Cell line for expressing porcine epidemic diarrhea virus S1 protein as well as construction method, application and ELISA (Enzyme-Linked Immunosorbent Assay) detection method thereof

Abstract

The invention relates to the technical field of biology, and discloses a construction method of a cell line for expressing porcine epidemic diarrhea virus S1 protein.The method comprises the steps that a nucleotide sequence of the PEDV S1 protein is inserted into a plasmid, and the plasmid carrying nucleotide of the PEDV S1 protein is obtained; then transfecting the plasmid carrying the nucleotide of the PEDV S1 protein into CHO-K1, and culturing to obtain a cell line for expressing the PEDV S1 protein; the nucleotide sequence of the PEDV S1 protein is shown as SEQ ID NO: 1, the cell line capable of stably expressing the PEDV S1 protein is obtained through the design, and in addition, the invention further discloses the cell line capable of expressing the PEDV S1 protein, application of the cell line and an ELISA detection method.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a cell line expressing porcine epidemic diarrhea virus S1 protein, a construction method thereof, uses thereof, and an ELISA detection method. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the genus Coronavirus in the family Coronaviridae. It can cause intestinal villus atrophy and loss, leading to acute gastroenteritis with clinical symptoms such as diarrhea, vomiting, and dehydration. Based on the S protein gene sequence, PEDV can be divided into two genotypes: GⅠ and GⅡ. GⅠ includes two subtypes, GⅠ-a and GⅠ-b, while GⅡ includes two subtypes, GⅡ-a and GⅡ-b. Currently, the GⅡ variant of PEDV has become the main pathogen causing diarrhea and high mortality in piglets, causing significant economic losses to the pig industry.

[0003] The full genome of PEDV is about 28Kb in length. It is a single-stranded positive-sense RNA virus, containing ORF1a and ORF1b encoding non-structural proteins, spike protein S with receptor binding domain (RBD) and membrane fusion domain, single accessory protein ORF3, envelope protein (E protein), membrane protein (M protein) and nucleocapsid protein (N protein) that are mainly involved in virus assembly, with a total of 7 open reading frames (ORFs). In addition to the open reading frames, there are also 5′-non-coding regions (UTRs) and 3′-non-coding region poly (A) structures.

[0004] The S gene is approximately 4161 base pairs long and encodes the 1387-amino acid S protein, a spike protein located on the viral surface and a type I membrane glycoprotein. Most coronavirus S proteins have a protease cleavage site that separates the S protein into its S1 and S2 domains. However, the PEDV S protein lacks this protease site and is often artificially divided into an N-terminal S1 domain and a C-terminal S2 domain. The S1 domain epitopes, located externally on the protein structure, are mostly exposed. During host infection, they can recognize and bind to specific cell receptors. These epitopes contain an N-terminal signal peptide, a sialic acid binding region, and a core neutralizing epitope region (COE). With technological advancements, S proteins have been experimentally expressed in insect cells and Escherichia coli expression systems, tobacco mosaic virus, Lactobacillus, and Salmonella, resulting in strong immune responses in immunized animals. PEDV entry into cells is mediated by the binding of its spike protein (S protein) to host cell receptors. The S1 domain contains multiple receptor binding sites and is therefore often the preferred target for developing vaccines and diagnostic reagents.

[0005] Chinese hamster ovary cells (CHO cells), a type of fibroblast isolated from the ovary of a Chinese hamster by Theodore Puck in 1957, possess the ability to proliferate indefinitely and are one of the most widely used mammalian cell lines for the production of biotherapeutics. Compared to other expression systems, mammalian cell expression systems can accurately guide protein folding, phosphorylation, glycosylation, disulfide bond modification, and other post-translational processes during the protein translation phase, ensuring that the recombinant protein closely resembles its native structure in terms of molecular structure, physicochemical properties, and biological function. In addition to these advantages, the CHO cell expression system also offers the advantages of low endogenous protein secretion, efficient recombinant gene amplification and expression, stable integration of exogenous proteins, and the ability to culture in both adherent and suspension cultures.

[0006] Commonly used recombinant protein expression systems include the Escherichia coli prokaryotic expression system, the baculovirus eukaryotic expression system, the yeast eukaryotic expression system, and mammalian expression systems. The E. coli prokaryotic expression system is the most widely used due to its maturity, low cost, and high yield. However, eukaryotic proteins expressed using this system lack post-translational modifications and are easily expressed as inclusion bodies. Protein denaturation and renaturation often lead to inactivation of the recombinant protein. Traditional mammalian expression systems have long experimental cycles for screening stable cell lines, low success rates, and high expression costs. Currently, there are few published reports on the successful expression of PEDV S1 protein in CHO cell lines.

[0007] The problem to be solved in this scheme is: how to provide a method for constructing a CHO cell line expressing PEDV S1 protein. Summary of the Invention

[0008] The purpose of this application is to provide a method for constructing a CHO cell line expressing PEDV S1 protein to further enrich the expression system of PEDV S1 recombinant protein.

[0009] To achieve the above objectives, this protocol provides a method for constructing a cell line expressing porcine epidemic diarrhea virus S1 protein, comprising the following steps:

[0010] Step 1: inserting a nucleotide sequence directing the synthesis of PEDV S1 protein into a plasmid to obtain a plasmid carrying the nucleotide sequence of PEDV S1 protein;

[0011] Step 2: Transfect the plasmid carrying the nucleic acid of PEDV S1 protein into CHO-K1 and culture it to obtain a cell line expressing porcine epidemic diarrhea virus S1 protein;

[0012] The nucleotide sequence of the PEDV S1 protein is shown in SEQ ID NO: 1.

[0013] Preferably, a KOZAK sequence is added before the start codon of the nucleotide sequence of the PEDV S1 protein; the nucleotide sequence of the KOZAK sequence is shown in SEQ ID NO: 2.

[0014] Preferably, the nucleotide sequence of the PEDV S1 protein is also connected to an IgGκ signal peptide sequence, and the step 1 specifically comprises: inserting the nucleotide sequence of the PEDV S1 protein into a plasmid to obtain a plasmid carrying the nucleotide sequence of the PEDV S1 protein; then using the plasmid carrying the nucleotide sequence of the PEDV S1 protein as a vector, amplifying using homologous recombination primers homologous arm IgGκ-S1-F and homologous arm IgGκ-S1-R to obtain an amplified product;

[0015] The amplified product is then homologously recombined with the plasmid to obtain a plasmid carrying the IgGκ signal peptide sequence and the nucleotide sequence of the PEDV S1 protein.

[0016] In addition, the present application also discloses a cell line expressing porcine epidemic diarrhea virus S1 protein, which is prepared by the above-mentioned method for constructing a cell line expressing porcine epidemic diarrhea virus S1 protein.

[0017] In addition, the present application also discloses the use of the cell line expressing porcine epidemic diarrhea virus S1 protein as described above to express PEDV S1 protein.

[0018] In addition, the present application also discloses an ELISA detection method, wherein the coating antigen in the kit is the PEDV S1 protein expressed by the cell line expressing the porcine epidemic diarrhea virus S1 protein.

[0019] The beneficial effects of the present application are as follows: the present application provides a CHO cell line capable of stably expressing PEDV S1 recombinant protein, further enriching the categories of existing PEDV S1 recombinant protein expression systems. At the same time, the CHO cell line expressing PEDV S1 recombinant protein prepared by the present application has the advantages of low endogenous protein secretion, efficient amplification and expression of recombinant genes, stable integration of exogenous proteins, and can be cultured in both adherent and suspension forms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the plasmid map of the recombinant plasmid PXJ40-S1;

[0021] Figure 2 Lane 1 shows the PCR amplification results of the S1 gene carrying homology arms and IgGκ signal peptide, where M is Trans 2K Plus DNA Marker, and lane 1 shows the amplified product of the S1 gene carrying homology arms and IgGκ signal peptide;

[0022] Figure 3This is the plasmid map of the PXJ40-NeoR / KanR vector;

[0023] Figure 4 The results of double enzyme digestion of plasmid pXJ40-NeoR / KanR with EcoRI and PstI are shown, where M stands for Trans 2K Plus DNA Marker, and lanes 1 to 4 show the results of double enzyme digestion of pXJ40-NeoR / KanR with EcoRI and PstI;

[0024] Figure 5 The results of EcoRI and PstI double enzyme digestion of pXJ40-lgGκ-PEDV S1 plasmid are shown: M is Tr ans 2KPlus DNA Marker, lanes 1 to 4 are PCR products of EcoRI and PstI double enzyme digestion of pXJ40-lgGκ-PEDV S1 plasmid;

[0025] Figure 6 The results of PCR amplification of the bacterial solution after transformation with the pXJ40-lgGκ-PEDV S1 plasmid are shown: M represents Trans2K Plus DNA Marker, and lanes 1-10 represent the PCR amplification products of the bacterial solution after transformation with the pXJ40-lgGκ-PEDV S1 plasmid.

[0026] Figure 7 is the plasmid map of pXJ40-lgGκ-PEDV S1;

[0027] Figure 8 Western Blot analysis of S1 protein expression in CHO-K1 cells 48 hours after recombinant expression plasmid transfection. 1 is the transfection of pXJ40-IgGκ-PEDV S1 plasmid, 2 is the precipitate of transfection of pXJ40-IgGκ-PEDV S1 plasmid, 3 is the transfection of empty pXJ40-NeoR / KanR plasmid, and 4 is the negative control.

[0028] Figure 9 The cytopathic effect graph is the optimal concentration of G418 for CHO-K1 cells;

[0029] Figure 10 This is a graph showing the growth of CHO-K1 cells transfected with pXJ40-IgGκ-PEDV S1;

[0030] Figure 11 The figure shows the growth of CHO-K1 cells transfected with pXJ40-NeoR / KanR;

[0031] Figure 12 The pathological changes of CHO-K1 cells were not transfected with plasmids.

[0032] Figure 13This is the electrophoresis diagram of the first limiting dilution screening of different monoclonal cells, where M is a 180kDa Prestained Protein Marker, and 2, 10, 14, 16, 19, 20, 21, 27, and 37 are the supernatants of 9 monoclonal cell lines respectively;

[0033] Figure 14 This is a comparison of the grayscale values of Western Blots from the first limiting dilution screening of different monoclonal cell lines. M represents the 180 kDa Prestained Protein Marker, and 2, 10, 14, 16, 19, 20, 21, 27, and 37 represent the supernatants of nine monoclonal cell lines, respectively.

[0034] Figure 15 This is the electrophoresis diagram of the second limiting dilution screening of different monoclonal cells. M is the 180kDa Prest ained Protein Marker, and 5, 6, 8, 14, 15, 16, 20, 24, 25, 26, 27, 28, 29, and 30 are the supernatants of 14 monoclonal cells respectively;

[0035] Figure 16 This is a comparison of the grayscale values of Western Blots of different monoclonal cell lines screened by the second limiting dilution method. M is a 180 kDa prestained protein marker, and 5, 6, 8, 14, 15, 16, 20, 24, 25, 26, 27, 28, 29, and 30 are the supernatants of 14 monoclonal cell lines, respectively.

[0036] Figure 17 These are the Western Blot results of the supernatants of recombinant suspension cells S1 CHO-K1 from passages 0 to 15 after 48 h of culture. M is a 180 kDa prestained protein marker, and P1 to 4 represent passages 0, 5, 10, and 15 of S1 CHO-K1 cells.

[0037] Figure 18 Coomassie staining of the supernatant of suspended cells: 1, 2, and 3 are supernatants from three parallel groups, M represents 180 kDa Prestained Protein Marker;

[0038] Figure 19 The results of the BCA protein standard curve are shown;

[0039] Figure 20This is the WB image of purified protein. M represents 180 kDa Prestained Protein Marker, + represents S1 CHO-K1 supernatant, FT represents loading flow-through, W1 to W5 represent wash buffer 1 to 5 times, and E1 to E5 represent eluate 1 to 5 times;

[0040] Figure 21 Coomassie plot of purified protein, M represents 180 kDa Prestained Protein Marker, + represents S1CHO-K1 supernatant, FT represents loading flow-through, W1 to W5 represent wash buffer 1 to 5 times, and E1 to E5 represent eluate 1 to 5 times;

[0041] Figure 22 The P / N ratio graph is for different coating concentrations and serum dilutions;

[0042] Figure 23 This is the P / N ratio diagram for different blocking solutions and blocking times;

[0043] Figure 24 OD450 and P / N values measured during the determination of the optimal serum incubation time;

[0044] Figure 25 This is a test chart of the difference in P / N values measured during the determination of the optimal serum incubation time;

[0045] Figure 26 This is the P / N ratio graph for different dilutions and incubation times of the secondary antibody;

[0046] Figure 27 This is the P / N ratio diagram for different color development times;

[0047] Figure 28 OD measured during the determination of the optimal incubation temperature 450 value;

[0048] Figure 29 This is the difference test diagram of P / N value at different incubation temperatures;

[0049] Figure 30 This is a graph showing the results of a specificity detection test. DETAILED DESCRIPTION

[0050] The present invention will be described clearly and completely below in conjunction with the examples of the present invention. In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0051] Example 1

[0052] Construction of recombinant expression plasmid pXJ40-IgGκ-PEDV S1

[0053] The primers involved in this experiment are shown in Table 1:

[0054] Table 1

[0055]

[0056] It should be noted that the bold letters in Table 1 are homologous sequences;

[0057] The S1 gene sequence of the PEDV GⅡ-a strain isolated in this experiment was analyzed, and the designed nucleotide sequence was optimized according to the codon preference of CHO cells. The sequence is shown in SEQ ID NO: 1. A KOZAK sequence (SEQ ID NO: 2) was added before the start codon ATG to improve the translation efficiency of the S1 protein. A 6×His (SEQ ID NO: 3) and a SpyTag tag (SEQ ID NO: 4) were added to the C-terminus of the sequence. Finally, the modified gene sequence was ligated into the eukaryotic expression vector pXJ40 and synthesized by Qingke Biotechnology Co., Ltd. The newly synthesized recombinant plasmid was named PXJ40-S1. The plasmid map is shown in the figure. Figure 1 shown.

[0058] Using PXJ40-S1 as the vector, the homologous recombination primers homology arm lgGκ-S1-F and homology arm lgGκ-S1-R (as shown in Table 1) were designed, and the high-fidelity enzyme 2× Flash Master Mix (Dye Plus) was used to PCR amplify the S1 protein gene containing the mouse IgGκ chain signal peptide (SEQ ID NO: 5). The sequence is shown in SEQ ID NO: 6. The system is shown in Table 2:

[0059] Table 2

[0060]

[0061] The standard reaction procedure is: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, Tm annealing for 15 seconds, 72℃ extension at 10 seconds / kb for 30 cycles; 72℃ extension for 1 minute. The amplified product was identified and recovered by 1.0% agarose gel electrophoresis, and a 2576 bp band was obtained as the homologous recombination fragment. The results are as follows Figure 2 shown.

[0062] The PXJ40-NeoR / KanR vector preserved in the laboratory was used. The plasmid map is as follows: Figure 3 As shown, double enzyme digestion with EcoRI and PstⅠ was used, and the system is shown in Table 3:

[0063] Table 3

[0064]

[0065]

[0066] The reaction was carried out in a metal bath at 37°C for 2 hours. The product was identified by 1% agarose gel electrophoresis and completely linearized and recovered from the gel. As a vector for homologous recombination, a 4591 bp band was obtained. The results are as follows: Figure 4 The vector was homologously recombined with the PCR-amplified IgGκ-S1 gene. The recombinant plasmid was identified by double enzyme digestion with EcoRⅠ and PstⅠ, and four bands of 4591bp and 2536bp were obtained. Figure 5 The recombinant plasmid was transformed and identified by PCR and a 3854bp band was obtained. Figure 6 The plasmid was sent for sequencing and the correctly identified plasmid was named pXJ40-lgGκ-PEDV S. The plasmid map is shown in Figure 7 shown.

[0067] Example 2

[0068] PEDV-S1 protein was expressed in CHO-K1 cells

[0069] 2.1 Transfection of recombinant expression plasmid into CHO-K1 cells

[0070] CHO-K1 cells in the logarithmic growth phase were trypsinized and seeded into 6-well plates. They were cultured in a 37°C, 5% CO2 incubator until the cell confluence reached approximately 80%. Transfection was then initiated. The specific steps are as follows:

[0071] Discard the culture medium in the 6-well plate, wash twice with PBS, and then add 1.5 mL of Opti-MEM;

[0072] Prepare Solution A: Add 2.5 μg of plasmid to 250 μL of Opti-MEM and vortex to mix.

[0073] Prepare Solution B: Dilute 9 μL of transfection reagent Nulen PlusTrans™ with 250 μL of Opti-MEM and incubate at room temperature for 2-5 minutes.

[0074] Mix solution A and solution B, incubate at room temperature for 20 minutes, then add the mixture to a 6-well plate and shake gently to mix;

[0075] After culturing in a 37° C., 5% CO 2 incubator for 6 h, the cells were replaced with a basal medium containing 5% fetal bovine serum (FBS), and one well was reserved as a negative control.

[0076] The expression of S1 protein in CHO-K1 cells was detected by Western Blot experiment: the primary antibody was Anti-His Tag monoclonal antibody diluted 1:5000, and the secondary antibody was diluted 1:10000. 680RD Goat-anti-MouseAntibody, imaged using the Azure Biosystems C600 multifunctional molecular imaging system, the results are as follows Figure 8 As shown in the WB figure, it can be seen that the S1 protein is mainly expressed in the supernatant, that is, secretory expression, with a size of 180 kDa.

[0077] 2.2 Determination of the optimal concentration of G418 for CHO-K1 cells

[0078] Normally growing CHO-K1 cells were digested into single-cell suspensions and plated into 24-well plates. G418 was added at concentrations of 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, and 900 μg / mL per well. A control group was set up and each concentration was repeated for three wells to observe the cell growth status.

[0079] Results: 6 days after adding G418, it was found that 700 μg / mL was the lowest concentration that caused all CHO-K1 cells to die. Therefore, the optimal concentration for G418 screening of CHO-K1 cells was 700 μg / mL. The results are as follows Figure 9 shown.

[0080] 2.3 Drug Screening

[0081] 48 hours after plasmid transfection, G418 drug screening was started: the supernatant was discarded and 2 mL of complete culture medium (Ham's F-12K + 10% FBS + 1% penicillin-streptomycin + 700 μg / mL G418) was added. The pathological changes and growth of the cells were observed daily. Due to the influence of cell metabolism, the complete culture medium was replaced every 2 days until the CHO-K1 cells in the pXJ40-IgGκ-PEDV S1 group appeared in clusters, while the cells in the negative control group without plasmid transfection were completely dead. This process took about 2 weeks. The results are shown in the figure. Figure 10-12 shown.

[0082] 2.4 Screening of high-expressing monoclonal cell lines

[0083] Surviving cells were seeded into 96-well plates by limiting dilution method for screening of monoclonal cell lines. The culture medium did not contain G418. Figure 13-16Repeat the above steps twice to screen for single clones. After culturing single cells for one week, seed them into 24-well plates and continue culturing. Take a portion of the supernatant and cell samples for Western blot analysis. Cell lines that show specific target protein bands are further expanded and cryopreserved, designated CHO-PEDV / S1 cells.

[0084] 2.5 Acclimation of suspension cell lines

[0085] The cells were inoculated into 30 mL of Hanteng 031 medium to a final concentration of 5 × 10 5 Place the cell culture flask in a 37°C, 5% CO2 cell culture incubator on an orbital shaker at 120 rpm. -1 Incubate overnight, and check the cell density and viability every 24 hours using a cell counter. During the acclimation period, the content of serum added to the culture medium should be gradually reduced. Cells cultured in a culture medium containing 10% serum are called first-generation cells; cells cultured in a culture medium containing 8% serum are the second generation; and the content is reduced by 2% successively until the sixth generation, when no serum is added to the culture medium. When the survival rate of the first-generation cells reaches 94% to 97%, the second-generation culture is carried out. When the survival rate of the cells obtained from the second-generation culture is greater than 95%, the third-generation culture is carried out; after three cultures from the third to the sixth generation, the survival rate of the cells is greater than 95%. After 7 weeks, the cells have grown for three generations 3 days after inoculation, and the density reaches 1×10 6 / mL, and the cell viability reached 95%, and the cells were considered to have adapted to suspension culture.

[0086] 2.6 Stability evaluation of CHO-K1 cell lines expressing high levels of PEDV S1 protein

[0087] The suspension cell line in the above steps was expanded and frozen, and the recombinant cells were named S1 CHO-K1. At the same time, the stability test was performed: the supernatant of 0, 5, 10, and 15 generations of S1 CHO-K1 cells was taken for Western Blot detection. CHO-K1 cells without plasmid transfection were used as negative control. It was found that different generations could stably express S1 protein. The results are as follows Figure 17 shown.

[0088] 2.7 Determination of supernatant protein expression concentration

[0089] The S1 CHO-K1 cells that have been suspended and acclimated were expanded and the supernatant was taken for SDS-PAGE electrophoresis. The gel after electrophoresis was stained with Beyotime Coomassie Brilliant Blue. The results are as follows: Figure 18 The staining results were analyzed using ImageJ software to analyze the PEDV S1 band ratio. The results are shown in Table 4. At the same time, the protein concentration of the collected supernatant was detected. According to the instructions of the BCA protein concentration determination kit, a protein standard curve was prepared. The results are shown in Figure 19As shown, the total protein A562 diluted 5 times is 0.479. Substituting into the formula y = 1.4251x-0.1813, the total protein concentration of the 5-fold dilution is calculated to be 0.501 mg / ml, and the total protein concentration is 2.5 mg / ml. According to the proportion of PEDV S1 bands, the PEDV S1 protein concentration in the supernatant is calculated to be 0.24 mg / ml.

[0090] Table 4

[0091]

[0092] Purification of 2.8S1 protein

[0093] After the suspension-acclimated S1 CHO-K1 cells were expanded and cultured, the supernatant was taken and purified using the His-tag protein purification kit from Beyotime. The purified S1 protein was detected by Western Blot. The results were as follows: Figure 20-21 shown.

[0094] Example 3

[0095] Establishment of an indirect ELISA method for detecting PEDV

[0096] 3.1 Establishment of the optimal coating concentration of recombinant protein and serum dilution ratio

[0097] The purified recombinant PEDV S1 protein prepared in Example 2 was used as the coating antigen, and the optimal coating concentration and serum dilution ratio of the recombinant protein were determined by plate titration.

[0098] (1) Coating: Dilute the purified eukaryotically expressed S1 protein with coating solution to 2.5 μg / mL, 2.0 μg / mL, 1.5 μg / mL, 1.0 μg / mL, 0.8 μg / mL, 0.6 μg / mL, 0.4 μg / mL, and 0.2 μg / mL. Add 100 μL per well and coat overnight at 4°C.

[0099] (2) Washing: Wash the ELISA reaction wells using a plate washer. Set the wash program to 200 μL PBST per well, repeat three times, soaking for 5 seconds each time. After each wash, pat dry on paper.

[0100] (3) Blocking: Add 100 μL of PBST solution containing 2% BSA to each well and block at 37°C for 1 h. After blocking, wash the plate with a plate washer and pat dry.

[0101] (4) Serum dilution and incubation: Dilute the porcine epidemic diarrhea positive and negative sera with sample diluent at dilutions of 1:20, 1:40, 1:80, and 1:160, with 100 μL per well, and incubate at 37°C for 1 h. After incubation, wash the samples with a plate washer and pat dry.

[0102] (5) Dilution and incubation of enzyme-labeled secondary antibody: dilute rabbit anti-pig HRP-IgA with enzyme-labeled diluent at a dilution of 1:10,000, add 100 μL per well, and incubate at 37°C for 1 h. After incubation, wash the plate with a plate washer and pat dry.

[0103] (6) Color development, termination and determination: Add color development solution, 100 μL per well, incubate at 37°C in the dark for 10 min. Then add stop solution to terminate the reaction, 100 μL per well, and quickly determine the OD 450 Calculate the positive OD value in each reaction well 450 Value and negative OD 450 The ratio between the two values is the P / N value. The larger the P / N value, the better the antigen coating concentration and serum dilution. The value with the largest P / N value is selected as the optimal recombinant protein coating concentration and serum dilution.

[0104] The results are as follows Figure 22 As shown in the figure, the optimal coating concentration of recombinant S1 protein is 1.0 μg / mL, the optimal dilution of serum is 1:80, and the antigen coating concentration between 0.4 and 1.0 μg / ml has a good effect.

[0105] 3.2 Determination of the optimal blocking solution and blocking time

[0106] ELISA plates and diluted serum samples were coated with the optimal protein coating concentration and serum dilution, and PBST containing 1% BSA, 2% BSA, 5% BSA, and 5% skim milk powder was used as blocking solution. The plates were blocked at 37°C for 0.5 h, 1 h, 1.5 h, and 2 h, respectively. OD was measured. 450 When the P / N value is the highest, it is the best blocking solution and blocking time. Figure 23 As shown in the figure, the P / N value is the highest when the blocking solution is 5% skim milk powder and the blocking time is 0.5h. Therefore, the optimal blocking solution is PBST containing 5% skim milk powder, and the optimal blocking time is 0.5h.

[0107] 3.3 Determination of the optimal serum incubation time

[0108] The ELISA plate was coated and blocked according to the above optimal conditions and incubated at 37°C for 30 min, 45 min, and 60 min, respectively. 450 When the P / N value is the highest, it is the optimal serum incubation time; there is no significant difference in P / N value between incubation at 37℃ for 45min and incubation for 60min, so the serum incubation time is 45min. The results are as follows Figures 24-25 shown.

[0109] 3.4 Determination of the optimal secondary antibody dilution ratio and incubation time

[0110] Keeping the above optimal conditions unchanged, HRP-labeled rabbit anti-pig IgA was diluted with enzyme-labeled antibody diluent at dilutions of 1:5000, 1:10000, and 1:20000, and incubated at 37°C for 30 min, 45 min, and 60 min, respectively. Measure OD 450 When the P / N value is the highest, it is the optimal secondary antibody dilution ratio and incubation time. The results are as follows: Figure 26 As shown in the figure, when the secondary antibody dilution is 1:5000 and the incubation time is 45 minutes, the P / N value is the largest. Therefore, the optimal secondary antibody dilution ratio is 1:5000, and the optimal secondary antibody incubation time is 45 minutes.

[0111] 3.5 Determination of the optimal color development time

[0112] Keeping the above optimal conditions unchanged, add the color developing solution and incubate at 37℃ for 5min, 10min, and 15min respectively. Then add 100μL / well of 2mol / L H2SO4 stop solution to terminate the reaction. The results are as follows: Figure 27 As shown in the figure, when the substrate reaction time is 15 min, the P / N value is the largest, that is, 15 min is the optimal color development reaction time.

[0113] 3.6 Determination of the optimal incubation temperature

[0114] Keeping the above optimal conditions unchanged, the primary antibody, secondary antibody and color development incubation were performed at 37°C and room temperature respectively. The results are as follows Figures 28-29 As shown in the figure, there is no significant difference in P / N value between incubation at 37℃ and room temperature. For the convenience of operation, room temperature incubation was selected.

[0115] 3.7 Determination of critical value for positive and negative determination

[0116] According to the optimized ELISA conditions, 31 PEDV-negative sera were tested and the OD 450 According to statistical analysis, OD values of 30 negative serum samples were calculated. 450 average value and standard deviation (SD). and The critical value and suspicious value of positive and negative determination are calculated, that is, When , it is judged as positive; The result was negative; The samples were judged as suspicious. The results were statistically analyzed and 31 negative serum OD 450 Average of the values is 0.231, and the standard deviation SD is 0.050. and As the critical value and suspicious judgment standard of ELISA, the critical value was calculated to be 0.381, that is, the OD of the sample to be tested 450When the value is ≥0.381, it is PED V antibody positive, and the OD of the sample to be tested is 450 When the value is less than 0.331, it is negative for PEDV antibodies. 450 Samples with values between 0.331 and 0.381 were considered suspicious, and IBV-negative serum OD 450 The values are shown in Table 5:

[0117] Table 5

[0118]

[0119]

[0120] 3.8 Specificity test

[0121] The established ELISA method was used to test 6 known PEDV, classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRSV), porcine parvovirus (PPV), pseudorabies virus (PRV) positive sera, and Japanese encephalitis virus (JEV) three times under the optimal conditions to analyze the specificity of the kit. Figure 30 As shown in the figure, except for the positive PEDV serum test, the positive sera of other pathogens were all negative, indicating that the specificity of the kit was good and there was no cross reaction with the positive sera of other common swine disease pathogens.

[0122] 3.9 Repeatability test

[0123] Select the same batch of coated ELISA plates and test 8 serum samples according to the established indirect ELISA method. Set up 3 parallels for each serum sample to test the intra-batch reproducibility; select different batches of coated ELISA plates and test the same 8 samples to test the inter-batch reproducibility. Calculate the OD of each serum sample within and between batches respectively. 450 Value nm average value Standard deviation (SD) and coefficient of variation (CV, The test results are shown in Tables 6 and 7 (Table 6 is the intra-batch repeatability test data of PEDV S1 indirect ELISA, and Table 7 is the inter-batch repeatability test data of PEDV S1 indirect ELISA). The intra-batch repeatability coefficient of variation was between 0.947% and 3.444%, and the inter-batch repeatability coefficient of variation was between 0.000% and 9.871%, both of which were less than 10%.

[0124] Table 6

[0125]

[0126] Table 7

[0127]

[0128]

[0129] 3.10 Compliance Evaluation

[0130] The serum was tested using the established indirect ELISA antibody detection method and the PED VELISA antibody detection kit of Zhaoqing Dahuanong Biological Products Co., Ltd., and the compliance rate was calculated. The results are shown in Table 8.

[0131] Relative sensitivity (%) = {number of positive results / (number of positive results + number of false negative results)} × 100%;

[0132] Relative specificity (%) = {number of negative results / (number of negative results + number of false positive results)} × 100%;

[0133] Total compliance rate (%) = {(positive number + negative number) / total number of tests} × 100%.

[0134] Table 8

[0135]

Claims

1. A method for constructing a cell line expressing porcine epidemic diarrhea virus S1 protein, characterized in that: The following steps are involved: Step 1: inserting a nucleotide sequence directing the synthesis of PEDV S1 protein into a plasmid to obtain a plasmid carrying the nucleotide sequence of PEDV S1 protein; Step 2: transfecting the plasmid carrying the nucleic acid of PEDV S1 protein into CHO-K1 and culturing the cell line to obtain a cell line expressing porcine epidemic diarrhea virus S1 protein; The nucleotide sequence of the PEDV S1 protein is shown in SEQ ID NO:

1.

2. The method for constructing a cell line expressing porcine epidemic diarrhea virus S1 protein according to claim 1, characterized in that: A KOZAK sequence is added before the start codon of the nucleotide sequence of the PEDV S1 protein; the nucleotide sequence of the KOZAK sequence is shown in SEQ ID NO:

2.

3. The method for constructing a cell line expressing porcine epidemic diarrhea virus S1 protein according to claim 1, characterized in that: The nucleotide sequence of the PEDV S1 protein is also connected to an IgGκ signal peptide sequence. The step 1 specifically comprises: inserting the nucleotide sequence of the PEDV S1 protein into a plasmid to obtain a plasmid carrying the nucleotide sequence of the PEDV S1 protein; then using the plasmid carrying the nucleotide sequence of the PEDV S1 protein as a vector, amplifying using homologous recombination primers homologous arm IgGκ-S1-F and homologous arm IgGκ-S1-R to obtain an amplified product; The amplified product is then homologously recombined with the plasmid to obtain a plasmid carrying the IgGκ signal peptide sequence and the nucleotide sequence of the PEDV S1 protein.

4. A cell line expressing porcine epidemic diarrhea virus S1 protein, characterized in that: The cell line is prepared by the method for constructing a cell line expressing porcine epidemic diarrhea virus S1 protein according to any one of claims 1 to 3.

5. Use of the cell line expressing porcine epidemic diarrhea virus S1 protein according to claim 4 for expressing PEDV S1 protein.

6. An ELISA detection method, characterized in that, The coating antigen in the kit is the PEDV S1 protein expressed by the cell line expressing porcine epidemic diarrhea virus S1 protein according to claim 4.

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