人肿瘤蛋白D52重组融合蛋白的原核表达及特性鉴定

Expression and characterization of human tumor protein D52(hTPD52) protein with prokaryotic expression vectors in E.coli

目的:表达人肿瘤蛋白D52(human tumor proteinD52,hTPD52)的重组融合蛋白,并对该蛋白的生物学特性进行鉴定。方法:RT-PCR扩增乳腺癌MCF-7细胞中的hTPD52基因并克隆于pMD18-T载体中,测序鉴定序列正确后,分别插入pET-32a、pGEX-4T-2和pET-28a3种表达载体中。重组质粒经酶切鉴定、序列分析正确后转化大肠杆菌BL21(DE3),选择最佳表达载体和表达条件进行大量表达,表达产物经His柱亲和纯化和超滤浓缩后,应用Dot blot、Western blot鉴定纯化蛋白的特性。结果:正确扩增出了hTPD52基因,鉴定其为isoform3型。用pET-32a-hTPD52重组表达载体能够大量表达可溶性的hTPD52-Trx融合蛋白,Dot blot显示表达的融合蛋白能和抗hTPD52的抗体结合,SDS-PAGE和Western blot显示表达的融合蛋白分子质量约为44ku,去除Trx后仍能够与特异性抗体结合。结论:成功制备hTPD52重组融合蛋白,并证明原核表达的hTPD52保留了原有的抗原结合活性。

Objective：To express and characterize human tumor protein D52 （hTPD52） protein using the proper prokaryotic expression vector in E.coli. Methods：The hTPD52 gene was amplified by RT-PCR from MCF-7 cell line,and cloned into vector pMD18-T. After sequenced, the correct fragment of hTPD52 was inserted into three different prokaryotic expression vectors, which were then transformed into E.coli.BL21 （DE3）,respectively. The optimal expression condition was selected. After induced with IPTG, the recombinant protein was purified with His Trap affinity chromatography, and characterized by Dot blot and Western blot. Results ： The hTPD52 with isoform 3 was amplified and sequenced correctly, and the recombinant expression plasmid was constructed successfully. SDS-PAGE analysis showed that the molecular weight of hTPD52-Trx fusion protein was about 44ku. The purified protein could be used as antigen to detect the specific anti-hTPD52 antibody by Dot blot and Western blot. Conclusion：The recombinant fusion protein of hTPD52 could be expressed with high performance in E.coli,and the antigenicity of hTPD52 protein is retained for the research in the future.