鸭圆环病毒Cap基因酵母双杂交诱饵载体的构建及鉴定

Construction and Identification of the Bait Vector Containing Duck Circovirus Cap Gene for the Yeast Two-hybrid System

为构建鸭圆环病毒Cap基因酵母双杂交诱饵载体,以本实验室分离鉴定的DuCV GH01株Cap基因为模板,对其进行酵母密码子优化后,连接到pGBKT7载体上构建诱饵质粒,经菌落PCR、酶切鉴定以及测序后,转化酵母菌株Y2HGold感受态,检测诱饵蛋白表达以及诱饵蛋白对酵母细胞毒性和自激活现象。结果表明,优化后的Cap基因全长774bp,成功连接到诱饵载体pGBKT7中,重组质粒pGBKT7-Cap转入酵母细胞后,Western blot检测到50kD左右的蛋白条带,诱饵蛋白对酵母细胞既无毒性,又没有自激活现象。为进一步利用酵母双杂交技术筛选与Cap蛋白互作的蛋白奠定了基础。

To construct a bait expression vector containing the duck circovirus Cap gene for use in the yeast two-hybrid system,the whole cap codon-optimized gene was inserted into pGBKT7 vector and confirmed by PCR,restriction enzyme digestion,and sequence analysis.After transformation into a Y2 HGold yeast strain,the expression of Cap protein was analyzed by Western blotting.Toxicity and self-activation of the bait protein were detected using different dropout minimal base.PCR reaction,restriction enzyme digestion,and sequencing analyses indicated that the duck circovirus Cap gene was correctly inserted into pGBKT7.Western blotting showed that the whole Cap protein was expressed.The recombinant bait protein had no toxicity and self-activation.Therefore,the bait vector with the Cap gene was constructed successfully,providing a foundation for future screening for interacting proteins in the yeast two-hybrid system.