黏着斑蛋白Supervillin的抗体制备及纯化

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。ＷｅｓｔｅｒｎＢｌｏｔ（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。

The preparation and purification of Supervillin antibody

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.