黏着斑蛋白Supervillin的抗体制备及纯化  

The preparation and purification of Supervillin antibody

作　　者：张佟佟 钱积成 朱长军[1] 董智雄[1] 

机构地区：[1]天津师范大学分子细胞系统生物学重点实验室,天津300387

出　　处：《山东大学学报（理学版）》2014年第1期25-30,共6页Journal of Shandong University(Natural Science)

基　　金：国家自然科学基金资助项目(31271485);2011年教育部"新世纪优秀人才支持计划"资助项目(NCET-11-1066);天津市应用基础与前沿技术研究计划重点资助项目(12JC2DJC21400);国家自然科学基金青年基金资助项目(31301138);天津师范大学中青年教授学术创新推进计划资助项目(52XC1001);天津师范大学博士基金资助项目(52XB1104);天津师范大学"渤海学者"基金资助项目

摘　　要：黏着斑蛋白Supervillin(SVIL)是一个细胞膜结合蛋白分子,定位于细胞与细胞外基质接触面的黏着斑。为了进一步研究SVIL蛋白分子在细胞内的功能,应用分子克隆技术构建原核细胞表达质粒pGEXKG-SVILC302和pHIS8-SVILC302,并在细菌BL-21(DE3)中用IPTG分别诱导表达GST-SVILC302和HIS-SVILC302融合蛋白。用谷胱甘肽琼脂糖凝胶柱亲和层析纯化GST-SVILC302蛋白,然后免疫新西兰大白兔制备SVIL多克隆抗体,并用Ni-NTA柱子结合HIS-SVILC302蛋白,对其进行交联,纯化抗体。Western Blot(WB)和Immunofluorescence(IF)实验证明该抗体可以识别外源的GFP-SVIL和内源的SVIL蛋白,并且可以用于蛋白免疫沉淀实验。表明此SVIL多克隆抗体具有较高的特异性和灵敏度,为进一步研究黏着斑蛋白SVIL在细胞内的功能奠定了坚实的基础。Gelling spot protein Supervillin（SVIL） is a membrane protein molecule located in cells and extracellular ma- trix interface adhesive spots. To further study the cellular function of SVIL protein, we constructed plasmids of pGEXKG-SVILC302 and pH1S8-SVILC302 to express recombinant protein GST-SVILC302 and HIS-SVILC302 in E. coli BL21-DE3, respectively. The GST-SVILC302 protein was purified by Glutathione affinity chromatography and used to immune New Zealand white rabbits to generate the polyclonal antibody of SVIL. The HIS-SVILC302 protein was crosslinked with Ni-TNA Beads for affinity purification of the antibody. The results of Western Blot and Immun- oflurescence experiments demonstrated that the antibody can specific recognize exogenous GFP-SVIL and endogenous SVIL protein. Our results showed that anti-SVIL polyclonal antibody we generated has high specificity and sensitivity enough to study the cellular functions of SVIL protein.

关 键 词：SUPERVILLIN 抗体 纯化 

分 类 号：Q78[生物学—分子生物学]