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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。
Objective To construct recombinant prokaryotic expression vector pGEX-4T-1 / SH3 and test the biological activity of prokaryotic expression of SH3 protein. Methods HCK SH3 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The prokaryotic expression plasmid pGEX-4T-1 / SH3 was constructed and transformed into E. coli DH5α for double enzyme digestion and sequencing . Positive plasmids were screened and transformed into E. coli BL21 (DE3) for expression. SDS-PAGE and western blotting were used to detect the expression products, and SH3 protein was also purified. HIV-1 Nef protein was expressed and purified, and the binding activity of SH3 protein to Nef protein was detected by GST pull-down method. Results The recombinant plasmid pGEX-4T-1 / SH3 was successfully obtained and the SH3 protein was expressed and purified efficiently and correctly. GST pull-down results showed that SH3 has good binding activity with HIV-1 Nef protein. Conclusion The GST-SH3 protein was successfully cloned, expressed and purified. SH3 and Nef protein had specific binding activity in vitro, which provided the experimental basis for further screening of targeting drugs targeting Nef and SH3 binding.