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利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性
The fusion expression vector of human hepatic stimulating factor (hHSS) and glutathione transferase (GST) was constructed by gene recombination technique and transformed into E.coli BL21 (DE3). The recombinant protein was purified by His Tag Affinity Chromatography. FactorXa HHSS monomers were excised and tested for their biological activity. The results showed that hHSS existed in both pET 4 2a and soluble forms of inclusion bodies. The expression of GST hHSS accounted for 30% of the bacterial soluble protein. Factor Xa cut the peptide between GST and hHSS, 15kD two protein bands confirmed by Western blot 3 3kD band GST, and 15kD band molecular weight and hHSS gene sequence conformed that the protein results. HHSS monomer was purified by His / Tag purification again, and preliminary confirmed that recombinant hHSS can promote the proliferation of hepatocellular carcinoma cells