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.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
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)~~
