小反刍兽疫病毒酵母双杂交诱饵载体pGBKT7-tH的构建、表达和鉴定

Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-tH of peste des petits ruminants virus

利用PCR技术扩增获得PPRV-tH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AH109中,检测其在酵母中有无渗漏、自我激活作用和毒性。利用Western blotting分析诱饵蛋白在酵母中的表达情况,以鉴定其作为诱饵蛋白的可行性。结果表明,成功扩增到了PPRV-tH,并正确构建了pGBKT7-tH诱饵表达载体,此载体在酵母细胞AH109中无毒性、渗漏和自我激活能力,且能正确表达tH蛋白。

The fragment of tH was amplified by PCR,and was cloned into the bait expression vector pGBKT7.After being verified by restriction digestion and sequencing,the bait vector was transformed into yeast cells AH109.The toxicity,leakage and self-transcriptional activation of PPRV-tH protein were firstly detected.Then the expression of was analyzed by Western blotting.Results showed that PPRV-tH was amplified and cloned into pGBKT7 successfully.The bait protein vector was transformed into AH109 as well and the toxicity,leakage and self-transcriptional activation of PPRV-tH protein were not observed.The PPRV-tH protein could be effectively expressed in transformed yeast.It plays a critical part in works of screening receptor gene by yeast two-hybrid system.