Porcine delta coronavirus s1-ctd protein linear b cell epitope peptide and application thereof

Abstract

The application discloses a porcine delta coronavirus S1-CTD protein linear B cell epitope peptide and application thereof, and belongs to the technical field of biological medicines. 585 EYLQVQAEQVIVDCP 599 The linear B cell epitope peptide can be recognized and combined by a monoclonal antibody of the porcine delta coronavirus S1-CTD protein, and provides technical support for research and development of porcine delta coronavirus related vaccines, medicines and diagnostic reagents.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to a porcine delta coronavirus S1-CTD protein linear B cell epitope peptide and application thereof. BACKGROUND

[0002] Porcine delta coronavirus (PDCoV) is a newly discovered pathogenic coronavirus in pig intestines in recent years, mainly infecting piglets of about one week old, and the mortality rate is as high as 100%, and there is no corresponding commercial vaccine on the market. At present, the pathogenesis of PDCoV is not clear and there is a lack of effective detection and prevention means, and further in-depth exploration is still needed.

[0003] PDCoV belongs to the coronavirus subfamily of the coronavirus family, and 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 important roles in the process of virus invasion into host cells.

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

[0005] The application aims to provide a porcine delta coronavirus S1-CTD protein linear B cell epitope peptide and application thereof, so as to solve the problems in the prior art. The application screens a porcine delta coronavirus S1-CTD protein linear B cell epitope peptide 585 EYLQVQAEQVIVDCP 599 which can be recognized and combined by a porcine delta coronavirus S1-CTD protein monoclonal antibody, and provides 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 application provides the following scheme:

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

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

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

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

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

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

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

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

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

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

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

[0018] Figure 2 SDS-PAGE identification results of the S1-CTD recombinant protein after renaturation in Example 1; wherein, M is Marker; 1 is the S1-CTD recombinant protein after renaturation;

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

[0020] Figure 4 Western Blot identification results of the S1-CTD recombinant protein after renaturation in Example 1; wherein, M is Marker; 1 is the S1-CTD recombinant protein after renaturation and the reaction with the anti-PDCoV positive pig serum;

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

[0022] Figure 6 Indirect ELISA detection results of the positive hybridoma cell strain 2D1 in Example 3;

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

[0024] Figure 8 Titer detection results of the monoclonal antibody 2D1 in Example 3;

[0025] Figure 9 Truncation schematic diagram of the PDCoV S1-CTD protein in Example 4;

[0026] Figure 10 Results of Peptide-ELISA and Dot-ELISA experiments for monoclonal antibody 2D1 reacting with truncated proteins in Example 4;

[0027] Figure 11 Results of Peptide-ELISA and Dot-ELISA experiments for monoclonal antibody 2D1 reacting with truncated proteins in Example 4;

[0028] Figure 12 Results of Peptide-ELISA and Dot-ELISA experiments for epitope peptide e2-3 reacting with positive swine serum in Example 4; where A is the Dot-ELISA experiment and B is the Peptide-ELISA experiment. DETAILED DESCRIPTION

[0029] Various illustrative embodiments of the present application are now described in detail. The description made herein is not to be construed as limiting the present application, but rather merely as describing certain aspects, features, and embodiments of the present application.

[0030] It should be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to limit the present application. Additionally, for numerical ranges that are expressed in a range format, it is intended that any and every set of numbers that are separated by a

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather a representation that the reference is part of the state of the art.

[0032] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. For example, the specification can be used to design equivalent methods and materials for the practice of the application. Such equivalent methods and materials are intended to fall within the scope of the application. The specification and examples given herein are illustrative of the application and are not intended to be limiting, therefore, it will be readily understood by those skilled in the art that numerous changes can be made in the application as described without departing from the scope of the application as described herein.

[0033] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and / or methods.

[0034] Preparation of the immunogen of Example 1

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

[0036] 1. Primer design

[0037] According to the Spike gene sequence of PDCoV / USA / Ohio137 / 2014 strain published on NCBI (GI: 1272615995), the S1-CTD gene was optimized for species (CHO) according to the relevant literature, and SnapGene was used to design primers. Select BamH I and Xho I as the enzyme cutting site (underlined part), and add 3 protection bases, for amplifying PDCoV S1-CTD gene, the primer sequence is shown in Table 1.

[0038] Table 1 Primer sequence

[0039]

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

[0041] The S1-CTD gene optimized for 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 product

[0045] After amplification of the target gene, the PCR amplification product was verified by 1% agarose gel electrophoresis. The DNA purification and recovery kit was used to cut and recover the amplification product at the target band position, the concentration of the recovered DNA solution was determined using NanoDrop, and the label was made, and it was stored at -20℃ for standby.

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

[0047] (1) Double enzyme digestion of S1-CTD gene / pET-28a vector, double enzyme digestion reaction system as shown in Table 3.

[0048] Table 3 Double enzyme digestion reaction system

[0049]

[0050]

[0051] After the components are mixed evenly, place the mixture on a 37°C metal bath for double enzyme digestion for 4 hours.

[0052] 5. Recovery of the enzyme digestion product

[0053] Recover the double enzyme digestion product using a DNA purification recovery kit, measure the concentration of the recovered DNA by taking 1 μL of the recovered product, label it, and store it at -20°C for later use.

[0054] 6. Construction of the recombinant plasmid

[0055] (1) Connect S1-CTD gene and pET-28a vector: add the components listed in Table 4 to a 200 μL EP tube and mix thoroughly, then place the sample in a 16°C constant temperature metal bath for 14 hours to obtain the ligation product.

[0056] Table 4 Ligation 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 blow and suck to mix the sample, and then place it on ice for 30 minutes. After ice bath, quickly place the sample in a 42°C metal bath for heat shock for 90 seconds, then ice bath for 5 minutes. In the clean bench, gently add 0.8 mL of sterilized liquid LB medium (without resistance) to the heat-shocked mixture, and incubate at 37°C on a shaker at 220 r / min for 50 minutes. Centrifuge the bacterial solution at 8000 rpm for 3 minutes at room temperature, then discard 0.7 mL of the supernatant. Use the remaining 0.1 mL of the supernatant to resuspend the bacterial body, take an appropriate amount of solid LB medium plate (Kana resistance) and spread it on the plate, and then place the plate in a 37°C constant temperature incubator for culture. Observe the results after 14 hours.

[0059] (3) Positive monoclonal bacteria screening: randomly select a white round single colony on the plate, resuspend the colony with 1 mL of sterilized LB liquid medium (Kana resistance), and incubate it at 37°C for 4 hours. Perform colony PCR identification, and add the components listed in Table 5 to a 200 μL EP tube and mix thoroughly.

[0060] Table 5 Bacterial solution PCR reaction system

[0061]

[0062] Place the mixed sample in a PCR instrument for PCR amplification. After the bacterial solution PCR is completed, identify the product using a 1% agarose gel, and select the single colony strain with the same band position as the target gene position for sequencing at Anhui Chuzhou General Biotechnology Co., Ltd.

[0063] (4) Extraction of recombinant plasmid: select the positive strain with successful sequencing, expand the culture, and then use the Genorius plasmid extraction kit to extract the pET-28a-S1-CTD recombinant plasmid according to the instructions.

[0064] 7. Induction expression and identification of the target protein

[0065] (1) Expression of the target protein

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

[0067] b. Inoculate the constructed pET-28a-S1-CTD-BL21 expression bacteria into 200 mL of liquid LB medium (Kana antibody) at an inoculation amount of 1:50, and perform two lives.

[0068] c. After inoculation, place it 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 at 37°C in a shaker at 220 rpm for 5 hours.

[0069] d. Collect the bacteria and reserve 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, 12000 rpm for 10 min. Take 40 μL of the ultrasonic disruption supernatant and the ultrasonic disruption precipitate, respectively. Add 10 μL of 5x SDS loading buffer to each of the four samples, and boil for 10 min for SDS-PAGE identification. As shown in FIG. 1, the S1-CTD recombinant protein is expressed in the form of inclusion bodies. Figure 1

[0071] (2) Renaturation and identification of the target protein

[0072] a. Collect the ultrasonic disruption precipitate and wash it with the washing buffer three times, each time at room temperature with 200 rpm stirring for 30 min, and centrifuge at 4°C, 12000 rpm for 10 min. Then gently resuspend the precipitate with the solubilization buffer, stir at 200 rpm for 30 min at room temperature, and then transfer it to the 4°C refrigerator for overnight standing. The next day, centrifuge the sample at 4°C, 12000 rpm for 10 min, separate and collect the supernatant, and take a sample for identification.

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

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

[0075] Table 6 Protein denaturation buffer formula

[0076]

[0077]

[0078] Example 2 S1-CTD recombinant protein mouse immunization and immune effect analysis

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

[0080] 1. Take 2 Balb / c mice aged 6-8 weeks, and use the S1-CTD recombinant protein prepared in Example 1 as the immunogen to immunize the mice, 50 μg per mouse, a total of three times, every two weeks. The first immunization was mixed and emulsified with Freund's complete adjuvant and S1-CTD recombinant protein, and the second and third immunizations were mixed and emulsified with Freund's incomplete adjuvant and S1-CTD recombinant protein. 14d, 28d, 42d after the first immunization, the mouse tail vein was collected, and the serum titer of the immunized mouse was determined by indirect ELISA.

[0081] 2. Indirect ELISA determination of S1-CTD recombinant protein immune mouse serum titer

[0082] S1-CTD recombinant protein was diluted to 2 μg / mL with CBS and coated on 96-well reaction plate, 100 μL per well, 37°C for 2 h; the coating solution was discarded, washed with PBST for three times, and blocked with 5% skim milk at 37°C for 2 h; the coating solution was discarded, washed with PBST for three times, 1:100 diluted immune mouse serum was added to the first well, and un-immune mouse serum was set as negative control, gradient dilution from left to right, 37°C for 1 h; washed with PBST for three times, 1:5000 diluted Goat Anti-Mouse IgG / HRP was added, 37°C for 1 h; the secondary antibody was discarded, washed with PBST for three times, 100 μL TMB color developing solution was added to each well, color developed in dark for 5 min, 100 μL 2 mol / L H2SO4 was added to each well; OD value was determined, and immune effect was analyzed. As shown in 450 Table 1, good immune effect was obtained against S1-CTD recombinant protein. Figure 5

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

[0084] 1. Cell fusion

[0085] ​Four days before cell fusion, the mouse with the best serum titer was selected to extract 100 μg of S1-CTD recombinant protein for intraperitoneal injection to shock the mouse. Four days after shock immunization, cell fusion was performed. Sixteen hours before fusion, a healthy Kunming mouse was selected, killed by the neck, and immersed in 75% alcohol. The mouse was fixed on the foam board with its abdomen facing up. The fur on the mouse's abdomen was cut along the midline using sterilized scissors and forceps without damaging the peritoneum. A new forceps was used to hold the peritoneum, and a sterile syringe was used to extract 10 mL of HAT medium. The medium was slowly injected along the held peritoneum, avoiding fat accumulation, and gently pumped twice. The mouse's abdomen was gently massaged with forceps, and then the medium containing macrophages in the mouse's abdominal cavity was sucked out and added to the prepared 95 mL HAT medium. After vortexing, 100 μL per well was added to a 96-well cell culture plate. The mouse was taken for eye blood after shock immunization, and the blood sample was placed in a 37°C incubator for 2 h, then centrifuged at 3000 rpm for 10 min, and the serum was stored in a -20°C refrigerator.The mouse is killed by necking, soaked in 75% alcohol for 5 min, and then fixed on a special clean foam board and placed on the operating table. A 200-mesh nylon net is moistened with GNK solution. Sterilized scissors and forceps are used to cut the mouse's skin, exposing the mouse's abdominal cavity. A new set of scissors and forceps is used to cut the peritoneum on the right side of the mouse's abdomen. The mouse's complete spleen is removed using a new set of scissors and forceps. The spleen is placed on the moistened nylon net, and the mouse's spleen is slowly ground using scissors while preheated GNK solution is added to keep the spleen moist. After the spleen is ground, the nylon net and the cup wall are rinsed with GNK solution. The volume of the spleen cell suspension is adjusted to 40 mL, and the supernatant is discarded after centrifugation. The cell pellet is resuspended in 25 mL of GNK solution, and the suspension is counted. The volume of the SP2 / 0 cell suspension is calculated based on the ratio of spleen cells to SP2 / 0 cells, which is 8:1. The corresponding volume of SP2 / 0 cell suspension is added to the spleen cell suspension and vortexed, and then centrifuged. The supernatant is discarded after centrifugation, and the cell pellet is gently tapped. The centrifuge tube is placed in a 37°C water bath, and 1 mL of preheated PEG 1500 fusion agent is added evenly and slowly within 90 seconds. The centrifuge tube is gently shaken to ensure that the PEG 1500 and the cells are in contact. After the addition is complete, the mixture is left to stand for 90 seconds. The termination procedure is divided into three 30-second steps. In the first 30 seconds, 1 mL of GNK solution is added evenly and slowly. In the second 30 seconds, 3 mL of GNK solution is added evenly. In the third 30 seconds, 11 mL of GNK solution is added evenly. The cell suspension after termination of the fusion is transferred to a 37°C cell incubator and left to stand for 5 minutes. After the standing period is complete, GNK is added to the centrifuge tube to a total volume of 40 mL, and then centrifuged. The supernatant is discarded, and the cell pellet is resuspended in an appropriate amount of HAT medium. According to the cell suspension of 100 μL per well, the HAT medium is calculated and added, and then the feeder layer cell culture plate prepared the day before is added. The culture plate is placed in a 37°C cell incubator, and the next day the cell culture plate is observed for contamination. If there is no contamination, the cell culture plate is left to stand for more than 7 days, and the cell culture plate is not moved during this period.

[0086] 2. Screening of positive hybridoma cell lines

[0087] After 7-10 days of cell fusion, cell masses were observed under a microscope at a ten-fold field of view, and cell supernatants were detected by indirect ELISA. S1-CTD recombinant protein was diluted with CBS to 2 μg / mL, and 100 μL was added to each well of a 96-well reaction plate, and the plate was incubated at 37°C for 2 h. The coating solution was discarded, and the plate was washed with PBST three times, and then blocked with 5% skim milk at 37°C for 2 h. The coating solution was discarded, and the plate was washed with PBST three times. The first well was added with 1:100 diluted serum of immunized mice, and serum of non-immunized mice was used as a negative control, and serum of the eyes of the fusion mice was used as a positive control. The serum was diluted in gradient ratios from left to right, and the plate was incubated at 37°C for 1 h. The plate was washed with PBST three times, and 1:5000 diluted Goat Anti-Mouse IgG / HRP was added, and the plate was incubated at 37°C for 1 h. The primary antibody was discarded, and the plate was washed with PBST three times. 100 μL TMB color developing solution was added to each well, and the plate was developed in the dark for 5 min. Then, 100 μL 2 mol / L H2SO4was added to each well. The OD value was determined. 450 The OD 450 value was screened for positive hybridoma cells by comparing the OD 450 value. The number of cell masses in the target well was required to be less than 2, and the OD 450 value was required to be greater than 1. After two or more reviews, a cell strain with good specificity, high and stable OD 4 value was screened. The positive hybridoma cell well was subcloned by dilution method. The hybridoma cell strain stably secreting monoclonal antibodies was obtained by multiple subcloning. The results are shown in Table 1. One positive hybridoma cell strain was screened and named 2D1. Figure 6

[0088] 3. Immunoperoxidase monolayer assay (IPMA)

[0089] To promote cell adhesion, 1x polylysine solution was added to the 96-well plate at 50 μL per well, and the plate was left to stand for 10 min. The polylysine was washed away with sterile ddH2O 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 2.5x10 4HEK-293T cells were cultured in treated 96-well cell culture plates at a density of one cell; when the cells reached 60% confluence, pcDNA3.1-CTD (constructed as in Example 1-6) was mixed with polyplus transfection reagent and transiently transfected into HEK-293T cells. After 6 hours of transfection, the original culture medium in the wells was gently aspirated, and the same volume of Opti-MEM medium was slowly added along the well wall. The cell plate was returned to the 37°C cell culture incubator for continued culture for 48 hours. The old culture medium was discarded, and 100 μL / well of 4% tissue cell fixative was added. The plate was placed at room temperature for 15 minutes to achieve the purpose of fixing the cells. After fixation was complete, the fixative was discarded, and sterile PBS buffer was used to gently wash the plate. 100 μL of 5% skim milk was gently added to each well along the well wall, and the plate was placed in a 37°C constant temperature incubator for blocking for 2 hours. The blocking solution was discarded, and the plate was washed three times with PBS buffer. The aspirating and adding of the liquid should be gentle to avoid blowing the cells on the plate bottom and affecting the experimental results. 50 μL of HEK-293T cell supernatant was added to each well, and incubation was performed at 37°C for 40 minutes. To the HEK-293T cell supernatant, 2D1 cell supernatant was added, and a positive control (PC) of 1:500 diluted immune mouse serum and a negative control (NC) of non-immune mouse serum (1:500 dilution) were set up. A blank control group (BC) of pcDNA3.1 empty plasmid transfection was also set up. The secondary antibody was Goat Anti-Mouse IgG / HRP diluted 1:5000. The secondary antibody was discarded, and the plate was washed three times with PBST. 100 μL of AEC color developing solution was added to each well, and color development was performed for 10 minutes in the dark. Then, 100 μL of ddH2O was added to each well to terminate the reaction. The experimental results were observed and photographed under a microscope. The results are shown in Figure 7 Figure 2, indicating that the monoclonal antibody 2D1 can react with the eukaryotic expressed PDCoV S1-CTD protein.

[0090] 4. Mass production of monoclonal antibodies and purification of ascites

[0091] After subcloning, indirect ELISA was also used for screening, and the positive hybridoma cell strain 2D1 selected was expanded and cultured. Seven to ten days before injection, 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 2 x 106 cells per mouse were injected intraperitoneally. The total injection volume was 500 μL, and the volume of culture medium required for resuspension of the cells was calculated and injected intraperitoneally into the mice. About 10 days later, the mice were sacrificed by the neck after the abdominal swelling, and the ascites was collected. The light yellow liquid in the middle layer was collected after centrifugation. Protein A affinity chromatography was used to purify the ascites.

[0092] 5. Monoclonal antibody titer determination

[0093] The titer of the monoclonal antibody was determined by indirect ELISA. The S1-CTD recombinant protein was diluted to 2 μg / mL with CBS, 100 μL per well, coated at 37°C for 2 h. After discarding the coating solution, the plate was washed with PBST for 3 times, 200 μL of 5% skim milk was added to each well for blocking at 37°C for 2 h. After discarding the blocking solution, the plate was washed with PBST for 3 times, the purified monoclonal antibody 2D11 was diluted to 1:500, added to the first well, and then gradiently diluted from left to right, with the non-immune mouse serum as a negative control, 100 μL per well, incubated at 37°C for 1 h. After discarding the primary antibody, the plate was washed with PBST for 3 times, 100 μL of 1:5000 diluted Goat Anti-Mouse IgG / HRP was added to each well, incubated at 37°C for 1 h. After discarding the secondary antibody, the plate was washed with PBST for 3 times, 100 μL of TMB color developing solution was added to each well, color developed in the dark for 5 min, 100 μL of 2 mol / L H2SO4 was added to each well to stop the reaction, and the OD was read 450 , and the results are shown in Figure 8 .

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

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

[0096] The PDCoV S1-CTD protein was truncated by the overlapping polypeptide method, the truncation is shown in Figure 9 , a total of 8 segments were truncated, the primer sequences shown in Table 7 were used to construct the truncated fragments between the BamHI and Xho I enzyme cutting sites of the pET-32a vector according to the method of Example 1, the E. coli Rosetta competent cells were transformed, and the same method was used for induction expression to obtain truncated proteins A, B, C, D, E, e1, e2, and e3.

[0097] Table 7 Primer series for each truncated protein

[0098]

[0099] Dot-ELISA was used to preliminarily screen the B cell epitopes recognized by the monoclonal antibody. An agar puncher was used to punch holes of appropriate size and spacing on the NC membrane. After the NC membrane was soaked in PBS buffer, the NC membrane was transferred to a 37°C oven for drying. Each truncated fragment expressed protein was spotted on the NC membrane, with S1-CTD protein spotting holes as positive controls and pET-32a-Rosetta empty expression protein spotting holes as negative controls. Each hole was spotted with 1 μL of the protein, and the protein was naturally dried before being spotted again. The NC membrane was placed in 20 mL of 5% skim milk at 37°C for 2 h of blocking. After the blocking solution was discarded, the membrane was washed with PBST three times. The membrane was transferred to 20 mL of 1:1000 diluted monoclonal antibody 2D1 diluent, and incubated at 37°C for 1 h. After the primary antibody was discarded, the membrane was washed with PBST three times. The membrane was transferred to 20 mL of 1:5000 diluted Goat Anti-Mouse IgG / HRP, and incubated at 37°C for 1 h. After the secondary antibody was discarded, the membrane was washed with PBST three times. AEC display solution was added for reaction. After 5 min, ddH2O was added to terminate the reaction, and the experimental results were recorded by taking pictures.

[0100] Peptide-ELISA was used to screen the B cell epitopes recognized by the monoclonal antibody. The purified truncated proteins and pET-32a-BL21 empty proteins were diluted to 2 μg / mL with CBS, and 100 μL of each was coated at 37°C for 2 h. After the coating solution was discarded, the plate was washed with PBST three times. The plate was blocked with 200 μL of 5% skim milk at 37°C for 2 h. After the blocking solution was discarded, the plate was washed with PBST three times. 50 μL of 1:1000 diluted monoclonal antibody 2D1 was added to each well as the primary antibody, and incubated at 37°C for 1 h. After the primary antibody was discarded, the plate was washed with PBST three times. 50 μL of 1:5000 diluted Goat Anti-Mouse IgG / HRP was added to each well as the secondary antibody, and incubated at 37°C for 1 h. After the secondary antibody was discarded, the plate was washed with PBST three times. 50 μL of TMB color developing solution was added to each well, and color was developed in the dark for 5 min. 50 μL of 2 mol / L H2SO4 was added to each well to terminate the reaction, and the OD 450 The results of the Peptide-ELISA and Dot-ELISA experiments are shown in Figure 10 Finally, it was determined that the monoclonal antibody 2D1 recognized the region of 571-599 amino acids.

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

[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 (Jieren Bioengineering Co., Ltd.). The sequence information of each truncated polypeptide is shown in Table 8.

[0103] Table 8 Amino acid sequences of truncated polypeptides

[0104]

[0105] The synthesized 3-segment polypeptides were diluted with PBS to 4 mg / mL, each polypeptide was coated at 4 g per well, S1-CTD recombinant protein was coated as a positive control (PC), PBS was coated as a negative control (NC), 1:1000 diluted monoclonal antibody 2D1 was used as a primary antibody, and 1:5000 diluted Goat Anti-Mouse IgG / HRP was used as a secondary antibody. The results are shown in Figure 11 Peptide-ELISA and Dot-ELISA showed that monoclonal antibody 2D1 reacted with polypeptide e2-3, and the reaction was consistent with 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, 1:1000 diluted anti-PDCoV positive pig serum was used as a primary antibody, and 1:5000 diluted Goat Anti-Mouse IgG / HRP was used as a secondary antibody. The results are shown in Figure 12 Peptide-ELISA and Dot-ELISA showed that e2-3 could react with anti-PDCoV positive pig serum.

[0108] The above examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

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

21.

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

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

4. A kit for detecting porcine delta coronavirus, characterized by, The kit takes the porcine delta coronavirus S1-CTD protein linear B cell epitope peptide according to claim 1 as a coating antigen.

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