人Bcl-2蛋白的基因克隆、表达及初步活性分析

目的：以特定形式对高度疏水性的人Bcl-2(hBcl-2)蛋白进行可溶性表达，获得非疏水性hBcl-2蛋白并具备生物学结合活性，为进一步研究hBcl-2三维结构并在此基础上研发特异性小分子药物提供充足的蛋白样品。方法：用PCR方法对hBcl-2基因进行克隆重组，插入原核表达载体pET28a( )中。用Western Blot和MALDI-TOF生物质谱鉴定，采用荧光极化方法进行活性测定。结果：重组hBcl-2在E．Coli中实现了高效、可溶性的融合表达，重组蛋白经纯化至电泳纯，具备了与Bcl-2家族Bak蛋白BH3功能域特异结合的能力，表明原核表达的重组hBcl-2具有较好的结合活性。结论：成功地对重组hBcl-2蛋白进行了可溶性表达和活性测定。

Gene cloning,expression and preliminary activity analysis of hBcl-2

Objective:Through gene cloning,expression and activity analysis of hBcl 2 to provide enough proteins for the development of new drugs on the basis of 3 D structue of hBcl 2 protein.Methods:Gene cloning using PCR amplification,identification with Western Blot and MALDI TOF MS.Results:hBcl 2 gene was cloned,inserted into pET28a(+) and solublely expressed in E.Coli.Its molecule weight was confirmed through MALDI TOF MS,which fits exactly with its theoretical value.After purification to reach electrophoresis homogeneity,it showed the ability of combining specifically with BH 3 domain of Bak,and then provide the basis for the further research on small chemical compounds which could specifically bind hBcl 2 protein.Conclusion:In this work hBcl 2 was successfully expressed and recovered its binding activity in a soluble form. [