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Recombinant cell line for stably expressing classical swine fever virus E2 protein, and applications of the same in preparation of subunit vaccines and diagnosis reagents of classical swine fever

A swine fever virus, stable expression technology, applied in antiviral agents, cells modified by introducing foreign genetic material, applications, etc., can solve problems such as increased difficulty in production process, poor folding and modification, cell lysis, etc., to achieve sustained antibody persistence. Long time, easy to culture, fast proliferation effect

Active Publication Date: 2014-04-30
HARBIN WEIKE BIOTECH DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some differences with the natural antigenic protein structure of the virus, and the folding and modification of the protein after expression is not as good as that of the mammalian cell expression system
Moreover, when the system produces and prepares antigens, the cells will lyse and die after being infected by the virus, which will increase the difficulty of downstream purification and other production processes.

Method used

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  • Recombinant cell line for stably expressing classical swine fever virus E2 protein, and applications of the same in preparation of subunit vaccines and diagnosis reagents of classical swine fever
  • Recombinant cell line for stably expressing classical swine fever virus E2 protein, and applications of the same in preparation of subunit vaccines and diagnosis reagents of classical swine fever
  • Recombinant cell line for stably expressing classical swine fever virus E2 protein, and applications of the same in preparation of subunit vaccines and diagnosis reagents of classical swine fever

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction and detection of a recombinant cell line stably expressing classical swine fever virus E2 protein

[0044] 1 Materials and methods

[0045] 1.1 Plasmids, strains and cells

[0046] The eukaryotic expression vector plasmid pCAG-neo, DH5α competent cells and BHK-21 cells are preserved by the State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The plasmid extraction kit and RNA extraction kit are products of QIAGEN, DNA Gel recovery kit was purchased from Shanghai Huashun Biotechnology Co., Ltd., G418 was purchased from Gibco, trypsin was purchased from Hyclone, Reverse Transcriptase M-MLV﹑PrimeSTAR TM HS DNA Polymerase ﹑ Sal I ﹑ Xho I ﹑ BamH I ﹑ T4 DNA ligase was purchased from TaKaRa Company, the anti-CSFV E2 protein monoclonal antibody was prepared by our research group, the classical swine fever virus antigen ELISA detection kit was the product of Median Diagnostics, ...

Embodiment 2

[0072] Example 2 Cell line expressing recombinant protein to the immune protection test of pig

[0073]Vaccine preparation: When the recombinant mammalian cell line BCSFV-E2 cells (CGMCC No.7719) constructed and screened in Example 1 grow to 90% full after normal passage, replace the medium with low serum (serum content is 1-2%) Continue to culture for 4-6 days, harvest the cell culture supernatant and store it at 4°C. The cell supernatant is concentrated by ultrafiltration with a molecular weight cut-off of 50kD until the E2 antigen ELISA titer is 1:160 to 1:320, and the final concentration is 0.02% After thimerosal is the vaccine antigen solution. The vaccine antigen solution and MONTANIDE ISA206VG adjuvant were mixed and fully emulsified at a weight ratio of 1:1 to prepare a water-in-oil-in-water vaccine. The attenuated vaccine in the control group was a commercially available swine fever cell vaccine (derived from primary bovine testicular cells).

[0074] Animal groupin...

Embodiment 3

[0084] Embodiment 3 uses recombinant cell line to express antigen indirect ELISA method to detect swine fever virus antibody

[0085] The BCSFV-E2 cells were expanded and cultured, and the cell culture supernatant was harvested. The cell supernatant was centrifuged at low speed to remove impurities such as cell debris, and then filtered through a 0.45 μm filter membrane for clarification. The clarified supernatant was then concentrated by ultrafiltration with a molecular weight cut-off of 50kD. After being concentrated by about 40 times of volume, it is centrifuged at 12000rpm to remove insoluble impurities. After concentration, the supernatant was dialyzed with pH 8.0 Tris-HCl buffer, bound to a DEAE anion resin chromatography column, washed and eluted with pH 8.0 Tris-HCl, 250mM NaCl buffer itd, and the activity was collected. peak protein. After being concentrated by ultrafiltration, it was chromatographed by molecular sieve chromatography to collect the active peak prot...

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Abstract

The present invention discloses a strain of a recombinant cell line for stably expressing classical swine fever virus E2 protein, and applications of the recombinant cell line in preparation of subunit vaccines and diagnosis reagents of classical swine fever, wherein specifically the recombinant cell line is BCSFV-E2, is preserved in the China General Microbiological Culture Collection Center, and has the preservation number of CGMCC No.7719. The classical swine fever subunit vaccine prepared by using the recombinant cell line has characteristics of high safety, good immunization effect, easy mass production, less being susceptible to exogenous virus pollution or influence of antibodies, and no influence of the maternal antibody on immunization of swine, and can induce and produce high level classical swine fever virus neutralization antibodies after the swine is immunized. In addition, the present invention further discloses a method for constructing the recombinant mammalian cell line, a method for preparing the classical swine fever subunit vaccine, and applications of the antigen expressed by the recombinant cell line in preparation of classical swine fever prevention vaccines and diagnosis reagents.

Description

technical field [0001] The present invention relates to a recombinant mammalian cell line stably expressing classical swine fever virus E2 protein and its application in the preparation of classical swine fever subunit vaccines and diagnostic reagents. Specifically, the recombinant cell line involved in the present invention is BCSFV-E2, which The deposit number is: CGMCC No.7719. The invention also discloses a method for preparing the recombinant cell line and the application of the recombinant cell line in preparing a vaccine for preventing swine fever and in preparing reagents for diagnosing or detecting swine fever virus infection. The invention belongs to the technical field of animal vaccines and veterinary biological products. Background technique [0002] Classical swine fever (CSF), also known as hog cholera (Hog cholera, HC), is an acute febrile fatal disease caused by swine fever virus (Hog Cholera virus, HCV or Classical swine fever virus, CSFV). Plague is high...

Claims

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Application Information

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IPC IPC(8): A61K39/187A61K9/107A61P31/14G01N33/68G01N33/569C12N15/85C12N15/40C12N5/10C12R1/91
Inventor 华荣虹步志高
Owner HARBIN WEIKE BIOTECH DEV
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