人朊蛋白相关蛋白Shadoo的表达、纯化及抗体的制备  

Expression and purification of human PrP-like protein Shadoo and preparation of antibody against Shadoo

作　　者：高永军[1] 高晨[1] 陈建明[1] 石琦[1] 郭燕[1] 张宝云[1] 董小平[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052

出　　处：《中国病原生物学杂志》2007年第3期161-164,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30500018;30571672);欧盟;中国及中东欧变异型克雅病监测项目(No.QL-RT200001441);国家科技攻关计划项目(No.2003BA712A04-02)

摘　　要：目的克隆、表达和纯化人朊蛋白相关Shadoo(SHO)蛋白并制备抗Shadoo的多克隆抗体。方法提取仓鼠各组织的mRNA,分析SHO基因在仓鼠各组织中转录水平的差异;提取人DNA,PCR方法获得人SHO基因,克隆至融合表达载体,在大肠埃希菌中表达人SHO蛋白并纯化;以纯化蛋白为抗原免疫家兔制备抗血清,用Western blot方法分别检测与重组及内源性SHO蛋白的免疫反应性。结果SHO基因在仓鼠各组织中的转录水平差异很大,脑组织转录水平高。在大肠埃希菌中表达了分子质量单位为37 ku的人SHO融合蛋白,制备的SHO蛋白抗体可与重组的SHO蛋白反应,可识别内源性的SHO。结论成功表达纯化人SHO融合蛋白并制备了抗SHO抗体,为研究SHO和正常朊蛋白之间的关系打下初步基础。Objective To clone, express and purify human PrP-like protein Shadoo （SHO）, and to prepare polyclonal antibody against SHO. Methods Total RNAs of different tissues of hamster were extracted and the transcription status of SHO gene was comparatively evaluated by a RT-PCR methodology. Full-length human SHO gene was amplified by specific PCR and cloned into a HIS-GST （fusion protein） expression vector after verified by sequence analysis. Human SHO fusion protein was expressed and purified from Escherichia coli SHO-specific antibody was prepared by immunizing rabbits with the purified SHO protein. The immuno-reactivities of the prepared antibody with recombinant and endogenous SHO protein were analyzed with Western blot. Results The transcription level of SHO gene in different tissues varied remarkably. The significant SHO transcripts were observed in brain tissues. SDS-PAGE assays yielded that a roughly 37 ku SHO protein was expressed and purified. The prepared polyclonal antibody against SHO protein reacted well with the genetic engineering expressed and endogenous SHO proteins. ConclusiOn SHO fusion protein has been expressed and purified successfully and this study supplies basis of studying the relationship between SHO and prion protein.

关 键 词：Shadoo 朊蛋白 蛋白纯化 多克隆抗体 

分 类 号：R373.9[医药卫生—病原生物学]