Cloning and prokaryotic expression of rat surfactant protein C in E.coli / 临床儿科杂志

ABSTRACT

Objectives To construct prokaryotic expression plasmid of rat surfactant protein C (sp-c) gene under hyperoxia and expression in E.coli. Methods Twenty-one-day-old SD premature rats were exposed to 85%hyperoxia 12 hours after birth. The rats were executed after 7 days and their RNA were extracted from lung and cDNA was synthesized and amplified. And then the cDNA was cloned into pMD18-T vector and confirmed by enzyme digestion and sequencing. After the prokaryotic expression vector pET-28a(+)-sp-c was constructed, the recombinant plasmid was induced by IPTG and expressed in E.coli BL21 strain. The fusion protein was analyzed by SDS-PAGE and Western blotting. Results The pET-28a(+)-sp-c plasmid was constructed and the fusion protein with relative molecule mass of 21000 was highly expressed. Conclusions SP-C is successfully expressed in E. coli, which can be used to study the mechanism of action between SP-C and endoplasmic reticulum, the expression of SP-C in lung epi-thelial cell A549 and the impact of SP-C on proliferation, differentiation and apoptosis of AECIIs in future.