BoHV-1 virus antigenic epitope polypeptide, its conjugate protein, enzyme-linked immunosorbent assay kit and application

Abstract

The present invention application discloses a BoHV-1 virus antigenic epitope polypeptide, its conjugate protein, an enzyme-linked immunosorbent assay kit and applications thereof, aiming to solve the technical problems of the scarcity of BoHV-1 virus immune kits, complex preparation processes and high costs. The present invention application first screens for dominant mimotopes using a phage library and BoHV-1 virus polyclonal antiserum; subsequently synthesizes corresponding polypeptides to identify the dominant B cell epitopes of the virus, and further screens for B cell epitopes suitable for antibody preparation; finally, develops an ELISA kit for detecting BoHV-1 virus based on the B cell epitopes. The B cell epitope polypeptide provided by the present invention application has high specificity and sensitivity, and the provided enzyme-linked immunosorbent assay kit has high specificity and good detection performance.

Description

Technical Field

[0001] This invention application relates to the field of bioimmunotechnology, specifically to a BoHV-1 viral antigen epitope polypeptide, its coupled protein, an enzyme-linked immunosorbent assay kit, and its applications. Background Art

[0002] Bovine herpesvirus type 1 (BoHV-1), also known as bovine infectious rhinotracheitis virus (IBRV), belongs to the Herpesviridae family and the Alphaherpesvirinae subfamily. The genome of this virus is between 135kb and 140kb in size and encodes at least 73 proteins. Its genome consists of a long unique region (UL, approximately 102kb–104kb), a short unique region (US, approximately 10.5kb–11kb), and two terminal inverted repeat sequences (approximately 24kb). As one of the most important pathogens causing bovine diseases, BoHV-1 infection can trigger various inflammatory diseases, damaging the respiratory tract, nasal cavity, and ocular mucosa of cattle. This makes them susceptible to secondary infections with other pathogens, such as bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza virus-3 (PI3V), and bacteria such as Mansonia hemolyticus, Pasteurella multocida, and Histolytica acidophilus, leading to bovine respiratory disease syndrome (BRDC). BRDC treatment is lengthy and costly, can lead to calf mortality, and BoHV-1 is also a major pathogen causing abortion in pregnant cows, resulting in enormous economic losses to the global cattle industry. It is estimated that the annual losses due to BoHV-1 infection in the United States alone reach as high as $3 billion.

[0003] Currently, there are limited commercially available antibodies against BoHV-1 viral proteins, and they are expensive. For example, VMRD's gD and gC protein monoclonal antibodies cost as much as 4,600 yuan per 100 μL, and the price continues to rise, significantly increasing research costs for researchers in related fields. At present, my country has no independently developed antibody products against this virus with independent intellectual property rights available on the market.

[0004] Strengthening antibody detection is one of the key technical means for the prevention and control of BoHV-1. Currently, ELISA antibody detection kits, both domestically and internationally developed, generally use baculovirus or E. coli expression systems to prepare the coating antigen. However, this method has significant problems: the expressed protein needs to undergo a purification step, which is not only costly and complex, but also relies on expensive equipment. Furthermore, 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 section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] This invention discloses a BoHV-1 viral antigenic epitope polypeptide, its coupled protein, an enzyme-linked immunosorbent assay kit, and its applications, aiming to solve the technical problems of the scarcity of BoHV-1 viral immunosorbent assay kits and the complexity and high cost of their preparation process.

[0007] This invention first utilizes a phage library and BoHV-1 virus polyantiserum to screen for dominant mimic epitopes; then, corresponding peptides are synthesized to identify dominant B-cell epitopes of the virus, and further screens for B-cell epitopes suitable for antibody preparation; finally, based on these B-cell epitopes, an ELISA kit for detecting BoHV-1 virus is developed.

[0008] Specifically, this disclosure provides a BoHV-1 viral antigenic epitope polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] According to a second aspect of this disclosure, a conjugated protein is provided, which is formed by conjugating the above-mentioned BoHV-1 viral antigenic epitope polypeptide with hemocyanin.

[0010] According to a third aspect of this disclosure, the BoHV-1 viral antigenic epitope polypeptide or the conjugate protein is used in the preparation of immunological detection reagents.

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

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

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

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

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

[0016] In some embodiments of this 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 this application have at least one of the following technical effects or advantages:

[0018] 1. The B-cell antigenic epitope polypeptide of BoHV-1 virus gC protein was screened and coupled with hemocyanin to form pv1-KLH coupled protein. The serum of mice immunized with this protein can specifically recognize BoHV-1 virus protein, and the virus protein can still be detected even when the serum is diluted 12,000 times, which shows high specificity and sensitivity.

[0019] 2. A BoHV-1 antibody ELISA kit was constructed based on the B-cell antigen epitope peptide. The coefficient of variation of the kit in batch-to-batch repeatability experiments was between 2.9% and 12.6%, which showed high repeatability, good consistency and high specificity. It can accurately detect the target antigen and has a high concordance rate of 95.45% compared with existing commercial kits, demonstrating good detection performance. Attached Figure Description

[0020] Figure 1 This is the Dot-Blot identification result of phage screening in one embodiment of this application; where + represents BoHV-1 virus; - represents irrelevant phage.

[0021] Figure 2 This is an example of comparing a bacteriophage mimic epitope sequence with a viral protein sequence in one embodiment of this application.

[0022] Figure 3 This is the Dot-Blot detection result of the peptide-KLH coupled protein in one embodiment of this application; where + represents BoHV-1; - represents KLH.

[0023] Figure 4 This is the Western Blot result of serum from immunized mice in one embodiment of this application. Detailed Implementation

[0024] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] 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; and the detection, testing and preparation methods involved are all conventional methods unless otherwise specified.

[0026] Example 1: Screening BoHV-1 phage mimic epitopes using a phage random peptide library

[0027] This example utilizes a phage random peptide library combined with BoHV-1 virus polyantiserum to screen for dominant mimic epitopes of BoHV-1 phage, thereby identifying key regions on the viral surface that can be recognized by the immune system. The specific steps are as follows:

[0028] 1. Panning of phage random peptide libraries

[0029] (1) Antibody coating: In a clean bench, using a 60 mm tissue culture dish as the coating plate, dissolve BoHV-1 polyclonal antibody serum in 0.1 mol / L NaHCO3 (pH 8.6), 80 μg / mL, mix by pipetting and gently shaking the cell culture dish to completely submerge the bottom. Place in a humidified chamber and incubate overnight at 4 ℃.

[0030] (2) Blocking: Take out the cell culture dish coated with antibody, tilt it gently, use a pipette to remove all the liquid from the wall, then add 4 mL of the prepared 1% BSA blocking solution, put it back into the humidification chamber, and block at 4 ℃ for 2 h.

[0031] (3) Washing: Discard the blocking solution and wash 6 times with 0.1% TBST for 1-2 minutes each time. After each wash, pat the petri dish dry on a clean paper towel (the action should be quick to avoid drying out the petri dish).

[0032] (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 phages added is 1×10⁻⁶). 11 After adding PFU, add it to the cell culture dish, place it on a shaker, and incubate at room temperature for 1 h.

[0033] (5) Washing: Aspirate the above liquid and wash 10 times with 0.1% PBST to discard unbound phages.

[0034] (6) Elution: Add 1 mL of elution buffer (0.2 mol / L glycline-HCl, pH 2.2, 1 mg / mL BSA), place on a shaker, and shake at room temperature for 10-20 min to elute the specifically bound phage. Collect the eluent in a sterile centrifuge tube pre-filled with 150 μL Tris-HCl buffer (1 mol / L, pH 9.1) to neutralize the pH value, thus obtaining the first round of specifically bound phage. Store at 4 ℃, take 10 μL for determining the phage titer, and use the remainder for the next round of panning after amplification and purification.

[0035] 2. Amplification and purification of specific bacteriophages

[0036] (1) The bacteriophages obtained after panning were added to the single colony culture of Escherichia coli (E. coli ER2738), placed on a shaker, and cultured vigorously at 37 ℃ for 5 h, so that the bacteriophages infected E. coli and amplified in E. coli, and lysed E. coli.

[0037] (2) Centrifuge at 12,000 g for 10 min at 4℃ to remove unlysaturated E. coli precipitate from the solution. Transfer 80% of the supernatant to a new centrifuge tube, add 1 / 6 volume of 20% PEG solution to the tube, let stand at 4℃ for 2 h, then centrifuge at 12,000 g for 15 min at 4℃ and discard the supernatant.

[0038] (3) Take 1.5 mL of TBS to resuspend the precipitate, centrifuge briefly at 4 ℃ to remove the remaining cell debris precipitate, add 1 / 6 volume of 20% PEG solution to the supernatant, let stand for 2 h, centrifuge at 4 ℃ for 12,000 g for 15 min, take 200 μL of TBS to resuspend the phage precipitate, and obtain the amplified and purified panning phage.

[0039] 3. Phage titer determination

[0040] (1) The selected bacteriophages were cultured in LB liquid medium at 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 The phage was diluted 6 times at each of the 6 dilution ratios. Then, 10 μL of the diluted phage was added to 200 μL of E. coli culture and incubated at room temperature for 5 min.

[0041] (2) Add the incubated phage Escherichia coli mixture to 5 mL of top agar, mix well, spread evenly on LB solid medium plates, and invert them at 37 ℃ for overnight incubation after solidification.

[0042] (3) The next day, examine the plates, count the plaques on the plates, and calculate the titer units (pfu).

[0043] The above screening process was repeated three times. The phages eluted after the third screening did not need to be amplified. The phages were then serially diluted 10-fold according to the method described in Example 3, and the number of plaques was measured. The phage titer was then determined, and the results are shown in Table 1. The results indicate that when the phage dosage was consistent (1×10⁻⁶), the phages were effectively amplified. 11In the case of ), starting from the second round of panning, the concentration of Tween-20 in the washing buffer 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 onto the ligand increased in each round, indicating that the phages specifically binding to BoHV-1 antiserum were effectively selectively enriched after affinity screening.

[0044] Table 1. Enrichment of bacteriophages selected in the three-round affinity screening.

[0045] .

[0046] 4. Preparation of bacteriophage stock solution

[0047] Plates with approximately 100-150 plaques were selected. 150 single-clone plaques were randomly picked using a sterile pipette tip and placed in 1 mL of LB liquid medium containing E. coli ER2738 in the early logarithmic growth phase. The plates were incubated at 37 °C with vigorous shaking for 5 h. After centrifugation at 4 °C, 14,000 r / min for 30 s, the supernatant was transferred to a new centrifuge tube and centrifuged again. The upper 80% of the supernatant was collected as a storage solution for single phage clones for further analysis.

[0048] 5. Purification and sequencing of sequencing templates

[0049] (1) Take out 750 μL of phage supernatant, add 300 μL of 20% PEG (2.5 mol / L NaCl) solution, mix by inverting repeatedly, and let stand at room temperature for 10-20 min.

[0050] (2) Centrifuge at 4 ℃ and 14,000 r / min for 10 min, discard the supernatant, centrifuge again, and discard the supernatant.

[0051] (3) Add 100 μL of iodide buffer to the precipitate, resuspend and mix well, 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).

[0052] (4) Centrifuge at 4 ℃ and 14,000 r / min for 10 min, discard the supernatant, add 500 μL of 75% ethanol to wash the precipitate, centrifuge at 14,000 r / min for 10 min, discard the supernatant, and finally put the precipitate into a 37 ℃ oven to dry for 10 min.

[0053] (5) The precipitate was suspended in 30 μL TE buffer (pH 8.0) to obtain phage single-stranded DNA, which was then stored at -20 °C.

[0054] (6) The phage template DNA was sent to Genewiz Biotechnology Sequencing Co., Ltd., and DNA sequencing was performed using specific primers 96gⅢ: 5'-CCC TCATAG TTA GCG TAA CG-3'. The sequencing results were then translated into amino acid sequences using the free biological software DNAstar, thus obtaining the simulated epitopes.

[0055] 6. Dot-Blot identification of phage mimic epitopes

[0056] The monoclonal phages screened were identified using Dot-blot Analysis. The specific steps are as follows:

[0057] (1) Cutting the film: Take a nitrocellulose membrane (NC membrane) and cut it to the appropriate size.

[0058] (2) Spotting: Take 5 μL of each of the cultured BoHV-1 virus (positive control), irrelevant phage (negative control) and the phage storage solution obtained by screening and spot it on the corresponding position on the NC membrane and dry at room temperature.

[0059] (3) Sealing: Immerse the NC membrane in 5% skim milk, place it on a shaker, and seal it at room temperature for 1.5 h.

[0060] (4) Incubation of primary antibody: The sealed nitrocellulose membrane was rinsed with PBS, and after rinsing, BoHV-1 polyclonal antibody serum diluted with 1% horse serum (1:5000) was added. The membrane was placed on a shaker and incubated at room temperature for 3 h.

[0061] (5) Washing the membrane: Wash the incubated NC membrane with PBS for 1 h.

[0062] (6) Incubation of secondary antibody: After rinsing, add HRP-rabbit anti-sheep secondary antibody (1:8000) diluted with 5% skim milk and incubate at room temperature for 1 h.

[0063] (7) Washing the membrane: Wash the membrane three times with PBS after incubation.

[0064] (8) Development: Mix the A and B solutions of the ECL luminescent solution in a 1:1 ratio and then drop them onto the NC membrane for subsequent development.

[0065] The results of the identification are as follows Figure 1 As shown, the results indicated that a total of 15 phages capable of reacting with BoHV-1 polyclonal antibody serum were screened, labeled as p116, p108, p109, p49, p50, p71, p76, p104, p113, p59, p133, p62, p63, p65, and p93, respectively. Irrelevant phages did not react with polyclonal antibody serum. Therefore, it is preliminarily believed that these phages possess mimic epitopes.

[0066] Example 2: B-cell epitope analysis and identification

[0067] 1. Peptide synthesis

[0068] In this example, the sequencing results of the simulated epitopes screened in Example 1 were compared with the BoHV-1 viral protein sequence in Snapgene software. Based on these simulated epitopes, multiple viral protein epitope sequences were inferred, of which four viral protein epitope sequences were on the envelope protein. Figure 2 These viral epitope sequences were labeled pv1, pv2, pv3, and pv4 (SEQ ID NO. 1~4) for subsequent research in this case. The above viral epitope sequences were then used by Shanghai Jier Biochemical Co., Ltd. for peptide synthesis (Table 2).

[0069] Table 2. Nomenclature of synthesized peptides and their amino acid sequence list

[0070] .

[0071] 2. Peptide-coupled carrier protein hemocyanin KLH

[0072] B-cell epitopes are specific regions on an antigen molecule that can be recognized and bound by B cells. Based on immunological principles, these epitopes can be recognized by specific antibodies and therefore can serve as coating agents for establishing detection methods. Although B-cell epitopes themselves may only be haptens, they can potentially transform into complete antigens when conjugated with a specific carrier. This transformation allows them to function as immunogens for antibody preparation. Therefore, to enhance the immunogenicity of peptides, the peptides are conjugated with KLH using Sulfo-SMCC as the conjugation catalyst. The specific steps are as follows:

[0073] (1) Dissolve the 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.

[0074] (2) Take 100 μL of Sulfo-SMCC solution and slowly add it to 600 μL of KLH protein solution while stirring continuously to mix it evenly. React at room temperature for 2 h.

[0075] (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 solution was changed every two hours during dialysis, for a total of three changes. The dialysate was PBS (containing 3 mM EDTA). After dialysis, the concentration of SMCC-activated KLH protein was adjusted to 4 mg / mL.

[0076] (4) Dissolve the antigenic peptide in PBS (containing 3 mM EDTA) and adjust the concentration to 4 mg / mL. Slowly mix the SMCC-activated KLH solution with the antigenic peptide solution at a mass ratio of 6:5, let it stand at room temperature for 4 h, and then let it stand overnight at 4 ℃.

[0077] 3. Identification of the reactivity between antibody and peptide-KLH conjugate protein

[0078] To determine whether the conjugated peptide-KLH protein specifically reacts with the BoHV-1 antibody, the conjugated protein was identified using a Dot-Blot method with commercially available BoHV-1 antibody and clinically isolated BoHV-1-positive bovine serum.

[0079] (1) Cutting and spotting the membrane: Take a nitrocellulose membrane (NC membrane) and cut it to a suitable size. Spot the positive control BoHV-1 virus (7 μL), the negative control KLH (5 mg / mL, 7 μL), the KLH-phage mimic epitope peptide (5 mg / mL, 7 μL) and the KLH-virus epitope peptide (5 mg / mL, 7 μL) on the corresponding positions on the membrane from left to right. Allow the membrane to dry at room temperature for 20 min.

[0080] (2) Sealing: 5% skim milk sealed at room temperature for 1 h.

[0081] (3) Incubation of primary antibody: Discard the blocking solution, add primary antibody serum (commercial BoHV-1 polyclonal antibody serum / BoHV-1 positive bovine serum isolated and detected clinically), dilute with 1% horse serum, the dilution ratio is 1:5000, and incubate at room temperature for 3 h.

[0082] (4) Washing the membrane: Wash the membrane with PBS for 1 h.

[0083] (5) Incubation of secondary antibody: Add HRP-rabbit anti-goat secondary antibody, dilute with 5% skim milk, the dilution ratio is 1:8000, and incubate at room temperature for 1 h.

[0084] (6) Washing the membrane: Wash the membrane with PBS for 1 h.

[0085] (7) Development: Add ECL color developer to develop.

[0086] The results are as follows Figure 3As shown, all eight peptide-KLH conjugates (pp116-KLH, pv1-KLH, pp108-KLH, pv2-KLH, pp109-KLH, pv3-KLH, pp49-KLH, and pv4-KLH) specifically reacted with commercially available BoHV-1 polyclonal antibody serum. Furthermore, when other reaction conditions remained unchanged, but the primary antibody was replaced with clinically isolated and tested BoHV-1 positive bovine serum, the eight peptide-KLH conjugates also reacted with BoHV-1 positive bovine serum, but not with negative serum. Among these, pp116-KLH, pv1-KLH, pv2-KLH, pv3-KLH, pp49-KLH, and pv4-KLH showed the strongest reactions. This indicates that the peptides were successfully conjugated to KLH proteins and can specifically react with BoHV-1 viral antibodies.

[0087] 4. Immunogenicity identification of BoHV-1 viral antigenic epitope peptides

[0088] Using viral antigenic epitope peptides conjugated with hemocyanin (KLH) as immunogens, 6-8 week old SPF-grade BALB / c mice were used as immunization subjects (the purchased mice were raised for one week to allow them to adapt to the environment without adverse reactions). The immunogenicity of BoHV-1 viral antigenic epitope peptides 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.

[0089] For the first immunization, 50 μg of each immunogen was diluted with 300 μL of autoclaved PBS and mixed with an equal volume of Freund's complete adjuvant. The emulsifier was set to 25,000 r / min and emulsified for 5 min (successful emulsification is indicated when a small amount placed in water does not disperse or disperses slowly). Subcutaneous injections were administered at four points on the back of each mouse, 200 μL / mouse and 50 μL / point. The immunization procedure for the second and third immunizations was the same, but the emulsifier was replaced with Freund's incomplete adjuvant. The first to third immunizations were administered 2 weeks apart. Ten days after the third immunization, blood was collected from the tail of each mouse (10 μL). This blood was diluted with 490 μL of 1xPBS (serum:PBS = 1:50), centrifuged at 4 ℃, 5000 r / min for 10 min, and the supernatant was collected and stored at -20 ℃. This supernatant can be used to measure the immunization effect. The specific steps are as follows:

[0090] (1) Culture MDBK cells. Cell lysis buffer was added to MDBK cells that were infected with BoHV-1 virus for 6 h, 12 h, 24 h, 36 h and uninfected with BoHV-1 virus, respectively, to lyse them. The lysate was then collected, and 1 / 3 volume of loading buffer was added to it and boiled for 10 min.

[0091] A549 cells were cultured, and cell lysis buffer was added to A549 cells that had been infected with BoHV-1 virus for 24 h and 48 h and those that were not infected with BoHV-1 virus, respectively, to induce lysis. The lysis products were then collected, and 1 / 3 volume of loading buffer was added to them and the mixture was boiled for 10 min.

[0092] Take the BoHV-1 virus samples stored in the laboratory, add 1 / 3 volume of loading buffer to them, and boil for 10 minutes.

[0093] (2) SDS-PAGE electrophoresis: The above samples were subjected to SDS-PAGE electrophoresis.

[0094] (3) Transfer: After electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry transfer apparatus.

[0095] (4) Blocking: The above samples were used to perform Western Blot experiments to identify the immune status of mice. The blocking solution was 5% skim milk for 1 h.

[0096] (5) Primary antibody incubation: The primary antibody is the mouse serum obtained in the above steps, diluted with 1% horse serum, incubated at room temperature for 3 hours, and washed with PBS buffer for 1 hour.

[0097] (6) Secondary antibody incubation: The secondary antibody is HRP-rabbit anti-mouse secondary antibody, diluted with 5% skim milk, incubated at room temperature for 1 h, and washed with PBS buffer for 1 h.

[0098] (7) Development: Add ECL color developer to develop.

[0099] The results are as follows Figure 4 As shown, mouse serum immunized with pv1-KLH protein reacted with lysates of MDBK cells infected with the virus for 24 h, but not with lysates of uninfected MDBK cells, exhibiting only a single specific band at 130 kDa. Furthermore, viral proteins containing this B-cell epitope were detectable in MDBK cells infected at different time points, starting from 12 h. In virus-infected A549 cells, viral proteins containing this B-cell epitope were detectable at 48 h of infection. Even after a 12000-fold dilution of the serum, the viral protein remained detectable, appearing only in virus-infected cell samples, while the negative control did not show this band. Moreover, the band intensity gradually increased with increasing serum concentration, further demonstrating the high specificity and sensitivity of the antibody.

[0100] Serum from mice immunized with pv2-KLH, pv3-KLH, and pv4-KLH did not show specific reactions with either MDBK cell lysates infected with the virus for 24 hours or MDBK cell lysates not infected with the virus.

[0101] In summary, antibodies induced by the BoHV-1 virus gC protein B cell antigenic epitope peptide pv1 can be used in Western blot experiments to specifically recognize the BoHV-1 virus.

[0102] 5. Detection of BoHV-1 antibody levels in bovine serum from cattle farms

[0103] More than 100 serum samples were collected from different cattle farms, and antibody levels were detected using a commercially available BoHV-1 antibody detection kit. Twenty-two BoHV-1 antibody-positive sera and twenty-two BoHV-1 antibody-negative sera were selected for subsequent studies. The results of these 44 serum samples are shown in Table 3.

[0104] Table 3. Results of the detection using a commercial BoHV-1 antibody detection kit for serum collected from cattle farms.

[0105] .

[0106] Example 3: Establishment of a BoHV-1 antibody ELISA detection kit based on screened B-cell epitopes

[0107] This example establishes a BoHV-1 antibody ELISA detection kit based on the antigenic epitope peptide pv1 screened in the above embodiments, wherein the indirect ELISA detection method is as follows:

[0108] (1) Antigen coating: The coupled protein was diluted to a certain concentration in the coating solution using CBS carbonate buffer as the coating antigen. The diluted antigen was added to the ELISA plate in a volume of 100 μL and coated overnight at 4°C.

[0109] (2) Washing the plate: Discard the coating solution in the coated ELISA plate, wash the plate once with PBST and pat dry.

[0110] (3) Blocking: Add 300 μL of blocking solution to each well of the ELISA plate and block at room temperature for 1 h.

[0111] (4) Washing: After the blocking is completed, discard the blocking solution, wash the plate twice with PBST and pat dry.

[0112] (5) Incubation of primary antibody: Dilute the serum to be tested with primary antibody dilution buffer at a certain ratio, add 100 μL to the ELISA plate, and incubate at 37 °C for 3 h.

[0113] (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 stand for 5 min. After the 5th wash, place the ELISA plate on a shaker and shake for 3 s.

[0114] (7) Incubation of secondary antibody: Use secondary antibody dilution buffer 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 at room temperature in the dark and incubate for 1 h.

[0115] (8) Washing the plate: 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 stand for 5 min. After the 5th, 6th and 7th washes, place the ELISA plate on a shaker and shake for 3 s.

[0116] (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 using a microplate reader.

[0117] Based on the above detection method, the operation steps were optimized to determine the optimal antigen coating concentration, optimal serum dilution factor, optimal primary antibody dilution, optimal blocking solution, optimal secondary antibody dilution, and optimal secondary antibody dilution, in order to improve the accuracy of the BoHV-1 antibody ELISA detection kit.

[0118] 1. Determination of the optimal antigen coating concentration

[0119] Add 100 μL of antigen peptides diluted with CBS carbonate buffer to the wells of an ELISA plate, with the antigen concentration gradually increasing from left to right: 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 80 μg / mL. Each concentration of antigen is repeated in two wells. Coating is performed overnight at 4°C. After coating, discard the coating solution, wash the plate once with PBST buffer, and block with 300 μL of 5% skim milk for 1 h. Discard the blocking solution, wash the plate twice with PBST buffer, and add BoHV-1 positive and BoHV-1 negative bovine serum diluted 1% horse serum (1:100). Incubate at room temperature for 3 h. Discard the primary antibody serum, wash the plate six times with PBST buffer, and add HRP-rabbit anti-bovine secondary antibody diluted 5% skim milk (1:5000). Incubate at room temperature for 1 h. Discard the secondary antibody, wash the plate 7 times with PBST buffer, pat dry, add 100 μL of TMB substrate to each well, incubate at room temperature in the dark for 15 min, add 100 μL of 10% H2SO4 to stop the color development, and read the OD450 value of each well using a microplate reader.

[0120] The results are shown in Table 4. When the antigen protein concentration was 30 μg / mL, the ratio of the OD450 value of BoHV-1 positive bovine serum to that of negative bovine serum was the highest. Therefore, 30 μg / mL is the optimal antigen coating concentration.

[0121] Table 4. Effect of different antigen coating concentrations on the ratio of OD450 values ​​in BoHV-1 positive / negative bovine serum.

[0122] .

[0123] 2. Determination of the optimal serum dilution factor

[0124] Add 100 μL of antigenic peptide (40 μg / mL) diluted with CBS carbonate buffer to each well of an ELISA plate and incubate overnight at 4°C. Discard the coating solution, wash once with PBST buffer, and block for 1 h with 300 μL of 5% skim milk. Discard the blocking solution, wash twice with PBST buffer, and add BoHV-1 positive and BoHV-1 negative bovine serum diluted with 1% horse serum at different dilutions, from left to right: 1:800, 1:400, 1:200, 1:100, 1:50, and 1:10. Repeat each dilution in two wells and incubate at room temperature for 3 h. Discard the primary antibody serum, wash six times with PBST buffer, and add HRP-rabbit anti-bovine secondary antibody (1:5,000) diluted with 5% skim milk. Incubate at room temperature for 1 h. Discard the secondary antibody, wash the plate 7 times with PBST buffer, pat dry, add 100 μL of TMB substrate to each well, incubate at room temperature in the dark for 15 min, add 100 μL of 10% H2SO4 to stop the color development, and read the OD450 using a microplate reader.

[0125] 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 that of negative bovine serum is relatively high, and the serum requirement is moderate. Therefore, 1:50 is the optimal serum dilution factor.

[0126] Table 5. Effect of different serum dilution factors on the ratio of OD450 values ​​in lower BoHV-1 positive / negative bovine serum.

[0127] .

[0128] 3. Optimal dilution of primary antibody

[0129] Add 100 μL of antigenic peptide (40 μg / mL) diluted with CBS carbonate buffer to each well of an ELISA plate and incubate overnight at 4 °C. Discard the coating solution, wash once with PBST buffer, and block with 300 μL of 5% skim milk for 1 h. Discard the blocking solution, wash twice with PBST buffer, and add BoHV-1 positive and BoHV-1 negative bovine serum diluted with different primary antibody diluents (PBS, 1% horse serum, and 5% skim milk from left to right). Repeat each diluent in two wells and incubate at room temperature for 3 h. Discard the primary antibody serum, wash six times with PBST buffer, and add HRP-rabbit anti-bovine secondary antibody diluted 1:5,000 with 5% skim milk. Incubate at room temperature for 1 h. Discard the secondary antibody, wash the plate 7 times with PBST buffer, pat dry, add 100 μL of TMB substrate to each well, incubate at room temperature in the dark for 15 min, add 100 μL of 10% H2SO4 to stop the color development, and read the OD450 value of each well using a microplate reader.

[0130] The results are shown in Table 6. When the primary antibody diluent was 1% horse serum, the ratio of the OD450 value of BoHV-1 positive bovine serum to the OD450 value of negative bovine serum was the highest. Therefore, 1% horse serum was preferred as the optimal primary antibody diluent.

[0131] Table 6. Effect of different primary antibody dilutions on the ratio of OD450 values ​​in lower BoHV-1 positive / negative bovine serum.

[0132] .

[0133] 4. Determination of the optimal sealing solution

[0134] Add 100 μL of antigenic peptide (40 μg / mL) diluted with CBS carbonate buffer to each well of an ELISA plate and incubate overnight at 4 °C. Discard the coating solution and wash the plate once with PBST buffer. Add different blocking solutions to different wells for blocking: 5% skim milk, 5% OVA protein, and commercial rapid blocking solution. Each blocking solution is repeated twice for each experimental group, and the plate is blocked at room temperature for 1 h. Discard the blocking solution and wash the plate twice with PBST buffer. Add BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum (1:50) and incubate at room temperature for 3 h. Discard the primary antibody serum, wash the plate 6 times with PBST buffer, add HRP-rabbit anti-bovine secondary antibody diluted with 5% skim milk (1:5,000), and incubate at room temperature for 1 h. Discard the secondary antibody, wash the plate 7 times with PBST buffer, pat dry, add 100 μL of TMB substrate to each well, incubate at room temperature in the dark for 15 min, add 100 μL of 10% H2SO4 to stop the color development, and read the OD450 value of each well using a microplate reader.

[0135] 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 optimal blocking solution is 5% OVA protein solution.

[0136] Table 7. Effects of different blocking solutions on the ratio of OD450 values ​​in lower BoHV-1 positive / negative bovine serum.

[0137] .

[0138] 5. Determination of the optimal dilution for the secondary antibody

[0139] Add 100 μL of antigenic peptide (40 μg / mL) diluted with CBS carbonate buffer to each well of the ELISA plate and incubate overnight at 4 °C. Discard the coating solution, wash the plate once with PBST buffer, and block with 300 μL of 5% OVA protein solution at room temperature for 1 h. Discard the blocking solution, wash the plate twice with PBST buffer, and add BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum (1:50), and incubate at room temperature for 3 h. Discard the primary antibody serum, wash the plate 6 times with PBST buffer, and add HRP-rabbit anti-bovine secondary antibody (1:000) diluted with different diluents to different wells. From left to right, the secondary antibody diluents are: PBST, 1% horse serum, and 5% skim milk, and incubate at room temperature for 1 h. Discard the secondary antibody, wash the plate 7 times with PBST buffer, pat dry, add 100 μL of TMB substrate to each well, incubate at room temperature in the dark for 15 min, add stop solution, and then read the OD450 value of each well using a microplate reader.

[0140] The results are shown in Table 8. When the secondary antibody dilution solution was 5% skim milk, the ratio of the OD450 value of BoHV-1 positive bovine serum to the OD450 value of negative bovine serum was the highest. Therefore, the optimal secondary antibody dilution solution is 5% skim milk.

[0141] Table 8. Effect of different secondary antibody dilutions on the ratio of OD450 values ​​in BoHV-1 positive / negative bovine serum.

[0142] .

[0143] 6. Optimal dilution of secondary antibody

[0144] Add 100 μL of antigenic peptide (40 μg / mL) diluted with CBS carbonate buffer to each well of the ELISA plate and incubate overnight at 4 °C. Discard the coating solution, wash the plate once with PBST buffer, and block with 300 μL of 5% OVA protein solution at room temperature for 1 h. Discard the blocking solution, wash the plate twice with PBST buffer, and add BoHV-1 positive bovine serum and BoHV-1 negative bovine serum diluted with 1% horse serum (1:50), and incubate at room temperature for 3 h. Discard the primary antibody serum, wash the plate 6 times with PBST buffer, and add different dilutions of HRP-rabbit anti-bovine secondary antibody (1:2500, 1:5000, 1:7500, 1:10000) diluted with 5% skim milk to each well, and incubate at room temperature for 1 h. Discard the secondary antibody, wash the plate 7 times with PBST buffer, pat dry, add 100 μl of TMB substrate to each well, incubate at room temperature in the dark for 15 min, add stop solution, and then read the OD450 value of each well using a microplate reader.

[0145] 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 dilution of secondary antibody was 1:5,000.

[0146] Table 9. Effect of different secondary antibody dilutions on the ratio of OD450 values ​​in BoHV-1 positive / negative bovine serum.

[0147] .

[0148] 7. Determination of blocking value

[0149] Using the established testing conditions, 24 bovine serum samples that had been identified as negative using commercial testing kits were tested, and their OD450 values ​​were measured. The mean (X) and standard deviation (SD) of the OD450 values ​​of the negative bovine serum were calculated. The blocking value was set as X + 3SD. That is, serum with an OD450 value < X + 3SD was defined as BoHV-1 virus negative serum; serum with an OD450 value ≥ X + 3SD was defined as BoHV-1 virus positive serum.

[0150] The results are shown in Table 10. The mean OD450 value (X) of the negative serum was 0.2314125, the standard deviation (SD) was 0.042184216, and the blocking value was 0.357965. Therefore, serum OD450 values ​​< 0.357965 were defined as BoHV-1 virus negative serum; serum OD450 values ​​≥ 0.357965 were defined as BoHV-1 virus positive serum.

[0151] Table 10 Results of indirect ELISA testing on 24 negative serum samples.

[0152] .

[0153] 8. Intra-batch repeatability and inter-batch repeatability

[0154] Using the established experimental conditions and blocking values, intra-batch and inter-batch repeatability experiments were conducted on six bovine serum samples using ELISA plates coated with the same batch and different batches. Each sample was replicated three times, and the OD450 value was measured. The coefficient of variation (CV) for the same sample was calculated to verify the repeatability of the method.

[0155] The results are shown in Table 11. The coefficients of variation for intra-batch replicates of six different bovine serum samples ranged from 3.1% to 8.8%, and the coefficients of variation for inter-batch replicates ranged from 2.9% to 12.6%. This indicates that the ELISA plates coated with different batches showed low variability and good repeatability when detecting the same serum sample.

[0156] Table 11 Repeatability Tests of Detection Methods

[0157] .

[0158] 9. Compliance Rate Testing

[0159] Using the established experimental conditions and blocking values, 44 bovine serum samples identified by a commercial BoHV-1 antibody detection kit were tested, and the concordance rate between this detection method and the commercial BoHV-1 antibody detection kit was calculated.

[0160] The results are shown in Table 12. Using the established detection method, 20 serum samples were BoHV-1 positive and 24 serum samples were BoHV-1 negative. Compared with the commercial BoHV-1 virus antibody detection kit, only two serum samples showed inconsistent results, with a concordance rate of 95.45%, which is good.

[0161] Table 12 Results of bovine serum collection and testing

[0162] .

[0163] 10. Established ELISA detection method

[0164] The kit contains: blocking buffer (5% OVA, diluted with PBS), primary antibody dilution buffer (1% horse serum, diluted with PBS), secondary antibody dilution buffer (5% skim milk, diluted with PBS), HRP-rabbit anti-bovine secondary antibody, BoHV-1 negative control, TMB substrate, and antigen-coated ELISA plate.

[0165] Operating steps:

[0166] (1) Blocking: Add 300 μL of blocking solution (5% OVA) to each well of the ELISA plate and block at room temperature for 1 h.

[0167] (2) Washing: After the blocking is completed, discard the blocking solution, wash the plate twice with PBST and pat dry.

[0168] (3) Incubation of primary antibody: Dilute the serum to be tested with primary antibody dilution buffer (1% horse serum) at a ratio of 1:50, add 100 μL to the ELISA plate, and incubate at 37 °C for 3 h.

[0169] (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 each time and let stand for 5 min. After the fifth wash, place the ELISA plate on a shaker and shake for 3 s.

[0170] (5) Incubation of secondary antibody: Use secondary antibody dilution buffer (5% skim milk) to dilute HRP-rabbit anti-bovine secondary antibody at a ratio of 1:5,000, take 100 μL and add it to the ELISA plate, seal the plate at room temperature in the dark and incubate for 1 h.

[0171] (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 each time you wash the plate and let it stand for 5 minutes. After the fifth, sixth and seventh washes, place the ELISA plate on a shaker and shake for 3 seconds.

[0172] (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 using a microplate reader.

[0173] (8) Judgment: 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.

[0174] The detection method / kit has high specificity, and the detection concordance rate with commercial BoHV-1 antibody detection kits can reach 95.45%. Moreover, the degree of variation between different batches of detection is low, and the repeatability is good.

[0175] Although some preferred embodiments of this invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0176] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of its inventive concept. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include these modifications and variations.

Claims

1. A BoHV-1 virus antigenic epitope polypeptide, whose amino acid sequence is shown as SEQ ID NO.

1.

2. A conjugate protein, characterized in that, It is a pv1-KLH conjugate protein formed by conjugating the BoHV-1 virus antigenic epitope polypeptide described in claim 1 with hemocyanin KLH.

3. Use of the BoHV-1 virus antigenic epitope polypeptide described in claim 1 or the conjugate protein described in claim 2 in the preparation of an antibody or vaccine against BoHV-1 virus.

4. Use of the BoHV-1 virus antigenic epitope polypeptide described in claim 1 or the conjugate protein described in claim 2 in the preparation of a reagent for detecting BoHV-1 virus.

5. An enzyme-linked immunosorbent assay kit for detecting BoHV-1 virus, 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, characterized in that, The ELISA plate is coated with the BoHV-1 virus antigenic epitope polypeptide described in claim 1.

6. The enzyme-linked immunosorbent assay kit according to claim 5, wherein The blocking solution is 4-6% ovalbumin diluted with phosphate buffer.

7. The enzyme-linked immunosorbent assay kit according to claim 5, wherein The primary antibody diluent is 0.8-1.2% horse serum diluted with phosphate buffer.

8. The enzyme-linked immunosorbent assay kit according to claim 5, characterized in that The secondary antibody diluent is 4-6% non-fat milk diluted with phosphate buffer.

Images