CN113174407A - Screening method for stably expressing CSFV complete structural protein CHO-K1 cell line - Google Patents

Abstract

The invention discloses a screening method of a cell line for stably expressing a classical swine fever virus complete structural protein CHO-K1, which mainly comprises the steps of amplifying a complete structural protein (ShmEs) gene of the classical swine fever virus (Shimen strain) by a PCR method, inserting the complete structural protein (ShmEs) gene into a eukaryotic expression vector pcDNA3.4, and constructing a recombinant expression plasmid pcDNA3.4-ShmEs. The recombinant plasmid was transformed into CHO-K1 cells by transient transfection and screened using the antibiotic G418. The test result shows that the ShmEs gene is expressed in CHO-K1 cell and can be stably expressed by passage, and the antigen immune rabbit body produced by the stable cell line can stimulate the rabbit body to produce the swine fever specific antibody and can be maintained for at least 42 days. The successful expression of the ShmEs gene and the establishment of a stable cell line thereof lay a foundation for the development of novel gene recombinant vaccines of the swine fever.

Description

Screening method for stably expressing CSFV complete structural protein CHO-K1 cell line

Technical Field

The invention relates to the technical field of biomedicine, in particular to a screening method for stably expressing a CSFV complete structural protein (E0-E1-E2) CHO-K1 cell line.

Background

Classical Swine Fever Virus (CSFV) is a highly virulent Virus that causes a highly infectious disease in pigs, known as Classical Swine Fever (CSF). CSFV belongs to Flaviviridae (Flaviviridae), members of the pestivirus genus (Pestvirus), members of the generic genus also Bovine Viral Diarrhea Virus (BVDV) and ovine Border Disease Virus (BDV). The clinical symptoms and severity of CSF vary from most acute, acute and chronic to late onset or clinical symptoms of the disease, depending on the infected strain, age of the host and immune status of the herd [1,2 ]]. Despite the phenomena of virus storage and disease transmission, eradication of CSF remains challenging, particularly in countries where breeding is prevalent. Pestiviruses such as CSFV and Bovine Viral Diarrhea Virus (BVDV) can cross the blood-placental barrier and potentially infect developing fetuses, resulting in vertical transmission of persistently infected animals^[3]. CSF causes enormous economic losses to the pig industry worldwide, and due to its enormous socioeconomic significance, CSF is a recognized disease by the World organization for Animal Health. Studies have shown that^[4]Infected boars are the natural reservoir of CSFV and the virus can be transferred from boars to boar herds by food and direct or indirect contact, accounting for approximately 59% of farm outbreaks in the first infection.

CSFV is a enveloped, single-stranded, positive-strand RNA virus with a genome size of about 12.3kb and with only one ORF, encodes a polyprotein of about 3868 amino acids which, under the influence of the host and the autoprotease, eventually forms 12 mature viral proteins, in the order NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, respectively. Wherein Erns (E0), E1 and E2 are envelope structural proteins of CSFV: e0, also called Erns, is the only highly glycosylated surface membrane protein in CSFV virion that can be secreted outside the cell, and it participates in CSFV infection, induces the immune response of the organism, and is related to the persistent infection of virus to host; e1 is a transmembrane protein on CSFV particle, and during the course of pig infection by virus, no antibody against E1 can be detected, but E1 protein can stabilize the molecular conformation and virion structure stability of E0 and E2^[5](ii) a The E2 protein is the most important antigen protein on CSFV, can induce the generation of neutralizing antibody, has good immunogenicity, and is an important candidate antigen for developing recombinant vaccines and antibody detection methods.

CHO cells (Chinese hamster ovary cells) are the most representative mammalian producer cells for the production of therapeutic proteins, mainly due to several key factors that this cell line has^[6]: capable of robust growth in serum-free and chemical-free suspension media; has the ability to post-translationally modify the expressed protein; in addition, most importantly, the CHO cell line is easy to generate engineering cell clone which can stably express target genes, and the CHO cell line has high yield and good quality and can meet the production requirement. The cells currently used for production in CHO cells mainly include CHO-K1, CHO-S, CHO/DHFR-, etc., and CHO-K1 is more practically used because CHO-K1 forms a stably expressing cell line more easily than other CHO cell subtypes.

The research establishes the complete structural protein Cap-E0-E1-E2 gene (ShmEs) of the Shimen strain of the classical swine fever virus to a eukaryotic expression vector pCDNA-3.4, and the gene is expressed by CHO-K1, and a stable expression cell strain is screened out, so that the immunogenicity of the cell strain is researched, and a foundation is laid for developing a novel classical swine fever recombinant vaccine and producing the vaccine.

Disclosure of Invention

The invention provides a screening method of a cell line for stably expressing a complete structural protein CHO-K1 of hog cholera virus, wherein the complete structural protein E0-E1-E2 gene (ShmEs) of a Shimen strain of hog cholera virus is constructed on a eukaryotic expression vector pCDNA-3.4, and a stably expressed cell strain is screened out through the expression of CHO-K1, so that the immunogenicity of the cell strain is researched, and a foundation is laid for developing a novel hog cholera recombinant vaccine and producing the vaccine.

The technical scheme adopted by the invention is as follows:

a screening method for stably expressing a Classical Swine Fever Virus complete structural protein CHO-K1 cell line is characterized in that a PCR method is utilized to amplify Classical Swine Fever Virus, namely classic Swine river Virus, and a complete structural protein of CSFV, namely ShmEs gene, and the gene is inserted into a eukaryotic expression vector pcDNA3.4 to construct a recombinant expression plasmid pcDNA3.4-ShmEs; the recombinant expression plasmid is transferred into CHO-K1 cells by a transient transfection method, and is screened by using antibiotic G418 to obtain a cell line stably expressing the CSFV complete structural protein CHO-K1.

A screening method for stably expressing a classical swine fever virus complete structural protein CHO-K1 cell line comprises the following specific steps:

construction of a, pCDNA3.4-ShmEs expression plasmid

The whole gene of the Shimen strain of the classical swine fever virus published by GenBank: GenBank: a pair of primers is designed by selecting a Cap-E0-E1-E2 gene segment of AY 775178.2; respectively carrying out enzyme digestion on the vector pCDNA3.4 by using restriction endonucleases Xho I and BamH I, connecting the linear vector subjected to enzyme digestion and an ShmEs gene fragment through homologous recombination, transforming a DH5 alpha competent cell, and carrying out PCR identification on a bacterial solution and then sequencing a positive bacterial colony; after the sequencing comparison is successful, extracting pCDNA3.4-ShmEs plasmid for later use according to the specification of the endotoxin-free plasmid large-extraction kit; b, eukaryotic expression of pCDNA3.4-ShmEs

Uniformly paving CHO-K1 in a six-hole plate, transfecting CHO-K1 and HEK293 cells by using Lipo 3000 for pCDNA3.4-ShmEs plasmids extracted in the step a after the cells grow to the density of 60-80%, transfecting the cells by using Lipo 3000 for 5 mu g of pCDNA3.4-ShmEs plasmids per hole, replacing cell supernatant after 8h, and culturing for 48h after transfection to collect the cells for Western-blot;

c, screening of stable CHO-K1 cell line over-expressed by ShmEs

1) Sensitivity determination of CHO-K1 to G418

Uniformly spreading CHO-K1 into a 12-well plate, adding G418 with different concentrations when the cell fusion degree reaches 50-60%, and screening out the minimum lethal concentration of G418 to CHO-K1 at the concentration of 0, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 mu G/mL; after 14d of screening, the minimum lethal concentration of G418 to the screened target cells is 600 mug/mL;

2) screening of stably expressed ShmEs CHO-K1 Positive monoclonals

The CHO-K1 is evenly paved in a six-hole plate, when the cell fusion degree reaches 50%, the plasmid pCDNA3.4-ShmEs transfects cells, after 8h, the supernatant is changed into F12/DMEM of 10% FBS for continuous culture, after 24h, 600 mug/mL G418 is added, the cell growth state is observed and the solution is changed every 2-3d, after the cell death of a control group, the viable cell of the experimental group is expanded and cultured, the viable cell is paved in a 96-hole plate according to the density of 0.5 cell per hole, after 72h, G418 is added for screening, after every 2-3d, observation is carried out, and the screened single clone is expanded and cultured to obtain the CHO-K1 cell line which stably expresses the swine fever envelope structural protein ShmEs.

Western blot and IFA test results show that the gene ShmE3 is expressed in CHO-K1 cells and can be stably expressed by passage, and the antigen produced by the stable cell line is used for immunizing rabbit bodies, and the immune serum of the rabbit bodies is detected by using an IDEXX swine fever antibody detection kit, so that the antigen can stimulate the rabbit bodies to produce swine fever specific antibodies and can last for at least 42 days. The successful expression of the ShmE3 gene and the establishment of a stable cell line lay a foundation for the development of novel gene recombinant vaccines of swine fever.

Drawings

FIG. 1 is a diagram showing the result of PCR identification of the ShmE3 gene;

FIG. 2 shows the results of Wesstern-Blot detecting the expression of ShmEs antigen in CHO-K1;

FIG. 3 shows the result of IFA detection of the expression of ShmE3 antigen in eukaryotic cells;

in fig. 3: a: expression of ShmE3 in CHO-K1; b: expression of ShmE3 in HEK 293;

FIG. 4 shows the IFA detection of secondary screening of CHO-K1 monoclonal cells stably expressed by ShmEs;

FIG. 5 shows the RT-PCR detection of the CHO-K1 monoclonal cell stably expressed by ShmE 3;

in fig. 5: m: marker; 1: 2-A1-7F 10; 2: CHO-K1

FIG. 6 shows that ShmE3 stably expresses CHO-K1 monoclonal cell passage IFA detection;

FIG. 7 is a schematic diagram showing the growth of antibodies in ShmE3 immunized rabbits; in the figure: ShmE3-1 and ShmE3-3 are experimental groups to immunize ShmEs antigen rabbit body serum; the control is immune blank CHO-K1 cell lysis supernatant rabbit body serum; MOCK is untreated rabbit serum; the dotted line represents the blocking rate cutoff value (40%).

Detailed Description

The present invention and its effects will be described in detail below by way of specific embodiments.

The detailed implementation process of the screening method for stably expressing the CSFV complete structural protein (E0-E1-E2) CHO-K1 cell line comprises the following steps:

1 materials and methods

1.1 vectors, cells and strains

The pCDNA3.4 vector, the CHO-K1 cell and the HEK-293 cell are all stored in laboratories of Lanzhou veterinary research institute of Chinese academy of agricultural sciences; DH5 α competent cells were purchased from Nanjing Novozapine Biotech GmbH; the genome of the Shimen strain was stored in the laboratory.

1.2 Primary reagents

Xho I and BamH I restriction enzymes are products of NEB corporation of America; the homologous recombination kit is a product of Nanjing Novozan Co.Ltd; the antibiotic G418, Lipofectamine 3000 and ampicillin are products of Beijing Soilebao science and technology Limited; F12/DMEM and Opti-MEM medium are products of Gibco company, and a DNA gel recovery kit, a plasmid DNA small-extraction medium-extraction kit and an endotoxin-free plasmid large-extraction kit are purchased from Omega company.

1.3 construction and expression identification of pCDNA3.4-ShmE3 plasmid

1.3.1 construction of pCDNA3.4-ShmE3 plasmid

A pair of primers is designed by selecting E0-E1-E2 gene segments of Shimen strain complete genes (GenBank: AY775178.2) of classical swine fever virus published by GenBank, and is synthesized by Xian engine Biotechnology limited company, as shown in Table 1. The vector pCDNA3.4 is digested by restriction endonucleases Xho I and BamH I respectively, the digested linear vector is connected with the ShmE3 fragment through homologous recombination, DH5 alpha competent cells are transformed, and a positive colony is sequenced after being identified by bacterial liquid PCR. After sequencing comparison is successful, extracting plasmids for later use according to the specification of the endotoxin-free plasmid macroextraction kit.

TABLE 1 ShmE3 PCR amplification primer information

1.3.2 eukaryotic expression of pCDNA3.4-ShmE3 plasmid

And (2) uniformly paving CHO-K1 in a six-hole plate, transfecting CHO-K1 and HEK293 cells with the designed plasmid by using Lipo 3000 after the cells grow to the density of 60-80%, transfecting the cells by using pCDNA3.4-ShmE3 plasmid 5 mu g per hole according to the Lipo 3000 transfection mode, replacing cell supernatant for 8h, and culturing for 48h after transfection to collect the cells for Western-blot.

1.4 screening of ShmE3 overexpressing Stable CHO-K1 cell line

1.4.1 measurement of sensitivity of CHO-K1 to G418

CHO-K1 was plated evenly into 12-well plates and G418(0, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100. mu.g/mL) was screened for minimal lethal concentrations of G418 to CHO-K1 at varying concentrations until cell confluence reached 50-60%. The minimal lethal concentration of G418 to the cells of interest for the screen was determined to be 600. mu.g/mL after the 14d screen.

1.4.2 screening of stably expressed ShmE3 CHO-K1 Positive monoclonals

The CHO-K1 is evenly paved in a six-hole plate, when the cell fusion degree reaches 50%, the plasmid pCDNA3.4-ShmE3 transfects cells, after 8h, the supernatant is changed into F12/DMEM of 10% FBS for continuous culture, after 24h, 600 mu G/mL G418 is added, the cell growth state is observed and the solution is changed every 2-3d, after the cells of a control group die, the living cells of the experimental group are expanded and cultured, the living cells of the experimental group are paved in a 96-hole plate according to the density of 0.5 cell per hole, after 72h, G418 is added for screening, and after every 2-3d, the screened monoclone is expanded and cultured.

1.5 identification of Stable cell lines

1.5.1 Indirect Immunofluorescence (IFA)

A small fraction of the selected cells was seeded into 12-well plates. After the cells were confluent in a monolayer, they were fixed by incubation with 4% neutral paraformaldehyde at room temperature for 20 min. Fixed cells were washed 3 times with PBS buffer (pH 7.4) and incubated for 30min with 0.05% Triton X-100(PBS formulation) for membrane permeation. 5% BSA (in PBS buffer) was blocked for 1h at room temperature with shaking. The swine fever positive serum is diluted 1: 100 and incubated overnight at 4 ℃. The cells were washed 3 times with PBS buffer, diluted 1: 300 with FITC-labeled goat anti-porcine secondary antibody, and incubated at room temperature for 1 h. PBS was washed 3 times, stained by addition of DAPI, and incubated at room temperature for 5 min. And (3) washing with PBS for 3 times, observing the protein expression condition under a fluorescence microscope, and observing the target protein subcellular localization under a laser confocal microscope.

1.5.2 RT-PCR

The transcription of ShmEs gene mRNA of the monoclonal cell line is identified by an RT-PCR method. Respectively extracting 5, 10 and 20 generations of subcellular total RNA, and reverse transcription system according to TAKARA PrimeScript^TMRT reagent Kit with gDNA Eraser (Perfect Real Time) instructions. PCR was performed using primers designed according to Table 1.

1.6 exploration of the immunogenicity of ShmE3

Culturing the screened monoclone, collecting cells and supernatant, repeatedly freezing and thawing and performing ultrasonic treatment, performing differential centrifugation to remove cell debris and insoluble substances, concentrating the supernatant by using an ultrafiltration tube, and performing double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) on the concentrated (200 x) protein mixed solution to determine the target antigen concentration; the antigen mixture with determined concentration is emulsified and immunized with 206 adjuvant to study the immunogenicity.

2 results

2.1 construction and expression identification of pCDNA3.4-ShmE3

2.1.1 construction of pCDNA3.4-ShmE3

2.1.1.1 amplification and identification of the ShmE3 Gene

After amplification of the target gene according to the primers shown in Table 1, a band as shown in FIG. 1 was obtained, and a band corresponding to the size of the target gene was found at the 3000bp size of 2000-. Cutting the target strip, recovering, connecting with vector, transforming, sequencing the obtained positive colony

2.1.1.2 pCDNA3.4-ShmE3 recombinant product sequencing

After homologous recombination of a target gene fragment ShmEs and a double-restriction enzyme linearized vector and transformation of competent DH5 alpha, the obtained bacterial colony is identified by colony PCR and sent to a company for sequencing, the obtained result is that the bacterial colony containing the plasmid with the same design as the expected design exists, the bacterial colony with the correct sequencing is subjected to amplification culture, and endotoxin-free plasmid is greatly extracted.

2.1.2 pCDNA3.4-ShmE3 eukaryotic expression characterization

Western-Blot validation of 2.1.2.1 ShmEs

We carried out Western-Blot on cells transiently expressing the target protein and cells without any treatment, and the results are shown in FIG. 2, from which we can see that there is a band at 100-140 kDa, while the control CHO-K1 blank cell has no band at this size, and the size of the band is consistent with the expected size.

IFA validation of 2.1.2.2 Shmes

After the transfected cells were treated, they were observed under a fluorescence microscope by an indirect immunofluorescence assay (IFA), and the following results were obtained (FIG. 3). Since the transfected cells will show green fluorescence if the target gene is expressed. As shown in the figure, green fluorescence appears in CHO-K1 cells, which indicates that the target gene can be successfully expressed in CHO-K1 cells.

2.2 screening and identification of Stable cell lines

2.2.1 IFA identification of Stable cell lines

After the primary screening of section 1.4.2, we finally obtained 8 monoclonal cells in total by secondary screening, and the eight monoclonal cells were identified by indirect Immunofluorescence (IFA), and the final result is shown in FIG. 4, in which FITC positive is positive, and thus from FIG. 4, we finally obtained 6 CHO-K1 cell lines stably expressing ShmE3, which are 2-A1-1, 2-A1-2, 2-A1-3, 2-A1-5, 2-A1-6, and 2-A1-7, respectively.

2.2.2 RT-PCR identification of Stable cell lines

After passaging the monoclonal cell line (2-A1-7), we collected the total RNA of the monoclonal cells passed to the 10 th generation, and amplified the target gene after reverse transcription, and we obtained the following results (FIG. 5), which show that a band at about 3000bp is identical to the band obtained in FIG. 1, indicating that the target gene is integrated into the cell gene and can be expressed by transcription, and that the gene still exists and is expressed after passaging.

2.2.3 Stable passage assay of Stable cell lines

And selecting a monoclonal strain capable of stably expressing the ShmEs protein to carry out cell passage test (the cell strain selected in the experiment is 2-A1-7). Cells were transferred to passage 5 and passage 10 and IFA identification was performed, and the following results were obtained (fig. 6). As can be seen from FIG. 6, after passage, the monoclonal cells obtained by the test still can express our target antigen ShmE3, and the positive rate is still high.

2.3 exploration of the immunogenicity of ShmE3

After the ShmE3 is concentrated and immunized with rabbit bodies, the immunized serum is collected and the antibody production is detected by using an IDEXX swine fever detection kit. And from the IDEXX calculation, we obtained the following results (table 2). In the case of antibody depletion of ShmE3 as shown in FIG. 7, we can see that the blocking rate of the detected antibodies against swine fever after 14 days of immunization is close to the positive threshold of 40%, and the antibody level after the second booster immunization reaches 65% or more, in two immunized experimental rabbits, the antibodies in one of the two rabbits began to drop at 42d and then to 56d or so and then to 126d or so and then to the threshold, and the antibodies in the blank Control group and the Control group are negative.

TABLE 2 IDEXX hog cholera detection kit detection results

Table 2 The tested results by IDEXX classical swine fever Ab test kit

3 analysis of the present invention

One of the main challenges facing China in the prevention and treatment of swine fever is to prevent sporadic outbreaks of CSF in medium and small-scale pig farms^[7]. The coverage rate of the immunity rate of large pig farms is high because all pigs (including boars, sows and fattening pigs) are immunized, but the coverage rate of the immunity rate of small and medium-sized pig farms is insufficient and sometimes the immunization program is not reasonable, including: (1) gilts that have not eliminated immune tolerance after entry into the herd; (2) the immunization program is not standard and does not follow the curve of maternal antibody growth, therefore, piglets may not gain sufficient immunity; (3) antibody titers were not monitored annually. In this case, even if the swine herd is immunized, the effect of the immunization is not ideal^[8,9]. Clearly, there is a need to develop better preventive and control measures to complete a program for eradication of swine fever, supported by various veterinary regulatory segments. In addition, co-infection of CSFV and other pathogens complicates the diagnostic, therapeutic and prophylactic regimens of swine herds, leading to possible increases in morbidity and mortality, and currently in china, common CSFV co-infections include Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), Swine Influenza Virus (SIV), and some common secondary infections: such as haemophilus parasuis, swine pasteurellosis, streptococcosis, swine enzootic pneumonia, paratyphoid fever, Escherichia coli disease, toxoplasmosis, etc.

At present, Chinese mainly takes the measure of immunization to prevent the swine fever. In the 50 s of the 20 th century, scientists in China continuously transmitted hog cholera virulent virus to rabbits for over 480 generations to form a currently commonly used attenuated strain, namely hog cholera lapinized attenuated vaccine. The hog cholera lapinized virus vaccine is also called C strain or HCLV (Chinese hog cholera lapinized virus), which is the hog cholera prevention in China at presentThe most commonly used vaccine selected in control. The C strain vaccine has the characteristics of safety and good immunogenicity, can simultaneously induce humoral immunity and cellular immune response, and can protect pigs of different ages against the attack of swine fever virulent viruses^[10]The vaccine is also the most safe and effective attenuated vaccine internationally recognized at present, and the current attenuated vaccine comprises a GPE cell attenuated vaccine in Japan and a Thiverval cold variant attenuated strain in France besides the C strain. Since C strain does not have a marker function, vaccination and field virus infection (DIVA) cannot be distinguished, which makes the use of C strain a huge challenge in swine fever decontamination work. According to the national guidelines for swine fever prevention and treatment (2012-2020), the aim of "all pig farms and partial areas in China reach the purification standard of swine fever by the end of 2020 on the basis of continuously improving the epidemic prevention capability of farms (households), and further expanding the purification area range of swine fever" is required. Therefore, the development of a novel vaccine which is safe and efficient and can distinguish between wild virus infection and immunity is a hot research focus of CSFV vaccines in recent years.

In order to eradicate swine fever infection in China, development of DIVA vaccine is also needed, and subunit vaccine developed around swine fever E2 protein so far has been greatly developed: as early as the nineties of the last century, it was discovered that E2 glycoprotein purified from CSFV can induce the body to generate protective immunity^[11-15]Whereas the concept of labeling is based on the positive response of virus-infected animals in a core protein-specific antibody ELISA, whereas vaccinated animals only respond with specific antibodies to E2, but the immunogenicity is limited, so far only one E2 subunit vaccine, oil-adjuvanted E2(CSFV strain Alfort/Tubingen) glycoprotein (C.sub.D.)

Pest, MSD Animal Health); in addition, one member of a new generation of marker vaccines based on the BVDV cell strain "CP 7" has been licensed from European Medicines (EMA) — CP7_ E2ALF "(Suvaxyn CSF marker, Zoetis)"^[16]The infectious cDNA clone of (A) constructed pestivirus chimera "CP 7_ E2 ALF" in this boneIn the frame, the coding region of E2 was replaced with the CSFV strain "Alfort/187"^[17]And after the design and initial testing of "CP 7_ E2 alf", the innocuity of the vaccine to the target vaccinated animals as well as non-target vaccinated species was confirmed by vaccination of domestic pigs, boars, calves, goats, sheep and rabbits^[18]Experiments have shown that these limited replications occur mainly in tonsils, where the tonsils genome is detectable by polymerase chain reaction within weeks^[19-21]Furthermore, the vaccine is not transmitted via urine, feces or semen^[21]. Studies of genetic stability targeting "CP 7_ E2 ALF" also demonstrated that the virus is highly stable in vitro and in vivo^[17,22,23]. Although CP7_ E2alf performs well in various ways, it has been found to be quite cross-reactive to both BVDV and BDV, as well as to be specific to multiple vaccinations and limited sample quality degradation^[24]. For this reason, more vaccine combination strategies and vaccine development are also needed. In the test, a CHO-K1 cell is used to express the CSFV Shimen strain complete envelope structural protein ShmE3 and establish a stable over-expression cell line, and after an antigen produced by the stable cell line obtained in the test is used to immunize a rabbit, the ShmEs antigen with insufficient purity can induce the organism to generate specific antibody, and the specific antibody can last at least 42 days, and can reach hundreds of days long, but the antibody level of the ShmEs is lower than that of the C strain vaccine, because the subunit vaccine is weak and the antigen is not complete, the antibody level is different, and in addition, the experiment can be related to the insufficient effective antigen amount used in the test.

Therefore, the invention preliminarily explores the immunogenicity of the antigen ShmEs, and lays a foundation for the research and development of the swine fever marker vaccine. The list of documents to which the present invention refers is as follows:

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[7] the research on the genome structure and functions of Luzongji, Shangchun, Yuxinglong and classical swine fever virus has been advanced [ J ]. Guangdong veterinary science and technology, 2001, (04):8-12.

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[10] Kogyo Yuying, Li Yongfeng, Xileibao, et al, hog cholera lapinized attenuated vaccine (strain C) -10 years looking back [ J ]. Chinese veterinary medical bulletin, 2019, (041) (006): 654) 659.

[11]van Zijl M,Wensvoort G,de Kluyver E,et al.Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera[J].J Virol, 1991,(65)(5):2761-5.

[12]van Rijn PA,Bossers A,Wensvoort G,et al.Classical swine fever virus(CSFV)envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge[J].J Gen Virol,1996, (77(Pt 11)):2737-45.

[13]Rümenapf T,Stark R,Meyers G,et al.Structural proteins of hog cholera virus expressed by vaccinia virus:further characterization and induction of protective immunity[J].J Virol,1991,(65)(2):589-97.

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M,Lengsfeld T,Pauly T,et al.Classical swine fever virus:independent induction of protective immunity by two structural glycoproteins[J].J Virol,1995,(69)(10):6479-86.

[15]Hulst MM,Westra DF,Wensvoort G,et al.Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera[J].J Virol,1993,(67)(9):5435-42.

[16]Meyers G,Tautz N,Becher P,et al.Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs[J].J Virol,1996,(70)(12):8606-13.

[17]Reimann I,Depner K,Trapp S,et al.An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus[J].Virology,2004,(322)(1):143-57.

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P,Blome S,Gabriel C,et al.Innocuousness and safety of classical swine fever marker vaccine candidate CP7_E2alf in non-target and target species[J].Vaccine,2011,(30)(1):5-8.

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Claims (2)

1. A screening method for stably expressing a Classical Swine Fever Virus complete structural protein CHO-K1 cell line is characterized in that the Classical Swine Fever Virus, namely classic Swine Fever Virus, is amplified by a PCR method, and the complete structural protein of CSFV, namely ShmEs gene is inserted into a eukaryotic expression vector pcDNA3.4 to construct a recombinant expression plasmid pcDNA3.4-ShmEs; the recombinant expression plasmid is transferred into CHO-K1 cells by a transient transfection method, and is screened by using antibiotic G418 to obtain a cell line stably expressing the CSFV complete structural protein CHO-K1.

2. The screening method for the cell line stably expressing the classical swine fever virus complete structural protein CHO-K1 according to claim 1, comprising the following steps:

construction of a, pCDNA3.4-ShmEs expression plasmid

The whole gene of the Shimen strain of the classical swine fever virus published by GenBank: GenBank: a pair of primers is designed by selecting a Cap-E0-E1-E2 gene segment of AY 775178.2; respectively carrying out enzyme digestion on the vector pCDNA3.4 by using restriction endonucleases Xho I and BamH I, connecting the linear vector subjected to enzyme digestion and an ShmEs gene fragment through homologous recombination, transforming a DH5 alpha competent cell, and carrying out PCR identification on a bacterial solution and then sequencing a positive bacterial colony; after the sequencing comparison is successful, extracting pCDNA3.4-ShmEs plasmid for later use according to the specification of the endotoxin-free plasmid large-extraction kit;

b, eukaryotic expression of pCDNA3.4-ShmEs

Uniformly paving CHO-K1 in a six-hole plate, transfecting CHO-K1 and HEK293 cells by using Lipo 3000 for pCDNA3.4-ShmEs plasmids extracted in the step a after the cells grow to the density of 60-80%, transfecting the cells by using Lipo 3000 for 5 mu g of pCDNA3.4-ShmEs plasmids per hole, replacing cell supernatant after 8h, and culturing for 48h after transfection to collect the cells for Western-blot;

c, screening of stable CHO-K1 cell line over-expressed by ShmEs

1) Sensitivity determination of CHO-K1 to G418

Uniformly spreading CHO-K1 into a 12-well plate, adding G418 with different concentrations when the cell fusion degree reaches 50-60%, and screening out the minimum lethal concentration of G418 to CHO-K1 at the concentration of 0, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 mu G/mL; after 14d of screening, the minimum lethal concentration of G418 to the screened target cells is 600 mug/mL;

2) screening of stably expressed ShmEs CHO-K1 Positive monoclonals

The CHO-K1 is evenly paved in a six-hole plate, when the cell fusion degree reaches 50%, the plasmid pCDNA3.4-ShmEs transfects cells, after 8h, the supernatant is changed into F12/DMEM of 10% FBS for continuous culture, after 24h, 600 mug/mL G418 is added, the cell growth state is observed and the solution is changed every 2-3d, after the cell death of a control group, the viable cell of the experimental group is expanded and cultured, the viable cell is paved in a 96-hole plate according to the density of 0.5 cell per hole, after 72h, G418 is added for screening, after every 2-3d, observation is carried out, and the screened single clone is expanded and cultured to obtain the CHO-K1 cell line which stably expresses the swine fever envelope structural protein ShmEs.

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