人GRP78蛋白的原核表达、纯化及抗原活性鉴定  

Prokaryotic expression, purification and antigenicity identification of human GRP78 protein

作　　者：宋慧娟[1] 李宏丹[1] 魏嘉[1] 苏荣健[1] 

机构地区：[1]辽宁医学院辽宁省高校分子细胞生物学与新药开发重点实验室,辽宁锦州121001

出　　处：《细胞与分子免疫学杂志》2011年第10期1079-1082,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：辽宁省科技攻关项目(2008225010-17)

摘　　要：目的:构建原核表达质粒pGEX-4T-1-GRP78,诱导表达、纯化后检测GRP78蛋白的抗原活性。方法:利用PCR方法从本实验室已构建的真核表达载体pcDNA3.1(+)上扩增GRP78基因,将其克隆至原核表达载体pGEX-4T-1,构建重组载体pGEX-4T-1-GRP78。转化至大肠杆菌BL21(DE3)中诱导表达,再将所表达的融合蛋白进行纯化。经SDS-PAGE分析后,将所得产物用Thrombin切割;进一步包板后用ELISA法对其抗原活性进行评价。结果:酶切和测序结果均证实GRP78原核表达载体构建正确,可诱导表达;ELISA检测显示纯化后的人GRP78抗原具有免疫原性。结论:成功构建了人GRP78基因的原核表达载体,获得了纯化的GRP78蛋白,该蛋白具有良好的抗原活性,为进一步研究以GRP78为基础的肝细胞癌的血清学检测提供了抗原。AIM： To construct a recombinant plasmid pGEX-4T-1-GRP78, express it and purify human glucose regulated protein 78 （GRP78）. METHODS： GRP78 gene was amplified by PCR from the recombinant vector constructed in our laboratory. The PCR product was inserted into pGEX-4T-1 prokaryotic expression vector to generate pGEX-4T-1-GRP78. The pGEX-4T-1-GRP78 was then transformed into BL21 and GRP78 was expressed on induction of IPTG. After purification, GRP78 was released by thrombin cleavage, and its antigenicity was identified by ELISA. RESULTS： The GRP78 prokaryotic expression vector was successfully constructed as confirmed by enzyme digestion and DNA sequencing. ELISA demonstrated the antigenicity of the purified GRP78 protein. CONCLUSION： The recombinant prokaryotic expression vector pGEX-4T-1-GRP78 has been constructed successfully. The purified GRP78 has been obtained with good antigenicity, which will be used for GRP78-based serum diagnosis of hepatocellular carcinoma.

关 键 词：GRP78 原核表达 蛋白纯化 肝细胞癌 

分 类 号：R392.1[医药卫生—免疫学]