pＥＴ３c衍生载体的构建及应用研究

对原核表达载体pＥＴ３c进行改建，消去载体上非必要的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.coliBL21(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３０.０４％。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，重蛋白具有与参照品一致的促有丝分裂活性。

Constrution andApplication of A pET3c Derivative

The prokaryote expression vector pET3c was modified by destroyingthe EcoRⅠ,HindⅢ restriction enzyme sites and inserting LacZ fragment in the BamH Ⅰ site .The derivative, named as pJN982,not only retained the ability of pET3 c to express exogenous gene efficiently but also had an increased number of fus ion cloning sites from 1 to 7,as well as gained the ability to identity the rec ombinant 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-activit y with the standard bFGF.