人肝脏再生增强因子cDNA的克隆、表达及纯化

克隆人肝脏再生增强因子（ｈＡＬＲ）ｃＤＲＮＡ，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ｈＡＬＲｃＤＮＡ，克隆于载体ｐＧＥＭ－Ｔ，酶切鉴定后亚克隆至表达载体ｐＧＥＸ－４Ｔ－３，测序证实序列正确并转化大肠杆菌ＢＬ２１（ＤＥ３），ＩＰＴＧ诱导表达融合蛋白ＧＳＴ－ｈＡＬＲ，表达物通过谷胱甘肽Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化后进行Ｔｈｒｏｍｂｉｎ酶切，

Cloning,expression and purification of the human augmenter of liver regeneration (hALR) cDNA

Objective: To clone human augmenter of liver regeneration (hALR) cDNA, construct the recombinant expression vector, express and purify its product. Methods: The hALR cDNA was obtained by using RT PCR method with total RNA extracted from the fetal hepatic tissue. Then it was cloned into the pGEM T vector, and subcloned into expression vector pGEX 4T 3.After proved to be correct by sequencing, recombinant expression plasmid pGEX 4T 3(hALR) was transformed into E.coli BL21(DE3). The fusion protein GST hALR was produced by IPTG induction, isolated by affinity chromatography glutathione Sepharose 4B and cleaved by Thrombin. Results and Conclusion: Recombinant expression plasmid pGEX 4T 3(hALR) is constructed. The hALR is highly expressed in E.coli . The fusion protein in the plasma is resoluble. The procedure of purification and cleavage of fusion protein is easy and simple.