人内皮活化相关新基因eola1的原核表达及鉴定

Identification of prokaryotic expression of a human eola1 gene in E. coli

目的 获得内皮高表达脂多糖相关因子 1(eola1)表达蛋白并对其进行分析鉴定。方法 采用反转录聚合酶链反应 (RT PCR)的方法从人ECV 3 0 4细胞中扩增出eola1基因开放阅读框 ,构建重组质粒 ,测序鉴定后转化大肠杆菌 ,IPTG诱导表达 ,裂解细菌抽提蛋白 ,目的蛋白进行聚丙烯酰胺凝胶电泳 (SDS PAGE)、Western免疫印迹鉴定。结果 测序鉴定eola1开放阅读框成功插入PET 46 EK LIC质粒 ;重组质粒在大肠杆菌中成功表达人eola1蛋白 ,主要以包涵体形式存在 ,聚丙烯酰胺凝胶电泳 (SDS PAGE)测定其分子质量约 2 0× 10 3,Westernblot验证出目的蛋白特异表达。结论 应用原核表达系统能够成功获得目的蛋白 ,为下一步制备eola1单克隆抗体及基因功能研究打下基础。

Objective To obtain and identify the expression product of human eola1 gene in E. coli. Methods The open reading frame of human eola1 was obtained by RT-PCR from human ECV-304 cells, and then inserted into PET-46-EK/LIC vector. Recombinant plasmid was identified by colony PCR and transformed into BL21 (DE3) pLysS and induced by IPTG for expressing protein. The expression product was analyzed by SDS-PAGE and Western blotting. Results The recombinant plasmid was successfully constructed and identified by sequencing. The expressed protein in E. coli mainly existed in the form of inclusion body. SDS-PAGE analysis and Western blotting revealed that the molecular weight of eola1 was about 20×103. Conclusion We have successfully obtained the eola1 protein by PET system, which lays the foundation for the preparation of monoclonal antibodies and for the functional studies of eola1.