BoHV-1 virus antigen epitope polypeptide, coupling protein thereof, enzyme-linked immunosorbent assay kit and application of BoHV-1 virus antigen epitope polypeptide

Abstract

The invention discloses a BoHV-1 virus antigen epitope polypeptide, a coupling protein thereof, an enzyme-linked immunosorbent assay kit and application, and aims to solve the technical problems that a BoHV-1 virus immunosorbent assay kit is scarce, the preparation process is complex and the cost is high. The method comprises the following steps: firstly, screening dominant mimic epitopes by utilizing a phage library and BoHV-1 virus multi-antibody serum; then synthesizing corresponding polypeptides to identify dominant B cell epitopes of viruses, and further screening B cell epitopes suitable for preparing antibodies; finally, the ELISA kit for detecting the BoHV-1 virus is researched and developed on the basis of the B cell epitope. The B cell epitope polypeptide provided by the invention has high specificity and sensitivity, and the provided enzyme linked immunosorbent assay kit has relatively high specificity and good detection performance.

Description

Technical Field

[0001] The present invention relates to the field of biological immunology technology, and specifically to a BoHV-1 virus antigen epitope polypeptide, its coupling protein, enzyme-linked immunosorbent assay kit and application. Background Art

[0002] Bovine herpesvirus type 1 (BoHV-1), also known as bovine infectious rhinotracheitis virus (IBRV), belongs to the family Herpesviridae and the subfamily Alphaherpesvirinae. The genome size of this virus is between 135kb and 140kb, which can encode at least 73 proteins. Its genome consists of a long unique region (UL, about 102kb~104kb), a short unique region (US, about 10.5kb~11kb), and inverted repeat sequences (about 24kb) at both ends. As one of the most important pathogens in cattle diseases, BoHV-1 infection can cause a variety of inflammatory diseases, destroy the respiratory, nasal and ocular mucosa of cattle, and then easily be infected with other pathogens, such as bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza virus-3 (PI3V), and bacteria such as Mannheimia hemolytica, Pasteurella multocida and acidophilus tissue bacillus, leading to bovine respiratory disease syndrome (BRDC). BRDC has a long treatment cycle and high cost, which may cause calf death. In addition, BoHV-1 is also the main pathogen that causes abortion in pregnant cows, causing huge economic losses to the global cattle industry. It is estimated that the annual losses caused by BoHV-1 infection in the United States alone are as high as $3 billion.

[0003] Currently, there are limited commercial antibodies targeting BoHV-1 viral proteins on the market, and they are expensive. For example, the gD and gC protein monoclonal antibodies provided by VMRD are priced at 4,600 yuan per 100 μL, and the price continues to rise, significantly increasing the research costs of researchers in related fields. At present, there are no antibody products targeting the virus that are independently developed and have independent intellectual property rights in my country and are on the market.

[0004] Strengthening antibody detection is one of the key technical means to prevent and control BoHV-1. At present, ELISA antibody detection kits developed domestically or abroad generally use baculovirus or Escherichia coli expression systems to prepare coating sources. However, this method has significant problems: the expressed protein needs to undergo a purification step, which is not only costly and complex, but also requires expensive equipment, and the purity and activity of the protein vary greatly between batches, making it difficult to control product quality.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0006] The present invention discloses a BoHV-1 virus antigen epitope polypeptide, a coupling protein, an enzyme-linked immunosorbent assay kit and an application thereof, aiming to solve the technical problems that the BoHV-1 virus immunoassay kit is scarce, has a complex preparation process and is costly.

[0007] The present invention first uses a phage library and BoHV-1 virus polyantisera to screen for dominant mimetic epitopes; then synthesizes the corresponding polypeptides to identify the dominant B cell epitopes of the virus, and further screens the B cell epitopes suitable for preparing antibodies; finally, an ELISA kit for detecting BoHV-1 virus is developed based on the B cell epitopes.

[0008] Specifically, the present disclosure provides a BoHV-1 viral antigen epitope polypeptide, whose amino acid sequence is shown in SEQ ID NO.1.

[0009] According to a second aspect of the present disclosure, a coupled protein is provided, which is formed by coupling the above-mentioned BoHV-1 viral antigen epitope polypeptide with hemocyanin.

[0010] According to a third aspect of the present disclosure, the BoHV-1 viral antigen epitope polypeptide or the coupled protein is used in the preparation of an immunological detection reagent.

[0011] According to a fourth aspect of the present disclosure, the BoHV-1 viral antigen epitope polypeptide or the coupled protein is used in the preparation of antibodies or vaccines against BoHV-1 virus.

[0012] According to a fifth aspect of the present disclosure, the BoHV-1 viral antigen epitope polypeptide or the coupled protein is used in the preparation of a reagent for detecting the BoHV-1 virus.

[0013] According to a sixth aspect of the present disclosure, an enzyme-linked immunosorbent assay kit for detecting BoHV-1 virus is provided, comprising a blocking solution, a primary antibody diluent, a secondary antibody diluent, an HRP-rabbit anti-bovine secondary antibody, a BoHV-1 negative control, a TMB substrate, and an ELISA plate, wherein the ELISA plate is coated with the BoHV-1 virus antigen epitope polypeptide.

[0014] In some embodiments of the present disclosure, the blocking solution is 4-6% ovalbumin diluted with phosphate buffer.

[0015] In some embodiments of the present disclosure, the primary antibody diluent is 0.8-1.2% horse serum diluted with phosphate buffer.

[0016] In some embodiments of the present disclosure, the secondary antibody diluent is 4-6% skim milk diluted with phosphate buffer.

[0017] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. The B cell antigen epitope peptide of BoHV-1 virus gC protein was screened and coupled with hemocyanin to form pv1-KLH coupling protein. The mouse serum immunized with this protein can specifically recognize BoHV-1 virus protein, and the virus protein can still be detected when the serum is diluted 12,000 times, which has high specificity and sensitivity.

[0018] 2. A BoHV-1 antibody ELISA detection kit was constructed based on the B cell antigen epitope peptide. The coefficient of variation of the batch-to-batch repeated experiments of the kit was between 2.9% and 12.6%. It has high repeatability, good consistency and high specificity, can accurately detect the target antigen, and has a higher compliance rate than existing commercial kits, reaching 95.45%, with good detection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the Dot-Blot identification result of screening phage in one embodiment of the present application; wherein, + represents BoHV-1 virus; - represents unrelated phage.

[0020] Figure 2 This is an alignment of the phage mimetic epitope sequence and the viral protein sequence in one embodiment of the present application.

[0021] Figure 3 This is the Dot-Blot detection result of the polypeptide-KLH coupling protein in one embodiment of the present application; wherein, + represents BoHV-1; - represents KLH.

[0022] Figure 4 This is the Western Blot test result of the immune mouse serum in one embodiment of the present application. DETAILED DESCRIPTION

[0023] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0024] Unless otherwise specified, the biochemical instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the biochemical reagents involved are all commercially available conventional reagents unless otherwise specified; the detection, test and preparation methods involved are all conventional methods unless otherwise specified.

[0025] Example 1: Screening of BoHV-1 phage mimetic epitopes using phage random peptide library In this case, a phage random peptide library was combined with BoHV-1 virus polyantibody to screen the BoHV-1 phage dominant mimic epitopes to identify the key areas on the virus surface that can be recognized by the immune system. The specific steps are as follows: 1. Panning of Phage Random Peptide Library (1) Antibody coating: In a clean bench, use a 60 mm tissue culture dish as the coating plate, dissolve BoHV-1 polyclonal antiserum in 0.1 mol / L NaHCO3 (pH 8.6), 80 μg / mL, pipette and mix, and gently shake the tissue culture dish to completely immerse the bottom. Place in a wet box at 4°C overnight.

[0026] (2) Blocking: Take out the antibody-coated cell culture dish, tilt it gently, use a pipette to suck out all the liquid along the wall, then add 4 mL of the prepared 1% BSA blocking solution, put it back into the wet box device, and block it at 4°C for 2 h.

[0027] (3) Washing: Discard the blocking solution and wash six times with 0.1% TBST, each time for 1-2 min. After each wash, pat the culture dish dry on a clean paper towel (move quickly to avoid drying the culture dish).

[0028] (4) Phage binding: Take 10 μL of phage random peptide library and dilute it with 1 mL TBST (1:100, so that the number of phage added is 1×10 11 After adding 100 pfu, the cells were placed in a shaker and incubated at room temperature for 1 h.

[0029] (5) Washing: Aspirate the above liquid and wash 10 times with 0.1% PBST to discard unbound phages.

[0030] (6) Elution: Add 1 mL of elution buffer solution (0.2 mol / L glycline-HCl, pH 2.2, 1 mg / mL BSA) and place on a shaker. Shake at room temperature for 10-20 min to elute the specifically bound phages. Collect the eluate in a sterile centrifuge tube pre-added with 150 μL Tris-HCl buffer (1 mol / L, pH 9.1) to neutralize the pH value to neutral. This is the first round of specifically bound phages. Store at 4 °C. Take 10 μL to determine the phage titer. The rest is used for the next round of panning after amplification and purification.

[0031] 2. Amplification and purification of specific phage (1) The phages obtained after panning were added to the Escherichia coli culture (E. coli ER2738) in a single colony culture, placed on a shaker, and cultured vigorously at 37°C for 5 h, so that the phages can infect the E. coli and amplify in the E. coli, thereby lysing the E. coli.

[0032] (2) Centrifuge at 4℃ and 12,000 g for 10 min to remove the unlysed E. coli precipitate in the solution. Transfer 80% of the upper part of the supernatant to a new centrifuge tube, add 1 / 6 of the supernatant volume of 20% PEG solution, let stand at 4℃ for 2 h, then centrifuge at 4℃ and 12,000 g for 15 min, and discard the supernatant.

[0033] (3) Resuspend the precipitate in 1.5 mL of TBS and centrifuge briefly at 4 °C to remove the remaining cell debris. Add 1 / 6 volume of 20% PEG solution to the supernatant. Let it stand for 2 h and then centrifuge at 12,000 g for 15 min at 4 °C. Resuspend the phage precipitate in 200 μL of TBS to obtain the amplified and purified panning phage.

[0034] 3. Phage titer determination (1) The selected phages were cultured in LB liquid medium at 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The phages were diluted 6 times at 6 dilution ratios, and then 10 μL of the diluted phages were added to 200 μL of E. coli solution and incubated at room temperature for 5 min.

[0035] (2) Add the incubated phage-E. coli mixture to 5 mL of top agar, mix well and spread evenly on the LB solid culture medium plate. After it solidifies, invert it and place it in a 37°C incubator for overnight culture.

[0036] (3) Check the plates the next day, count the plaques on the plates, and calculate the titer units (pfu).

[0037] The above panning process was repeated three times. The eluted phages after the third screening did not need to be amplified. The phages were diluted 10 times according to the method in Example 3, the number of plaques was determined, and the phage titer was titrated. The results of the phage titer determination are shown in Table 1. The results show that when the phage dosage is consistent (1×10 11), starting from the second round of panning, the concentration of Tween-20 in the washing solution TBST was increased from 0.1% to 0.5%. Although the coating concentration of the ligand decreased in each round of panning, the number of phages selectively adsorbed to the ligand increased in each round, indicating that the phages specifically binding to the BoHV-1 antiserum were effectively selectively enriched after affinity screening.

[0038] Table 1 Enrichment of phages in three rounds of affinity panning .

[0039] 4. Preparation of phage stock solution Select a plate with about 100-150 plaques, randomly pick 150 monoclonal plaques with a sterilized pipette tip, and place them in 1 mL of LB liquid culture medium of E. coli ER2738 in the early logarithmic growth period. Culture with vigorous shaking at 37 °C for 5 h, centrifuge at 4 °C and 14,000 r / min for 30 s, transfer the supernatant to a new centrifuge tube, centrifuge again, and take 80% of the liquid on the top of the supernatant as the storage liquid of a single phage clone for further analysis.

[0040] 5. Purification and sequencing of sequencing template (1) Take out 750 μL of phage supernatant and add 300 μL of 20% PEG (2.5 mol / L NaCl) solution to each aliquot. Invert and mix several times and let stand at room temperature for 10-20 min.

[0041] (2) Centrifuge at 4°C and 14,000 r / min for 10 min, discard the supernatant, and centrifuge again, discarding the supernatant.

[0042] (3) Add 100 μL of iodide buffer to the resulting precipitate, resuspend and mix, then add 250 μL of anhydrous ethanol and incubate at room temperature for 10-20 min (so that the single-stranded phage DNA is precipitated, while the phage protein remains in the supernatant).

[0043] (4) Centrifuge at 4°C and 14,000 r / min for 10 min, discard the supernatant, add 500 μL of 75% ethanol to wash the precipitate, centrifuge again at 14,000 r / min for 10 min, discard the supernatant, and finally place the precipitate in a 37°C incubator to dry for 10 min.

[0044] (5) Suspend the precipitate in 30 μL TE buffer (pH 8.0) to obtain the phage single-stranded DNA, which was then stored at -20 °C.

[0045] (6) The phage template DNA was sent to Genewise Biosequencing Company for DNA sequencing using the specific primer 96gⅢ: 5'-CCC TCATAG TTA GCG TAA CG-3'. The sequencing results were translated into amino acid sequences using the free biological software DNAstar to obtain the mimetic epitope.

[0046] 6. Dot-Blot Identification of Phage Mimic Epitopes Dot-blot Analysis was used to identify the selected monoclonal phage. The specific steps are as follows: (1) Cutting the membrane: Take a piece of nitrocellulose membrane (NC membrane) and cut it into a suitable size.

[0047] (2) Spotting: Take 5 μL of the cultured BoHV-1 virions (positive control), irrelevant phages (negative control), and the phage stock solution of the screened monoclonality and spot them on the corresponding positions on the NC membrane and dry them at room temperature.

[0048] (3) Blocking: Soak the NC membrane in 5% skimmed milk, place it on a shaker, and block it at room temperature for 1.5 h.

[0049] (4) Incubation with primary antibody: Rinse the blocked nitrocellulose membrane with PBS, add BoHV-1 polyclonal antibody diluted with 1% horse serum (1:5000), place on a shaker, and incubate at room temperature for 3 h.

[0050] (5) Washing the membrane: Wash the incubated NC membrane with PBS for 1 h.

[0051] (6) Incubation with secondary antibody: After rinsing, add HRP-rabbit anti-goat secondary antibody (1:8000) diluted in 5% skim milk and incubate at room temperature for 1 h.

[0052] (7) Washing the membrane: After incubation, wash the membrane three times with PBS.

[0053] (8) Development: Mix the A and B solutions of the ECL luminescent solution in a ratio of 1:1 and drop them onto the NC film for subsequent development.

[0054] The identification results are as follows Figure 1 As shown, the results showed that a total of 15 phages that could react with BoHV-1 polyantiserum were screened, marked as: p116, p108, p109, p49, p50, p71, p76, p104, p113, p59, p133, p62, p63, p65, p93, while unrelated phages did not react with polyantiserum. Therefore, it was preliminarily believed that these phages had mimicking epitopes.

[0055] Example 2: Analysis and identification of B cell epitopes 1. Peptide Synthesis In this example, the sequencing results of the mimetic epitopes screened in Example 1 were compared with the BoHV-1 viral protein sequence in the Snapgene software, and multiple viral protein epitope sequences were inferred based on these mimetic epitopes, of which four viral protein epitope sequences were on the envelope protein ( Figure 2 ), respectively marked as pv1, pv2, pv3, pv4 (SEQ ID NO. 1~4), for subsequent research in this case. The above viral epitope sequences were commissioned to Shanghai Jier Biochemical Company for peptide synthesis (Table 2).

[0056] Table 2 Nomenclature and amino acid sequence of synthesized peptides .

[0057] 2. Peptide-coupled carrier protein hemocyanin KLH B cell antigen epitopes are specific regions on antigen molecules that can be recognized and bound by B cells. According to the principles of immunology, these epitopes can be recognized by specific antibodies and can therefore be used as coatings for the establishment of detection methods. Although B cell epitopes themselves may only be haptens, they may be converted into complete antigens when coupled to specific carriers. This conversion enables them to be used as immunogens for antibody preparation. Therefore, in order to enhance the immunity of the polypeptide, the polypeptide is coupled with KLH, and Sulfo-SMCC is used as a coupling catalyst. The specific steps are as follows: (1) Dissolve KLH carrier protein in PBS (containing 3 mM EDTA) and adjust the protein concentration to 5 mg / mL. Dissolve Sulfo-SMCC in ultrapure water to a concentration of 5 mg / mL.

[0058] (2) Take 100 μL of Sulfo-SMCC solution and slowly add it into 600 μL of KLH protein solution while stirring continuously to mix well. React at room temperature for 2 h.

[0059] (3) After the reaction, the solution was added to the dialysis membrane and dialyzed overnight at 4°C to remove the remaining unreacted Sulfo-SMCC coupling agent. The dialysis solution was changed every two hours for a total of three times. The dialysis solution was PBS (containing 3 mM EDTA). After the dialysis, the concentration of SMCC-activated KLH protein was adjusted to 4 mg / mL.

[0060] (4) Dissolve the antigen peptide in PBS (containing 3 mM EDTA) and adjust the concentration to 4 mg / mL. Slowly mix the SMCC-activated KLH solution with the antigen peptide solution at a mass ratio of 6:5, and let it react at room temperature for 4 h and at 4 °C overnight.

[0061] 3. Identification of the reactivity between antibodies and peptide-KLH conjugated proteins In order to identify whether the coupled polypeptide-KLH protein and BoHV-1 antibody produce specific reactions, commercial BoHV-1 antibodies and BoHV-1 positive bovine serum detected by clinical isolation were used to identify the coupled proteins using the Dot-Blot method: (1) Cutting and spotting: Cut a nitrocellulose membrane (NC membrane) into an appropriate size, take the positive control BoHV-1 virus (7 μL), the negative control KLH (5 mg / mL, 7 μL), the KLH-phage mimetic epitope peptide (5 mg / mL, 7 μL) and the KLH-virus epitope peptide (5 mg / mL, 7 μL) and spot them on the corresponding positions on the membrane from left to right, and dry the membrane at room temperature for 20 min.

[0062] (2) Blocking: Block with 5% skim milk at room temperature for 1 h.

[0063] (3) Incubation with primary antibody: discard the blocking solution, add primary antibody serum (commercial BoHV-1 polyclonal antibody serum / BoHV-1 positive bovine serum tested by clinical isolation), diluted with 1% horse serum, the dilution ratio is 1:5000, and incubate at room temperature for 3 h.

[0064] (4) Washing the membrane: Wash the membrane with PBS for 1 h.

[0065] (5) Incubation with secondary antibody: Add HRP-rabbit anti-goat secondary antibody, dilute with 5% skim milk at a dilution ratio of 1:8000, and incubate at room temperature for 1 h.

[0066] (6) Washing the membrane: Wash the membrane with PBS for 1 h.

[0067] (7) Development: Add ECL color developing solution for development.

[0068] The results are as follows Figure 3 As shown, eight polypeptide-KLH coupling proteins (pp116-KLH, pv1-KLH, pp108-KLH, pv2-KLH, pp109-KLH, pv3-KLH, pp49-KLH, pv4-KLH) can react specifically with commercial BoHV-1 polyantibodies. And when other reaction conditions remain unchanged and the primary antibody becomes BoHV-1 positive bovine serum detected by clinical isolation, the eight polypeptide-KLH coupling proteins can also react with BoHV-1 positive bovine serum, but not with negative serum, among which pp116-KLH, pv1-KLH, pv2-KLH, pv3-KLH, pp49-KLH, and pv4-KLH react more strongly. This shows that the polypeptide is successfully coupled to the KLH protein and can react specifically with BoHV-1 virus antibodies.

[0069] 4. Identification of immunogenicity of BoHV-1 virus antigen epitope peptides The viral antigen epitope peptide coupled to hemocyanin (KLH) was used as the immunogen, and 6-8 week-old SPF-grade BALB / C mice were used as the immunization objects (the purchased mice were raised for one week to adapt to the environment without adverse reactions), and the immunogenicity of the BoHV-1 viral antigen epitope peptide was identified. A total of 12 mice were used for immunization and divided into 4 groups (pv1-KLH group, pv2-KLH group, pv3-KLH group, and pv4-KLH group), with 3 mice in each group.

[0070] For the first immunization, take 50 μg of each immunogen, dilute it with 300 μL autoclaved PBS, and mix it with an equal volume of Freund's complete adjuvant. Set the emulsifier to 25,000 r / min, emulsify for 5 min (when a little bit of it is put into the water and it does not disperse, or the dispersion speed is slow, it is considered that the emulsification is successful), and inject subcutaneously at four points on the back of each mouse, 200 μL / mouse, 50 μL / point; the immunization steps for the 2nd to 3rd immunization are the same as above, but the emulsifier is replaced with Freund's incomplete adjuvant. There is an interval of 2 weeks between the first and third immunizations. 10 days after the third immunization, the mice are tail-cut and blood is collected. 10 μL of blood is collected from the tail of each mouse, diluted with 490 μL 1xPBS (serum: PBS = 1:50), 4 ℃, 5000 r / min, centrifuged for 10 min, and the supernatant is taken and stored at -20 ℃. It can be used to measure its immune effect. The specific steps are as follows: (1) Cultivate MDBK cells, add cell lysis buffer to MDBK cells infected with BoHV-1 virus for 6 h, 12 h, 24 h, 36 h and uninfected with BoHV-1 virus for lysis, then collect the lysate, add 1 / 3 volume of loading buffer to it and boil it for 10 min.

[0071] A549 cells were cultured, and cell lysate was added to A549 cells infected with BoHV-1 virus for 24 h, 48 h and uninfected with BoHV-1 virus for lysis. The lysates were then collected, 1 / 3 volume of loding buffer was added thereto, and then boiled for 10 min.

[0072] Take the BoHV-1 virions stored in the laboratory, add 1 / 3 volume of loding buffer to it and boil it for 10 minutes.

[0073] (2) SDS-PAGE electrophoresis: The above samples were subjected to SDS-PAGE electrophoresis.

[0074] (3) Transfer: After electrophoresis, use a semi-dry transfer apparatus to transfer the protein to the PVDF membrane.

[0075] (4) Blocking: The above samples were used for Western Blot experiments to identify the immune status of mice. The blocking solution was 5% skim milk for 1 h.

[0076] (5) Primary antibody incubation: The primary antibody is the mouse serum obtained in the above step, diluted with 1% horse serum, incubated at room temperature for 3 h, and washed with PBS buffer for 1 h.

[0077] (6) Secondary antibody incubation: The secondary antibody was HRP-rabbit anti-mouse, diluted with 5% skim milk, incubated at room temperature for 1 h, and washed with PBS buffer for 1 h.

[0078] (7) Development: Add ECL color developing solution for development.

[0079] The results are as follows Figure 4 As shown in the figure, the serum of mice immunized with pv1-KLH protein can react with the lysate of MDBK cells infected with virus for 24 hours but not with the lysate of MDBK cells not infected with virus, and only shows a specific band at 130 kDa. In MDBK cells infected with virus at different time periods, viral proteins containing this B cell epitope can be detected from 12 hours onwards, and in A549 cells infected with virus, viral proteins containing this B cell epitope can be detected at 48 hours of virus infection. Even if the serum is diluted 1,2000 times, the viral protein can still be detected, and it only appears in the virus-infected cell samples, while the negative control does not show this band. In addition, with the increase of serum concentration, the intensity of the band gradually increases, further proving the high specificity and sensitivity of the antibody.

[0080] However, the sera of mice immunized with pv2-KLH, pv3-KLH, and pv4-KLH could not react specifically with the lysate of MDBK cells infected with the virus for 24 hours or the lysate of MDBK cells not infected with the virus.

[0081] In summary, the antibodies induced by the B cell epitope polypeptide pv1 of the BoHV-1 virus gC protein can be used in Western blot experiments to specifically identify the BoHV-1 virus.

[0082] 5. Detection of BoHV-1 antibody levels in cattle serum on cattle farms More than 100 sera were collected from different cattle farms, and the antibody levels were detected using a commercial BoHV-1 antibody detection kit. 22 BoHV-1 antibody-positive sera and 22 BoHV-1 antibody-negative sera were selected for subsequent studies. The results of these 44 serum tests are shown in Table 3.

[0083] Table 3 Test results of commercial BoHV-1 antibody detection kit for serum collected from cattle farms .

[0084] Example 3: Establishment of BoHV-1 antibody ELISA detection kit based on screened B cell epitopes This example establishes a BoHV-1 antibody ELISA detection kit based on the antigen epitope polypeptide pv1 screened in the above embodiment, wherein the indirect ELISA detection method is as follows: (1) Antigen coating: Dilute the coupled protein to a certain concentration in the coating solution using CBS carbonate buffer as the coating antigen. Add the diluted antigen in a volume of 100 μL to the ELISA plate and coat it at 4°C overnight.

[0085] (2) Washing: Pour away the coating solution from the ELISA plate, wash the plate once with PBST and pat dry.

[0086] (3) Blocking: Add 300 μL of blocking solution to each well of the ELISA plate and block for 1 h at room temperature.

[0087] (4) Washing: After blocking, discard the blocking solution, wash the plate twice with PBST and pat dry.

[0088] (5) Incubation with primary antibody: Dilute the test serum in a certain ratio using primary antibody diluent, add 100 μL of the aliquot to the ELISA plate, and incubate at 37 °C for 3 h.

[0089] (6) Washing: After the primary antibody incubation is completed, discard the primary antibody serum, wash the plate 6 times with PBST and pat dry. Add PBST each time and let it stand for 5 min. After the fifth wash, place the ELISA plate on a shaker and shake it for 3 s.

[0090] (7) Incubation with secondary antibody: Use secondary antibody diluent to dilute HRP-rabbit anti-bovine secondary antibody at a certain ratio, take 100 μL and add it to the ELISA plate, seal the plate and incubate for 1 h at room temperature in the dark.

[0091] (8) Washing: After the secondary antibody incubation is completed, discard the secondary antibody, wash the plate 7 times with PBST and pat dry. Add PBST each time and let it stand for 5 min. After the 5th, 6th, and 7th washes, place the ELISA plate on a shaker and shake it for 3 s.

[0092] (9) Color development: Add 100 μL of TMB substrate to each well of the ELISA plate and develop the color for 10 min at room temperature in the dark. Add 100 μL of 10% H2SO4 to stop the color development and measure the OD450 value of each well with an ELISA reader.

[0093] Based on the above detection method, its operating steps were optimized, and the optimal antigen coating concentration, optimal serum dilution multiple, optimal primary antibody dilution, optimal blocking solution, optimal secondary antibody dilution, and optimal secondary antibody dilution were determined to improve the accuracy of the BoHV-1 antibody ELISA detection kit.

[0094] 1. Determination of the optimal antigen coating concentration 100 μL of antigen peptides of different concentrations diluted with CBS carbonate buffer were added to the wells of the ELISA plate. The antigen concentration gradually increased from left to right, and the concentrations were: 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 80 μg / mL. Two wells were repeated for each concentration of antigen, and the coating was carried out at 4°C overnight. After the coating was completed, the coating solution was discarded, the plate was washed once with PBST buffer, and 300 μL of 5% skim milk was added for blocking for 1 h. The blocking solution was discarded, the plate was washed twice with PBST buffer, and BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum (1:100) were added, and incubated at room temperature for 3 h. The primary antibody serum was discarded, the plate was washed 6 times with PBST buffer, and HRP-rabbit anti-bovine secondary antibody diluted with 5% skim milk (1:5000) was added, and incubated at room temperature for 1 h. The secondary antibody was discarded, and the plate was washed 7 times with PBST buffer. After patting dry, 100 μL of TMB substrate was added to each well. The color was developed at room temperature in the dark for 15 min. 100 μL of 10% H2SO4 was added to stop the color development, and the OD450 value of each well was read with an enzyme reader.

[0095] The results are shown in Table 4. When the antigen protein concentration is 30 μg / mL, the ratio of the OD450 value of BoHV-1 positive bovine serum to the OD450 value of negative bovine serum is the highest. Therefore, 30 μg / mL is preferably used as the optimal antigen coating concentration.

[0096] Table 4 Effect of different antigen coating concentrations on the ratio of BoHV-1 positive / negative bovine serum OD450 values .

[0097] 2. Determination of the optimal serum dilution factor 100 μL of antigen peptide (40 μg / mL) diluted with CBS carbonate buffer was added to the wells of the ELISA plate, and coated overnight at 4°C. The coating solution was discarded, the plate was washed once with PBST buffer, and 300 μL of 5% skim milk was added for blocking for 1 h. The blocking solution was discarded, the plate was washed twice with PBST buffer, and BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum were added at different dilutions. The serum dilutions gradually decreased from left to right, and the dilutions were: 1:800, 1:400, 1:200, 1:100, 1:50, 1:10, and each dilution was repeated in two wells, and incubated at room temperature for 3 h. The primary antibody serum was discarded, the plate was washed 6 times with PBST buffer, and HRP-rabbit anti-bovine secondary antibody (1:5,000) diluted with 5% skim milk was added and incubated at room temperature for 1 h. The secondary antibody was discarded, and the plate was washed 7 times with PBST buffer. After patting dry, 100 μL of TMB substrate was added to each well, and color was developed at room temperature in the dark for 15 min. 100 μL of 10% H2SO4 was added to stop color development, and OD450 was read with a microplate reader.

[0098] The results are shown in Table 5. When the serum dilution is 1:50, the ratio of the OD450 value of BoHV-1 positive bovine serum to the OD450 value of negative bovine serum is relatively high, and the serum demand is moderate. Therefore, 1:50 is preferably the optimal serum dilution multiple.

[0099] Table 5 Effect of different serum dilution multiples on the ratio of BoHV-1 positive / negative bovine serum OD450 values .

[0100] 3. Optimal dilution of primary antibody 100 μL of antigen peptide (40 μg / mL) diluted with CBS carbonate buffer was added to the wells of the ELISA plate, and coated overnight at 4 °C. The coating solution was discarded, the plate was washed once with PBST buffer, and 300 μL of 5% skim milk was added for blocking for 1 h. The blocking solution was discarded, the plate was washed twice with PBST buffer, and BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with different primary antibody diluents were added. From left to right, the serum dilutions were: PBS, 1% horse serum, and 5% skim milk. Each dilution was repeated in two wells and incubated at room temperature for 3 h. The primary antibody serum was discarded, the plate was washed 6 times with PBST buffer, and HRP-rabbit anti-bovine secondary antibody (1:5,000) diluted with 5% skim milk was added and incubated at room temperature for 1 h. The secondary antibody was discarded, and the plate was washed 7 times with PBST buffer. After patting dry, 100 μL of TMB substrate was added to each well. The color was developed at room temperature in the dark for 15 min. 100 μL of 10% H2SO4 was added to stop the color development, and the OD450 value of each well was read with an enzyme reader.

[0101] The results are shown in Table 6. When the primary antibody diluent is 1% horse serum, the ratio of the BoHV-1 positive bovine serum OD450 value to the negative bovine serum OD450 value is the highest. Therefore, 1% horse serum is preferably used as the optimal primary antibody dilution.

[0102] Table 6 Effect of different primary antibody dilutions on the ratio of BoHV-1 positive / negative bovine serum OD450 values .

[0103] 4. Determination of the Optimal Blocking Solution 100 μL of antigen peptide (40 μg / mL) diluted with CBS carbonate buffer was added to the wells of the ELISA plate, and coated overnight at 4 °C. The coating solution was discarded and the plate was washed once with PBST buffer. Different blocking solutions were added to different wells for blocking, including 5% skim milk, 5% OVA protein, and commercial rapid blocking solution. Each blocking solution experimental group was repeated twice and blocked at room temperature for 1 h. The blocking solution was discarded and the plate was washed twice with PBST buffer, and BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum (1:50) were added, and incubated at room temperature for 3 h. The primary antibody serum was discarded, the plate was washed 6 times with PBST buffer, and HRP-rabbit anti-bovine secondary antibody diluted with 5% skim milk (1:5,000) was added, and incubated at room temperature for 1 h. The secondary antibody was discarded, and the plate was washed 7 times with PBST buffer. After patting dry, 100 μL of TMB substrate was added to each well. The color was developed at room temperature in the dark for 15 min. 100 μL of 10% H2SO4 was added to stop the color development, and the OD450 value of each well was read with an enzyme reader.

[0104] The results are shown in Table 7. When the blocking solution is 5% OVA, the ratio of the OD450 value of BoHV-1 positive bovine serum to the OD450 value of negative bovine serum is the highest. Therefore, the preferred best blocking solution is 5% OVA protein solution.

[0105] Table 7 Effect of different blocking solutions on the ratio of OD450 values ​​of BoHV-1 positive / negative bovine serum .

[0106] 5. Determination of the optimal dilution of the secondary antibody 100 μL of antigen peptide (40 μg / mL) diluted with CBS carbonate buffer was added to the wells of the ELISA plate, and coated overnight at 4 °C. The coating solution was discarded, the plate was washed once with PBST buffer, and 300 μL of 5% OVA protein solution was added for blocking at room temperature for 1 h. The blocking solution was discarded and the plate was washed twice with PBST buffer, and BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum (1:50) were added, and incubated at room temperature for 3 h. The primary antibody serum was discarded, the plate was washed 6 times with PBST buffer, and HRP-rabbit anti-bovine secondary antibody (1:000) diluted with different diluents was added to different wells. From left to right, the secondary antibody diluents were: PBST, 1% horse serum, 5% skim milk, and incubated at room temperature for 1 h. The secondary antibody was discarded, and the plate was washed 7 times with PBST buffer. After patting dry, 100 μL of TMB substrate was added to each well, and color was developed at room temperature in the dark for 15 min. After adding the stop solution, the OD450 value of each well was read with an ELISA reader.

[0107] The results are shown in Table 8. When the secondary antibody dilution is 5% skim milk, the ratio of the BoHV-1 positive bovine serum OD450 value to the negative bovine serum OD450 value is the highest. Therefore, the optimal secondary antibody dilution is preferably 5% skim milk.

[0108] Table 8 Effect of different secondary antibody dilutions on the OD450 value ratio of BoHV-1 positive / negative bovine serum .

[0109] 6. Optimal dilution of secondary antibody 100 μL of antigen peptide (40 μg / mL) diluted with CBS carbonate buffer was added to the wells of the ELISA plate, and coated overnight at 4 °C. The coating solution was discarded, the plate was washed once with PBST buffer, and 300 μL of 5% OVA protein solution was added for blocking at room temperature for 1 h. The blocking solution was discarded and the plate was washed twice with PBST buffer, and BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum (1:50) were added, and incubated at room temperature for 3 h. The primary antibody serum was discarded, the plate was washed 6 times with PBST buffer, and HRP-rabbit anti-bovine secondary antibody (1:2500, 1:5000, 1:7500, 1:10000) diluted with 5% skim milk was added to different wells, and incubated at room temperature for 1 h. The secondary antibody was discarded, and the plate was washed 7 times with PBST buffer. After patting dry, 100 μl of TMB substrate was added to each well, and color was developed at room temperature in the dark for 15 min. After adding the stop solution, the OD450 value of each well was read with an ELISA reader.

[0110] The results are shown in Table 9. When the dilution of HRP-rabbit anti-bovine secondary antibody was 1:5,000, the ratio of BoHV-1 positive bovine serum OD450 value to negative bovine serum OD450 value was the highest. Therefore, the optimal secondary antibody dilution was 1:5000.

[0111] Table 9 Effect of different secondary antibody dilutions on the ratio of BoHV-1 positive / negative bovine serum OD450 values .

[0112] 7. Determination of blocking value Using the above-mentioned determined detection conditions, 24 bovine sera that have been identified as negative by commercial detection kits were tested, and their OD450 values ​​were detected. The mean value X and standard deviation SD of the OD450 value of negative bovine serum were calculated. The blocking value was set to X+3SD. That is, when the serum OD450 value is <X+3SD, it is defined as BoHV-1 virus negative serum; when the serum OD450 value is ≥X+3SD, it is defined as BoHV-1 virus positive serum.

[0113] The results are shown in Table 10. The mean value X of the negative serum OD450 value is 0.2314125, the standard deviation SD is 0.042184216, and the blocking value is 0.357965. Therefore, when the serum OD450 value is <0.357965, it is defined as BoHV-1 virus negative serum; when the serum OD450 value is ≥0.357965, it is defined as BoHV-1 virus positive serum.

[0114] Table 10 Results of indirect ELISA testing of 24 negative sera .

[0115] 8. Intra-assay and inter-assay reproducibility Using the above-mentioned experimental conditions and blocking values, intra-batch and inter-batch repeatability experiments were carried out on 6 bovine sera using ELISA plates coated with the same batch and different batches. Three replicates were set for each sample, and the OD450 value was measured. The coefficient of variation (CV) of the same sample was calculated to test the repeatability of the method.

[0116] The results are shown in Table 11. The coefficient of variation of repeated experiments on 6 different bovine sera within the batch was between 3.1% and 8.8%, and the coefficient of variation of repeated experiments between batches was between 2.9% and 12.6%, indicating that ELISA plates coated with different batches had low variation and good repeatability when detecting the same sample serum.

[0117] Table 11 Repeatability experiment of detection method .

[0118] 9. Compliance rate detection Using the above-mentioned determined experimental conditions and blocking values, 44 bovine sera identified by commercial BoHV-1 virus antibody detection kits were tested, and the detection compliance rate of this detection method and the commercial BoHV-1 antibody detection kit was calculated.

[0119] The results are shown in Table 12. According to the established detection method, 20 sera were BoHV-1 positive and 24 sera were BoHV-1 negative. Compared with the commercial BoHV-1 virus antibody detection kit, only two serum test results were inconsistent, with a compliance rate of 95.45%, which was good.

[0120] Table 12 Test results of collected bovine serum .

[0121] 10. ELISA test method after establishment The kit contains: blocking solution (5% OVA, diluted in PBS), primary antibody diluent (1% horse serum, diluted in PBS), secondary antibody diluent (5% skim milk, diluted in PBS), HRP-rabbit anti-bovine secondary antibody, BoHV-1 negative control, TMB substrate, and antigen-coated ELISA plate.

[0122] Steps: (1) Blocking: Add 300 μL of blocking solution (5% OVA) to each well of the ELISA plate and block for 1 h at room temperature.

[0123] (2) Washing: After blocking, discard the blocking solution, wash the plate twice with PBST and pat dry.

[0124] (3) Incubation with primary antibody: Dilute the test serum 1:50 using primary antibody diluent (1% horse serum), add 100 μL of the aliquot to the ELISA plate, and incubate at 37 °C for 3 h.

[0125] (4) Washing: After the primary antibody incubation is completed, discard the primary antibody serum, wash the plate six times with PBST and pat dry. Add PBST and let it stand for 5 min each time. After the fifth wash, place the ELISA plate on a shaker and shake for 3 s.

[0126] (5) Incubation with secondary antibody: Dilute HRP-rabbit anti-bovine secondary antibody at a ratio of 1:5,000 using secondary antibody diluent (5% skim milk), add 100 μL to the ELISA plate, seal the plate and incubate for 1 h at room temperature in the dark.

[0127] (6) Washing: After the secondary antibody incubation is completed, discard the secondary antibody, wash the plate seven times with PBST and pat dry. Add PBST and let it stand for 5 min each time. After the fifth, sixth and seventh washes, place the ELISA plate on a shaker and shake it for 3 seconds.

[0128] (7) Color development: Add 100 μL of TMB substrate to each well of the ELISA plate and develop the color for 10 min at room temperature in the dark. Add 100 μL of 10% H2SO4 to stop the color development and measure the OD450 value of each well with an ELISA reader.

[0129] (8) Judgment: When the serum OD450 value is less than 0.357965, it is defined as BoHV-1 virus negative serum; when the serum OD450 value is ≥ 0.357965, it is defined as BoHV-1 virus positive serum.

[0130] This detection method / test kit has high specificity, and the detection consistency rate with the commercial BoHV-1 antibody detection kit can reach 95.45%. The degree of variation between different batches of detection is low and the repeatability is good.

[0131] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0132] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A BoHV-1 viral antigen epitope polypeptide, whose amino acid sequence is shown in SEQ ID NO.

1.

2. A coupling protein, characterized in that It is formed by coupling the BoHV-1 virus antigen epitope polypeptide described in claim 1 with hemocyanin.

3. the application of the BoHV-1 viral antigen epitope polypeptide described in claim 1 or coupled protein described in claim 2 in preparing immunological detection reagents.

4. the BoHV-1 viral antigen epitope polypeptide described in claim 1 or the application of coupled protein described in claim 2 in preparing antibodies or vaccines against BoHV-1 viruses.

5. the application of the BoHV-1 viral antigen epitope polypeptide described in claim 1 or the coupled protein described in claim 2 in the preparation of the reagent detecting BoHV-1 virus.

6. An enzyme-linked immunosorbent assay kit for detecting BoHV-1 virus, containing a blocking solution, a primary antibody diluent, a secondary antibody diluent, an HRP-rabbit anti-bovine secondary antibody, a BoHV-1 negative control, a TMB substrate, an ELISA plate, characterized in that, The ELISA plate is coated with the BoHV-1 viral antigen epitope polypeptide according to claim 1.

7. The ELISA kit according to claim 6, characterized in that: The blocking solution is 4-6% ovalbumin diluted with phosphate buffer.

8. The enzyme-linked immunosorbent assay kit according to claim 6, characterized in that: The primary antibody diluent is 0.8-1.2% horse serum diluted with phosphate buffer.

9. The enzyme-linked immunosorbent assay kit according to claim 6, characterized in that: The secondary antibody diluent is 4-6% skim milk diluted with phosphate buffer.

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