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目的在酿酒酵母AH109中表达柯萨奇病毒B3型VP1基因,并检测VP1蛋白对报告基因的转录自激活作用。方法用乙酸锂法将重组柯萨奇B3病毒VP1基因的细菌-酵母菌穿梭载体pGBKT7-VP1转化酵母菌。通过表型鉴定、PCR法验证质粒导入宿主菌后,Westernblotting验证VP1蛋白的表达,最后经营养缺陷培养基和酶底物X--αGal反应检测VP1基因对报告基因的转录自激活作用。结果表型鉴定和PCR法均证实pGBKT7-VP1质粒转入酵母菌成功,Westernblotting证实AH109能表达VP1蛋白,营养缺陷型培养基和酶底物X--αGal反应结果显示VP1蛋白对3种报告基因均无转录自激活作用。结论柯萨奇病毒B3型VP1蛋白可作为酵母双杂交系统的诱饵蛋白,为筛选与其相互作用的宿主细胞蛋白提供了基础。
Objective To express Coxsackievirus B3 VP1 gene in Saccharomyces cerevisiae AH109 and detect the transcriptional activation of VP1 protein. Methods Saccharomyces cerevisiae shuttle vector pGBKT7-VP1 of VP1 gene of recombinant Coxsackie virus B3 was transformed into yeast by lithium acetate method. The expression of VP1 protein was verified by Western blot after the plasmid was introduced into host bacteria by PCR and the transcriptional activation of VP1 gene was detected by X - αGal reaction of auxotrophic medium and enzyme substrate. Results The results of phenotyping and PCR confirmed the successful transformation of pGBKT7 - VP1 into yeast. Western blotting confirmed that AH109 could express VP1 protein. The results of auxin - deficient medium and enzyme substrate X - αGal showed that VP1 protein had no effect on the three reporter genes No transcriptional activation. Conclusion Coxsackievirus B3 VP1 protein can be used as bait protein in yeast two-hybrid system, which provides a basis for screening host cell proteins that interact with it.