禽网状内皮组织增生症病毒蛋白酶PR酵母双杂交诱饵载体的构建及鉴定

Construction and Identification of the Bait Vector of Reticuloendotheliosis virus PR using Yeast Two-hybrid System

为研究禽网状内皮组织增生症病毒(Reticuloendotheliosis virus,REV)蛋白酶PR与宿主细胞的相互作用,将REV PR基因克隆入酵母双杂交诱饵载体p GBKT7,构建诱饵载体p GBK-PR,经菌落PCR、酶切鉴定及测序验证正确后,将重组诱饵质粒转化酵母菌株Y2H Gold感受态,进行重组诱饵载体在酵母中的自激活活性和毒性检测。结果表明,成功构建诱饵载体p GBK-PR,此载体在酵母细胞Y2H Gold中无自激活活性和毒性。研究结果为进一步利用酵母双杂交技术筛选与REV蛋白酶PR互作的宿主蛋白奠定了基础。

To investigate the interaction between Reticuloendotheliosis virus （REV） PR and the host ceils, PR eDNA was ampli- fied and subcloned into the bait vector pGBKT7 of yeast two-hybrid system, and the recombinant bait vector pGBK-PR was construc- ted. After verifying by PCR amplification, enzymatic detection and sequencing validation, the yeast bait plasmid was transformed into Yeast strain Y2H Gold competent cells. Then the self - activation and toxicity of the bait vector were tested using the Matchmaker Gold Yeast Two-Hybrid System. Results showed that the bait vector pGBK-PR was successfully constructed with no self-activation and toxicity to Y2H Gold yeast cells. This study laid the foundation for further researches on the effect and mechanism of the interaction between REV PR and its host cells.