HCK SH3结构域与HIV-1 Nef蛋白体外结合活性的检测

目的构建重组原核表达质粒pGEX-4T-1/SH3,检测原核表达的SH3蛋白的生物学活性。方法利用PCR方法扩增HCK SH3基因,并将其克隆到原核表达载体pGEX-4T-1中,构建原核表达质粒pGEX-4T-1/SH3,转化E.coli DH5α,进行双酶切和测序鉴定。筛选阳性质粒,转化至E.coli BL21(DE3)中进行表达,对表达产物进行SDS-PAGE和蛋白质印迹检测,同时纯化SH3蛋白。表达并纯化HIV-1 Nef蛋白,利用GST pull-down方法检测SH3蛋白与Nef蛋白的结合活性。结果成功获得重组质粒pGEX-4T-1/SH3,测序正确,高效表达并纯化了SH3蛋白。GST pull-down实验结果显示SH3与HIV-1 Nef蛋白具有良好的结合活性。结论成功地克隆、表达和纯化了GST-SH3蛋白,SH3与Nef蛋白具有体外特异性结合活性,为进一步研究针对Nef与SH3结合的靶向药物的筛选提供了实验基础。

In vitro binding activity between HCK SH3 domain and HIV-1 Nef protein

ObjectiveTo construct prokaryotic expression plasmid pGEX-4T-1/SH3 and to examine the biological activity of the expressed SH3 protein. MethodsHCK SH3 gene was amplified by PCR and was cloned into the vector pGEX-4T-1 to construct prokaryotic expression plasmid pGEX-4T-1/SH3. The recombinant plasmid was identified by double enzyme digestion and sequencing, the positive plasmid was transformed into E. coli BL21 (DE3),and the expressed product was identified by SDS-PAGE electrophoresis and Western blotting analysis. HCK SH3 and HIV-1 Nef proteins were purified and their binding activity was detected by GST pull-down assay. ResultsThe recombinant plasmid pGEX-4T-1/SH3 was correctly constructed. SH3 protein was expressed and purified.GST pull-down assay showed that SH3 protein had a satisfactory binding activity to HIV-1 Nef protein.ConclusionWe have successfully expressed and purified GST-SH3 protein. Nef protein and SH3 proteins have a specific binding activity, which paves a way for screening drugs targeting Nef and SH3.