FL基因的克隆及在大肠杆菌中表达

Molecular cloning of human FL gene and its expression in E.coli

目的获得人FLcDNA膜外序列 ,构建表达人FL蛋白的重组体并实现其有效表达 ,更深入地探讨FL的生物学功能。方法提取胎肝细胞总RNA经RT PCR扩增目的cDNA片段 ,并将其克隆至pUC 18T载体中 ,测定其DNA序列 ,将该基因重组于GST融合蛋白表达载体pEGX 4T 1并进行表达。结果从胎肝细胞总RNA中扩增得到 5 46bp片段 ,序列测定结果与文献报道一致 ,表达的FL蛋白占菌体总蛋白的 10 %左右。结论人FL基因在大肠杆菌DH5α中获得了有效表达 ,为进一步的基础研究和临床应用奠定基础。

PurposeThe aim is to obtain the cDNA sequence of encoding extramembrane human FL gene with high level expression in E.coli. MethodsThe 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.ResultsThe 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.ConclusioncDNA was cloned successfully. This study provided a basis for the further fundamental research and clinical application of FL.