人类X盒结合蛋白1的融合表达、纯化及多克隆抗体的制备
Fusion expression of human X-box binding protein 1 and preparation of polyclonal antibody against protein
林建炜 1李婧 1刘平 1郭风劲2
作者信息
- 1. 重庆医科大学,分子医学与肿瘤研究中心,重庆,400016
- 2. 重庆医科大学,分子医学与肿瘤研究中心,重庆,400016;重庆医科大学,细胞生物学及遗传学教研室,重庆,400016
- 折叠
摘要
目的:将以构建的未剪接型x盒结合蛋白1(XBP1u)的原核表达,纯化及人XBPI多克隆抗体的制备.方法:重组质粒pET32a-XBP1u转化入宿主菌E.coli BL21(DE3),在IPTG诱导下表达,经SDS-PAGE鉴定正确后,用Ni~(2+)-NTA柱式亲和层析法纯化蛋白,将纯化后的目的蛋白经透析袋复性后进行蛋白质定量,再以此蛋白作为抗原免疫新西兰大白兔,末次免疫后心脏取血,采用ELISA法检测抗血清效价;免疫印迹和免疫组化法检测兔抗XBP1u血清的特异性.结果:XBP1u蛋白为包涵体表达,包涵体裂解后经过纯化、复性进行免疫,得到抗血清的效价大于1:64 000,免疫印迹结果出现特异Mr 33×10~3 XBPIU条带,免疫组化法在兔肝细胞中成功检测到XBP1u的表达.结论:成功表达和纯化人XBPIu蛋白,并得到高特异性、高效价兔抗XBP1u多克隆抗体.
Abstract
AIM: To express prokaryotically and purify the unspliced human X-box binding protein1 (XBP1u) recombinant plasmid and prepare polyclonal antibody against protein. METHORDS: We transformed the recombinant plasmid pET32aXBPIu into host bacterium E. coli BL21 (DE3), induced its expression with IPTG, and purified the protein with Ni~(2+) -NTA collumn after identified by SDS-PAGE, then immunized two New Zealand rabbits with the purified protein as antigen after renaturation with dialytic bag, then collected blood from the heart after the last immunization, detected the titer of antiserum by ELISA, detected the antiserum specificity by Western Blot and immunohistochemistry. RESULTS : XBP1u expressed in the inclusion body of E. coli BL21, the titer of antiserum was beyond 1:64 000 after immunize rabbit with purification and renaturation XBP1 u protein. Besides, it is confirmed that the antiserum reacted specifically to the XBPlu protein which is Mr 33 x 10~3 by Western Blot. It is successful to detect the expression in hepatocyte with immunohistochemistry. CONCLUSION: It is successful to induce XBPIu expression and purification. The highly specific and titer polycional antibody have been obtained on basis of this purified protein.
关键词
未剪接型X盒结合蛋白1/效价/纯化/多克隆抗体/免疫印迹Key words
XBPiu/titer/purification/polyclonal antibody/Western Blotting引用本文复制引用
基金项目
出版年
2009
NETL
NSTL
维普
万方数据