肝素结合生长因子Midkine基因在大肠杆菌中的克隆、表达和纯化

The cloning,expression and purification of cDNA in E.coli

目的:用基因工程的方法构建、表达和纯化人肝素结合生长因子humanMidkine(hMK),并进行活性测定。方法:利用RTPCR技术从胎儿肾组织中扩增MK,将去除信号肽序列的hMK插入pET30a,构建成表达载体pEThMK,经表达和亲和层析、纯化获得目的蛋白,3HTdR法测定活性。结果:hMK序列与Genebank发表的人MK基因编码序列一致,SDS-PAGE电泳显示表达出目的蛋白,3HTdR法测定有良好的活性。结论:人MK已被转入表达载体中,并在大肠杆菌中诱导表达,获得了hMK的表达菌株。

Objective：The human heparin-binding growth factor Midkine（hMK） gene was constructed and expressed in E. coli, and the activity of MK protein was tested. Methods ：The MK gene was amplificated from the tissue of human embryo kidney by RTPCR techniques. The MK fragment without signal peptide was inserted into plasmid pET30a. The gene was expressed in E. coli, the protein was purificated by affinity chromatography, and the activity was tested by 3^H-TdR method. Results：The sequence of hMK in our experiment was as same as the sequence published in Genebank. The activity assay was shown high activity of MK protein in our experiment. Conclusion ： The expression cartier of hMK was established successful and it can expression high activity hMK protein in E. coli.