人肌纤生成调节因子1融合蛋白在大肠杆菌的表达和抗体制备  

Expression of a Human Myofibrillogenesis Regulator 1 Gene in E.coli and Its Immunogenicity

作　　者：李天伯[1] 胡洋[1] 冯爽[1] 左增艳[1] 王以光[1] 

机构地区：[1]中国医学科学院中国协和医科大学医药生物技术研究所微生物代谢工程室,北京100050

出　　处：《中国医学科学院学报》2005年第1期42-47,共6页Acta Academiae Medicinae Sinicae

基　　金：北京市科技计划重大项目(H020220020310)~~

摘　　要：目的研究人肌纤生成调节因子1(MR-1)的表达,获得MR-1蛋白,制备MR-1抗体,为MR-1生物功能研究提供基础。方法利用大肠杆菌质粒pGEX-5X-1、pET30a(+)及pET24a(+),分别构建MR-1及其两端与不同标签序列融合的表达载体。在大肠杆菌BL21(DE3)和BL21-CodonPlus(DE3)-RIL中比较N端和/或C端融合标签序列对该基因表达的影响。通过凝胶蛋白电泳及电洗脱制备目的蛋白,免疫家兔。酶联免疫吸咐试验(ELISA)和Westernblot检测所制备抗体的滴度和免疫原性。结果利用GST或T7-tag序列在其N端融合,使MR-1在大肠杆菌BL21-CodonPlus(DE3)-RIL得到表达。利用所表达获得的MR-1-T融合蛋白,制备了针对此蛋白的多克隆抗体。ELISA检测所制备抗体滴度达到1∶105,Westernblot显示所制备的多克隆抗体可用于检测天然细胞中的MR-1蛋白。结论MR-1蛋白需在N端与GST或T7-tag序列融合方可实现表达。利用在大肠杆菌表达纯化的蛋白所制备的抗体可用于MR-1生物学功能的研究。Objective To study the expression of human myofibrillogenesis regulator 1(MR-1)gene in E.coli and obtain the MR-1 protein and its antibody for further investigation of its biological function. Methods Expression vectors pGEX-5X-1, pET30a(+), and pET24a(+), as well as host strain E.coli BL21(DE3)and BL21-CodonPlus(DE3)-RIL were used for expression of MR-1. MR-1 N-terminal with GST or T7-tag or C-terminal with His-tag, separately, or N terminal with T7-tag and C terminal with His-tag, simultaneously, were fused in plasmids pGEX-5X-1, pET30a(+), and pET24a(+). The expressed MR-1-T protein, separated and purified by preparative SDS-PAGE, was applied to immunize the rabbits. The titer of the antibody was assayed by ELISA and its immunogenicity was tested by Western blot with pcDNA3/MR-1 transfected human breast cancer cell MCF7. Results The MR-1 protein was successfully expressed as inclusion body by fusing its N-terminal with T7-tag in E.coli BL21-CodonPlus(DE3)-RIL. MR-1 protein was purified by electro-elution from SDS-PAGE gel. Using this purified protein, polyclonal antibody in rabbit against MR-1 was essentially generated. ELISA and Western blot showed the titer of this antibody was about 1∶105 with high immunogenicity. Conclusions The N-terminal fusion tag is the most important mechanism for MR-1 expression. The polyclonal antibody of the generated MR-1 protein in E.coli may be applied for its further biological function studies.

关 键 词：人肌纤生成调节因子1 融合表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]