Pig delta coronavirus S1-CTD protein linear B cell epitope peptide and application thereof

Abstract

The invention discloses a linear B-cell epitope peptide of porcine delta coronavirus S1-CTD protein and application thereof, and belongs to the technical field of biological medicine. According to the invention, the linear B cell epitope peptide 585EYLQVQAEQVIVDCP599 of the porcine delta coronavirus S1-CTD protein is screened, and can be recognized and combined by a monoclonal antibody of the porcine delta coronavirus S1-CTD protein, so that a technical support is provided for research and development of vaccines, medicines and diagnostic reagents related to the porcine delta coronavirus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein and an application thereof. Background Art

[0002] Porcine deltacoronavirus (PDCoV) is a newly discovered porcine enteropathogenic coronavirus that mainly infects piglets around one week old, with a mortality rate of up to 100%. There is currently no corresponding commercial vaccine on the market. The pathogenic mechanism of PDCoV is still unclear and there is a lack of relevant effective detection and prevention methods, which still needs further exploration.

[0003] PDCoV belongs to the genus Deltacoronavirus of the subfamily Coronavirus of the family Coronaviridae. Its genome encodes four important main structural proteins: Spike protein (S), Nucleocapsid protein (N), Membrane glycoprotein (M), and Envelope protein (E). The main structural proteins play their respective important roles in the process of virus invasion of host cells.

[0004] The PDCoV S protein is composed of S1 subunits and S2 subunits, which are closely related to the virus tissue tropism. The C-terminal domain (CTD) of the S1 subunit close to the carboxyl side exerts the function of the receptor domain (RBD) by binding to specific receptors. The currently reported S1-CTD receptor is aminopeptidase-N (APN). S1-CTD is the main neutralizing antigen epitope region on the S protein, which plays an indispensable and important role in the process of virus recognition and binding to receptors. Therefore, it is expected to be used as a target protein for vaccine development. The preparation of anti-S1-CTD monoclonal antibodies and the screening of their B cell epitopes can provide important technical support for the development of PDCoV-related diagnostic reagents and the research of therapeutic drugs, and lay the foundation for protein structure analysis and epitope vaccine preparation. Summary of the invention

[0005] The purpose of the present invention is to provide a linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein and its application to solve the problems existing in the above-mentioned prior art. The present invention screened a linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein 585 EYLQVQAEQVIVDCP 599 , which can be recognized and bound by monoclonal antibodies against the S1-CTD protein of porcine delta coronavirus, providing technical support for the research and development of porcine delta coronavirus-related vaccines, drugs and diagnostic reagents.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein, the amino acid sequence of which is shown in SEQ ID NO.21.

[0008] The present invention also provides a nucleotide fragment encoding the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein.

[0009] The present invention also provides the use of the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein in the preparation of a drug for preventing and / or treating porcine delta coronavirus.

[0010] The present invention also provides the use of the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein in the preparation of a vaccine for preventing porcine delta coronavirus.

[0011] The present invention also provides the use of the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein in the preparation of a reagent or a kit for detecting porcine delta coronavirus.

[0012] The present invention also provides a vaccine for preventing porcine delta coronavirus, which is prepared by using the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein as an antigen and adding a pharmaceutically acceptable carrier.

[0013] The present invention also provides a kit for detecting porcine delta coronavirus, wherein the kit uses the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein as a coating antigen.

[0014] The present invention discloses the following technical effects:

[0015] The present invention utilizes molecular biology, virology, immunology and other related technologies to screen and identify a linear B cell epitope peptide of PDCoVS1-CTD protein 585 EYLQVQAEQVIVDCP 599 It has been verified that the monoclonal antibody against PDCoV S1-CTD protein can specifically recognize the 585 EYLQVQAEQVIVDCP 599 The sequence region reacts with the epitope peptide, providing technical support for the application of PDCoV S1-CTD protein in vaccine development. The screening and identification of the linear B cell epitope peptide of PDCoV S1-CTD protein also enriched the immunological function of PDCoV S1-CTD protein, providing a reference for subsequent research on the structural characteristics and antigenic characteristics of S1-CTD protein, and can be applied to the development of antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The SDS-PAGE identification results of the pET-28a-S1-CTD-BL21 expression bacterial solution sample in Example 1; wherein M is a marker; 1-4 are the whole bacteria before induction, the whole bacteria after induction, the ultrasonic supernatant and the ultrasonic precipitate, respectively;

[0018] Figure 2 is the SDS-PAGE identification result of the renatured S1-CTD recombinant protein in Example 1; wherein, M is a marker; 1 is the renatured S1-CTD recombinant protein;

[0019] Figure 3 The Western Blot identification results of the S1-CTD recombinant protein before and after renaturation in Example 1; wherein M is a marker; 1 is the S1-CTD recombinant protein before renaturation; 2 is the S1-CTD recombinant protein after renaturation;

[0020] Figure 4 1 is the Western Blot identification result of the renatured S1-CTD recombinant protein in Example 1; wherein M is a marker; 1 is the reaction between the renatured S1-CTD recombinant protein and anti-PDCoV positive pig serum;

[0021] Figure 5 The results of serum titer detection of mice immunized with S1-CTD recombinant protein in Example 2;

[0022] Figure 6 The indirect ELISA test results of the positive hybridoma cell line 2D1 in Example 3;

[0023] Figure 7 The IPMA experimental verification results of the specific binding of PDCoV S1-CTD protein to monoclonal antibody 2D1 in Example 3;

[0024] Figure 8 The titer test result of monoclonal antibody 2D1 in Example 3;

[0025] Fig. 9 This is a schematic diagram of the truncation of the PDCoV S1-CTD protein in Example 4;

[0026] Fig.10 The results of the Peptide-ELISA and Dot-ELISA experiments of the monoclonal antibody 2D1 reacting with the truncated protein in Example 4;

[0027] Fig.11 The results of the Peptide-ELISA and Dot-ELISA experiments of the monoclonal antibody 2D1 reacting with the truncated polypeptide in Example 4; wherein A is the result of the Dot-ELISA experiment, and B is the result of the Peptide-ELISA experiment;

[0028] Fig.12 These are the results of Peptide-ELISA and Dot-ELISA experiments of the reaction of epitope peptide e2-3 with positive pig serum in Example 4; wherein A is the result of Dot-ELISA experiment, and B is the result of Peptide-ELISA experiment. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1 Preparation of immunogen

[0035] Expression of PDCoV S1-CTD protein using prokaryotic expression system:

[0036] 1. Primer design

[0037] According to the Spike gene sequence of the PDCoV / USA / Ohio137 / 2014 strain published on NCBI (GI: 1272615995), the S1-CTD gene was optimized for species (CHO) with reference to relevant literature, and primers were designed using SnapGene. BamH I and Xho I were selected as restriction sites (underlined parts), and 3 protective bases were added to amplify the PDCoV S1-CTD gene. The primer sequences are shown in Table 1.

[0038] Table 1 Primer sequences

[0039]

[0040] 2. PCR amplification of S1-CTD gene

[0041] The S1-CTD gene optimized and modified by species (CHO) was used as a template for PCR amplification. The PCR amplification system is shown in Table 2.

[0042] Table 2 PCR amplification system

[0043]

[0044] 3. Purification and recovery of PCR products

[0045] After the target gene amplification is completed, the PCR amplification product is verified by 1% agarose gel electrophoresis. The amplification product at the target band position is excised and recovered using a DNA purification and recovery kit, and the concentration of the recovered DNA solution is determined using NanoDrop, marked, and stored at -20°C for future use.

[0046] 4. Double restriction enzyme digestion of target gene and vector

[0047] (1) The S1-CTD gene / pET-28a vector was double-digested with enzymes. The double-digestion reaction system is shown in Table 3.

[0048] Table 3 Double enzyme digestion reaction system

[0049]

[0050]

[0051] After all components were mixed evenly, they were placed in a 37°C metal bath for double enzyme digestion for 4 h.

[0052] 5. Recovery of enzyme digestion products

[0053] Use a DNA purification and recovery kit to recover the double-digestion product, aspirate 1 μL of the recovered product to determine the recovered DNA concentration, mark it, and store it at -20°C for later use.

[0054] 6. Construction of recombinant plasmid

[0055] (1) Ligation of S1-CTD gene with pET-28a vector: Add the components listed in Table 4 to a 200 μL EP tube and mix thoroughly. Place the sample in a 16° C. constant temperature metal bath for 14 h to obtain a ligation product.

[0056] Table 4 Connection system

[0057]

[0058] (2) Transformation: Slowly add 10 μL of the ligation product to 50 μL of DH5α competent cells in a clean bench, gently pipette and evenly mix the sample solution, and place it on ice for 30 minutes. After the ice bath, quickly place the sample in a 42°C metal bath for heat shock for 90 seconds. After the heat shock, place it on ice for 5 minutes. Gently add 0.8 mL of sterile liquid LB medium (no resistance) to the heat-shocked mixture in a clean bench, and culture it at 37°C on a shaker at 220 r / min for 50 minutes; centrifuge the bacterial solution at room temperature at 8000 rpm for 3 minutes, discard 0.7 mL of the supernatant, use the remaining 0.1 mL of the supernatant in the Ep tube to resuspend the bacteria, take a solid LB medium plate (Kana resistance) and apply an appropriate amount of the resuspended bacterial solution on it, invert the plate and culture it in a 37°C constant temperature incubator, and observe the results for about 14 minutes.

[0059] (3) Screening of positive monoclonal bacteria: Randomly select milky white round colonies on the plate, resuspend the colonies in 1 mL of sterile LB liquid medium (Kana resistance), and expand and culture on a shaker at 37°C for 4 h. Perform colony PCR identification, use the turbid bacterial solution after expansion culture as the template, add the components listed in Table 5 to a 200 μL EP tube and mix thoroughly.

[0060] Table 5 Bacterial liquid PCR reaction system

[0061]

[0062] The mixed sample was placed in a PCR instrument for PCR amplification. After the bacterial solution PCR was completed, the product was identified using 1% agarose gel, and the monoclonal strain whose band position matched the target gene position was selected and sent to Anhui Chuzhou General Biological Company for sequencing.

[0063] (4) Extraction of recombinant plasmid: Select the positive strain that has been successfully sequenced, expand the culture, and use the Tiangen Plasmid Extraction Kit to extract the pET-28a-S1-CTD recombinant plasmid according to the instructions.

[0064] 7. Induced expression and identification of target protein

[0065] (1) Expression of target protein

[0066] a. Transform the pET-28a-S1-CTD recombinant plasmid into E. coli BL21 competent cells to construct the pET-28a-S1-CTD-BL21 expression bacteria.

[0067] b. The constructed pET-28a-S1-CTD-BL21 expression bacteria were inoculated into 200 mL of liquid LB medium (Kana antibody) at an inoculum size of 1:50 for secondary activation.

[0068] c. After inoculation, culture the cells in a 37°C shaker at 220 rpm for about 2 hours. When the OD value is between 0.6 and 0.8, take 40 μL of the whole bacterial sample before induction and add the inducer IPTG (1 mmol / L). Continue to culture the cells in a 37°C shaker at 220 rpm for 5 hours.

[0069] d. Collect the bacteria and keep 40 μL of the whole bacterial sample after induction. Resuspend the remaining bacterial solution with pre-cooled PBS buffer and perform ultrasonic disruption.

[0070] e. Centrifuge at 4°C and 12,000 rpm for 10 min, take 40 μL of the ultrasonic supernatant and ultrasonic precipitate, add 10 μL of 5×SDS loading buffer to each of the four samples, boil for 10 min, and perform SDS-PAGE identification. Figure 1 As shown, the S1-CTD recombinant protein was expressed in the form of inclusion bodies.

[0071] (2) Denaturation and identification of target protein

[0072] a. Collect the ultrasonically broken precipitate and wash it three times with washing buffer, stirring at 200 rpm for 30 minutes at room temperature and centrifuging at 4°C and 12,000 rpm for 10 minutes each time. Then gently resuspend the precipitate with dissolution buffer, stir at 200 rpm for 30 minutes at room temperature, and transfer to a 4°C refrigerator to stand overnight. The next day, take out the sample and centrifuge it at 4°C and 12,000 rpm for 10 minutes, separate and collect the supernatant, and take samples for identification.

[0073] b. The supernatant was dialyzed at low temperature gradient in inclusion body urea-free refolding solution containing different concentrations of urea (6, 3, 2, 1.5, 1 mol / L) to allow the target protein to refold slowly. After refolding, samples were taken and detected by SDS-PAGE and Western Blot. Figure 2 , Figure 3 As shown in Figure 2, the concentration of the recombinant S1-CTD protein after renaturation reached more than 90%. Figure 4 As shown, the S1-CTD recombinant protein reacted well with anti-PDCoV positive pig sera.

[0074] The buffer formula used in the denaturation and renaturation process of the target protein is shown in Table 6.

[0075] Table 6 Protein denaturation buffer formula

[0076]

[0077]

[0078] Example 2 Immunization of mice with S1-CTD recombinant protein and analysis of immune effects

[0079] Mouse immunization and preliminary evaluation of immune effects:

[0080] 1. Take two 6-8 week old Balb / c mice, and immunize the mice with the S1-CTD recombinant protein prepared in Example 1 as the immunogen, 50 μg per mouse, and immunize three times in total, once every two weeks. The first immunization is mixed and emulsified with Freund's complete adjuvant and S1-CTD recombinant protein, and the second and third immunizations are mixed and emulsified with Freund's incomplete adjuvant and S1-CTD recombinant protein. Collect the tail vein of the mice 14d, 28d, and 42d after the first immunization, and determine the titer of the mouse serum after immunization by indirect ELISA.

[0081] 2. Indirect ELISA to determine the titer of serum from mice immunized with S1-CTD recombinant protein

[0082] The S1-CTD recombinant protein was diluted to 2 μg / mL with CBS and then coated on a 96-well reaction plate, 100 μL per well, and coated at 37°C for 2 h; the coating solution was discarded, washed three times with PBST, and blocked with 5% skim milk at 37°C for 2 h; the coating solution was discarded, washed three times with PBST, 1:100 diluted immunized mouse serum was added to the first well, and non-immunized mouse serum was set as a negative control, and gradient dilutions were made from left to right, and incubated at 37°C for 1 h; washed three times with PBST, 1:5000 diluted Goat Anti-Mouse IgG / HRP was added, and incubated at 37°C for 1 h; the secondary antibody was discarded, washed three times with PBST, 100 μL TMB colorimetric solution was added to each well, and after coloring for 5 min in the dark, 100 μL 2 mol / L H2SO4 was added to each well; OD was measured 450 Value, analyze the immune effect. Figure 5 As shown, the S1-CTD recombinant protein produced a better immune effect.

[0083] Example 3 Preparation and identification of monoclonal antibodies against S1-CTD recombinant protein

[0084] 1. Cell fusion

[0085] Four days before cell fusion, select mice with the best serum titer and absorb 100 μg S1-CTD recombinant protein by intraperitoneal injection to superimmunize the mice. Cell fusion was performed 4 days after shock immunization. 16 hours before fusion, select a healthy Kunming mouse, kill it by cervical scissoring, and immerse it in 75% alcohol, with the abdomen of the Kunming mouse facing up, and fix its limbs on a foam board; use sterilized scissors and tweezers to cut the mouse's abdominal fur along the midline of the Kunming mouse's abdomen, and fully expose the Kunming mouse's abdominal cavity without damaging the Kunming mouse's peritoneum; use new tweezers to clamp the Kunming mouse's peritoneum, use a sterile syringe to absorb 10mL HAT culture medium, insert the needle along the clamped peritoneum to avoid the fat accumulation, slowly inject the culture medium and gently aspirate twice, gently massage the Kunming mouse's abdomen with tweezers, then aspirate the culture medium containing macrophages in the Kunming mouse's abdominal cavity, suspend it in the air and add it to the 95mL HAT culture medium prepared in advance, vortex and mix well, and add 100μL per well to the 96-well cell culture plate. After the shock immunization, blood was collected from the eyeballs of the mice. The blood samples were placed in a 37°C incubator for 2 hours and then centrifuged at 3000 rpm for 10 minutes. The serum was collected and stored in a -20°C refrigerator.The mice were killed by cervical dislocation, and then fixed on a special clean foam board after soaking in 75% alcohol for 5 minutes, and then placed on the operating table; the 200-mesh nylon mesh was moistened with GNK solution; the mouse epidermis was cut open with sterilized scissors and tweezers to fully expose the mouse abdominal cavity, and a new set of scissors and tweezers were used to cut the peritoneum on the right side of the mouse abdomen, and the scissors and tweezers were replaced again to remove the complete spleen of the mouse; the spleen was placed on the moistened nylon mesh, and the mouse spleen was slowly ground with scissors while dripping with preheated GNK solution to keep the spleen moistened at all times. After the spleen was ground, the nylon mesh and the cup wall were rinsed with GNK solution, and the volume of the spleen cell suspension was filled to 40 mL, and the supernatant was discarded after centrifugation, and 25 mL Resuspend the cell pellet with GNK solution, mix well and count the cells in the suspension. Calculate the volume of SP2 / 0 cell suspension added according to the ratio of spleen cells to SP2 / 0 cells of 8:1. Then add the corresponding volume of SP2 / 0 cell suspension to the spleen cell suspension and vortex and centrifuge. After centrifugation, discard the supernatant and flick the cell pellet. Place the centrifuge tube in a 37°C water bath. Add 1 mL of preheated PEG 1500 fusion agent at a uniform speed in 90 seconds. Gently shake the centrifuge tube while adding to facilitate full contact between PEG 1500 and cells. After the addition is completed, let it stand for 90 seconds. The termination procedure is divided into three 30 seconds. In the first 30 seconds, 1 mL of GNK solution is added at a uniform speed. In the second 30 seconds, 3 mL of GNK solution is added at a uniform speed. In the third 30 seconds, 11 mL GNK solution; after terminating the fusion, the cell suspension was transferred to a 37°C cell culture incubator and allowed to stand for 5 minutes; after standing, GNK was added to the centrifuge tube to a total volume of 40 mL and then centrifuged. After centrifugation, the supernatant was discarded and the cell pellet was resuspended with an appropriate amount of HAT culture medium; according to 100 μL of cell suspension per well, HAT culture medium was calculated and added to the feeder cell culture plate prepared the day before, and the culture plate was placed in a 37°C cell culture incubator. The cells were observed for contamination the next day. If there was no contamination, the cells were allowed to stand for more than 7 days without moving the cell culture plate during this period.

[0086] 2. Screening of positive hybridoma cell lines

[0087] 7-10 days after cell fusion, the cell clusters were observed under a microscope at 10x magnification, and the cell supernatant was detected by indirect ELISA. The S1-CTD recombinant protein was diluted to 2 μg / mL with CBS and then coated on a 96-well reaction plate, 100 μL per well, and coated at 37°C for 2 h; the coating solution was discarded, washed three times with PBST, and blocked with 5% skim milk at 37°C for 2 h; the coating solution was discarded, washed three times with PBST, and 1:100 diluted immunized mouse serum was added to the first well. At the same time, the tail serum of unimmunized mice was set as a negative control and the eye serum of fused mice was set as a positive control. The plates were diluted from left to right in a gradient ratio and incubated at 37°C for 1 h: washed three times with PBST, and 1:5000 diluted GoatAnti-Mouse IgG / HRP was added, and incubated at 37°C for 1 h; the primary antibody was discarded, washed three times with PBST, 100 μL TMB colorimetric solution was added to each well, and after coloring for 5 min in the dark, 100 μL 2 mol / L H2SO4 was added to each well; OD was measured 450 By comparing OD 450 The positive hybridoma cells were screened by the OD value, which required that the number of cell clusters in the target well was less than 2 and the OD 450 The value is greater than 1. After more than two retests, the samples with better specificity and OD 450 The positive hybridoma cell wells were subcloned by dilution method, and the hybridoma cell lines that stably secreted monoclonal antibodies were obtained through multiple subcloning. Figure 6 As shown, a positive hybridoma cell line was screened out and named 2D1.

[0088] 3. Immunoperoxidase monolayer assay (IPMA)

[0089] To promote cell adhesion, 1× poly-lysine solution was added to the 96-well plate, 50 μL per well, and the plate was left to stand for 10 min. Sterile ddH2O was used to wash away the poly-lysine three times. The plate was left to stand until the bottom of the plate was completely dry. HEK-293T cells were cultured in advance at 2.5×10 4HEK-293T cells were cultured in a treated 96-well cell culture plate at a density of 100 cells; when the cell confluence reached 60%, pcDNA3.1-CTD (the construction process was the same as 1-6 in Example 1) was mixed with polyplus transfection reagent to transiently transfect HEK-293T cells. After 6 hours of transfection, the original culture medium in the well was gently aspirated, and the same volume of Opti-MEM medium was slowly added along the wall of the well, and the cell plate was returned to the 37°C cell culture incubator for further culture for 48 hours; the old culture medium was discarded, 100 μL / well was added with 4% tissue cell fixative, and the culture plate was allowed to stand at room temperature for 15 minutes to achieve the purpose of fixing the cells. After the fixation was completed, the fixative was discarded, and the plate was gently washed with sterile PBS buffer; 100 μL of 5% skim milk was gently added to each well along the wall of the well, and it was placed in a 37°C constant temperature incubator for 2 hours; the blocking solution was discarded, and the plate was washed three times with PBS buffer, and attention was paid to the gentle action when aspirating and adding liquid to avoid blowing up the cells at the bottom of the plate and affecting the experimental results. Add 50 μL of HEK-293T cell supernatant to each well and incubate at 37°C for 40 min. Add 2D1 cell supernatant to the HEK-293T cell supernatant, set up a positive control (Positive control, PC) with 1:500 dilution of immune mouse serum, a negative control (Negative control, NC) with non-immunized mice (1:500 dilution), and a blank control group (Blank control, BC) transfected with pcDNA3.1 empty plasmid. Use GoatAnti-Mouse IgG / HRP with a 1:5000 dilution as the secondary antibody, discard the secondary antibody, wash three times with PBST, add 100 μL AEC color development solution to each well, protect from light for 10 min, then add 100 μL ddH2O to each well to terminate the reaction; observe under a microscope and take pictures to record the experimental results. The results are as follows Figure 7 As shown, monoclonal antibody 2D1 can react with eukaryotically expressed PDCoV S1-CTD protein.

[0090] 4. Large-scale preparation of monoclonal antibodies and purification of ascites

[0091] After subcloning, indirect ELISA was also used for screening, and the screened positive hybridoma cell line 2D1 was expanded and cultured. 7-10 days before injecting the cells, sterile Freund's incomplete adjuvant was selected as an immunosuppressant, and the mice were injected intraperitoneally at a dose of 500 μL per mouse. On the day of intraperitoneal injection, the positive hybridoma cells were counted, and the volume of culture medium required to resuspend the cells was calculated based on the injection of 2×106 cells per mouse and a total injection of 500 μL, and the mice were injected intraperitoneally. About 10 days later, the mice were killed by cervical distension after their abdomens were swollen, and the ascites were collected. The middle layer of light yellow liquid was collected after centrifugation. The ascites was purified using Protein A affinity chromatography.

[0092] 5. Monoclonal antibody titer determination

[0093] The titer of monoclonal antibodies was determined by indirect ELISA. The S1-CTD recombinant protein was diluted to 2μg / mL with CBS, 100μL per well, and coated at 37℃ for 2h. After discarding the coating solution, the plate was washed 3 times with PBST, and 200μL of 5% skim milk was added to each well and blocked at 37℃ for 2h. After discarding the blocking solution, the plate was washed 3 times with PBST, and the purified monoclonal antibody 2D was diluted 11:500 and added to the first well, and then diluted in a gradient from left to right. At the same time, the mouse serum was used as a negative control, 100μL per well, and incubated at 37℃ for 1h. After discarding the primary antibody, the plate was washed 3 times with PBST, and 100μL of 1:5000 diluted GoatAnti-Mouse IgG / HRP was added to each well and incubated at 37℃ for 1h. After discarding the secondary antibody, wash the plate 3 times with PBST, add 100 μL TMB colorimetric solution to each well, color for 5 min in the dark, add 100 μL 2 mol / L H2SO4 to each well to terminate the reaction, and read the OD 450 , the results are as follows Figure 8 shown.

[0094] Example 4 Localization of linear B cell epitopes of PDCoV S1-CTD protein

[0095] 1. Truncated expression, purification and identification of the amino acid sequence of PDCoV S1-CTD protein

[0096] The PDCoV S1-CTD protein was truncated using the overlapping peptide method. The truncation status is shown in Fig. 9 As shown, a total of 8 segments were truncated. The primer sequences shown in Table 7 were used, and the truncated fragments were constructed between the BamHI and Xho I restriction sites of the pET-32a vector according to the method of Example 1. Escherichia coli Rosetta competent cells were transformed, and the same method was used to induce expression to obtain truncated proteins A, B, C, D, E, e1, e2, and e3.

[0097] Table 7 Primer series for truncated proteins

[0098]

[0099] Dot-ELISA was used to preliminarily screen the B cell epitopes recognized by monoclonal antibodies. Use an agar puncher to punch holes of appropriate size and spacing on the NC membrane. After soaking the NC membrane with PBS buffer, transfer the NC membrane to a 37°C oven and wait for it to dry. Spot the expression proteins of each truncated fragment on the NC membrane. At the same time, set the S1-CTD protein spotting well as the pET-32a-Rosetta empty protein spotting well as the negative control. Spot 1 μL per well each time, and spot twice after natural drying. Put the NC membrane in 20mL 5% skim milk at 37°C for 2h. After discarding the blocking solution, wash the membrane 3 times with PBST, transfer the membrane to 20mL of 1:1000 dilution of monoclonal antibody 2D1 dilution, and incubate at 37°C for 1h. After discarding the primary antibody, wash the membrane 3 times with PBST, transfer the membrane to 20mL of 1:5000 dilution of GoatAnti-Mouse IgG / HRP, and incubate at 37°C for 1h. After discarding the secondary antibody, the membrane was washed three times with PBST, and AEC display solution was added for reaction. After 5 minutes, ddH2O was added to terminate the reaction, and photos were taken to record the experimental results.

[0100] The B cell epitopes recognized by the monoclonal antibody were screened by Peptide-ELISA. The purified truncated protein and pET-32a-BL21 empty protein were diluted to 2μg / mL with CBS, and 100μL was coated in each well at 37℃ for 2h. After discarding the coating solution, the plate was washed 3 times with PBST, and each well was blocked with 200μL 5% skim milk at 37℃ for 2h. After discarding the blocking solution, the plate was washed 3 times with PBST, and 50μL of 1:1000 diluted monoclonal antibody 2D1 was added to each well as the primary antibody, and incubated at 37℃ for 1h. After discarding the primary antibody, the plate was washed 3 times with PBST, and 50μL of 1:5000 diluted GoatAnti-Mouse IgG / HRP was added to each well as the secondary antibody, and incubated at 37℃ for 1h. After discarding the secondary antibody, wash the plate 3 times with PBST, add 50 μL TMB colorimetric solution to each well, color for 5 min in the dark, add 50 μL 2 mol / L H2SO4 to each well to terminate the reaction, and read the OD 450 The results of Peptide-ELISA and Dot-ELISA experiments are shown in Fig.10 , and finally determined that the recognition region of monoclonal antibody 2D1 was the 571-599 amino acid region.

[0101] 2. Synthesis and identification of PDCoV S1-CTD protein peptide

[0102] Based on the above experimental results, the truncated fragment e2 (aa 571-599) was further truncated to synthesize polypeptides e2-1, e2-2, and e2-3 (Gill Biochemical Co., Ltd.). The sequence information of each truncated polypeptide is shown in Table 8.

[0103] Table 8 Amino acid sequence of truncated polypeptide

[0104]

[0105] The three synthesized peptides were diluted with PBS to 4 mg / mL, and 4 g of each peptide was coated per well. The S1-CTD recombinant protein was used as the positive control (PC), PBS was used as the negative control (NC), the monoclonal antibody 2D1 was used as the primary antibody at a dilution of 1:1000, and the Goat Anti-Mouse IgG / HRP was used as the secondary antibody at a dilution of 1:5000. Fig.11 As shown, Peptide-ELISA and Dot-ELISA showed that monoclonal antibody 2D1 reacted with peptide e2-3, and the reaction was consistent with that of S1-CTD recombinant protein.

[0106] 3. Identification of PDCoV S1-CTD protein epitope peptides

[0107] The screened epitope peptides were coated using the above method, with 1:1000 dilution of anti-PDCoV positive pig serum as the primary antibody and 1:5000 dilution of GoatAnti-Mouse IgG / HRP secondary antibody. Fig.12 As shown, Peptide-ELISA and Dot-ELISA showed that e2-3 could react with anti-PDCoV positive pig serum.

[0108] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

21.

2. A nucleotide fragment encoding the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein according to claim 1.

3. Use of the linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein according to claim 1 in the preparation of a drug for preventing and / or treating porcine delta coronavirus.

4. Use of the linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein according to claim 1 in the preparation of a vaccine for preventing porcine delta coronavirus.

5. Use of the linear B cell epitope peptide of porcine delta coronavirus S1-CTD protein according to claim 1 in the preparation of a reagent or kit for detecting porcine delta coronavirus.

6. A vaccine for preventing porcine delta coronavirus, characterized in that: The vaccine is prepared by using the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein as claimed in claim 1 as an antigen and adding a pharmaceutically acceptable carrier.

7. A kit for detecting porcine delta coronavirus, characterized in that: The kit uses the linear B cell epitope peptide of the porcine delta coronavirus S1-CTD protein according to claim 1 as the coating antigen.

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