柯萨奇病毒B3型VP1基因在酿酒酵母中的表达  

Expression of VP1 gene of Coxsackievirus B3 in Saccharomyces cerevisiae

作　　者：黄孝天[1] 陈宏新[1] 陈洪[1] 吴斌贝 刘晶星[1] 

机构地区：[1]上海第二医科大学病原生物学教研室

出　　处：《中国人兽共患病杂志》2005年第7期558-560,565,共4页Chinese Journal of Zoonoses

摘　　要：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。To express VP1 gene of Coxsackievirus B3 in Saccharomyces cerevisiae AH109, and test DNA-BD/VP1 protein for transcriptional activation of reporter genes,the recombinant plasmid pGBKT7-VP1 containing Coxsackievirus B3 VP1 gene was transformed into yeast strain by the LiAc-mediated method. Phenotype identification, PCR and Western blotting were respectively used to detect VP1 gene and protein. Nutrition-deficient medium and substrate of enzyme X-α-Gal were used to test DNA-BD/VP1 protein for transcriptional activation of reporter genes. Phenotype identification and PCR confirmed that recombination plasmid was successfully transformed into the yeast strain AH109, and western blotting also verified that VP1 protein was successfully expressed in AH109. Nutrition-deficient medium and X-α-Gal assay showed that DNA-BD/VP1 protein can not autonomously activate 3 reporter genes(ADE2, HIS3 and MEL1). CVB3 VP1 protein can be used as a bait protein for the yeast two-hybrid system, which provided a basis for screening proteins of host cells that interact with VP1 protein.

关 键 词：柯萨奇病毒 B3 VP1基因 表达 

分 类 号：R373.22[医药卫生—病原生物学]