Porcine pseudorabies virus antibody detection kit and application thereof

Abstract

The invention provides a porcine pseudorabies virus antibody detection kit and application thereof, a PRV gD protein monoclonal antibody hybridoma cell strain and a monoclonal antibody 10G12 secreted by the PRV gD protein monoclonal antibody hybridoma cell strain are prepared and screened by taking a PRV gD protein as an immunogen, and a variable region sequence of the monoclonal antibody is determined. The monoclonal antibody can be used for IFA and PRV recognition; the latex chromatographic test strip can be used for detecting ASFV antigen, can also be used for western blotting, and can be used for preparing the latex chromatographic test strip for detecting ASFV antigen. An indirect ELISA method is established on the basis, and compared with a neutralization test, it is expected that an antibody detection method which is good in relevance with a neutralizing antibody and suitable for large-scale rapid breeding farms is established, and a foundation is laid for further establishment of a specific, sensitive and rapid PRV detection method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a porcine pseudorabies virus antibody, and a detection kit prepared on the basis of the antibody and application thereof. Background Art

[0002] Pseudorabies (PR) is an acute infectious disease of pigs caused by pseudorabies virus (PRV). The disease is one of the major infectious diseases that endanger the pig industry. It can cause miscarriage and stillbirth in pregnant sows, infertility in boars, mass death of newborn piglets, breathing difficulties and growth stagnation in fattening pigs, etc. Once the disease becomes prevalent, it is difficult to completely eradicate it, causing serious economic losses to the pig industry.

[0003] PRV belongs to the family of Herpesviridae. Bartha-K61 attenuated vaccine is a commonly used PRV vaccine, which provides protection by inducing neutralizing antibodies through immunization. Since 2011, mutant PRV strains have appeared in my country, and the commonly used Bartha-K61 attenuated vaccine cannot provide complete protection. Most of the ELISA kits available on the market are relatively expensive and do not clearly state the relationship with neutralizing antibodies, which are the key to immune protection. Therefore, there is an urgent need for a detection kit with a higher correlation with neutralizing antibodies to evaluate the immune effect of vaccines. Recent studies have found that one of the main antigens of PRV is glycoprotein gD. The gD protein plays a key role in the fusion and invasion of viruses and host cells, and is also the main antigen that induces host cells to produce neutralizing antibodies. Antibodies against gD can inhibit viral penetration and protect pigs from fatal infection with PRV. Subunit vaccines targeting gD protein have shown significant immune protection effects in both mice and piglets, and are expected to become a new option for preventing PRV infection. At present, the detection technologies for pseudorabies in pigs include virus isolation, immunoperoxidase monolayer cell assay (IPMA), indirect immunofluorescence (IFA), RT-PCR and other methods, but the detection takes a long time and is expensive. In the ELISA detection of pseudorabies in pigs, gB and gE proteins are usually used as target antigens, while there are few ELISA detection methods for gD protein. Summary of the invention

[0004] The purpose of the present invention is to provide a monoclonal antibody against porcine pseudorabies virus gD protein and its application.

[0005] Furthermore, the present invention provides a hybridoma cell line 10G12 that can secrete monoclonal antibodies against the gD protein of pseudorabies virus.

[0006] The present invention also provides a monoclonal antibody against the gD protein of pseudorabies virus of swine, wherein the amino acid sequence of the heavy chain variable region (VH) of the antibody is as follows:

[0007] QVQLQQSGPELVHPGASVKMSAKASGVITYTFTDYWVKQRGLEWIGEIYPGIGSKYNGCTDQYNDKFKTAYLQLSS TLTADKSSLTSEADSAVAFYGKANWGQGTATVSS(SEQ ID NO.1)

[0008] The amino acid sequence of the light chain variable region (VL) is as follows:

[0009] DVLMTQTPLGSLFSGSGSGTPVSLGDSQSIAHSSGNTYLAEDLQWYLKLLIYKVSHRFSGVPDRPPDFTLAISRVE GVYYCYQGHVTLAQKPGQSPSISCRSFGGGTSLEIK (SEQ ID NO. 2).

[0010] Furthermore, the present invention provides a use of the above-mentioned monoclonal antibody against porcine pseudorabies virus gD protein in the preparation of a kit for detecting porcine pseudorabies virus.

[0011] The detection method includes indirect immunofluorescence detection, antigen capture enzyme-linked immunosorbent assay, immunohistochemistry detection or test strip detection.

[0012] The test strip detection method includes colloidal gold detection method or enzyme-labeled detection method.

[0013] In the immunohistochemical detection method, the enzyme-labeled antibody is goat anti-mouse horseradish peroxidase, the color development uses an AEC enzyme substrate color development kit, and the sample is suspected PRV pig disease tissue such as lungs, tonsils, lymph nodes, etc.

[0014] Furthermore, the present invention provides a PRV detection test strip, characterized in that the test strip is coated with the above-mentioned monoclonal antibody against the gD protein of pseudorabies virus.

[0015] Beneficial Effects

[0016] In this study, a PRV gD protein monoclonal antibody hybridoma cell line and its secreted monoclonal antibody 10G12 were screened using PRV gD protein as an immunogen, and the variable region sequence of the monoclonal antibody was determined. This monoclonal antibody can be used for IFA to identify PRV; it can also be used for western blotting and can be used to prepare latex chromatography test strips for detecting ASFV antigens. On this basis, an indirect ELISA method was established and compared with the neutralization test. It is expected to establish an antibody detection method that has a good correlation with neutralizing antibodies and is suitable for large-scale rapid use in farms, laying the foundation for further establishing a specific, sensitive and rapid PRV detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of amplification and purification of the gD protein of pseudorabies virus of pigs. In Figure A, M is a marker, and 1 is the gD fragment after PCR amplification and purification. In Figure B, M is a marker, and 1 is a schematic diagram of SDS-PAGE identification of the gD protein after purification.

[0018] Figure 2 The antibody titer of the immunized mice was much higher than 1:12800 in the experimental group, and the mice with the highest titer were selected for cell fusion;

[0019] Figure 3 This is the Western-Blot test result of 10G12 antibody;

[0020] Figure 4 Antibody subclass identification results of 10G12 monoclonal antibody. DETAILED DESCRIPTION

[0021] The experimental methods used in the following examples are all conventional methods unless otherwise specified. PK-15 cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd.; PRV strain YN-6 was isolated and stored by this laboratory; Chlamydia, foot-and-mouth disease, porcine epidemic diarrhea, porcine circovirus disease, porcine encephalitis B, porcine parvovirus disease, classical swine fever, porcine reproductive and respiratory syndrome and pseudorabies positive serum were isolated and stored by this laboratory for daily monitoring. Other materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0022] Example 1: Amplification and purification of porcine pseudorabies virus gD protein

[0023] According to the PRV sequence information published in GenBnak, specific primers were designed for gD protein using DNAStar software: upstream primer: CGGGATCGCATGCTGCTTGCAAGCGCTATTAGCGG;

[0024] Downstream primer: CCAAGCATCTAAGGGATGGTGCTGGTGATGGCGGGGCGCGAGC. Take 200uL of PRV strain YN-6 virus liquid and extract the PRV genome according to the instructions of the Viral Nucleic Acid Extraction Kit. Use the extracted PRV DNA as a template to amplify the gD gene. The PCR reaction system and conditions refer to the instructions of Prime STARHSDNA Polymerase. The PCR product was identified by 10g / L agarose gel electrophoresis, and the target fragment was recovered using the OMEGA gel recovery kit. After double enzyme digestion, the target gene was recovered and purified and inserted into the prokaryotic expression vector pET-28. After identification, the target recombinant plasmid pET-28-gD was obtained.

[0025] Take 5 μl of the correctly sequenced recombinant bacteria BL21 (DE3) and inoculate it into 5 ml LB (Amp + ) and cultured on a shaker at 37°C for 10 hours, then expanded to 1L. When the OD600 value was about 0.6, 1mmmol / L IPTG was added to induce expression. After 5 hours of culture, the cells were collected by centrifugation, resuspended in an appropriate volume of PBS, and ultrasonically treated on ice until the cell solution was clear. Centrifuged at 12000r / min for 15 minutes in a low-temperature centrifuge, the supernatant was collected and purified by Ni-NTA affinity chromatography. The specific operation steps refer to the molecular cloning manual. After protein purification, SDS-PAGE was used for identification to obtain a single-band target protein ( Figure 1 ).

[0026] Example 2 Preparation of Monoclonal Antibodies to Pseudorabies Virus gD Protein and Determination of Biological Characteristics

[0027] Select female BALB / c mice aged 6-8 weeks. Emulsify the purified gD protein with an equal volume of Freund's complete adjuvant (Sigma, catalog number F5881) and inject BALB / c mice at multiple points in the abdominal cavity and back subcutaneously, 30 μg / mouse; emulsify the purified virus with an equal volume of Freund's incomplete adjuvant (Sigma, catalog number F5506) for the second and third immunizations in the second and fourth weeks after the first immunization; collect blood 2 weeks after the last immunization and measure the serum antibody titer with indirect ELISA. When the ELISA titer reaches 1:10 5 Cell fusion was performed at the above time. Three days before fusion, 100 μg of antigen solution without adjuvant was injected into the tail vein or peritoneum for booster immunization. Blood was collected from the tail on the 7th day after the immunization to determine the antibody titer.

[0028] PK15 cells were plated in T75 cell culture flasks and inoculated with 0.1 MOI of PRV after a monolayer was grown. After 60 hours of inoculation, the cells were placed at -80℃ and thawed three times to completely release the virus in the cells. The cells were centrifuged at 12,000 r / min for 10 minutes in a cryocentrifuge, and the supernatant was collected and inactivated in a 56℃ water bath for 30 minutes. The inactivated virus was mixed with the coating solution in a ratio of 1:1 as an antigen and then coated on the ELISA plate. For specific operations, refer to: Duan Yueyue, Kong Xiangyu, Yuan Cong, et al. Prokaryotic expression of membrane protein of porcine acute diarrhea syndrome coronavirus and preparation of its polyclonal antibody. Chinese Journal of Veterinary Science, 2022, 52(1): 62-69. In this process, negative and positive sera were diluted with PBST in multiple ratios (1:100 to 1:12,800), of which the positive serum was gD protein serum and the negative serum was serum from unimmunized healthy mice.

[0029] Take one mouse with a higher antibody titer detected by ELISA ( Figure 2 ), the spleen was taken, ground and mixed with SP / 20 cells (5:1), the spleen cells and SP / 20 cells were fused with the help of the fusion agent PEG, and then hybridoma cells were obtained under pressure screening in HAT medium. The supernatant of the hybridoma cells was detected by the above-mentioned ELISA method. The cells in the ELISA-positive wells were subcloned three times by limiting dilution method and a single monoclonal antibody that could specifically react with PRV gD protein was obtained under pressure screening in HT medium, which was named 10G12.

[0030] PK15 cells were cultured at 1×10 6 100 μg / well were plated on a 6-well cell plate. The next day, the cells were inoculated with 0.1 MOI of PRV, and the uninfected cells were used as negative controls. 48 hours after virus infection, the cells were lysed with RIPA lysis buffer, and SDS PAGE, membrane transfer and blocking were performed. 5% skim milk was selected as the blocking buffer, and the blocking condition was shaking at room temperature for 1 hour. The primary antibodies were the supernatant of hybridoma 10G12 cells and the internal reference GAPDH; the secondary antibody was HRP-labeled goat anti-mouse IgG (10,000-fold dilution), and the action condition was shaking at room temperature for 1 hour. After each action, it was washed 4 times with PBST. Finally, the ECL chemiluminescence imager was used for color development. The results showed ( Figure 3 ), the 10G12 antibody can effectively recognize the PRV gD protein in infected cells.

[0031] According to the instructions of the mouse monoclonal antibody Ig class / subclass identification kit of Biodragon, the antibody subclass of 10G12 was identified. The results showed that the heavy chain constant region of 10G12 was Ig2a type, and the light chain constant region was Kappa type ( Figure 4 ).

[0032] Ten female BALB / c mice aged 10 to 12 weeks were selected, and 0.5 mL of ascites adjuvant was aseptically aspirated and injected into the mouse peritoneal cavity. After 12 to 15 days, hybridoma cells 1C10 were inoculated at 5×10 per mouse. 5 The mouse abdominal cavity was injected with a dose of 1:1000 to 1:256000, and the abdominal cavity was significantly enlarged around 10 days. The ascites was extracted with a 5mL syringe. The collected ascites was purified using a ProteinA / G affinity chromatography column, and then the purity was identified by SDS PAGE electrophoresis. The purified ascites was diluted in multiples (1:1000 to 1:256000), and then its titer was detected by ELISA. The results confirmed that when diluted 1:128000, its D450 value was still higher than that of the control group, indicating that the antibody titer can reach more than 1:128000.

[0033] The hybridoma cell line against PRV gD protein obtained above was continuously passaged for 3 months and frozen in liquid nitrogen before recovery. The culture supernatant was taken and its antibody ELISA titer was detected according to the above method, and the normal culture fluid of PK15 cells was used as a negative control. The ELISA results showed that the antibody titer was not significantly different from the previous results, indicating that the secretion stability of the monoclonal antibody was high.

[0034] After the supernatant of 10G12 hybridoma cells cultured in a 6-well plate was removed, 1 mL of Trizol was added to each well to lyse the cells. The lysed samples were collected in 1.5 mL centrifuge tubes and temporarily stored at -80°C. The samples were submitted to a biotechnology company for sequencing of the variable regions of mouse hybridoma monoclonal antibodies. The sequencing results are as follows:

[0035] The amino acid sequence of the heavy chain variable region (VH) is as follows:

[0036] QVQLQQSGPELVHPGASVKMSAKASGVITYTFTDYWVKQRGLEWIGEIYPGIGSKYNGCTDQYNDKFKTAYLQLSS TLTADKSSLTSEADSAVAFYGKANWGQGTATVSS(SEQ ID NO.1)

[0037] The amino acid sequence of the light chain variable region (VL) is as follows:

[0038] DVLMTQTPLGSLFSGSGSGTPVSLGDSQSIAHSSGNTYLAEDLQWYLKLLIYKVSHRFSGVPDRPPDFTLAISRVE GVYYCYQGHVTLAQKPGQSPSISCRSFGGGTSLEIK (SEQ ID NO. 2).

[0039] The indirect ELISA method (following the steps in the instructions of the commercial kit, the secondary antibody is a goat anti-mouse enzyme-labeled secondary antibody) was used to determine the OD value of the reaction between the monoclonal antibody (ascites) obtained and porcine parvovirus (PPV, kit from CEDI, the Netherlands), porcine reproductive and respiratory syndrome virus (PRRSV, kit from IDEXX, the United States), classical swine fever virus (HCV, kit from IDEXX, the United States), and porcine circovirus type 2 (PCV-2, kit from CEDI, the Netherlands), using negative PK15 cell ascites as a control.

[0040] The results show that the monoclonal antibody (ascites) does not react with PPV, PRRSV, HCV, and PCV-2. The above experimental data show that the monoclonal antibody against PRV gD protein of the present invention has strong specificity, high sensitivity, high titer, and stability, and is particularly suitable for preparing a detection kit for PRV virus.

[0041] Example 3 Application of monoclonal antibodies

[0042] Preparation of colloidal gold conjugate pad: colloidal gold particles were prepared according to conventional methods, purified monoclonal antibody 10G12 was labeled on the surface of colloidal gold particles, the colloidal gold particles were diluted evenly with colloidal gold diluent, sprayed on the cut glass fiber membrane, and freeze-dried.

[0043] On the same nitrocellulose membrane, use a Bio-Dot membrane sprayer to spray purified monoclonal antibodies (test line) and goat anti-mouse IgG (control line). The control line and the test line are separated by 3-7 mm. Place in an incubator to dry, take out, seal and store for later use.

[0044] Assemble the test strips: stick the sample absorption pad (glass fiber membrane), the conjugate pad, the nitrocellulose membrane sprayed with the test line (T line) and the control line (C line), and the absorption pad (water-absorbing pad) on the PVC bottom plate (PVC or other hard thin plates) in order, cut the assembled test paper into 3-6 mm wide strips with a Bio-Dot cutter, fix them on the plastic support plate, put them into an aluminum foil bag with a desiccant, seal them, and store them at room temperature to obtain colloidal gold rapid detection test strips.

[0045] Slowly add 3-5 drops of the sample to be tested (suspected serum, plasma or tissue grinding fluid) to the sample well of the test strip and observe the results within 20 minutes.

[0046] Result determination: Positive: a purple-red line appears in the control line area (C) and the test line area (T) of the test strip; Negative: a purple-red line appears only in the control line area (C) of the test strip; Invalid: no purple-red line appears on the test strip.

[0047] The inactivated PRV virus and the cell culture supernatant of PPV, PRRSV, HCV, and PCV-2 were diluted with sample diluent and added dropwise to the sample injection port of the test strip. The test strip was left to stand at room temperature for 5-10 minutes. The results showed that only the test strip with the inactivated PRV virus showed a positive result, and the test strip with the cell culture supernatant of PPV, PRRSV, HCV, and PCV-2 showed a negative color development result, indicating that the antigen detection test strip has good specificity.

[0048] The purified PRV gD protein was diluted to a concentration of 512 ng / mL, 256 ng / mL, 128 ng / mL, 64 ng / mL, 32 ng / mL, 16 ng / mL, 8 ng / mL, 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, and 0.125 ng / mL, respectively, and added dropwise to the sample addition port of the antigen detection test strip and allowed to stand at room temperature for 5-10 min. The results showed that when the antigen concentration in the sample was ≥1 ng / mL, the T line of the test strip was colored, and when the antigen concentration in the test sample was 0.5 ng / mL, the T line of the test strip was not colored, indicating that the detection limit of the antigen detection test strip was 1 ng / mL.

[0049] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A monoclonal antibody against the gD protein of pseudorabies virus, characterized in that: The amino acid sequence of the heavy chain variable region (VH) of the antibody is as follows: QVQLQQSGPELVHPGASVKMSAKASGVITYTFTDYWVKQRGLEWIGEIYPGIGSKYNGCTDQYNDKFKTAYLQLSS TLTADKSSLTSEADSAVAFYGKANWGQGTATVSS(SEQ ID NO.1) The amino acid sequence of the light chain variable region (VL) is as follows: DVLMTQTPLGSLFSGSGSGTPVSLGDSQSIAHSSGNTYLAEDLQWYLKLLIYKVSHRFSGVPDRPPDFTLAISRVE GVYYCYQGHVTLAQKPGQSPSISCRSFGGGTSLEIK (SEQ ID NO. 2).

2. Use of the monoclonal antibody against porcine pseudorabies virus gD protein as claimed in claim 1 in the preparation of a kit for detecting porcine pseudorabies virus.

3. Use of the monoclonal antibody against the gD protein of pseudorabies virus according to claim 1 in preparing a colloidal gold rapid test strip for detecting pseudorabies virus.

4. A PRV test strip, characterized in that: The test strip is coated with the monoclonal antibody against the gD protein of the pseudorabies virus of claim 1.

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