帕金森病相关蛋白α-共核蛋白的可溶性表达、纯化及抗体制备  

Soluble expression and purification of human neuroprotein α-synuclein related to Parkinson's disease and its antibody preparation

作　　者：黄银霞[1] 韩俊[1] 张晓光[1] 姚海兰[1] 王小凡[1] 张瑾[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：《中国老年学杂志》2006年第2期201-204,共4页Chinese Journal of Gerontology

基　　金：国家自然科学基金委项目(30070038);国家自然科学基金委重点项目(30130070);国家高科技研究发展计划(863计划;2001AA215391);欧盟项目(QLRT200001441);国家科技攻关计划项目(2003BA712A04-02)

摘　　要：目的利用分子克隆技术在原核细胞中表达α-共核蛋白(SNCP),并制备兔多克隆抗体。方法从人神经母细胞瘤SH-SY5Y细胞中提取总RNA,逆转录成cDNA。利用PCR技术扩增获得SNCP的cDNA序列,测序正确后克隆至pQE-30-GST表达载体上,在大肠埃希菌中表达GST-SNCP融合蛋白。融合蛋白纯化后免疫新西兰大白兔,制备特异性抗血清,并对该抗体进行检测。结果琼脂糖凝胶电泳显示获得的SNCP cD-NA序列为420 bp。SDS-PAGE和W estern b lot分析,表达的融合蛋白呈可溶性,相对分子质量约为45 000。以其为抗原制备的SNCP抗血清的ELISA效价达到1:32000,并且该抗体可与BALB/c小鼠脑组织中内源性SNCP发生特异性反应。结论人SNCP可在原核细胞中可溶性表达。用表达的蛋白制备获得特异性较好的SNCP抗血清,为进一步了解SCNP的结构和功能以及在中枢神经系统退行性疾病的作用提供了必要的实验基础。Objective To express human α-synuclein protein （SNCP） in prokaryotic cells with genetic engineering technique and prepare SNCP specific rabbit polyelonal antibody.Methods Total RNA of a human neuroblastoma cell line SH-SY5Y was extracted and the single-stranded cDNA was synthesizedo With PCR technique, the cDNA sequences encoding SNCP was amplified. After being confirmed by DNA sequence analysis, the full-length SNCP cDNA was cloned into glutathione S transferase （GST） fusion protein expression vector pQE-30-GST, and GST-SNCP fusion protein was expressed in E-coli. New Zealand rabbits were immunized by the purified GST-SNCP proteins to prepare specific antiserum aud detect its antibody. Results A 420 bp α-syuaclein cDNA has been amplified and verified by sequence assay. SDS-PAGE and Western blot assays revealed that the GST-SNCP protein was expressed in soluble form with a rough 45000 relative molecule weight. The immunoreactive titer of the prepared α-syuuclein rabbit＇s aatiserum was evaluated up to 1：32 000 with ELISA. Western blot confirmede that this antibody could specifically recognize the endogennus SNCP in the brain tissues of BALR/c mice. Conclusions Human SNCP has been successfully expressed in soluble form with the prokaryotic expression eystem and the SNCP polyclonal antibody with reliable immunospecificity has been elicited by immunization of the purified SNCP, which provides foundation for further study of SNCP＇s biological functions and pathogenesis of nenrodegeuerative disorders.

关 键 词：Α-共核蛋白 神经退行性变 原核表达 抗体制备 

分 类 号：Q42[生物学—神经生物学] Q786[生物学—生理学]