Molecular cloning of human FL gene and its expression in E.coli
- VernacularTitle:FL基因的克隆及在大肠杆菌中表达
- Author:
Huaiping ZHU
;
Zimin SUN
;
Jian WANG
;
Haiming DAI
;
Jingsheng WU
- Publication Type:Journal Article
- From:
Chinese Journal of Biochemical Pharmaceutics
2001;22(3):109-111
- CountryChina
- Language:Chinese
-
Abstract:
Purpose The aim is to obtain the cDNA sequence of encoding extramembrane human FL gene with high level expression in E.coli. Methods The primers were designed based on the known FL cDNA sequence. The total RNA was isolated from fetal liver cells , and then RT-PCR was performed. The fragment was cloned into pUC-18T vector, and further sequenced by automatic sequence analyzer. The gene was inserted into GST fusion expression vector between BamH Ⅰ and EcoR Ⅰ sites. The recombinant plasmid was transformed into E.coli strain DH5 α and induced with 1mmol/L IPTG.Results The 546bp DNA fragment was amplified by RT-PCR method from fetal liver cells and its sequence was identical to the published sequence encoding human FL. The expressed fusion protein, with molecular weight of about 22kD, was about 10% of the total bacteria protein by SDS-PAGE and densitometry analysis.Conclusion cDNA was cloned successfully. This study provided a basis for the further fundamental research and clinical application of FL.
