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目的 :克隆人肝脏再生增强因子 (h AL R) c DNA,构建重组表达载体并对其诱导表达、纯化。 方法 :提取胎儿肝组织总 RNA,利用 RT- PCR技术扩增出 h AL R c DNA,克隆于载体 p GEM- T,酶切鉴定后亚克隆至表达载体 p GEX- 4T- 3,测序证实序列正确并转化大肠杆菌 BL 2 1(DE3) ,IPTG诱导表达融合蛋白 GST- h AL R,表达产物通过谷胱甘肽 Sepharose 4B亲和层析纯化后进行 Throm bin酶切。 结果和结论 :构建成融合蛋白 GST- h AL R的重组表达质粒 ,h AL R在大肠杆菌高效表达 ,重组蛋白主要以可溶形式存在于胞质中 ,融合蛋白的分离纯化简单易行。
OBJECTIVE: To clone h AL R c DNA of human liver and construct a recombinant expression vector for its expression and purification. Methods: Total RNA was extracted from fetal liver tissue. H AL R c DNA was amplified by RT-PCR and cloned into vector p GEM-T. After restriction analysis, the recombinant plasmid was subcloned into expression vector pGEX-4T-3. Sequencing The recombinant plasmid was transformed into E. coli BL21 (DE3). The fusion protein GST-h AL R was induced by IPTG. The expressed product was purified by glutathione Sepharose 4B affinity chromatography and digested with Thrombin. RESULTS AND CONCLUSION: The recombinant expression plasmid hST constructed by fusion protein GST-h AL R was highly expressed in E. coli. The recombinant protein was mainly existed in soluble form in the cytoplasm. The isolation and purification of the fusion protein was simple and easy.