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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 prokaryotic expression vector pET3c was modified to eliminate the unnecessary EcoRI and HindIII restriction sites on the vector, and then the LacZ fragment was introduced into the BamHI site of the clone. After two-step transformation, the vector was named as pJN982. This vector retains the ability of pET3c to express foreign genes efficiently and at the same time increases its fusion cloning site from 1 to 7 and screened recombinant for visualization. The vector was used for the fusion expression of bovine basic fibroblast growth factor in E. coli BL21 (DE3) pLysS, which accounted for 30 0 4% of the total bacterial proteins. The supernatant of the broken cell was subjected to cation exchange and heparin affinity two-step chromatography to obtain a recombinant protein with a purity of 95%. The activity test showed that the recombinant protein has the same mitogenic activity as the reference product.