Construction of eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA4Ig gene and expression in COS-7 cells
- VernacularTitle:人CTLA-4Ig融合基因减毒鼠伤寒沙门菌真核表达载体的构建及表达
- Author:
Chunli ZHOU
- Publication Type:Journal Article
- Keywords:
CTLA-4Ig;
attenuated Salmonella typhimurium;
eukaryotic expression vector;
gene expression
- From:Journal of Third Military Medical University
1983;0(03):-
- CountryChina
- Language:Chinese
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Abstract:
Objective To construct eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA-4Ig cDNA and identify its expression in COS-7 cells for the further study of function in SLE models. Methods Touchdown PCR was used to amplify hCTLA-4Ig cDNA.The PCR product was ligated into the multiple clone site of eukaryotic expression vector pcDNA3.1(+) by gene recombination technique.Then the recombinated plasmid pcDNA3.1(+)-CTLA-4Ig was transfected into COS-7 cells using DOTAP.The expression of interest protein in the supernatant of the cell disruption was detected with SDS-PAGE and Western blotting.Results Restriction analysis and DNA sequence analysis showed that the CTLA-4Ig cDNA had been successfully inserted into pcDNA3.1(+) eukaryotic expression vector.The interest protein could be detected in the supernatant of cell disruption 48h after the transfection of pcDNA3.1(+)-CTLA-4Ig.This protein specifically bound with human CTLA-4 monoclonal antibody.Conclusion The eukaryotic expression vector containing hCTLA-4Ig gene was successfully constructed and bioactive interest protein could be successfully expressed in mammalian cells.
