Porcine epidemic diarrhea virus protein S1 cell line, construction method and application

Abstract

The invention provides a porcine epidemic diarrhea virus protein S1 cell line as well as a construction method and application thereof. The construction method comprises the following steps: modifying an S1 gene of a porcine epidemic diarrhea virus PEDV; human H11 site homologous arms are added to the two ends of the modified S1 protein gene, and the modified S1 protein gene is cloned to a # imgabs0 #-Blunt Zero vector; the method comprises the following steps: designing a gRNA sequence of a targeted 293F cell genome H11 site, and cloning the gRNA sequence of the targeted 293F cell genome H11 site to a PX400 vector; the first plasmid and the second plasmid are co-transfected to a 293F cell, and the 293F cell line for stable exocrine expression of the porcine epidemic diarrhea virus S1 protein is obtained through screening, so that the 293F cell line for stable exocrine expression of the porcine epidemic diarrhea virus S1 protein is successfully constructed, and a foundation is laid for further development of porcine epidemic diarrhea virus detection reagents and subunit vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein expression and gene editing, and particularly relates to a cell line of porcine epidemic diarrhea virus protein S1, a construction method and an application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), with high infectivity. Its typical symptoms include watery diarrhea, vomiting, dehydration, etc. It can infect pigs of different ages, especially neonatal piglets within 7 days after birth are most severely affected, and its lethality rate can be as high as 100%.

[0003] The genome of porcine epidemic diarrhea virus is about 28 Kbp in length, containing 7 open reading frames, encoding a total of 16 non-structural proteins, 4 major structural proteins (spike glycoprotein S, membrane protein M, envelope protein E, nucleocapsid protein N) and 1 auxiliary protein ORF3. The spike glycoprotein S has two regions, S1 and S2. The S1 protein participates in the process of virus binding to host cell receptors, and the S2 protein participates in the process of virus envelope fusing with the host cell membrane. In current research, most antibodies bind to the S1 protein, so the S1 protein has become a very important candidate for developing PEDV subunit vaccines and detection reagents.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a sequence within the prokaryotic genome. Bacteria have evolved the CRISPR / Cas system to resist the invasion of foreign genetic material. The CRISPR / Cas9 system is a ribonucleoprotein formed by the binding of sgRNA and Cas9 endonuclease. The sgRNA can specifically recognize and bind to the target DNA sequence through base complementary pairing, and guide Cas9 to specifically cleave the target genomic DNA to form a double-strand break. When the DNA double-strand break occurs, if a DNA repair template enters the cell, the broken part of the genome will perform homologous recombination repair according to the repair template, thereby realizing gene knock-in. The repair template consists of the target gene to be introduced and the homologous sequences (homologous arms) upstream and downstream of the target sequence. Using the CRISPR / Cas9 technology, precise site-directed knock-in of foreign genes can be achieved, making the genetic background simpler and the experimental operation more accurate and efficient.

[0005] The Hipp11 (H11) locus, initially an exogenous gene-friendly locus (Safe Harbor) isolated and identified by Simo Hippenmeyer et al. from Stanford University on chromosome 11 of mice in 2010, is located in the intergenic region between EIF4ENIF1 and DRG1 genes, with high safety and no gene silencing effect. Therefore, the gene inserted at the H11 locus can be highly expressed under the initiation of an exogenous promoter, and this locus has been successfully applied in transgenic research of humans, mice, and pigs.

[0006] Human embryonic kidney cells HEK293F have a high growth efficiency and usually produce exogenous proteins for biomedical and pharmaceutical research. The exogenous protein products expressed by mammalian cell expression systems can undergo correct post-translational modification and transformation, including polypeptide chain folding, disulfide bond formation, post-translational cleavage of proteins, and glycosylation, etc. Therefore, the recombinant protein expressed using HEK293F cells has basically the same structure and function as the natural protein, and the expression product can be secreted into the culture medium, facilitating the separation and purification of this recombinant protein.

[0007] Therefore, there is a need to provide a cell line that stably secretes and expresses the S1 protein of porcine epidemic diarrhea virus for corresponding detection of porcine epidemic diarrhea virus. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a cell line of porcine epidemic diarrhea virus protein S1, a construction method, and an application to solve the problems in the above background technology.

[0009] On the one hand, the invention provides the following technical solution, a construction method of a cell line of porcine epidemic diarrhea virus protein S1, including:

[0010] S1. Modify the S1 gene of porcine epidemic diarrhea virus PEDV to obtain a modified S1 protein gene;

[0011] S2. Add human H11 locus homologous arms to both ends of the modified S1 protein gene and clone it into -Blunt Zero vector to obtain the first plasmid;

[0012] S3. Design a gRNA sequence targeting the H11 locus of the 293F cell genome, and clone the gRNA sequence targeting the H11 locus of the 293F cell genome into the PX400 vector to obtain the second plasmid;

[0013] S4. Co-transfect the first plasmid and the second plasmid into 293F cells, and screen to obtain a 293F cell line that stably secretes and expresses the S1 protein of porcine epidemic diarrhea virus.

[0014] The construction method of the porcine epidemic diarrhea virus protein S1 cell line proposed by the present invention has the following beneficial effects:

[0015] Based on the analysis of the S1 protein of porcine epidemic diarrhea virus, it is partially modified to facilitate the secretory expression of the S1 protein. Then, the modified S1 protein gene is directionally knocked into the H11 site of the 293F cell genome by using the CRISPR / Cas9 technology, and the information of this cell line can be accurately understood. The present invention has successfully constructed a 293F cell line that stably secretes and expresses the porcine epidemic diarrhea virus S1 protein, laying a foundation for the further development of porcine epidemic diarrhea virus detection reagents and subunit vaccines. In addition, through the indirect ELISA test of the 293F cell line of porcine epidemic diarrhea virus S1 protein on the purified PEDV S1 protein, it can specifically recognize the PEDV S1 antibody and achieve the discrimination of PEDV antibody-positive serum and negative serum.

[0016] In addition, according to the construction method of the porcine epidemic diarrhea virus protein S1 cell line provided by the present invention, the following additional technical features may also be included:

[0017] Preferably, the step S1 is specifically as follows:

[0018] Add a Kozak sequence before the start codon ATG of the S1 gene sequence of porcine epidemic diarrhea virus PEDV, use the CMV promoter, mutate its signal peptide into the IL-2 secretion signal peptide, add a 6xHis tag sequence at the C-terminus of the peptide chain, and connect the EGFP tag protein sequence with a T2A cleavage peptide sequence after the His tag sequence to obtain the modified S1 protein gene.

[0019] Preferably, the sequence of the S1 protein gene is as shown in SEQ ID NO.1.

[0020] Preferably, the sequences of the human-derived H11 site homologous arms are as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0021] Preferably, the gRNA sequence targeting the H11 site of the 293F cell genome is as shown in SEQ ID NO.4.

[0022] Preferably, the sequence of the first plasmid is as shown in SEQ ID NO.5.

[0023] Preferably, the sequence of the second plasmid is as shown in SEQ ID NO.6.

[0024] Preferably, the step S4 is specifically as follows:

[0025] The first plasmid and the second plasmid were co-transfected into 293F cells. After 48 hours, the 293F cells were seeded into cell culture dishes by limited dilution method for adherent culture. Monoclonal green fluorescent cell clusters were observed and marked under a fluorescence microscope, and then collected using a cell cloning ring to obtain monoclonal cell lines. The monoclonal cell lines were passaged and expanded for suspension culture to obtain a 293F cell line that stably secreted and expressed the S1 protein of porcine epidemic diarrhea virus.

[0026] On the other hand, the present invention provides the following technical solution. A cell line of porcine epidemic diarrhea virus protein S1, characterized in that the cell line of porcine epidemic diarrhea virus protein S1 is constructed by using the construction method of the cell line of porcine epidemic diarrhea virus protein S1 as described above.

[0027] On yet another aspect, the present invention provides the following technical solution. The application of the cell line of porcine epidemic diarrhea virus protein S1 as described above in the preparation of porcine epidemic diarrhea virus reagents or subunit vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 The recombinant vector - PEDV S1-EGFP provided in Example 1 of the present invention;

[0030] Figure 2 The map of the expression vector PX400-sgRNA provided in Example 1 of the present invention;

[0031] Figure 3 In Example 2 of the present invention - Fluorescence image of 293F cells after co-transfection of PEDV S1-EGFP and PX400-sgRNA;

[0032] Figure 4 Fluorescence image of monoclonal cells of 293F-PEDV S1-EGFP provided in Example 2 of the present invention;

[0033] Figure 5 Western Blot detection of the expression of PEDV S1 protein in monoclonal cells of 293F-PEDV S1-EGFP provided in Example 3 of the present invention;

[0034] Figure 6This is the Coomassie brilliant blue staining detection result diagram of the purified PEDV S1 protein of the 293F-PEDV S1-EGFP monoclonal cells in Example 3 of the present invention;

[0035] Figure 7 This is the agarose gel electrophoresis diagram of the PCR products of the H11 site of the 293F-PEDV S1-EGFP monoclonal cells in Example 4 of the present invention;

[0036] Figure 8 This is the cell morphology and cell fluorescence diagram of different passages of the 293F-PEDV S1-EGFP monoclonal cells in Example 5 of the present invention;

[0037] Figure 9 This is the growth curve diagram of the 293F-PEDV S1-EGFP monoclonal cells and normal 293F cells in Example 5 of the present invention;

[0038] Figure 10 This is the protein expression of different passages of the 293F-PEDV S1-EGFP monoclonal cells in Example 5 of the invention.

[0039] The present invention will be further described below in conjunction with the accompanying drawings and their descriptions. Specific Embodiments

[0040] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the embodiments of the present invention and should not be construed as limiting the present invention.

[0041] The present invention provides a porcine epidemic diarrhea virus protein S1 cell line, a construction method and an application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] For the construction method of the porcine epidemic diarrhea virus protein S1 cell line of the present invention, it includes:

[0043] S1. Modify the S1 gene of the porcine epidemic diarrhea virus PEDV to obtain a modified S1 protein gene;

[0044] S2. Add human-derived H11 site homologous arms to both ends of the modified S1 protein gene and clone it into -On the Blunt Zero vector to obtain the first plasmid;

[0045] S3. Design a gRNA sequence targeting the H11 site of the 293F cell genome, and clone the gRNA sequence targeting the H11 site of the 293F cell genome into the PX400 vector to obtain the second plasmid;

[0046] S4. Co-transfect the first plasmid and the second plasmid into 293F cells, and screen to obtain a 293F cell line that stably secretes and expresses the S1 protein of porcine epidemic diarrhea virus.

[0047] Among them, the specific step S1 is as follows:

[0048] Add a Kozak sequence before the start codon ATG of the S1 gene sequence of porcine epidemic diarrhea virus PEDV, use the CMV promoter, mutate its signal peptide into the IL-2 secretion signal peptide, add a 6xHis tag sequence at the C-terminus of the peptide chain, and connect the EGFP tag protein sequence with a T2A cleavage peptide sequence after the His tag sequence to obtain the modified S1 protein gene.

[0049] Among them, the sequence of the S1 protein gene is as shown in SEQ ID NO.1, the sequences of the human-derived H11 site homologous arms are as shown in SEQ ID NO.2 and SEQ ID NO.3, the gRNA sequence targeting the H11 site of the 293F cell genome is as shown in SEQ ID NO.4, the sequence of the first plasmid is as shown in SEQ ID NO.5, and the sequence of the second plasmid is as shown in SEQ ID NO.6;

[0050] Among them, the specific step S4 is as follows:

[0051] Co-transfect the first plasmid and the second plasmid into 293F cells. After 48 hours, inoculate the 293F cells into a cell culture dish by limited dilution method for adherent culture. Observe and mark the monoclonal green fluorescent cell clusters through a fluorescence microscope, and collect them using a cell cloning ring to obtain monoclonal cell strains. Passage and expand the suspension culture of the monoclonal cell strains to obtain a 293F cell line that stably secretes and expresses the S1 protein of porcine epidemic diarrhea virus.

[0052] For the porcine epidemic diarrhea virus protein S1 cell line, the porcine epidemic diarrhea virus protein S1 cell line is constructed by the construction method of the porcine epidemic diarrhea virus protein S1 cell line as described above.

[0053] For the application of the porcine epidemic diarrhea virus protein S1 cell line in the present invention, such as the application of the porcine epidemic diarrhea virus protein S1 cell line in the preparation of porcine epidemic diarrhea virus reagents or subunit vaccines as described above.

[0054] All the reagents and consumables used in the present invention are ordinary commercially available products. The present invention will be further described below in conjunction with the examples:

[0055] Example 1 Design and construction of the first plasmid and the second plasmid

[0056] 1. Based on the published sequence of the PEDV S1 protein (GenBank accession number: QCG74450.1), the PEDV S1 gene was modified and optimized, and other components were added. The specific modifications are as follows: A Kozak sequence was added before the start codon ATG of the S1 gene sequence of the porcine epidemic diarrhea virus PEDV, the CMV promoter was used, its signal peptide was mutated to the IL-2 secretion signal peptide, a 6x His tag sequence was added to the C-terminus of the peptide chain, and an EGFP tag protein sequence was connected with a T2A cleavage peptide sequence after the His tag sequence to obtain the modified S1 protein gene.

[0057] 2. Homologous arms of the human H11 site were added to both ends of the modified S1 protein gene and cloned into the -BluntZero vector to obtain the recombinant vector -PEDV S1-EGFP, that is, the first plasmid. The map of the recombinant vector -PEDVS1-EGFP is as shown in Figure 1 Figure.

[0058] 3. A gRNA sequence targeting the H11 site of the 293F cell genome was designed, and the gRNA sequence targeting the H11 site of the 293F cell genome was cloned into the PX400 vector to obtain the expression vector PX400-sgRNA, that is, the second plasmid. The map of the expression vector PX400-sgRNA is as shown in Figure 2 Figure.

[0059] Example 2 Obtaining of a 293F cell line stably secreting and expressing the porcine epidemic diarrhea virus S1 protein

[0060] 1. Resuscitate the suspension cell 293T using the SMM 293-TⅡ medium (producer: Sino Biological Inc.) containing 2% fetal bovine serum, and perform suspension culture passage at a ratio of 1:5 (37°C, 5% CO2, 120 rpm / min). After the cells resume normal growth, take 3x 10 6Cells were seeded into 100-mm cell culture dishes and allowed to adhere by static culture. When the cell confluence reached 70%, the recombinant vectors were co-transfected with Lipofectamine 3000 (Invitrogen). -PEDV S1-EGFP (the first plasmid) and the expression vector PX400-sgRNA (the second plasmid), and cultured in an incubator at 37 °C for 48 h. As Figure 3 shown, cells successfully co-transfected would show green fluorescence under a fluorescence microscope.

[0061] 2. The successfully transfected cells were digested with trypsin and seeded into 100-mm cell culture dishes at a density of 100 cells, followed by static adherent culture for 2 - 6 days. Every 2 days, cell clusters showing green fluorescence and being monoclonal were observed and marked under a fluorescence microscope. Then, the selected monoclonal cell clusters were picked using a cell cloning ring. As Figure 4 shown, the cells were successively expanded in 96-well plates, 48-well plates, 24-well plates, 12-well plates, and 6-well plates, and the fluorescence expression of the cells was continuously observed. Finally, 293F-PEDV S1-EGFP monoclonal cells, namely monoclonal cells of the 293F cell line stably secreting and expressing porcine epidemic diarrhea virus S1 protein, were obtained.

[0062] Example 3 Expression, purification, and immunoblot analysis of S1 protein of 293F-PEDV S1-EGFP monoclonal cells

[0063] 1. After adherent expansion culture of the obtained 293F-PEDV S1-EGFP monoclonal cells, they were seeded into a conical culture flask at a density of 5 × 10 5 / mL and cultured in an oscillating cell incubator at 37 °C, 5% CO2, and 120 rpm / min.

[0064] 2. When the cell density reached 2 × 10 6When it reaches [X] cells / mL, take 1 mL of the cell suspension, centrifuge at 3000 g, 4 °C for 5 min, collect the cells and the supernatant of the cell culture medium respectively. Use 200 μL of RIPA lysis buffer containing 1% protease inhibitor to lyse the cells, lyse on ice for 15 min, centrifuge at 12000 g, 4 °C for 10 min to collect the supernatant of the cell lysate; the supernatant of the cell culture medium is also centrifuged at 12000 g, 4 °C for 10 min to collect the supernatant. Then add 5X protein denaturation buffer to the supernatant of the cell lysate and the supernatant of the cell culture medium, heat and denature at 98 °C for 8 min, perform SDS-PAGE electrophoresis, electrotransfer the fusion recombinant S1 protein to the NC membrane by wet transfer, block with 5% skim milk TBST blocking solution at 37 °C for 1 h, dilute His-Tag mouse antibody and PEDV S1 mouse antibody with TBST at 1:3000, incubate the antibody overnight at 4 °C and then wash thoroughly with TBST, dilute horseradish peroxidase-labeled goat anti-mouse secondary antibody with TBST at 1:4000, incubate at 37 °C for 1 h and then wash thoroughly with TBST, and then develop using a developer. It can be seen that the band size is consistent with the expected size, indicating that the obtained S1 protein is successfully expressed, as specifically shown in Figure 5 shown (the original 293F cells were used as negative controls);

[0065] 3. When the cell density reaches 2x10 6 / mL, take 100 mL of the cell suspension, centrifuge at 3000 g, 4 °C for 5 min, collect the supernatant of the cell culture medium, and then centrifuge at 12000 g, 4 °C for 10 min to collect the supernatant; use the nickel ion affinity chromatography kit of Beyotime Institute of Biotechnology {BeyoGold TM His-tag Purification Resin (reduction-resistant chelating type)} for separation and purification. First, mix the supernatant and the nickel medium in a ratio of 8:1, incubate overnight at 4 °C on a shaker to allow the His-tag protein of the PEDV S1 protein to fully bind to the nickel medium. Then load the above mixture into a chromatography empty column tube, collect the flow-through liquid, wash the column 5 times with the washing solution (0 / 2 / 5 / 10 / 20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0), collect the washing solution, and finally perform gradient elution of the protein with the elution solution (50 / 100 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Take protein samples for SDS-PAGE electrophoresis and Coomassie brilliant blue staining. It can be seen that the purification effect of the S1 protein is good, as specifically shown in Figure 6 shown. Finally, the protein concentration of 6.228 mg / mL was collected, with a total of 3 mL.

[0066] Example 4 Sequencing of the DNA sequence at the H11 site of the 293F-PEDV S1-EGFP monoclonal cell

[0067] 1. Suspend the cells obtained in step 1 of Example 3 in culture until the cell density reaches 2 x 10 6 / mL. Take 1 mL of the cell suspension, centrifuge at 3000 g, 4 °C for 5 min, collect the cells, extract the DNA of monoclonal cells as a template, design highly specific primers near the H11 locus of 293F, and perform PCR amplification. Use 2xPhanta Flash Master Mix (Dye Plus) (Vazyme) for PCR amplification. Reaction system: 25 μL of 2xPhanta Flash Master Mix, 2 μL of H11-F, 2 μL of H11-R, 2 μL of cell DNA, 19 μL of ddH2O; Reaction program: 98 °C for 30 s, 98 °C for 10 s, 60 °C for 5 s, 72 °C for 25 s (× 34 cycles), 72 °C for 2 min, 12 °C for 5 min; Perform agarose gel electrophoresis on the obtained PCR product. Using the standard DL 10000 DNA Marker as a reference, the expected size of the amplified fragment is 4500 bp, as specifically shown in Figure 7 (The original 293F cells are used as a negative control, and the expected size of the PCR product is 400 bp). Refer to the instructions of the gel extraction kit (Omega) for operation to obtain the purified PCR product. Among them, the sequence of H11-F is as shown in SEQ ID NO.7, and the sequence of H11-R is as shown in SEQ ID NO.8.

[0068] 2. Perform gene sequencing on the purified PCR product. The PEDV S1 gene sequence was successfully inserted into the H11 locus of the 293F-PEDV S1-EGFP monoclonal cells. The sequence of the 293F-PEDV S1-EGFP monoclonal cells is as shown in SEQ ID NO.9.

[0069] Example 5 Evaluation of the stability of different passages of the 293F-PEDV S1-EGFP monoclonal cell line

[0070] 1. Continuously passage and cryopreserve the cells obtained in step 1 of Example 3. After resuscitating the cells of different passages (F5, F10, F15, F20), suspend them in culture. When the cell density reaches 2 x 10 6 / mL, take 1 mL of the cell suspension and place it in a 60 mm culture dish, supplement the medium to 4 mL, and statically culture it in an incubator. When the cell density reaches 80% - 90%, observe it under a fluorescence microscope, as specifically shown in Figure 8 It can be seen that there are no obvious differences in the cell morphology and fluorescence expression intensity of different passages, and the cell morphology has no obvious difference from that of normal 293F cells.

[0071] 2. Suspend the cells obtained in step 1 of Example 3 in a medium containing 2% serum and culture them. Use normal 293F cells as a control. At time points of 1d, 2d, 3d, 4d, 5d, and 6d, use CCK-8 to detect cell proliferation. Specifically, as Figure 9 shown, by comparing the results, it was found that there was no significant difference in the growth curves between the 293F-PEDV S1-EGFP monoclonal cell line and the normal 293F cell line.

[0072] 3. Resuscitate and suspend the cells of different passages (F5, F10, F15, F20) for culture, and perform immunoblotting according to the method in step 2 of Example 3. The results are as Figure 10 shown. It can be seen that there is no significant difference in the expression level of PEDV S1 protein among the cell lines of different passages, indicating that the protein expressed by the cell line is stable.

[0073] Example 6 Application of the S1 protein of 293F-PEDV S1-EGFP monoclonal cells

[0074] 1. Take 1 mL of each of the 4 purified PEDV S1 protein samples in step 3 of Example 3. Name the four samples Sample 1, Sample 2, Sample 3, and Sample 4 respectively. Dilute each sample with 15 mL of PBS, and remove the high-concentration imidazole during the purification process of the protein by centrifugation (4000 g, 4 °C, 10 - 30 min) through a protein ultrafiltration tube with a cut-off molecular weight of 50 KDa. Measure the protein concentration by the BCA protein quantification method;

[0075] 2. Dilute the purified PEDV S1 protein above to 0.5 μg / mL and 0.25 μg / mL with the coating solution, add 100 μL per well to the enzyme-linked immunosorbent assay (ELISA) plate, and incubate overnight at 4 °C. The next day, wash the plate 3 times with PBST solution, soak and shake for 5 s each time, and pat dry the moisture; Add 2% BSA-PBST solution to the coated ELISA plate at 100 μL per well, block at 37 °C for 1 h, then wash 3 times with PBST solution, soak and shake for 5 s each time, and pat dry the moisture; Add the diluted PEDV antibody positive serum and negative serum, with a dilution factor of 1:40, 100 μL per well, incubate at 37 °C for 1 h, then wash 3 times with PBST solution, soak and shake for 5 s each time, and pat dry the moisture; Dilute the enzyme-labeled antibody (goat anti-pig secondary antibody labeled with HRP) with PBST solution at 1:12000, add 100 μL per well, incubate at 37 °C for 1 h, then wash 3 times with PBST solution, soak and shake for 5 s each time, and pat dry the moisture; Add 100 μL of TMB chromogenic solution to each well, develop color in the dark at 37 °C for 15 min; Add 100 μL of stop solution to each well to terminate the reaction, and measure the OD 450 value with an enzyme-labeled immunosorbent assay reader. Calculate the positive OD 450 value and negative OD 450The ratio between values (P / N value), select the antigen coating concentration and serum dilution ratio when the P / N value is the largest as the optimal working concentration, and the test results are shown in Table 1:

[0076] Table 1

[0077]

[0078] Based on the preliminary selection according to the test results, the PEDV S1 protein is coated at a concentration of 0.5 μg / mL and the serum is diluted at a ratio of 1:40.

[0079] 3. Conduct an ELISA test according to the reaction conditions determined above, and detect 30 PEDV antibody-negative sera. The results are shown in Table 2:

[0080] Table 2

[0081] Sample number <![CDATA[OD 450 > Sample number OD450 Sample number <![CDATA[OD 450 > 1 0.173 11 0.152 21 0.179 2 0.166 12 0.152 22 0.18 3 0.158 13 0.213 23 0.162 4 0.218 14 0.241 24 0.187 5 0.176 15 0.178 25 0.222 6 0.159 16 0.179 26 0.166 7 0.175 17 0.15 27 0.168 8 0.181 18 0.152 28 0.186 9 0.158 19 0.19 29 0.156 10 0.147 20 0.177 30 0.157

[0082] According to Table 2, the average value (Mean) calculated from the OD 450 value is 0.175, and the standard deviation (SD) is 0.023. Thus, the positive and negative critical value is determined as Mean + 3 * SD = 0.244, that is, when the OD 450 value of the serum to be tested is equal to or higher than 0.244, it is judged as positive, and when it is lower than 0.244, it is judged as negative.

[0083] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention are all equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing a cell line of porcine epidemic diarrhea virus protein S1, characterized in that, Comprising: S1. Modify the S1 gene of porcine epidemic diarrhea virus (PEDV) to obtain a modified S1 protein gene; S2. Add human H11 site homologous arms to both ends of the modified S1 protein gene and clone it into -BluntZero vector to obtain the first plasmid; S3. Design a gRNA sequence targeting the H11 site of the 293F cell genome, and clone the gRNA sequence targeting the H11 site of the 293F cell genome into the PX400 vector to obtain a second plasmid; S4. Co-transfect the first plasmid and the second plasmid into 293F cells, and screen to obtain a 293F cell line that stably secretes and expresses the S1 protein of porcine epidemic diarrhea virus.

2. The method for constructing a porcine epidemic diarrhea virus protein S1 cell line according to claim 1, wherein The specific step S1 is as follows: Add a Kozak sequence before the start codon ATG of the S1 gene sequence of porcine epidemic diarrhea virus (PEDV), use the CMV promoter, mutate its signal peptide into an IL-2 secretion signal peptide, add a 6xHis tag sequence at the C-terminus of the peptide chain, and connect an EGFP tag protein sequence with a T2A cleavage peptide sequence after the His tag sequence to obtain a modified S1 protein gene.

3. The method for constructing a porcine epidemic diarrhea virus protein S1 cell line according to claim 2, characterized in that, The sequence of the S1 protein gene is shown as SEQ ID NO.

1.

4. The method for constructing a porcine epidemic diarrhea virus protein S1 cell line according to claim 1, characterized in that, The sequences of the human-derived H11 site homologous arms are shown as SEQ ID NO.2 and SEQ ID NO.

3.

5. The method for constructing a cell line of porcine epidemic diarrhea virus protein S1 according to claim 1, characterized in that, The gRNA sequence targeting the H11 site of the 293F cell genome is shown as SEQ ID NO.

4.

6. The method for constructing a porcine epidemic diarrhea virus protein S1 cell line according to claim 1, characterized in that, The sequence of the first plasmid is shown as SEQ ID NO.

5.

7. The method for constructing a porcine epidemic diarrhea virus protein S1 cell line according to claim 1, characterized in that, The sequence of the second plasmid is shown as SEQ ID NO.

6.

8. The method for constructing a porcine epidemic diarrhea virus protein S1 cell line according to claim 1, characterized in that, The specific step S4 is as follows: Co-transfect the first plasmid and the second plasmid into 293F cells. After 48 h, inoculate the 293F cells into a cell culture dish by the limiting dilution method for adherent culture. Observe and label monoclonal green fluorescent cell clusters through a fluorescence microscope, and collect them using a cell cloning ring to obtain monoclonal cell strains. Subculture and expand the suspension culture of the monoclonal cell strains to obtain a 293F cell line that stably secretes and expresses the S1 protein of porcine epidemic diarrhea virus.

9. A porcine epidemic diarrhea virus protein S1 cell line, characterized in that, The porcine epidemic diarrhea virus protein S1 cell line is constructed by using the construction method of the porcine epidemic diarrhea virus protein S1 cell line according to any one of claims 1-8.

10. Use of the porcine epidemic diarrhea virus protein S1 cell line according to claim 9 in the preparation of porcine epidemic diarrhea virus reagents or subunit vaccines.

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