Cloning and expression of MHC class I chain-related gene A in E. coli.
- Author:
Yan-Ming HE
1
;
Su-Dan TAO
;
Yan-Ling YING
;
Fa-Ming ZHU
;
Hang-Jun LÜ
;
Li-Xing YAN
Author Information
1. Institute of Blood Transfusion, Blood Center of Zhejiang Province, Key Laboratory for Blood Safety Research Subordinated to Ministry of Health, Hangzhou 310006, Zhejiang Province, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Escherichia coli;
metabolism;
Genetic Vectors;
Histocompatibility Antigens Class I;
genetics;
metabolism;
Humans;
Recombinant Proteins;
genetics;
metabolism
- From:
Journal of Experimental Hematology
2010;18(5):1256-1259
- CountryChina
- Language:Chinese
-
Abstract:
In order to construct prokaryotic expression system of MHC classI chain-related gene A (mica) and purify MICA protein, RNAs were extracted from the peripheral blood samples and mica cDNA fragments were obtained by RT-PCR method. The cDNA for mica was ligated with cloning vector by TOPO method. The recombinant cloning vector and prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct pET-28a-MICA recombinant expression vector, then the pET-28a-MICA vector was transformed and expressed in E. coli BL21 DE3. The recombinant protein was purified by Ni-NTA Spin method. The results showed that the recombinant MICA protein expressed with soluble form in host with pET-28a-MICA vector after IPTG induction. The recombinant target protein was obtained by Ni-NTA spin purification. In conclusion, this study has constructed prokaryotic expression system of mica gene and has purified MICA protein which would help to explore the interaction between MICA and transplantation immunology.
