CAJ | 학술논문

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。
점착반단백Supervillin（ SVIL）시일개세포막결합단백분자，정위우세포여세포외기질접촉면적점착반。위료진일보연구SVIL단백분자재세포내적공능，응용분자극륭기술구건원핵세포표체질립pGEXKG-SVILC302화pHIS8-SVILC302，병재세균BL-21（DE3）중용IPTG분별유도표체GST-SVILC302화HIS-SVILC302융합단백。용곡광감태경지당응효주친화층석순화GST-SVILC302단백，연후면역신서란대백토제비SVIL다극륭항체，병용Ni-NTA주자결합HIS-SVILC302단백，대기진행교련，순화항체。 Western Blot（WB）화Immunofluorescence （ IF）실험증명해항체가이식별외원적GFP-SVIL화내원적SVIL단백，병차가이용우단백면역침정실험。표명차SVIL다극륭항체구유교고적특이성화령민도，위진일보연구점착반단백SVIL재세포내적공능전정료견실적기출。
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 pHIS8-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.