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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
引用本文:林建炜,李婧,刘平,郭风劲.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[J].第四军医大学学报,2009(22):2533-2536.
作者姓名:林建炜  李婧  刘平  郭风劲
作者单位:重庆医科大学分子医学与肿瘤研究中心;重庆医科大学细胞生物学及遗传学教研室;
基金项目:重庆市科委自然科学基金(CSTC2009BB5062); 重庆市教委基金(KJ070314); 重庆医科大学重点项目(XBZD200803)
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XBP1多克隆抗体的制备.方法:重组质粒pET32a-XBP1u转化入宿主菌E.coliBL2l(DE3),在IPTG诱导下表达,经SDS-PAGE鉴定正确后,用Ni2+-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∶64000,免疫印迹结果出现特异Mr33×103XBP1u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XBP1u蛋白,并得到高特异性、高效价兔抗XBP1u多克隆抗体.

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

Fusion expression of human X-box binding protein 1 and preparation of polyclonal antibody against protein
LIN Jian-Wei,LI Jing,LIU Ping,GUO Feng-Jin, Molecular Medicine & Cancer Research Center.Fusion expression of human X-box binding protein 1 and preparation of polyclonal antibody against protein[J].Journal of the Fourth Military Medical University,2009(22):2533-2536.
Authors:LIN Jian-Wei  LI Jing  LIU Ping  GUO Feng-Jin  Molecular Medicine & Cancer Research Center
Institution:LIN Jian-Wei1,LI Jing1,LIU Ping1,GUO Feng-Jin1,2 1Molecular Medicine & Cancer Research Center,2Department of Cell Biology and Genetics,Chongqing Medical University,Chongqing 400016,China
Abstract:AIM:To express prokaryotically and purify the unsplicedhuman X-box binding protein1(XBP1u)recombinant plasmid and prepare polyclonal antibody against protein.METHORDS:We transformed the recombinant plasmid pET32a-XBP1u into host bacterium E.coli BL21(DE3),induced its expressionwith IPTG,and purified the protein with Ni2+-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...
Keywords:XBP1u  titer  purification  polyclonal antibody  Western Blotting  
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