猪瘟病毒酵母双杂交pGBKT7-NS3诱饵载体的构建与鉴定

Construction and Identification of CSFV pGBKT7-NS3 Bait Vector in Yeast Two-hybrid System

利用RT-PCR技术扩增获得猪瘟病毒NS3基因片段,将其克隆至酵母双杂交系统诱饵载体pGBKT7中,经酶切,测序验证其正确插入后,将重组诱饵质粒转化酵母菌Y2HGold,检测其在酵母中毒性和自我激活作用,并利用Western blot分析诱饵载体在酵母中的转化情况。结果表明,pGBKT7-NS3诱饵载体在Y2HGold酵母细胞中可以表达,此载体在酵母细胞Y2HGold中无毒性和自我激活能力,研究结果为使用酵母双杂交技术筛选相互作用蛋白奠定了基础。

The NS3 gene of classical swine fever virus was amplified by RT-PCR and confirmed by sequencing and enzyme digestion.The NS3 gene was then subcloned into pGBKT7 to construct the bait vector according to the reading frame.The reconstructed bait vector pGBKT7-NS3 was transformed into Y2 HGold yeast strain,its self-activation and toxic effect were tested by the phenotype assay.The expression of pGBKT7-NS3 was analysed by Western blot.As a result,the successfully reconstructed bait vector pGBKT7-NS3 would not self-activitate the reporter gene and had no toxic effect to the yeast,which laid the foundation for using yeast two-hybrid assay to screen interaction proteins.