Cloning and Expression of Human sTNFR1 in E.Coli JM109
- VernacularTitle:人可溶性肿瘤坏死因子受体1基因的克隆及原核表达
- Author:
Lei FU
;
Deming TAN
;
Shifang PENG
- Publication Type:Journal Article
- Keywords:
Human sTNFR1;
Clone;
Prokaryotic expression
- From:
Journal of Chinese Physician
2001;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant plasmid carrying human sTNFR1 cDNA, and express sTNFR1 in E. Coli JM109. Methods Total RNA was extracted from Hela cells, and used as a template to amplify human sTNFR1 cDNA by RT-PCR. The PCR products were cloned into T vector, and then sTNFR1 cDNA fragment was subcloned into a prokaryotic expression plasmid pMAL-c2x. The recombinant plasmid was transferred into E. Coli JM109, and induced by IPTG to express fusion protein sTNFR1-MBP. sTNFR1-MBP was purified by amylose resin affinity chromatography(ARAC), and analyzed by SDS-PAGE. Results A 558 bp human sTNFR1 cDNA was amplified by RT-PCR, and successfully inserted into plasmid pMAL-c2x. sTNFR1-MBP was produced in E.Coli after IPTG induction, and a 66 KD sTNFR1-MBP was purified by ARAC. [WTHZ]Conclusion Recombinant plasmid carrying human sTNFR1 cDNA was successfully constructed and epxressed in E. Coli JM109.
