The concentrations of antibodies MB004, MB005, MB028 and MB032 were 0.55 mg / mL, 0.97 mg / mL, 0.059 mg / mL and 0.15 mg / mL respectively. There were no obvious bands for MB022 and MB023. The binding activity of the single-chain antibody was detected by indirect ELISA, see Figure 8 . The OD450 value of the single-chain antibody was (P), the OD450 of D1×2 was the negative control (N), and P / N>2 was determined as the single-chain antibody binding to P.grHA2. All 4 single-chain antibodies bound to P.grHA2, and among them, the MB004 antibody had the highest binding ability to HA2. When the antibody concentration was greater than 0.32 ng / mI, all 4 antibodies bound to HA2. The binding concentrations of antibodies MB004 and MB005 to HA2 were greater than or equal to 0.064 ng / mL, and the binding concentrations of MB028 and MB032 to HA2 were greater than or equal to 0.32 ng / mL.

[0163] Effect Example 8: Titer of single-chain antibody against P.g

[0164] The titer of the single-chain antibody against P.g was detected by ELISA, seeFigure 9 For the MB004 antibody, the anti-P.g titer is >1:25600. The MB005 antibody, MB028 antibody, and MB032 antibody do not recognize P.g.

[0165] Effect Example 9: Specificity of single-chain antibody in recognizing P.g strains

[0166] The antibody MB004 only recognizes P.gingivalis and has no cross-reaction with other bacteria such as P.intermedia, A.vicosus, A.actinomycetemcomitans, S.mutans, and F.nucleatum. The MB005 antibody, MB028 antibody, and MB032 antibody cannot specifically recognize P.g, as shown in Figure 10 .

[0167] Effect Example 10: Sensitivity of single-chain antibody MB004 in recognizing P.g

[0168] The antibody MB004 can recognize P.gingivalis at a concentration >5×10 6 CFU / ml, as shown in Figure 11 .

[0169] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limitations on the present invention. The protection scope of the present invention should be defined by the scope of the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A single-chain antibody (scFv) that specifically recognizes Porphyromonas gingivalis, characterized in that: The DNA sequence of the anti-Porphyromonas gingivalis single-chain antibody (scFv) is shown in SEQ ID No.

1.

2. A single-chain antibody (scFv) that specifically recognizes Porphyromonas gingivalis according to claim 1, characterized in that: The amino acid sequence of the anti-Porphyromonas gingivalis single-chain antibody (scFv) is shown in SEQ ID No.

2.

3. The single-chain antibody according to claim 1 or 2, characterized in that A single-chain antibody obtained by substituting, adding or deleting one or more amino acids.

4. The single-chain antibody according to claims 1 to 3, characterized in that The antibodies are transformed into dual variable region antibodies, triple variable region antibodies, multi-variable region antibodies, bivalent tandem antibodies, trivalent tandem antibodies, multivalent tandem antibodies, complete antibodies containing Fc fragments, etc.

5. Use of the antibody according to any one of claims 1 to 4 in the preparation of prevention and treatment tools, diagnostic monitoring tools and scientific research for diseases caused by Porphyromonas gingivalis.

6. The use according to claim 5, characterized in that The Porphyromonas gingivalis-related diseases include, but are not limited to, infectious diseases caused by P. gingivalis, gingivitis, periodontitis, chronic obstructive pulmonary disease, cardiovascular and cerebrovascular diseases, diabetes, premature birth, Alzheimer's disease, rheumatoid arthritis, oral cancer, intestinal cancer and other tumors.

7. The use according to claim 5, characterized in that The prevention and treatment tools include pharmaceutical compositions, pharmaceutical preparations, and the like.

8. A pharmaceutical composition and pharmaceutical preparation according to claim 7, characterized in that: The invention comprises the antibody according to any one of claims 1 to 4, a drug and a pharmaceutically acceptable excipient.

9. The pharmaceutical preparation according to claim 7, characterized in that Its dosage forms are injections, microcapsules, implants, lozenges, gargles, sprays, ointments, creams, plasters, films, granules, oral solutions and tablets.

10. The use according to claim 5, characterized in that The diagnostic and monitoring tools include test kits, test strips or chips.

11. The use kit according to claim 10, characterized in that: Comprising the antibody according to any one of claims 1 to 4.

12. The application test strip according to claim 10, characterized in that: Comprising the antibody according to any one of claims 1 to 4.

13. The application chip according to claim 10, characterized in that: Comprising the antibody according to any one of claims 1 to 4.

14. The use according to claim 5, characterized in that The scientific research fields include but are not limited to immunology research, biomedical research, protein research, etc.

15. The use according to claim 14, wherein the immunological research includes but is not limited to the use of antibodies for detecting and identifying Porphyromonas gingivalis, studying Porphyromonas gingivalis, etc. The research methods include but are not limited to ELISA, immunoblotting, flow cytometry and other techniques.

16. The use as claimed in claim 14, wherein biomedical research includes but is not limited to research on the pathogenesis, disease diagnosis and treatment of diseases caused by Porphyromonas gingivalis.

17. The use according to claim 14, wherein protein research includes but is not limited to ELISA, immunoblotting detection and purification of HA2 protein.

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