摘要
用Ncol Ⅰ和EcoRI双酶酶切,获得人酸性成纤维细胞生长因子haFGF基因,将该基因片断连接进入pBV220的EcoRI位点。连接物转化入大肠杆菌RR1,用菌落原位杂交的方法筛选出杂交阳性菌株,用酶切和Southern Blot的方法确定haFGF的插入与否以及插入方向的正反,结果我们得到了haFGF正向插入在pBV220 PLPR启动子下游的重组质粒pBV-αFGF。42℃热诱导使重组子表达,其菌体的裂解液对3T3细胞的DNA合成有较强的促进作用,说明重组菌株能够表达具有生物活性的hαFGF。SDS-PAGE结果表明,hαFGF表达产物约占菌体总蛋白的15％。hαFGF表达产物以包涵体的形式存在。
hαFGF gene was inserted into EcoRI site of plasmid pBV220. The recombinant plasmids were identif ed by in situ colony hybridization, enzyme digestion and Southern blot hybridization. Finally,the plasmid pBV220-αFGF in which haFGF gene was located immediately after the PLPR promoter in right direction has been obtained. The bacteria that contain the plasmid pBV-αFGF was treated by 42℃ heat induction, and its lysates had strong effect to stimulate the DNA synthesis in 3T3 cell. SDS-PAGE showed that the haFGF gene expression product is about 15% of the total bacterial proteins.
基金
国家自然科学基金389720200号
