Preparation and functional identification of human high mobility group box-1 protein
- VernacularTitle:人HMGB1的制备及功能鉴定
- Author:
Xiaoru XING
;
Fengtian HE
;
Zhaohui YANG
;
Rongfen LI
;
Yingru ZHENG
;
Huiguang GAO
;
Song LI
;
Yan ZHANG
;
Li ZHANG
- Publication Type:Journal Article
- Keywords:
HMGB1;
prokaryotic expression;
purification;
TNF-?
- From:Journal of Third Military Medical University
2003;0(19):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone the cDNA encoding human HMGB1, express it in E. coli, and identify its biological activity. Methods Human HMGB1 cDNA was amplified by RT-PCR and cloned into vector pUC19. After sequence analysis, the cDNA was ligated into prokaryotic expression vector pQE-80L and induced by IPTG to express HMGB1. The protein was purified with Ni~(2+)-NTA chromatography and polymyxin B affinity column. To identify the function of purified protein, the product was co-cultured with THP1 cells. Results Recombinant expression plasmid pQE-80L/HMGB1 was constructed successfully. After purification, the protein purity reached 96%. The recombinant HMGB1 stimulated THP1 to secrete TNF-? . Conclusion The highly purified HMGB1 was obtained successfully, which showed biological activity. These results lay the foundation for further research on the function of human HMGB1.
