人食管癌相关基因在大肠杆菌中的表达

Expression in E.coli of protein encoded by human esophageal cancer related gene-1

目的 研究食管癌相关基因 1(ECRG 1)的编码蛋白质在大肠杆菌中的表达 ,进一步制备抗ECRG 1编码蛋白质的多克隆抗体。方法 用逆转录多聚酶链式反应 (RT PCR)方法克隆ECRG 1基因蛋白编码序列。构建该基因的表达质粒 ,在大肠杆菌中诱导表达 ,以Westernblot鉴定。用聚丙烯酰胺凝胶电泳 (SDS PAGE电泳 )纯化的蛋白免疫BALB/C小鼠制备多抗。结果 RT PCR所分离的ECRG 1基因蛋白编码序列 ,经测序证明与 5′ RACE方法钓取的该基因序列相符。Westernblot证实该基因编码蛋白质在大肠杆菌中表达 ,并获得效价为 1∶32 0 0的多克隆抗体。结论 ECRG 1基因编码蛋白质能够在原核细胞中表达 ,并能获得抗ECRG 1基因编码蛋白质的多克隆抗体 。

Objective To express esophageal cancer related gene 1 (ECRG 1) in E.coli.Methods The human ECRG 1 was cloned by RT PCR. Expression plasmid of the ECRG 1/GST π fusion gene was constructed and introduced into E. Coli. The fusion protein was induced to express by ITPG. The recombinant protein was identified by Western blot. BALB/C mice were immunized with the protein purified by SDS PAGE for the preparation of polyclonal antibody.Results DNA sequencing confirmed that the sequence of ECRG 1 was identical to that of the previously obtained by 5′ RACE technique. Expression of the protein in E.coli was verified by Western blot. The polyclonal antibody obtained from immunized BALB/C mice had a title of 1∶3 200.Conclusion The antibody available is useful for the further study of structure and function of the esophageal cancer related protein.