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Recombinant BHK cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the same in preparation of vaccines and diagnosis reagents of classical swine fever

A technology of E0-E1-E2 and swine fever virus, which is applied in the fields of biomedical genetic engineering and immunology, can solve problems such as the inability to form VLP structures, and achieve the effects of easy mass production, increased antigen expression, and rapid proliferation

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

Whether the structural protein of CSFV can form a VLP structure after being expressed in mammalian cells has not yet been reported.

Method used

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  • Recombinant BHK cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the same in preparation of vaccines and diagnosis reagents of classical swine fever
  • Recombinant BHK cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the same in preparation of vaccines and diagnosis reagents of classical swine fever
  • Recombinant BHK cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the same in preparation of vaccines and diagnosis reagents of classical swine fever

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction and detection of a recombinant cell line stably expressing classical swine fever virus E0-E1-E2 protein

[0047] 1 Materials and methods

[0048] 1.1 Plasmids, strains and cells

[0049] 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. 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, anti-CSFV E2 protein and anti-CSFV E0 protein monoclonal antibodies were prepared by our research group, and the ELISA detection kit for classical swine fever virus antigen was Median Di...

Embodiment 2

[0080] Example 2 Cell line expressing recombinant protein to the immune protection test of pig

[0081] Vaccine preparation: When the recombinant cell line BCSFV-E012 (CGMCC No.7720) cells constructed and screened in Example 1 grow to 90% full after normal passage, change to low serum medium (serum content is 1-2%) and continue to culture 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 100kD, adjusted to the E2 antigen ELISA titer of 1:32 to 1:64, and then added to a final concentration of 0.02% After thimerosal is the vaccine antigen solution. The vaccine antigen solution and the oil adjuvant are mixed and fully emulsified at a volume ratio of 1:1.5. The attenuated vaccine in the control group was a commercially available swine fever cell vaccine (derived from primary bovine testicular cells).

[0082] Animal grouping and immunization: before immunization, the pigs...

Embodiment 3

[0090] Embodiment 3 uses recombinant cell line to express antigen indirect ELISA method to detect swine fever virus antibody

[0091] The BCSFV-E012 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 100kD. After being concentrated by about 40 times of volume, it is centrifuged at 12000rpm to remove insoluble impurities. The supernatant was centrifuged through a continuous sucrose density gradient ranging from 10% to 50%. The 20%-30% concentration sucrose layer was collected, then desugared by ultrafiltration and concentrated to determine the protein content, and the purified antigen was stored at -70°C for future use. Purified CSFV-E012 protein was used as the coating antigen to coa...

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Abstract

The present invention discloses a recombinant cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the recombinant cell line in preparation of vaccines and diagnosis reagents of classical swine fever, wherein the recombinant cell line is BCSFV-E012, is preserved in the China General Microbiological Culture Collection Center, and has the preservation number of CGMCC No.7720. In addition, the present invention further discloses an establishment method for the cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and a method for preparing a classical swine fever prevention vaccine composition by using the cell line. The present invention further discloses applications of the E0-E1-E2 protein stably expressed by the recombinant cell line in preparation of classical swine fever prevention vaccines and diagnosis reagents. The classical swine fever 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 classical swine fever virus non-structural protein antibody production so as to identify the vaccinated animal and the virus infected animal.

Description

technical field [0001] The invention relates to a recombinant BHK cell line stably expressing the E0-E1-E2 protein of classical swine fever virus and its application in the preparation of classical swine fever vaccine and diagnostic reagent. More specifically, the present invention relates to a recombinant BHK cell line expressing classical swine fever virus E0-E1-E2 protein, which is BCSFV-E012. The invention also discloses a method for preparing the recombinant BHK cell line and the application of the recombinant cell line in preparing vaccines for preventing swine fever and diagnostic reagents for swine fever. It belongs to the field of biomedical genetic engineering and immunology. 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 highly contagious, widely prevalent, high in morbidit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/187A61K9/107A61P31/14C12N15/85C12N5/10C07K19/00G01N33/68
Inventor 华荣虹步志高
Owner HARBIN WEIKE BIOTECH DEV
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