关键词: 人角质细胞生长因子2; pBV220载体; pET30a(+)载体; 基因表达; 蛋白纯化

Abstract:

Objective To explore expressions of hKGF2 （human keratinocyte growth factor 2）in different prokaryotic vectors and host bacteria and their corresponding purification and bioactivity. Methods  The mature hKGF2 gene was amplified by RT-PCR from cultured human embryonic lung fibroblast. After DNA cloning and sequencing, the hKGF2 gene was ligated with pBV220 and pET-30a(+)， and then transformed into different host bacteria. The expressed protein was determined by SDS-PAGE and Western blot. The protein in different vectors was purified by cation-exchange chromatography and Ni-affinity chromatograph,  and determined by electrophoretic analysis, followed by bioactivity detection with MTT. Results The expression level of hKGF2 in pBV220 was highest when transformed into E.coli DH5α, which amounted to 15% of total bacterial proteins and the target protein level in pET-30a(+) transformed into E.coli BL21（DE3）reached 25%. The purity of the target protein was over 95% and all purified proteins could promote the proliferation of human embryonic kidney epithelial cells. Conclusions The purified protein yield of hKGF2 in E.coli BL21(DE3)/pET30a(+) is significantly higher than that in E.coli JM109/pBV220.

Key words: Human keratinocyte growth factor 2; pBV220 vector; pET30a(+)vector; Gene expression; Protein purification