pET3c衍生载体的构建及应用研究

Constrution and Application of A pET3c Derivative

对原核表达载体 pET3c进行改建 ,消去载体上非必需的EcoRⅠ、HindⅢ限制酶位点 ,然后在克隆位点BamHⅠ处引入LacZ片段 ,经此二步改建后的载体命名为 pJN982。该载体保留了 pET3c高效表达外源基因的能力 ,同时 ,其融合克隆位点由 1个增至 7个 ,并获得以目视法筛选重组子的能力。应用该载体在E coliBL2 1(DE3)pLysS中融合表达牛碱性成纤维细胞生长因子 ,表达量占菌体总蛋白 30 0 4%。破碎菌体上清经阳离子交换和肝素亲和两步层析 ,得纯度为 95 %的重组蛋白 ,活性检测显示 ,重组蛋白具有与参照品一致的促有丝分裂活性。

The prokaryote expression vector pET3c was modified by destroying the EcoRⅠ,HindⅢ restriction enzyme sites and inserting LacZ fragment in the BamHⅠ site The derivative, named as pJN982,not only retained the ability of pET3c to express exogenous gene efficiently but also had an increased number of fusion cloning sites from 1 to 7,as well as gained the ability to identity the recombinant by visual screening of E coli colonies Using pJN982,fused bovine basic fibroblast growth factor(B bFGF) was expressed in E coli BL21(DE3)pLysS to a level of 30 04% of total celluar protein The target protein was purified by cation exchange and heparin affinity chromatography from the supernant of bacteria lysate It was found that the recombinant B bFGF had an identical bio activity with the standard bFGF