摘要
为构建鸭肠炎病毒(DEV)核衣壳蛋白(NP)基因酵母双杂交诱饵载体,采用PCR技术扩增出DEV-NP基因,克隆至酵母双杂交诱饵质粒pGBKT7中,以PCR检测、限制性酶切和序列测定等方法进行鉴定;然后采用PEG/LiAc法将阳性质粒pGBKT7-NP转化酵母Y2HGold,在不同营养缺失型培养基进行自激活检测,结果显示,经PCR检测和EcoR I/BamHⅠ双酶切,可见到与预期相一致的目的片段;测序表明该片段含DEV NP基因全部序列;转化Y2HGold酵母菌后,在SD/-Trp/X-α-Gal固体培养基上长出蓝色菌落,而在SD/-Trp/X-α-Gal/AbA固体培养基上未见有菌落生长。无自激活作用的诱饵载体pGBKT7-NP的成功构建,为DEV NP宿主结合蛋白的筛选奠定了基础。
To construct a bait vector of nucleocapsid for duck enteritis virus and to evaluate autoactivation in yeast two-hybrid system.Full-length of NP gene was amplified by polymerase chain reaction（PCR） and confirmed by sequencing.A fragment of the gene was then subcloned into MCS of pGBKT7 vector to construct the bait vector according to the reading frame.The reconstructed bait vector pGBKT7-NP was transformed into the yeast strain Y2HGold by PEG/LiAc method and its self-activation was tested by the phenotype assay.The results showed that the PCR detection and EcoR I/BamH I double digestion observed,the recombinant plasmid pGBKT7-NP can be seen in line with the expected target fragment;sequencing showed that the fragment contains all DEV NP gene sequence;conversion Y2HGold after yeast in SD/-Trp/X-α-Gal blue colonies grown on solid medium,while in SD /-Trp / X-α-Gal / AbA solid medium there was no colony growth.The results showed that NP could act as a bait in the screening of cDNA library of host cell to trap the interaction proteins in yeast two-hybrid system.
出处
《生物技术通报》
CAS
CSCD
北大核心
2011年第3期196-199,共4页
Biotechnology Bulletin
基金
国家自然科学基金项目(30760182)
