草鱼胰岛素样生长因子-Ⅰ基因在大肠杆菌中的表达

 为构建草鱼 (Ctenopharyngodonidellus )胰岛素样生长因子 Ⅰ (IGF Ⅰ )大肠杆菌表达质粒 ,对已克隆到的草鱼IGF Ⅰ基因进行改造 .改造后的基因去除了原cDNA的信号肽和E区序列 ,并在基因的两端分别加入起始密码子和终止密码子 .将改造后的编码草鱼IGF Ⅰ成熟肽基因亚克隆到pBV 2 2 0中 ,构建成表达质粒pBVgIGF7.转化大肠杆菌 (Escherichiacoli)进行表达 .SDS PAGE显示 ,含重组表达质粒的菌株经热诱导后表达出一约 7 5kD的特异蛋白 .表达量占菌体总蛋白的2 0 0 3% ,表达产物主要以包涵体形式存在 .重组蛋白经纯化和复性后 ,采用MTT法测定其对草鱼吻端成纤维细胞PSF和草鱼卵巢细胞CO的促增殖作用 .结果表明 ,所获得的重组草鱼IGF Ⅰ具有生物活性

Expression of Grass Carp (Ctenopharyngodon idellus ) Insulin-like Growth Factor- Ⅰ in E. coli

In order to construct expression vector of grass carp( Ctenopharyngodon idellus )IGF Ⅰ, the cloned grass carp IGF Ⅰgene was modified. The signal peptide and E domain were deleted while one start code ATG and two stop codes were added. The modified gene encoding the mature peptide of grass carp IGF Ⅰwas subcloned into pBV220 to construct expression vector pBVgIGF 7. The vector was then transformed into E.coli. SDS PAGE analysis showed that the bacteria containing the recombinant vector produced a special protein band about 7 5 kD in molecular weight. The expression level reached up to 20 03% of the total soluble bacterial proteins. The recombinant protein was mainly in the form of inclusion body. The purified and renatured recombinant protein was used to study its effect on proliferation of both PSF and CO cell lines of grass carp. The results showed that the recombinant grass carp IGF Ⅰhad bioactivity.