Construction method and application of suspension HEK293 cell line for stably expressing classical swine fever virus vaccine strain E2 protein

Abstract

The invention provides a construction method of a suspension HEK293 cell line capable of stably expressing classical swine fever virus vaccine strain E2 protein. The invention constructs a suspension HEK293 cell line for expressing hog cholera virus vaccine strain E2 protein. A cell stable passage test proves that when an HEK-E2 cell line can be stably passed for 10 generations, the expression quantity of the E2 protein in a culture supernatant is stable, the yield is stably kept at 1.0-1.2 mg / ml, the purity of the E2 protein obtained by affinity chromatography purification reaches 95%, double-antibody sandwich ELISA determines that the E2 protein has good reactogenicity, a 206 adjuvant emulsified E2 protein immune pig can induce a high-level serum antibody, and the E2 protein can be used for immunizing the HEK-E2 cell line for immunizing the HEK-E2 cell line for immunizing the HEK-E2 cell line for immunizing the HEK-E2 cell line. And after the virulent virus is attacked, complete protection can be achieved. The HEK-293 cell line constructed by the invention can stably secrete and express the CSFV E2 protein, and has an application prospect of being developed into a recombinant E2 genetic engineering vaccine.

Description

Technical Field

[0001] The invention relates to a method for constructing a suspended HEK293 cell line stably expressing E2 protein of a swine fever virus vaccine strain and application thereof, and belongs to the technical field of cell biology and animal genetic engineering vaccines. Background Art

[0002] Classical swine fever rabbit-attenuated vaccine (HCLV) is the most effective vaccine for the prevention and control of classical swine fever virus (CSFV) infection. HCLV is a weakened live vaccine. After vaccination, the serum antibodies induced by the body are consistent with the antibodies produced by wild virus infection. Therefore, it is impossible to effectively distinguish whether the detected antibodies are from vaccine immunity or wild virus infection by detecting serum antibodies, which is not conducive to the prevention and control of classical swine fever in pig farms and the realization of the purification and eradication goals of classical swine fever in my country.

[0003] The envelope glycoprotein E2 is the most important immunogenic protein encoded by the CSFV genome and is the preferred antigen for the development of recombinant genetic engineering vaccines. Immunizing pigs with the E2 protein expressed in vitro by the eukaryotic system can effectively stimulate the pigs to produce neutralizing antibodies and protect them from attacks by strong strains of swine fever. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a suspension HEK293 cell line stably expressing the E2 protein of a classical swine fever virus vaccine strain and its application.

[0005] The method for constructing a suspension HEK293 cell line stably expressing the E2 protein of the swine fever virus vaccine strain of the present invention comprises the following steps: (1) Clone the CSFV vaccine strain E2 gene expression cassette into the PBHyB vector to obtain the recombinant expression plasmid pB-E2; The CSFV vaccine strain E2 gene expression cassette comprises a coding sequence for a secretion signal peptide, a polynucleotide sequence encoding an E2 gene, an amino acid linker sequence (5`-GGGGS-3`), and a polyhistidine tag sequence; the amino acid sequence of the E2 gene expression cassette is shown in SEQ ID No. 1; hIL-2 is used as a secretion signal peptide (amino acid sequence MYRMQLLSCIALSLALVTNS); Construction method of PBHyB vector: The target fragment Fra1 was amplified using plasmid PiggyBac Dual Promoter as a template and primers P1 and P2, the target fragment Fra2 was amplified using plasmid pTT5 as a template and primers P3 and P4, the target fragment Fra3 was amplified using plasmid pCEP4 as a template and primers P5 and P6, and the Fra1, Fra2 and Fra3 fragments were recombined using DNA homologous recombinase to obtain PBHyB; The sequence of P1, P2, P3, P4, P5, P6 is as follows: P1:5`-TTATGAGGGACAGCCGCGTACATTTATATTGGCTCATGTC-3` (SEQ ID No.3) P2: 5`-GGCTGTCCCTCATAAAAGTTTTG-3` (SEQ ID No. 4) P3:5`-TTATGAGGGACAGCCGCGTACATTTATATTGGCTCATGTCC-3` (SEQ ID No.5) P4: 5`-GCTAGTCGCATATGCTATTGAATTAGGGTTAGTCTGG-3` (SEQ ID No. 6) P5: 5`-GCATATGCGACTAGCTTGGCACG-3` (SEQ ID No. 7) P6: 5`-TATCAGGGACAGCCCGGCTGCTGGAGATGG-3` (SEQ ID No. 8).

[0006] (2) The recombinant expression plasmid pB-E2, the recombinant transposase auxiliary plasmid pTT5-chyPBase and the transfection reagent PEI were co-transfected into HEK-293 suspension cells using the PEI method; the molar ratio of the recombinant expression plasmid pB-E2, the recombinant transposase auxiliary plasmid pTT5-chyPBase and the transfection reagent PEI was 3:1:8.

[0007] Construction of the recombinant transposase auxiliary plasmid pTT5-chyPBase: After codon optimization, a transposase (chyPBase) gene fragment with high enzyme activity was obtained (the sequence of the transposase gene is shown in SEQ ID No. 2). Hind III and Not Ⅰ The synthesized transposase gene fragment was double-digested, purified and cloned into the pTT5 vector to obtain the recombinant transposase auxiliary plasmid pTT5-chyPBase.

[0008] (3) Through blasticidin resistance screening, positive cell clones expressing the recombinant expression plasmid were obtained. After multiple subcultures, a HEK-E2 recombinant cell line that can stably and sustainably secrete and express E2 protein was obtained.

[0009] The present invention also provides the use of the suspended HEK293 cell line in preparing a swine fever virus subunit vaccine.

[0010] In summary, the present invention uses mammalian cell HEK-293 as a host cell, which can glycosylate the envelope glycoprotein E2 protein, so that the structure and function of the recombinant E2 protein are almost the same as the E2 protein on the natural virus particle. In order to obtain a recombinant cell strain that can be stably propagated and can be used for large-scale industrial production and high-density fermentation, we constructed a matching transposon-constitutive auxiliary plasmid derived from insect cells, which can stably integrate the E2 gene into the transcriptional active region of the genome on the chromosome of HEK-293 cells, thereby achieving efficient translation, transcription and expression of the E2 gene; by adding a secretory signal peptide gene that enhances protein secretion into the cell culture supernatant and a polyhistidine tag for purifying the protein to the E2 gene, the purification procedure of the target protein is simplified. By co-transfecting HEK293 cells with plasmids, screening positive cell clones using blasticidin resistance, expanding reproduction and suspension culture, a HEK-293 recombinant cell line that can stably secrete and express E2 protein was obtained, named HEK-E2. In the stable cell passage test, it was confirmed that when the HEK-E2 cell line was passed to the 10th generation, the expression level of E2 protein in the culture supernatant was stable, and the yield was stably maintained at 1.0 mg~1.2 mg / ml, indicating that the E2 gene had been stably integrated into the chromosome of the host cell, the purity of the E2 protein obtained by affinity chromatography purification reached more than 95%, and the double antibody sandwich ELISA determined that it had good reactivity. The 206 adjuvant emulsified E2 protein immunized pigs can induce high levels of serum antibodies and can be fully protected after attacking strong toxins. The HEK-293 cell line constructed by the present invention can stably secrete and express CSFV E2 protein, and has the application prospect of being developed into a recombinant E2 genetic engineering vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Viability detection of HEK-293 cells expressing E2 protein for resistance screening; Figure 2 RT-PCR detection of the integration efficiency of E2 gene into HEK-293 cells, where M: DNA standard molecular weight; 5-1: fifth-generation E2 gene fragment; 5-2: fifth-generation 5'-end transposase gene + E2 gene fragment; 10-1: tenth-generation E2 gene fragment; 10-2: tenth-generation 5'-end transposase gene + E2 gene fragment; Figure 3 The purified E2 protein was detected by SDS-PAGE, where M: protein standard molecular weight, 1.200 mM imidazole to wash the E2 protein; 2.300 mM imidazole to wash the E2 protein; 3.400 mM imidazole; Figure 4 Immunoblotting was used to detect the purified E2 protein, where M is the standard molecular weight of the protein; 1. 200 mM imidazole was used to wash the E2 protein; 2. 500 mM imidazole was used to wash the E2 protein; Figure 5 is a double antibody sandwich detection of the binding activity of secreted expressed E2 protein; Figure 6 shows the trend of rectal body temperature changes in E2-immunized pigs after challenge. DETAILED DESCRIPTION

[0012] The present invention will be further described below through specific implementation modes.

[0013] Example 1 Construction of E2 protein HE-293 recombinant cell line 1. Construction of recombinant transposase auxiliary plasmid containing transposase gene (1) Construction of recombinant plasmid pTT5-chyPBase. After codon optimization, a transposase (chyPBase) gene fragment with high enzyme activity was obtained (transposase gene sequence SEQ ID No. 2). Hind III and Not Ⅰ The synthesized transposase gene fragment was double-digested and purified, then cloned into the pTT5 vector (purchased from Guangzhou Cuiben Biotechnology Co., Ltd.) and named pTT5-chyPBase.

[0014] 2. Construction of recombinant transposase plasmid (1) Construction of recombinant plasmid PBHyB. Using plasmid PiggyBac Dual Promoter (PBDP) (purchased from Guangzhou Cuiben Biotechnology Co., Ltd.) as a template and primers P1 and P2 to amplify the target fragment Fra1, using plasmid pTT5 as a template and primers P3 and P4 to amplify the target fragment Fra2, using plasmid pCEP4 as a template and primers P5 and P6 to amplify the target fragment Fra3, the Fra1, Fra2 and Fra3 fragments were recombined using DNA homologous recombinase to obtain plasmid PBHyB.

[0015] 3. Construction of expression vector for E2 protein (1) Synthesize the E2 gene expression cassette.

[0016] In order to obtain a high level of secretory expression of E2 protein, an expression cassette containing different secretory signal peptides was constructed to screen the signal peptide sequences with the highest secretion effect and expression level. The selected signal peptides include Human OSM (human oncostatin M), VSV-G (vesicular stomatitis virus G protein), Mouse Ig Kappa (mouse Ig light chain K), Mouse Ig H (mouse Ig heavy chain), Human IL-2 (human interleukin-2, hIL-2), Humanm Chymotrypsinogen (human trypsin collagenase), Human trypsinogen-2 (human trypsinogen-2), Albumin (albumin), and Human insulin (human insulin). The E2 expression cassette (5`-3` direction) includes the coding sequence of the secretory signal peptide, the polynucleotide sequence encoding the E2 gene, an amino acid linker sequence (5`-GGGGS-3`), and polyhistidine (purification tag). The gene fragment of the expression cassette was sent to a biological company for synthesis (Qingke Biotechnology Co., Ltd.). The expression cassettes containing different secretory signal peptides were respectively prepared by EcoR Ⅰ and Hind The fragment was subcloned into the pTT5 vector at site III.

[0017] Amino acid sequence of E2 gene expression cassette (SEQ ID No.1): MYRMQLLSCIALSLALVTNSASMRLACKEDYRYAISSTDEIGLLGAGGLTTTWKEYNHDLQLNDGTVKASCVAGSFKVTALNVVSRRYLASLHKKALPTSVTFELLFDGTNPSTEEMGDDFRSGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRRDK PFPHRMDCVTTIVENEDLFYCKLGGNWTCVKGEPVVYTGGVVKQCRWCGFDFDGPDGLPHYPIGKCILANETGCRIVDSTDCNRDGVVISTEGSHECLIGNTTVKVHASDERLGPMPCRLKEIVSSAGPVMKTSCTFNYTKTLKNRYYEPRDSYFQQYMLKGEYQYWFDLDATDCHSGGGGSHHHHHH.

[0018] (2) Screening for the best secretion signal peptide sequence. Eukaryotic expression vectors containing the above signal peptide sequences were constructed and transiently transfected into HEK-293 cells. The expression of E2 protein in the culture supernatant was detected, and sequences with good secretion effect and high expression were selected as signal peptide sequences. The results of the E2 protein expression test showed that hIL-2 achieved the expected purpose as a secretion signal peptide. Therefore, hIL-2 was used as a secretion signal peptide sequence to express E2.

[0019] (3) Construct an E2 expression vector containing the transposase gene.

[0020] The synthetic E2 gene expression cassette fragment was Kpn Ⅰ and Hind Ⅲ double restriction enzyme digestion and subcloning into PBHyB vector to obtain the recombinant plasmid, which was named PB-E2.

[0021] 4. Construction and screening of E2 protein expressing cell library (1) Transfection of target gene. PB-E2, pTT5-chyPBase and transfection reagent PEI were co-transfected into 1×10 6 HEK-293 suspension cells were transfected with 10ug / ml blasticidin S (BS) for resistance screening 24 hours after transfection. The cell number was counted every 48 hours, and the culture supernatant was collected for E2 protein detection. At the same time, the culture medium was replaced with new medium containing BS (10 ug / ml). The cells were blindly cultured 7-10 times continuously. When under antibiotic pressure, the cell number remained stable at 1.0×106 When the cell viability reached more than 95% at 400 μg / ml, the cell number and viability remained stable for 2-3 more passages (Figure 1), indicating that the primary cell seed bank was successfully established.

[0022] (2) Passaging experiment of recombinant HEK-293 cell stable E2 protein Dilute the primary cell seed stock to 0.5 × 10 6 / ml, and cultured in medium without antibiotics. The expression of E2 in the cell supernatant was detected once every 5 generations using double antibody sandwich ELISA. After 10 generations, the expression level of E2 protein did not change (Table 2), indicating that the E2 gene was stably integrated into the genes of HEK-293 cells and could be used as a seed bank for batch preparation of E2 protein. The cell line was named HEK-E2.

[0023] (3) RT-PCR detection of E2 gene integration efficiency The total genomic RNA of the fifth and tenth generation HEK-93 cells was extracted and reverse transcribed into cDNA with random primers. The target gene was amplified by using a specific primer pair for amplifying the E2 gene, an upstream primer for extending the 5' end part of the transposase gene, and a downstream primer for the complete E2 gene. As shown in Figure 2, the E2 gene fragment of about 1000 bp can be amplified in the genome of the fifth and tenth generation HEK-93 cells, including the transposase gene and the E2 gene fragment, indicating that the E2 gene is integrated into the transcriptional active region of the HEK-293 cell chromosome genome, and the E2 gene can be stably passed down as the cells divide, and the E2 protein is released into the cell culture supernatant in the form of continuous secretion, which is easy to purify the protein.

[0024] Example 2 E2 protein purification The cell culture supernatant was centrifuged at 10,000 rpm and 4°C for 30 minutes, the precipitate was discarded, and the supernatant was transferred to a nickel ion affinity resin chromatography column equilibrated with equilibration buffer Mc-30 (50mM Tris-HCl, 400mM NaCl, 30mM imidazole, 0.05% v / v Tween-20, pH=8.0). The column was bound for 30 minutes under ice bath conditions, and the flow-through was repeatedly bound 2-3 times; the impurities were washed with 10-15 column volumes of Mc-30 buffer, and then the target protein was eluted with 2-3 column volumes of Mc-300 solution (50mMTris-HCl, 400mM NaCl, 300mM imidazole, 0.05% v / v Tween-20, pH=8.0), the flow-through was collected, and samples were added to the loading buffer for boiling and then tested. Through SDS-PAGE detection (Figure 3) and immunoblotting detection (Figure 4), it was found that the size of E2 protein was about 55 kDa. Due to glycosylation, the protein band showed tailing phenomenon, and the purity was above 95%. The purified E2 protein was dialyzed with dialysis buffer Db-100 (0.1M PBS, 100mM reduced glutathione) to remove imidazole, and then the protein was quantified by ultraviolet absorption method and diluted to 60ug / ml as the raw material for vaccine preparation.

[0025] Example 3 Double Antibody Sandwich ELISA to Detect the Reactivity of E2 Protein The capture anti-swine fever virus monoclonal antibody was diluted 1:3000 with coating buffer (0.05 M carbonate buffer pH 9.6, NaHCO3 1.59 g NaHCO3 2.93 g, distilled water to 1000 ml), 100 μl / well was added to the wells of the ELISA plate, and incubated at 4°C overnight; the liquid in the wells was discarded and 220 μl / well of 1× PBST (0.1 M PBS, pH 7.4, 0.05% Tween-20), wash 3-4 times, and thoroughly pat dry the liquid in the wells for the last time; add 260μl / well of blocking solution (1×PBST, 5% skim milk powder, 1% gelatin, 5% trehalose), act at room temperature for 30 minutes, discard the liquid in the wells, wash the plate 4-5 times with 220μl / well of 1×PBST, and thoroughly pat dry the liquid in the wells for the last time; add the harvested cell culture supernatant to the wells at a dilution ratio of 1:50, 1:400, 1:800, 1:1600, 1:3200, and 1:6400 with 1×PBST, 100μl / well, do 2 wells for each dilution, incubate at 37℃ for 45min after sealing, and repeat the washing process; dilute HRP-labeled anti-classic swine fever virus E2 protein monoclonal antibody at 1:4000, 100μl / well, incubate at 37℃ for 45min after sealing min, repeat the washing process; add 100μl / well TMB, seal and incubate at 37℃ for 12min; add 100μl / well 2M H2SO4, shake and mix, and read the result by OD450 on a microplate reader. When the culture supernatant was diluted 1:50, the OD450 was 4.0, when it was diluted 1:400, the OD450 was about 3.2, and when it was diluted 1:6400, the OD450 was 2.0. This test result shows that the expressed E2 protein has good reactivity and can be specifically recognized by antibodies against classical swine fever virus ( Figure 5 ).

[0026] Example 4. E2 antigen immunization pig test (1) Vaccine antigen: E2 protein purified by affinity chromatography and emulsified with SA201 adjuvant to prepare 30 μg / ml; Classical swine fever rabbit-attenuated (C-strain) vaccine cell seedling (Zoetis), virus content ≥10 4.0 FAID 50 / head portion / ml.

[0027] (2) Experimental pigs: Eleven 90-day-old piglets that were negative for classical swine fever virus antibodies and foot-and-mouth disease virus serum antibodies and had not been vaccinated with porcine respiratory and reproductive syndrome live vaccine were divided into three groups: E2 antigen immunization group, 5 pigs; C-strain control group, 3 pigs; blank control (PBS) group: 3 pigs.

[0028] (3) Virus strain: Classical swine fever virus Shimen strain (CVCC: AV1411, 10 5 MLD 50 / ml) was purchased from the National Veterinary Microbiological Bacteria (Virus) Collection Center.

[0029] All experiments used pigs, and blood was collected before immunization to separate serum. Peripheral blood was collected every 7 days after immunization to separate serum for antibody detection until before virus attack.

[0030] (1) E2 antigen immunization group. 2 ml of vaccine (60 ug / head) was inoculated into the neck muscle. 21 days after the first immunization, a booster immunization with the same dose was given once, and the virus was challenged 14 days after the booster immunization.

[0031] (2) C-strain control group: 1 ml / head, 1 immunization.

[0032] (3) Serum antibody detection. The IDEXX Classical Swine Fever Virus Antibody Detection Kit was used to detect serum antibodies, and the results were judged by the blocking rate: antibody positive, blocking rate % ≥ 40%; suspicious: 30% < blocking rate % < 40%; negative: blocking rate % < 30%. The antibody of pig No. 131 in the E2 antigen immunization group turned positive 7 days after immunization (41.11%), and the antibodies of the five immunized pigs all turned positive 14 days after immunization. The highest blocking rate was pig No. 131 (67.51%), and the blocking rate of all pigs was greater than 55% 21 days after immunization. After the first immunization, the antibody level of the E2 antigen immunization group rose rapidly after the booster immunization 21 days after the first immunization. The antibodies of the C-strain control pigs all turned positive 7 days after immunization; 21 days after immunization, the antibody of pig No. 128 turned suspicious (32.61%), and the antibody turned negative (25.25%) until 35 days (Table 3).

[0033] Example 5. Immunized pig challenge test The pigs in the E2 antigen immunization group and the PBS control group were transferred to an animal biosafety level 3 laboratory (ABSL-3) for virus challenge research and were housed in two pens. All pigs were inoculated with 1 ml of the virulent blood virus of the Shimen strain of classical swine fever virus in the neck muscle, and the anorectal temperature was measured daily (normal body temperature 38-40℃, mild fever 40-41℃, moderate fever 41-42℃) and the clinical symptoms of the pigs were observed.

[0034] from Figure 6As shown in Table 4, the rectal temperature changes of the experimental groups showed that 3-5 days after the virus attack, the rectal temperature of the pigs in the PBS group rose to between 40.4 and 41.5. The pigs showed fear of cold, huddled and lethargic, bleeding spots on the abdominal skin, extreme depression, severe diarrhea, messy back hair, and basically no appetite. The blood from the anterior vena cava was collected and analyzed and found that the blood was watery and did not coagulate, which meant that severe viremia occurred. On the 6th day after the virus attack, the body temperature began to return to normal, but all pigs entered a state of near death. On the 7th day after the virus attack, the PBS control group was established, and all pigs were handled in accordance with animal welfare and ABSL-3 standardized animal handling procedures. On the 4th and 5th day after the virus attack, the E2 immunization group had a temperature reaction, the body temperature rose to between 40.2 and 40.6, the mental state was poor, and the feeding was normal. On the 6th day after the infection, the body temperatures of all pigs returned to the normal range. Until the 11th day after the infection, there were no recurrences of body temperature or clinical symptoms. The mental state and feed intake returned to normal, and all the pigs immunized with E2 vaccine were protected (Table 5).

Claims

1. A method for constructing a suspension HEK293 cell line that stably expresses the E2 protein of the classical swine fever virus vaccine strain. The following steps are involved: The CSFV vaccine strain E2 gene expression cassette is cloned into the PBHyB vector to obtain the recombinant expression plasmid pB-E2; the CSFV vaccine strain E2 gene expression cassette comprises a secretion signal peptide coding sequence, a polynucleotide sequence encoding the E2 gene, an amino acid linker sequence, and a polyhistidine tag sequence; the amino acid sequence of the E2 gene expression cassette is shown in SEQ ID No. 1; hIL-2 is used as the secretion signal peptide; (2) The recombinant expression plasmid pB-E2, the recombinant transposase helper plasmid pTT5-chyPBase and the transfection reagent PEI were co-transfected into HEK-293 suspension cells using the PEI method; (3) Through blasticidin resistance screening, positive cell clones expressing the recombinant expression plasmid were obtained. After multiple subcultures, a HEK-E2 recombinant cell line that can stably and sustainably secrete and express E2 protein was obtained.

2. The method for constructing a suspension HEK293 cell line stably expressing the E2 protein of the classical swine fever virus vaccine strain according to claim 1, Features: Construction method of PBHyB vector: The target fragment Fra1 was amplified using plasmid PiggyBac Dual Promoter as a template and primers P1 and P2, the target fragment Fra2 was amplified using plasmid pTT5 as a template and primers P3 and P4, the target fragment Fra3 was amplified using plasmid pCEP4 as a template and primers P5 and P6, and the Fra1, Fra2 and Fra3 fragments were recombined using DNA homologous recombinase to obtain PBHyB; The sequence of P1, P2, P3, P4, P5, P6 is as follows: P1:5`-TTATGAGGGACAGCCGCGTACATTTATATTGGCTCATGTC-3` P2:5`-GGCTGTCCCTCATAAAAGTTTTG-3` P3:5`-TTATGAGGGACAGCCGCGTACATTTATATTGGCTCATGTCC-3` P4:5`-GCTAGTCGCATATGCTATTGAATTAGGGTTAGTCTGG -3` P5:5`-GCATATGCGACTAGCTTGGCACG-3` P6: 5`-TATCAGGGACAGCCCGGCTGCTGGAGATGG-3`.

3. The method for constructing a suspension HEK293 cell line stably expressing the E2 protein of the classical swine fever virus vaccine strain according to claim 1, Features: Construction of the recombinant transposase auxiliary plasmid pTT5-chyPBase: After codon optimization, a transposase gene fragment with high enzyme activity was obtained. Hind III and Not Ⅰ The synthesized transposase gene fragment was double-digested, purified and cloned into the pTT5 vector to obtain the recombinant transposase auxiliary plasmid pTT5-chyPBase. The sequence of the transposase gene is shown in SEQ ID No.

2.

4. The method for constructing a suspension HEK293 cell line stably expressing the E2 protein of the classical swine fever virus vaccine strain according to claim 1, Features: The molar ratio of the recombinant expression plasmid pB-E2, the recombinant transposase auxiliary plasmid pTT5-chyPBase and the transfection reagent PEI is 3:1:

8.

5. The method for constructing a suspension HEK293 cell line stably expressing the E2 protein of the classical swine fever virus vaccine strain according to claim 1, Features: When the HEK-E2 recombinant cell line was stably propagated for 10 generations, the expression level of E2 protein in the culture supernatant was stable, the yield was stably maintained at 1.0-1.2 mg / ml, and the purity of E2 protein obtained by affinity chromatography purification reached 95%.

6. A suspension HEK293 cell line stably expressing the E2 protein of the classical swine fever virus vaccine strain constructed according to the construction method of claim 1.

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