细胞与分子免疫学杂志 , Chinese Journal of Cellular and Molecular Immunology, 编辑部邮箱 , 2011年10期 【作者】 宋慧娟; 李宏丹; 魏嘉; 苏荣健;
【Author】 SONG Hui-juan, LI Hong-dan, WEI Jia, SU Rong-jian* High Educational Key Laboratory of Molecular Cell Biology and Drug Research , Liaoning Medical University, Jinzhou 121001, China
【机构】 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;
【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。 更多还原
【Abstract】 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 antigeni... 更多还原 【关键词】 GRP78; 原核表达; 蛋白纯化; 肝细胞癌; 【Key words】 glucose regulated protein 78; prokaryotic expression; protein purification; HCC; 【基金】 辽宁省科技攻关项目(2008225010-17)【分类号】R392.1
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