鸡贫血病毒VP1、VP2和VP3基因酵母双杂交载体的构建及其自激活活性的鉴定

Construction of "bait vectors" with VP1,VP2 and VP3 genes of CAV and identification of its self-activation in yeast two-hybrid system

为构建鸡贫血病毒(CAV)VP1、VP2、VP3基因酵母双杂交载体并鉴定其自激活作用,从CAV细胞传代毒M9905毒株中提取基因组DNA,利用PCR分别扩增VP1、VP2和VP3基因,采用Gateway技术将VP1、VP2和VP3基因分别克隆到酵母双杂交载体pDEST32和pDEST22中,构建了诱饵载体及猎物载体,经酶切和PCR鉴定且测序正确后,用醋酸锂法转化酵母菌株Mav203,通过缺陷型平板SD/-Leu/-Trp/-His和SD/-Leu/-Trp/-Ura筛选以及LacZ报告基因检测载体有无自激活作用。结果表明,成功构建了诱饵载体pDEST32-VP1/VP2/VP3和捕获载体pDEST22-VP1/VP2/VP3,并证明其VP1、VP2和VP3蛋白在酵母双杂交系统中无自激活作用。研究结果为下一步利用酵母双杂交系统探索CAVVP1、VP2、VP3蛋白之间的相互作用奠定了基础。

To construct the yeast two-hybrid bait vectors with VP1,VP2 and VP3 genes of chicken anemia virus（CAV） and identify their self-activations,the VP1,VP2 and VP3 genes were amplified by PCR from the CAV M9905 strain total genomic DNA and then cloned them into pDEST32 and pDEST22 vectors using Gateway technology,respectively.The restriction enzyme digestion,PCR and sequence analysis were performed to confirm the fact that those genes had been inserted successfully into the vectors,then pDEST32-VP1/VP2/VP3,pDEST22,pDEST22-VP1/VP2/VP3 and pDEST32 were co-transformed into the yeast strain Mav203 by PE g/LiAC method and amplified by yeast in 3AT plates with SC/-Leu/-Trp/-His,SC/-Leu/-Trp/-Ura and YPAD with NC filter and their self-activations were tested by both selective plate and X-gal assay.The yeast two-hybrid tests showed that Mav203 transfected with the vectors did not grow in plates containing SD/-Leu/-Trp/-His with 3AT concentration higher than 30 mmol/L or in SD/-Leu/-Trp/-Ura plates,and the situation also happened in the X-gal color assay,indicating that the bait plasmid pDEST32-VP1/VP2/VP3 had no autonomous activation,therefore could serve as bait vector in researching the interactions of the three proteins of CAV.