人肝细胞生长因子cDNA的克隆及其在Pichia系统中的表达

Molecular Cloning of cDNA Gene Encoding Human Hepatocyte Growth Factor and Its Expression with Pichia

采用 RT- PCR方法以胎肝 m RNA为模板克隆人肝细胞生长因子全长 c DNA,核苷酸序列分析证实比文献报道多 2个氨基酸编码序列。经与 Pichia分泌型表达载体 p PIC9重组 ,表达质粒转化 GS115细胞 ,mut+ 表型筛选 ,经甲醇诱导实现了 h HGF在 Pichia Pastoris酵母系统中分泌型表达 ,SDS- PAGE电泳 ,银染法分析证实表达产物分子量为 80～ 85KD左右 ,具有刺激肝细胞增殖活性。

The cDNA encoding human hepatocyte growth factor(hHGF) was amplified by RT PCR using mRNA of fetal liver tissues as template, and the cDNA was cloned with pGEM 3Z. The result of hHGF cDNA sequencing was shown that it contained 6 nucleotides more than that as reported. The hHGF cDNA was inserted into pPIC9 plasmid and got a Pichia expression plasmid. The pPIC9/hHGF plasmid was transfected Pichia GS115 cells. The Mut + transformants were isolated from Mut + and Mut s phenotype screening. In the presence of inducer methanol, the recombinant hHGF was secreted into medium. Two bands with M.W. of 80～85KD were observed on SDS PAGE. The expression products could stimulate proliferation of hepatocytes.