论文部分内容阅读
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。
Supervillin (SVIL) is a membrane-bound protein molecule localized to the focal adhesions on the cell-extracellular matrix interface. In order to further study the function of SVIL protein in cells, molecular cloning techniques were used to construct prokaryotic expression plasmids pGEXKG-SVILC302 and pHIS8-SVILC302. IPTG was used to induce expression of GST-SVILC302 and HIS- SVILC302 fusion protein. GST-SVILC302 protein was purified by glutathione sepharose column affinity chromatography, and then immunized New Zealand white rabbits to prepare SVIL polyclonal antibody, and Ni-NTA column with HIS-SVILC302 protein, its cross-linking, purified antibodies . Western Blot (WB) and Immunofluorescence (IF) experiments show that the antibody can recognize exogenous GFP-SVIL and endogenous SVIL protein, and can be used in protein immunoprecipitation experiments. This SVIL polyclonal antibody has high specificity and sensitivity, which laid a solid foundation for further study of the function of the vascularized protein SVIL in the cell.