Cell line and method for stably expressing S protein of porcine epidemic diarrhea virus and application

Abstract

The invention discloses a cell line and method for stably expressing porcine epidemic diarrhea virus S protein and application, the cell line is an IPEC-PEDV-S stably transfected cell line, and is a cell line for stably expressing porcine epidemic diarrhea virus S protein constructed by a bioengineering technology. The cell line can be used for research and development of PEDV replication-deficient vaccines and PEDV antibody detection reagents, overcomes the defects of a traditional IFA antibody detection method for PEDV, and is safer, simpler, more convenient and more accurate.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to an IPEC-J2 cell line stably expressing the S protein of porcine epidemic diarrhea virus, a method and an application thereof. Background Art

[0002] A stable transfected cell line refers to integrating an exogenous gene into the genome of a host cell, enabling the exogenous gene to be stably expressed in the host cell for a long time. The principle is to clone the exogenous gene onto a vector with a certain resistance, transfect the host cell, integrate the exogenous gene into the host chromosome, and screen with the resistance gene contained in the vector to obtain cells that have successfully integrated the target gene. During the experiment, commonly used eukaryotic expression vector resistance screening markers include puromycin, neomycin, hygromycin, etc. Through screening, cell lines with stable high expression of the target protein meeting the experimental requirements or cell lines with stable silencing of the target gene can be obtained. Lentivirus can infect almost all types of cells and has the characteristic of infecting both dividing and non-dividing cells. Lentivirus belongs to retrovirus. After the viral genomic RNA enters the cell, it is reverse transcribed into DNA by reverse transcriptase in the cytoplasm. The formed pre-integration complex of DNA enters the nucleus and is integrated into the cell genome. The integrated DNA is transcribed into mRNA to express the target protein or produce small RNAs in the cytoplasm. The gene expression or small RNA interference mediated by lentivirus is continuous and stable and divides with the division of the cell genome. Lentiviral vectors are a type of viral vector modified from human immunodeficiency virus (HIV), with characteristics such as a wide host range and a large gene capacity, and are one of the most commonly used methods for constructing stable transfected cell lines. The obtained stable transfected cell lines can be used for the research and development of the biological functions of genes, new genetic engineering vaccines, and diagnostic reagents.

[0003] Porcine epidemic diarrhea (PED) is an acute and highly contagious porcine intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). Typical symptoms include watery diarrhea, dehydration, vomiting, etc. This disease is characterized by a high infection rate and a high mortality rate. Since PED was first reported in the UK in 1971, the disease has spread worldwide. PEDV can infect pigs of different ages, and it is most harmful to nursing piglets, with an incidence and mortality rate of over 90%. Since 2010, highly virulent mutant strains of PEDV have emerged, with higher infection rates and mortality rates, making it more difficult to prevent and control.

[0004] Porcine epidemic diarrhea virus (PEDV) is a single-stranded positive-sense RNA virus with a diameter of 95 - 190 nm and is a member of the genus Alphacoronavirus (α-CoV). The full-length genome of PEDV is approximately 28 kb and consists of 7 open reading frames (ORFs), namely ORF1a, ORF1b, ORF2 - 6 from the 5'→3' end. Among them, the ORF1a and ORF1b genes encode non-structural proteins, the ORF3 gene encodes the accessory protein ORF3, and ORF2 and ORF4 - 6 encode the spike protein (S), small membrane protein (E), membrane glycoprotein (M), and nucleocapsid protein (N), respectively. The S protein is a glycosylated protein containing epitopes that can induce neutralizing antibodies and has a high degree of genetic diversity. Based on the genetic diversity of the S gene, PEDV is divided into two major genotypes, GⅠ and GⅡ, and each genotype can be further divided into 2 subtypes, namely GⅠa, GⅠb and GⅡa, GⅡb. The GⅠa and GⅠb subtypes mainly include early prevalent low-pathogenicity strains such as CV777 and DR13, and both GⅡa and GⅡb belong to highly pathogenic variant strains, which are currently the dominant prevalent strains in China. The GⅡa and GⅡb subtype strains are highly pathogenic to suckling piglets, and there is serum cross-neutralization activity and cross-immune protection between them. However, there are significant differences in antigenicity and genetic evolutionary relationships between currently widely used PEDV GⅠ genotype vaccine strains such as CV777 and the clinically dominant prevalent strains in China, resulting in reduced protective efficacy against GⅡa and GⅡb subtype prevalent strains and frequent immune failure. Therefore, the research and development of vaccines against currently dominant prevalent strains are crucial for the prevention and control of PED. With the development of molecular tools and genome sequencing technologies, especially the emergence of some advanced technologies, special vectors and strategies, there have been many related studies on the establishment of coronavirus reverse genetics systems. Based on the infectious clone of PEDV clinical prevalent strains, the structural protein genes related to PEDV replication can be deleted, and a replication-defective PEDV vaccine can be developed using stable transfected cell lines that stably express the deleted PEDV-related proteins. This novel genetic engineering vaccine can replicate normally and has a high virus titer in the IPEC-J2 cell line that stably expresses PEDV structural proteins, and can only undergo one-time infection and replication in ordinary IPEC-J2 cell lines, which can solve the problems of poor immune effect of existing commercial PEDV inactivated vaccines and safety problems such as persistent virus carriage, intermittent virus excretion, and virulence reversion of PEDV attenuated vaccines. However, there is currently no IPEC-J2 cell strain that can stably express the PEDV S protein, and it needs to be constructed, screened and identified through experiments.

[0005] The diagnostic methods for PED are divided into two major categories: antigen detection and antibody detection. Antigen detection includes virus isolation, RT-PCR or quantitative real-time RT-PCR, indirect immunofluorescence (IFA), and enzyme-linked immunosorbent assay (ELISA). Antigen detection methods are the best diagnostic methods during PED outbreaks and epidemics. Antibody detection methods can determine whether an animal body has been infected with PEDV and can also provide the immune status of the pig herd. PED antibody detection methods mainly include virus neutralization test, indirect immunofluorescence (IFA), and ELISA antibody detection methods. The PEDV virus neutralization test plays a key role in evaluating vaccine efficacy, diagnosing virus infections, and studying the interaction between viruses and antibodies. However, this method is complex to operate, has a long detection cycle, requires special laboratory conditions and equipment, as well as professional technical personnel. The cost of the neutralization test is relatively high, there are certain biosafety risks, and the sensitivity of this method is relatively low. In particular, the PEDV virus neutralization test can only be carried out smoothly in Vero cells cultured in a serum-free medium containing a certain concentration of trypsin. The protein components in clinical milk and serum samples interfere with the activity of trypsin, and the presence of trypsin also affects the determination of cytopathic effects, making the operation of the virus neutralization test more complex and the results unstable, making it difficult to be widely promoted and applied on a large scale. Due to the advantages of simple and rapid operation, good sensitivity, repeatability, and stability, the ELISA antibody detection method has become a commonly used method for current PED epidemiological investigations and vaccine immunization effect evaluations. Currently, there are already many commercial ELISA antibody detection kits for measuring the IgA antibody level in sow milk and the IgG antibody level in serum on the market. However, improper handling of clinical samples to be tested, such as serum hemolysis, the presence of rheumatoid factors, complement, heterophilic antibodies, or bacterial contamination in the serum, will have a great impact on the accuracy of ELISA test results, resulting in false negatives or false positives. In addition, the coated antigens of ELISA antibody detection kits from different manufacturers are different, and there are also differences in the sensitivity of the kits, resulting in significant differences in the coincidence rates of test results from different kits. Compared with the virus neutralization test, the indirect immunofluorescence antibody detection method is simple to operate, has good specificity and high sensitivity, and is widely used in clinical diagnosis. It is often used as the gold standard for evaluating the accuracy of the ELISA antibody detection method and is also commonly used to verify samples with objections to the ELISA method. However, the traditional IFA method requires the preparation of antigen plates by infecting Vero cells or IPEC-J2 cells with PEDV virus, which poses a biosafety risk. Therefore, there is an urgent need to improve the traditional IFA method based on antigen plates of Vero or PEDV-J2 cells infected with PEDV. Summary of the Invention

[0006] The present invention solves the technical problems of poor immune effect of existing commercial inactivated PEDV vaccines, continuous virus carriage, intermittent virus excretion, and virus virulence reversion of PEDV attenuated vaccines, and overcomes the deficiencies of existing PEDV antibody detection methods, such as false positives, complex operations, and potential biosafety risks during the detection process. By constructing and screening IPEC-J2 cell lines stably expressing the PEDV S protein (IPEC-PEDV-S stable transfection cell lines), these cell lines can be used for the research and development of PEDV replication-defective vaccines and PEDV antibody detection reagents, solving the deficiencies of traditional PEDV IFA antibody detection methods and making them safer, simpler, and more accurate.

[0007] To solve the above technical problems, the present invention is achieved through the following technical solutions:

[0008] The present invention provides an IPEC-J2 cell line stably expressing the porcine epidemic diarrhea virus S protein, and the cell line is an IPEC-PEDV-S stable transfection cell line.

[0009] The present invention also provides a method for preparing the above cell line, which includes the following steps:

[0010] (1) Extract nucleic acids from the PEDV SD06 strain of genotype GⅡa, perform RT-PCR amplification and gene sequencing on the full sequence of the PEDV S gene, splice the sequencing results to obtain the complete sequence of the PEDV S gene, and optimize the codons of the PEDV S gene in order to improve the translation efficiency and protein expression level of the PEDV S protein in the stable transfection cell line.

[0011] (2) Clone the S protein DNA sequence of the codon-optimized PEDV SD06 strain into the lentiviral vector pLV-EF1-ZsGreen1-T2A-PuroR with puromycin resistance. This expression vector is co-transfected with the lentiviral packaging plasmid psPAX2 and the VSV-G envelope expression plasmid pMD2G into 293T cells. The harvested packaged lentivirus is used to infect IPEC-J2 cells, and the stable transfection cell line is passaged under the action of puromycin at an appropriate concentration to screen and obtain the IPEC-PEDV-S stable transfection cell line.

[0012] Preferably, the IPEC-PEDV-S stable transfection cell line that stably expresses the PEDV S protein is identified and evaluated for genetic stability through indirect immunofluorescence assay, Western-blot assay, and PCR.

[0013] Preferably, the concentration of puromycin is 4 μg / ml - 16 μg / ml.

[0014] Preferably, the nucleotide sequence of the PEDV S gene is as shown in Sequence 1 in the sequence listing, and the amino acid sequence of the PEDV S protein is as shown in Sequence 2 in the sequence listing.

[0015] In another aspect of the present invention, a recombinant lentiviral expression vector expressing the PEDV S protein is also provided, and the vector contains the gene sequence of the PEDV S structural protein.

[0016] In another aspect of the present invention, the above-mentioned IPEC-PEDV-S stable cell line is also provided for use in diagnostic test reagents.

[0017] In another aspect of the present invention, the above-mentioned IPEC-PEDV-S stable cell line is also provided for use in the preparation of vaccines for preventing or treating porcine epidemic diarrhea.

[0018] Preferably, the vaccine includes a replication-defective vaccine against porcine epidemic diarrhea virus.

[0019] Beneficial effects: The IPEC-PEDV-S stable cell line stably expressing the PEDV S protein of the present invention can be used for the research and screening of replication-defective PEDV vaccines. The replication-defective PEDV vaccines can replicate normally on these overexpressing cell lines and have a high virus titer, while they can only be infected and replicated once on ordinary IPEC-J2 cell lines. The preparation of the cell line does not require a negative pressure condition, and trypsin does not need to be added during the cell culture process.

[0020] Biological deposit description

[0021] Classification and naming: Porcine intestinal epithelial cell IPEC-PEDV-S expressing the S protein of porcine epidemic diarrhea virus was deposited at the China Center for Type Culture Collection on October 15, 2024, with the deposit number CCTCC NO: C2024344. The address of the deposit unit is Wuhan University, Wuhan, China. Description of the drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0023] Figure 1 It is the construction strategy diagram of the pLV-PEDV-S recombinant plasmid in Example 1 of the present invention;

[0024] Figure 2 It is the RT-PCR identification result diagram of the pLV-PEDV-S recombinant lentivirus in Example 2 of the present invention;

[0025] Figure 3It is the result diagram of observing the proportion of green fluorescent cells under a fluorescence microscope at different screening passages during the process of screening and obtaining the IPEC-PEDV-S stable cell line by adding drugs in Example 3;

[0026] Figure 4 It is the IFA identification diagram of the IPEC-PEDV-S stable cell line expressing the exogenous PEDV S protein in Example 3;

[0027] Figure 5 It is the Western-blot identification diagram of the IPEC-PEDV-S stable cell line expressing the exogenous PEDV S protein in Example 3;

[0028] Figure 6 It is the PCR identification diagram of the IPEC-PEDV-S stable cell in Example 3;

[0029] Figure 7 It is the detection result of the cell viability of the IPEC-PEDV-S stable cell line at different passages in Example 3;

[0030] Figure 8 It is the IFA detection diagram of the IPEC-PEDV-S stable cell line expressing the exogenous PEDV S protein at different passages in Example 4;

[0031] Figure 9 It is the PCR detection result of the IPEC-PEDV-S stable cell line at different passages in Example 4. Detailed implementation manners

[0032] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0033] The chemical reagents used in the embodiments of the present invention are all of analytical grade.

[0034] To make the present invention easier to understand, the present invention will be further elaborated below in conjunction with specific embodiments. The experimental methods described in the present invention are all conventional methods unless otherwise specified; the biological materials described are all commercially available unless otherwise specified.

[0035] Example 1 Construction of the recombinant lentiviral expression vector of PEDV-S gene

[0036] 1.1 Experimental materials and main reagents

[0037] 1.1.1 Cells, plasmids and strains

[0038] Cells and plasmids: The lentiviral expression vector pLV-EF1-ZsGreen1-T2A-PuroR, the lentiviral packaging plasmid psPAX2, and the VSV-G envelope expression plasmid pMD2G, 293T cells, and IPEC-J2 cells are all stored in the Henan Engineering Research Center for Animal Diseases and Public Health of Luoyang Vocational and Technical College. The competent E. coli DH5α cells are purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0039] Virus strain: The PEDV SD06 strain of genotype GⅡa was isolated from diseased pigs on a farm in Shandong Province in 2020 and was isolated, identified, and stored by Luoyang Zhongke Gene Detection and Diagnosis Center Co., Ltd.

[0040] 1.1.2 Main reagents

[0041] The Viral DNA / RNA Kit nucleic acid extraction kit is purchased from TransGen Biotech Co., Ltd.; the FastPure Endofree Plasmid Maxi Kit endotoxin-free plasmid extraction kit is purchased from Nanjing Novozymes Biotech Co., Ltd.; the MightyScript Plus First Strand cDNA Synthesis Master Mix (genomic DNA removed) reverse transcription kit, 2×FidCycle Fast high-fidelity PCR Mix (containing blue dye), column DNA gel recovery kit, T4 DNA ligase, Not I and EcoR I restriction endonucleases are all purchased from Sangon Biotech (Shanghai) Co., Ltd. OPTI-MEM, fetal bovine serum FBS, and DMEM (high glucose) medium are purchased from Gibco; Lipofectamine TM 3000 transfection reagent is purchased from Invitrogen; the cell proliferation and cytotoxicity detection kit (CCK-8, enhanced) is purchased from Dalian Meilun Biotechnology Co., Ltd. Other chemical reagents are all of analytical grade.

[0042] 1.2 Sequencing and codon optimization of the PEDV virus S protein gene

[0043] Add 20 μl of Proteinase K and 200 μl of BB5 into a sterile 1.5 ml centrifuge tube. After vortexing for 15 seconds, add 200 μl of the sample; vortex for 15 seconds and incubate in a water bath at 56 °C for 15 minutes. Add 250 μl of absolute ethanol (flocculent precipitate may appear at this time), vortex for 15 seconds, and let stand at room temperature for 5 minutes. Add the solution and precipitate together into the centrifugal column, centrifuge at 12,000 g for 1 minute, discard the effluent, add 500 μl of WB5, centrifuge at 12,000 g for 1 minute, discard the effluent, and repeat the rinsing once with WB5 using the same method; centrifuge at 12,000 g at room temperature for 1 minute to completely remove the residual ethanol; transfer the centrifugal column into a new 1.5 ml RNase-free centrifuge tube, add 50 μl of RNase-free Water to the center of the centrifugal column, let stand at room temperature for 1 minute, and centrifuge at 12,000 g at room temperature for 1 minute to elute DNA / RNA. Store the DNA / RNA at -70 °C.

[0044] Perform reverse transcription according to the MightyScript Plus First Strand cDNA Synthesis Master Mix (Genomic DNA Remover) reverse transcription kit instruction manual. In this experiment, it is not necessary to remove genomic DNA. The cDNA synthesis system is as follows: 15 μl of DNA / RNA nucleic acid template and 5 μl of 4×Ⅲ M-MLV RT Mix, with a total reaction system of 20 μl. Reaction conditions: 25 °C for 5 minutes, 55 °C for 15 minutes, 85 °C for 5 minutes (enzyme inactivation).

[0045] According to the conserved region sequences at the 5' and 3' ends of the PEDV NSP16, S, and ORF3 genes, oligonucleotide primers were synthesized. The synthesized PEDV cDNA was subjected to segmented PCR amplification of the PEDV NSP16-S-ORF3 gene according to the instructions of the 2×FidCycle Fast High-Fidelity PCR Mix (containing blue dye) kit. PCR amplification system and reaction conditions: 25 μl of 2×FidCycle Fast High-Fidelity PCR Mix (containing blue dye), 2 μl each of PEDV-NSP16-F1 / PEDV S-F2 / PEDV S-F3 / PEDV S-F4 (10 μM), PEDV S-R1 / PEDV S-R2 / PEDV S-R3 / PEDV ORF3-R4 (10 μM), 5 μl of cDNA, and 16 μl of ddH2O, for a total reaction system of 50 μl. Reaction conditions: pre-denaturation at 98°C for 30 Sec, denaturation at 98°C for 10 Sec, annealing at 60°C for 10 Sec, extension at 72°C for 2 min (denaturation, annealing, and extension for a total of 35 cycles), final extension at 72°C for 10 min, and the reaction ended at 25°C for 5 min. After detection of the PCR products by 1% agarose gel electrophoresis, the target gene fragments were excised for gel recovery, and the concentration after gel recovery was measured. The primer sequences are shown in Table 1.

[0046] Table 1 Primers for Amplifying the Full Gene of PEDV S

[0047]

[0048] The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were spliced and analyzed to obtain the complete full gene sequence of the PEDV S protein (4161 bp). After deleting the stop codon of the PEDV S gene, it was codon-optimized. The optimized PEDV S gene sequence is shown as Sequence 1 in the sequence list, and the amino acid sequence of the PEDV S protein is shown as Sequence 2 in the sequence list.

[0049] 1.3 Construction of the Recombinant Lentiviral Expression Vector of the PEDV-S Gene

[0050] 1.3.1 Synthesis of the Codon-Optimized PEDV S Gene

[0051] Not I and EcoR I restriction enzyme cleavage sites were introduced at the 5' and 3' ends of the optimized PEDV S gene sequence, respectively, and sent to Sangon Biotech (Shanghai) Co., Ltd. for full sequence synthesis and ligated onto the pET-28a plasmid. The pET28a recombinant plasmid containing the PEDV-S gene was transformed into E. coli DH5a competent cells. Single colonies were picked and cultured overnight in LB medium containing 100 μg / ml kanamycin. After plasmid extraction according to the kit instructions, sequencing analysis was performed. The correctly sequenced recombinant plasmid was named pET28a-PEDV-S.

[0052] 1.3.2 Construction of the recombinant lentiviral expression vector of PEDV-S gene

[0053] The pET28a-PEDV-S recombinant plasmid was double digested with NotI and EcoRI restriction enzymes. The double digestion system and conditions were as follows: 3 μg of plasmid, 3 μl each of NotI and EcoRI restriction enzymes, 3 μl of 10×Rapidcut Buffer, supplemented with ddH2O to 30 μl, and incubated in a water bath at 37°C for 1 h. The double digested products were electrophoresed. After gel recovery of the PEDV S gene according to the column type DNA gel recovery kit instructions, it was ligated with the pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro lentiviral expression vector that was also double digested with NotI and EcoRI restriction enzymes according to the T4 DNA ligase kit instructions. The recombinant plasmid construction strategy is shown in Figure 1 . The ligation system and reaction conditions were as follows: 5 μl (200 ng) of pLV vector, 10 μl (400 ng) of PEDV-S fragment, 1 μl of T4 DNA Ligase, 2 μl of 10×T4 DNA Ligase Buffer, 2 μl of ddH2O, for a total ligation system of 20 μl; ligated overnight at 4°C, and then heat inactivated at 65°C for 10 min.

[0054] 10 μl of the ligation product was taken to transform E. coli DH5a competent cells. After recovery, it was spread on an agarose plate containing 100 μg / ml ampicillin and cultured for 10 - 12 h. Single colonies were picked and cultured overnight (8 - 12 h) in LB medium containing 100 μg / ml ampicillin. After plasmid extraction, double digestion identification was performed using Not I and EcoR I restriction enzymes. The correctly enzyme digested recombinant plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for KBSeq sequencing. The correctly sequenced recombinant plasmid was named pLV-PEDV-S.

[0055] Example 2 Packaging and identification of recombinant lentivirus

[0056] 2.1 Packaging of recombinant lentivirus

[0057] Resuscitate 293T cells and culture and passage them in DMEM medium with 10% FBS. When the cells grow to a monolayer, digest them with 0.25% (containing EDTA) trypsin solution, centrifuge at 1000rpm for 10 minutes at room temperature, resuspend the cells and count the cells, and plate 6-well cell culture plates at 350,000 / well. 12-24 hours after 293T cells were seeded on 6-well plates, when the cell density reached 80%-90%, replace them with serum-free DMEM medium preheated in a 37°C water bath, 1ml / well, and start transfection. Follow the Lipofectamine TM Prepare the transfection system according to the instructions of 3000 transfection reagent for transfection. The specific method is as follows:

[0058] (1) Preparation system A: OPTI-MEM 125 μl / well + LP3000 6 μl / well.

[0059] (2) Preparation system B: OPTI-MEM 125 μl / well + pLV-PEDV-S recombinant plasmid 5 μg + psPAX2 plasmid 3 μg + pMD2G plasmid 1.5 μg + P3000 19 μl / well.

[0060] (3) Take the same volume of system A as that of system B, mix them evenly, and incubate them at room temperature for 10-15 minutes.

[0061] (4) Slowly add the incubated AB mixture dropwise into the 6-well plate (pay attention to add slowly and evenly to all parts of the well, and gently shake the plate while adding). Continue culturing for 4-6 hours after inoculation, and replace with 2% FBS DMEM maintenance medium.

[0062] The pLV-PEDV-S recombinant plasmid contains the ZsGreen1 green fluorescent tag. After transfection, the percentage of green fluorescent cells can be observed under a fluorescence microscope to assess transfection status. The percentage of green fluorescent cells should be observed 6, 12, 24, 48, and 72 hours after transfection. If the percentage of green-positive cells no longer increases 48-72 hours after transfection, collect the cell culture supernatant 72 hours after transfection, centrifuge at 1000 rpm for 10 minutes at room temperature to remove cell debris, and store in aliquots at -80°C to avoid repeated freeze-thaw cycles.

[0063] 2.2 Identification of recombinant lentivirus

[0064] Extract the harvested recombinant lentiviral RNA according to the method in Example 1. After reverse transcription using a cDNA synthesis kit, use the codon-optimized identification primers for the PEDV S gene, and identify the foreign gene fragment in the recombinant lentivirus according to the instructions of the 2×FidCycle Fast High-Fidelity PCR Mix (containing blue dye) kit. The PCR amplification system and reaction conditions refer to 1.2 of Example 1, and the sequences of the recombinant lentivirus identification primers are shown in Table 2. Detect the PCR products by 1% agarose gel electrophoresis. The sizes of each target gene fragment are as expected, and the pLV-PEDV-S recombinant lentivirus is successfully packaged. The results are as Figure 2 .

[0065] Table 2 Recombinant lentivirus identification primers

[0066]

[0067] Establishment of an IPEC-J2 cell line stably expressing PEDV-S in Example 3

[0068] 3.1 Determination of the optimal concentration of puromycin for IPEC-J2 cells

[0069] Seed IPEC-J2 cells at 150,000 - 200,000 cells per well in a 6-well plate. The next day, when the cell density reaches 50 - 60%, wash the cells 3 times with PBS and replace with 10% FBS DMEM medium containing different final concentrations of puromycin, 2 ml per well. Observe the cell survival of different puromycin experimental groups daily. Set the final concentrations of puromycin to 1 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml respectively, and at the same time set up a control group of cells without puromycin, with 3 replicates for each concentration. Observe the cell survival under a microscope after 48 h. 100% of the cells in the control group without puromycin survived, about 20% of the cells in the 1 μg / ml and 2 μg / ml puromycin experimental groups died, and 100% of the cells in the 4 μg / ml, 8 μg / ml, and 16 μg / ml puromycin experimental groups died. The optimal condition for screening the IPEC-J2 cell line stably expressing PEDV-S is a final concentration of 4 μg / ml puromycin.

[0070] 3.2 Infection with recombinant lentivirus and drug screening of stably transfected cell lines

[0071] Seed IPEC-J2 cells at a density of 300,000 - 350,000 cells per well in a 6-well plate. The next day, when the cell density reaches 80 - 90%, wash the cells 3 times with PBS and replace the medium with 2% FBS DMEM medium at 1 ml per well. Inoculate the recombinant lentivirus harvested in 2.1 of Example 2 at 1 ml per well. Discard the inoculum 24 h after inoculation. Repeat the inoculation of 1 ml per well of the recombinant lentivirus for each well of cells in the same manner and continue culturing for 48 h. Observe the proportion of green fluorescent cells expressing ZsGreen1 protein under a fluorescence microscope 60 h after the IPEC-J2 cells are infected with the recombinant lentivirus (counting from the first inoculation time), and the proportion is approximately 20%( Figure 3 ). During the period of 60 - 72 h after the recombinant lentivirus infection, the proportion of green fluorescent cells does not increase with the extension of the infection time. Transfer the cells from the 6-well plate (considered as the P0 generation) to a T25 cell culture flask using 10% FBS DMEM medium containing 4 μg / ml puromycin for subculture and drug screening. The subculture cycle for each generation of cells is 96 h. Continuously add the drug for subculture and screening until the proportion of green fluorescent cells expressing ZsGreen1 protein reaches 100%. When the IPEC-J2 cells are subcultured to the 3rd generation (P3 generation) using 10% FBS DMEM medium containing 4 μg / ml puromycin, the proportion of green fluorescent cells reaches 100%( Figure 3 ), and the IPEC-PEDV-S stable cell line is obtained.

[0072] 3.3 Identification of the stable cell line

[0073] 3.3.1 Identification by indirect immunofluorescence assay

[0074] Take IPEC-J2 and the 3rd generation IPEC-PEDV-S cell lines and seed the cells at a density of 300,000 - 350,000 cells per well in a 6-well plate respectively. The next day, when the cell density reaches 80 - 90%, wash the cells in the 6-well plate 3 times with PBS, add 2 ml per well of pre-cooled (-20 °C) acetone-methanol solution (acetone: methanol = 1:1) as the fixing liquid. After fixing for 10 min, discard the fixing liquid, wash 3 times with PBS, and store at 4 °C in the refrigerator for indirect immunofluorescence assay identification.

[0075] For indirect immunofluorescence assay, the PEDV-S monoclonal murine antibody was diluted with PBS at a ratio of 1:200. The diluted PEDV S monoclonal antibody was added to the surfaces of IPEC-J2 and IPEC-PEDV-S cells fixed in a 6-well plate, 500 μl per well. After incubation at room temperature for 1 h, the cells were washed three times with PBS to remove unbound primary antibody. CoraLite594-labeled goat anti-mouse IgG (H+L) secondary antibody diluted with PBS at a ratio of 1:200 was added, 500 μl per well. After incubation at room temperature for 1 h, the cells were washed three times with PBS to remove unbound secondary antibody; DAPI staining solution was added to stain the cell nuclei for 10 min. After staining, the cells were washed three times with PBS. Observation under a fluorescence microscope showed that the IPEC-PEDV-S stable cell line expressed 100% of the PEDV S protein, while the IPEC-J2 cell line did not express the PEDV S protein, as Figure 4 .

[0076] 3.3.2 Identification by Western-blot assay

[0077] IPEC-J2 and the 3rd generation IPEC-PEDV-S cell lines were seeded in a 6-well plate at 300,000 - 350,000 cells per well. When the cell density reached 90 - 100%, the cell culture supernatant was discarded. After washing the cells three times with PBS, RIPA cell lysis buffer with medium lysis intensity at 200 μL per well was added, and the cells were lysed on ice for 10 min. The cell lysate samples were scraped with a pipette tip and transferred to 1.5 mL EP tubes, then stored in a -80 °C refrigerator.

[0078] In the present invention, the C-terminus of the PEDV S protein in the IPEC-PEDV-S cell line was fused with a 3×Flag tag, and the predicted molecular weight of the fusion protein was 155.32 kDa. Therefore, the concentration of the separating gel of the SDS-PAGE gel was 12%, and the concentration of the stacking gel was 5%. The cell lysate samples stored in the -80 °C refrigerator were taken out, melted at room temperature, and then 50 μL / 200 μL of 5×SDS Loading Buffer was added. After boiling at 95 °C for 10 min, they were placed on ice for 2 min. 10 μL of the treated sample per well was loaded for electrophoresis. During SDS-PAGE electrophoresis, the voltage was first set at 80 V for 20 min, and then increased to 120 V for continued electrophoresis. After electrophoresis, the stacking gel was peeled off, and the separating gel and pre-prepared filter paper were immersed in the transfer buffer for 3 - 5 min. At the same time, the PVDF membrane was activated in methanol for 10 s and then equilibrated in the transfer buffer for 15 min.

[0079] On the transfer membrane apparatus, filter paper, PVDF membrane, gel, and filter paper were stacked in sequence from bottom to top. During the placement process, try to expel the air bubbles between each layer. At a constant voltage of 25 V and a current limit of 0.3 A, after transferring the membrane for 15 min, the membrane was blocked with 5% skim milk powder at room temperature for 2 h. After blocking, it was washed three times with TBS solution for 5 min each time. Then, the diluted PEDV S monoclonal antibody at 1:200 was added and incubated at room temperature for 1 h or overnight at 4 °C. The membrane was washed three times with TBST solution for 15 min each time. Then, the diluted HRP-labeled goat anti-mouse IgG (H+L) secondary antibody at 1:500 was added and incubated at room temperature for 1 h. After incubation, the membrane was washed three times with TBST solution for 15 min each time. Then, an appropriate amount of HRP luminescent substrate A and B solutions were mixed in equal volume and added to the surface of the membrane, and the luminescence signal was detected using a chemiluminescence detector. A specific PEDV S protein band with the expected molecular weight was detected in the IPEC-PEDV-S cell sample, and no target protein band was detected in the IPEC-J2 cell sample, as Figure 5 shown.

[0080] 3.3.3 PCR experiment identification

[0081] Take IPEC-J2 and the 3rd generation IPEC-PEDV-S cell lines, and seed the cells at 300,000 - 350,000 cells per well in a 6-well plate. When the cell density reaches 90 - 100%, discard the cell culture supernatant. After washing the cells three times with PBS, digest the cells using 0.25% trypsin cell digestion solution. When the cell gaps become wider and the cell morphology becomes round, terminate the digestion. Centrifuge the digested cells at 3000 rpm / min at room temperature for 10 min, discard the centrifuged supernatant, add 200 μL of PBS to resuspend the cells, and freeze-thaw the cells three times in a -80 °C refrigerator. Then, extract the cell DNA according to the method in 1.2 of Example 1. After extraction, use the 2×FidCycle Fast High-Fidelity PCR Mix (containing blue dye) kit to detect according to the method in 2.2 of Example 2. Use 1% agarose gel electrophoresis to detect the PCR products. The PEDV-S gene was detected in the IPEC-PEDV-S cell line, and no target gene was detected in the IPEC-J2 cell sample, as Figure 6 shown.

[0082] 3.3.4 Cell viability detection

[0083] IPEC-J2 cells in logarithmic growth phase and IPEC-PEDV-S stable transfected cell lines of P5, P10, P15, and P20 generations were made into single cell suspensions using 10% FBS DMEM complete medium. After cell counting for each generation, they were inoculated into 96-well cell culture plates at the cell numbers of 0, 2000, and 5000 cells / well, 100 μl / well, and each condition was set with 5 replicates. The 96-well plates were placed in a cell culture incubator at 37 °C and 5% CO2 for 48 h. The old medium was discarded, and the cells were washed 3 times with PBS. Fresh 10% FBS DMEM complete medium was added at 100 μl / well, and CCK-8 solution was added at a volume of 10 μL / well. Incubation was continued for 2 h, and the absorbance of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The well of the medium containing CCK-8 without cells was used as the blank control for detecting the background, and the cell proliferation rate was calculated. The cell proliferation rate % = (OD450 of IPEC-PEDV-S cell well - OD450 of blank control well) / (OD450 of IPEC-J2 cell well - OD450 of blank control well) × 100%. The results showed that there was no significant difference in the cell viability between the IPEC-PEDV-S stable transfected cell lines of P5, P10, P15, and P20 generations and the IPEC-J2 cell line in the present invention ( Figure 7 ), and the cell viabilities were comparable.

[0084] Example 4 Genetic Stability of IPEC-J2 Cell Line Stably Expressing PEDV-S

[0085] 4.1 Passage of Stable Transfected Cell Lines

[0086] The IPEC-J2 cell line stably expressing PEDV-S screened in 3.2 of Example 3 was regarded as the P0 generation, and it was continuously passaged to the P20 generation in a T25 cell culture flask using 10% FBS DMEM medium without puromycin. The passage cycle of each generation of cells was 72 h. Cells of P0, P5, P10, P15, and P20 generations were taken for detecting the genetic stability of the stable transfected cell lines.

[0087] 4.2 Genetic Stability of Stable Transfected Cell Lines

[0088] 4.2.1 Detection by Indirect Immunofluorescence Assay

[0089] According to the method in 3.3.1 of Example 3, indirect immunofluorescence assay was performed on IPEC-PEDV-S stable transfected cell lines of P0, P5, P10, P15, and P20 generations using PEDV S monoclonal antibody. The results showed that 100% of the IPEC-PEDV-S stable transfected cell lines of P5, P10, P15, and P20 generations expressed PEDV S protein, and there was no difference from the P0 generation cell line, indicating that the IPEC-PEDV-S stable transfected cell line had good genetic stability, as Figure 8 .

[0090] 4.2.2 PCR Experimental Detection

[0091] Select the primer pair pLV-F3 and pLV-R3 in Table 2 of Example 1. According to the method in 3.3.3 of Example 3, using the recombinant plasmid pLV-PEDV-S constructed in Example 1 as a positive control and the cellular DNA extracted from IPEC-J2 cells as a negative control, perform PCR experimental detection on the IPEC-PEDV-S stable cell lines of generations P0, P5, P10, P15, and P20. The results show that PEDV-S gene fragments with the same expected molecular weight size were detected in the samples of the IPEC-PEDV-S stable cell lines of generations P5, P10, P15, and P20, and the thickness of the target gene bands in each generation was the same, showing no difference from the P0 generation cell line, indicating that the IPEC-PEDV-S stable cell line has good genetic stability, as Figure 9 .

[0092] Example 5 Detection of PEDV IgG Antibodies Based on IPEC-J2 Cell Lines Stably Expressing PEDV-S

[0093] 5.1 Preparation of Cell Antigen Plates for Indirect Immunofluorescence Detection

[0094] Based on the IPEC-PEDV-S cell line, for the preparation method of the IFA antigen plate, seed IPEC-PEDV-S cells at a density of 100,000 - 120,000 cells per well in a 24-well plate. The next day, when the cell density reaches 80 - 90%, wash the cells in the 24-well plate 3 times with PBS, add 0.5 mL per well of pre-cooled (-20°C) acetone-methanol solution (acetone:methanol = 1:1) as the fixing liquid, fix for 10 min, then discard the fixing liquid, wash 3 times with PBS, and store at 4°C in the refrigerator for use in indirect immunofluorescence experiment identification.

[0095] For the traditional IFA antigen plate preparation method, seed Vero cells at a density of 100,000 - 120,000 cells per well in a 24-well plate. The next day, when the cell density reaches over 90%, wash the cells in the 24-well plate 3 times with PBS. According to the measured virus titer, dilute the PEDV SD06 strain of genotype GⅡa with serum-free DMEM containing 2 μg / ml trypsin to 200 TCID50 / 200 μl, and inoculate the Vero cells in the 24-well plate at 200 TCID50 / 200 μl per well. After cell inoculation, culture for 48 h, wash the cells in the 24-well plate 3 times with PBS, add 0.5 mL per well of pre-cooled (-20°C) acetone-methanol solution (acetone:methanol = 1:1) as the fixing liquid, fix for 10 min, then discard the fixing liquid, wash 3 times with PBS, and store at 4°C in the refrigerator for use in indirect immunofluorescence experiment identification, serving as the control antigen plate for detecting clinical PEDV serum with IPEC-PEDV-S cells.

[0096] 5.2 Indirect Immunofluorescence Assay for Detecting PEDV in Clinical Porcine Serum

[0097] Collect 215 clinical porcine sera with different immune backgrounds from different pig farms in Henan and Shandong provinces, and perform indirect immunofluorescence assays using the IPEC-PEDV-S cell line antigen plate prepared in 5.1 of Example 5 and the Vero cell antigen plate infected with PEDV respectively. Compare the detection results of the two methods and calculate the coincidence rate between them. The calculation method of the coincidence rate of the detection results of the two methods is as follows:

[0098] (1) Positive coincidence rate: It refers to the proportion of samples detected as positive by the first method and also detected as positive by the second method. Positive coincidence rate = a / (a + c)×100%, where a is the number of samples that are positive by both methods, and c is the number of samples detected as positive by the first method but negative by the second method.

[0099] (2) Negative coincidence rate: It refers to the proportion of samples detected as negative by the first method and also detected as negative by the second method. Negative coincidence rate = d / (b + d)×100%. Where d is the number of samples that are negative by both methods, and b is the number of samples detected as negative by the first method but positive by the second method.

[0100] (3) Total coincidence rate: It refers to the proportion of the number of samples with the same results of the two detection methods in the total number of samples. Total coincidence rate = (a + d) / n×100%. Where a is the number of samples that are positive by both methods, d is the number of samples that are negative by both methods, and n is the total number of samples.

[0101] For indirect immunofluorescence assay, the collected porcine serum was diluted 1:50 with PBS at pH 7.4, and 200 μl / well was added to two cell antigen plates, followed by incubation at room temperature for 1 h or overnight at 4 °C in a refrigerator. Then, it was washed three times with PBS at pH 7.4 to remove unbound porcine serum. Next, an enzyme-labeled secondary antibody (Texas Red-labeled rabbit anti-pig IgG antibody) diluted at 1:200 was added, 200 μl / well, and incubated at room temperature in the dark for 1 h. After that, it was washed three times with PBS at pH 7.4 to remove unbound secondary antibody. Finally, the cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) at a concentration of 10 μg / ml. After staining, the cells were washed three times again using the above method, and red positive cells were observed under a fluorescence microscope. The results of IFA detection of clinical porcine serum showed that: based on the IPEC-PEDV-S cell line, 139 PEDV-positive sera were detected by IFA, and 141 PEDV-positive sera were detected by traditional IFA; based on the IPEC-PEDV-S cell line, 76 PEDV-negative sera were detected by IFA, and 74 PEDV-negative sera were detected by traditional IFA. The coincidence rate of PEDV-positive sera detected by IFA based on the IPEC-PEDV-S cell line and traditional IFA was 98.6%, the coincidence rate of negative sera was 100%, and the total coincidence rate was 99.1%. The results are shown in Table 3. It indicates that the stably transfected IPEC-PEDV-S cell line constructed and screened in the present invention can be used for IFA detection of clinical samples.

[0102] Table 3 Comparison of Results of Detecting PEDV Antibodies in Clinical Porcine Serum by Different IFA Methods

[0103]

[0104] Sequence 1: The nucleotide sequence of the codon-optimized porcine epidemic diarrhea virus S protein gene is as follows: 4161 bp

[0105]

[0106] Sequence 2: The amino acid sequence of the S protein of porcine epidemic diarrhea virus is as follows: 151.7 kDa

[0107]

[0108] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art, without departing from the technical solution of the present invention, can make some changes or modifications to equivalent embodiments by using the above-disclosed technical content. However, as long as it does not depart from the technical content of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A cell line stably expressing the S protein of porcine epidemic diarrhea virus, characterized in that, The cell line is the IPEC-PEDV-S stable transfection cell line, and the deposit number is CCTCC NO: C2024344.

2. A method for preparing a cell line stably expressing the S protein of porcine epidemic diarrhea virus, comprising the following steps: (1) Extract nucleic acid from the PEDV SD06 strain of genotype GⅡa, perform RT-PCR amplification and gene sequencing on the full sequence of the PEDV S gene, splice the sequencing results to obtain the complete sequence of the PEDV S gene. In order to improve the translation efficiency and protein expression level of the PEDV S protein in the stable transfection cell line, codon optimization is performed on the PEDV S gene; (2) Clone the S protein DNA sequence of the PEDV SD06 strain after codon optimization into the lentiviral vector pLV-EF1-ZsGreen1-T2A-PuroR with puromycin resistance. This expression vector is co-transfected with the lentiviral packaging plasmid psPAX2 and the VSV-G envelope expression plasmid pMD2G into 293T cells. The harvested packaged lentivirus is used to infect IPEC-J2 cells, and the stable transfection cell line is passaged under the action of puromycin at an appropriate concentration to screen out the IPEC-PEDV-S stable transfection cell line.

3. The preparation method according to claim 2, characterized in that, The IPEC-PEDV-S stable transfection cell line that stably expresses the PEDV S protein obtained by screening is identified and evaluated for genetic stability by indirect immunofluorescence assay, Western-blot assay, and PCR.

4. The preparation method according to claim 2, characterized in that, The concentration of the puromycin is 4 μg / ml - 16 μg / ml.

5. The preparation method according to claim 2, wherein The nucleotide sequence of the PEDV S gene is as shown in Sequence 1 in the sequence listing, and the amino acid sequence of the PEDV S protein is as shown in Sequence 2 in the sequence listing.

6. A recombinant lentiviral expression vector expressing the PEDV S protein, characterized in that The vector contains the gene sequence of the PEDV S structural protein.

7. Use of the cell line stably expressing the S protein of porcine epidemic diarrhea virus according to claim 1 in diagnostic detection reagents.

8. Use of the cell line stably expressing the S protein of porcine epidemic diarrhea virus according to claim 1 in the preparation of a vaccine for preventing or treating porcine epidemic diarrhea.

9. The application according to claim 8, characterized in that The vaccine includes a replication-defective vaccine of porcine epidemic diarrhea virus.

10. A method for the production of the S protein of porcine epidemic diarrhea virus, characterized in that, By culturing the cell line according to claim 1, obtaining cell supernatant, and then purifying the cell supernatant to obtain the S protein of porcine epidemic diarrhea virus.

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