与猪圆环病毒2型基因组茎环结构相互作用的宿主细胞蛋白的筛选

Screening of host proteins interacting with porcine circovirus type 2 genome stem-loop structure

利用酵母单杂交技术对猪圆环病毒2型(PCV2)基因组复制起始处茎环结构序列与PK-15细胞中的互作蛋白进行了筛选和鉴定。首先将合成的PCV2复制起始处的茎环结构序列整合到酵母染色体中构建诱饵酵母菌株;采用SMART技术合成PK-15细胞cDNA文库,将总cDNA与pGADT7-Rec表达载体共同转化酵母诱饵菌株,通过同源重组在酵母细胞内同步进行cDNA文库的构建,以药物筛选阳性克隆;通过酵母菌落PCR得到阳性克隆中的cDNA插入片段,通过序列分析对此予以确认。本试验成功地构建了酵母单杂交文库,筛选出1.01×106个酵母克隆,其中196个为阳性克隆。经PCR产物测序,并通过NCBI Blast法进行序列比对,确定有4种蛋白可与PCV2基因组茎环序列发生作用。本研究从宿主细胞cDNA文库中筛选到4种可与PCV2基因组茎环序列发生作用的蛋白,为下一步阐明这些宿主蛋白对PCV2复制的调节作用奠定了基础。

To screen host proteins in the PK-15 cells that interact with the stem-loop structure of porcine circovirus type 2 by yeast one-hybrid system, the sequence of the ＂stem-loop＂ structure, which is the viral replication origin, was integrated into the yeast chromosome to construct a bait strain by means of synthesizing oligonucleotide method firstly, cDNA of PK-15 cells was synthesized by SMART technique and co-transformed into the bait yeast strain using pGADTT-Rec vector. One-hybrid cDNA library was simultaneously constructed in yeast as a result of plasmid recombination in vivo and positive clones were screed using drugs. The positive clones were identified by colony PCR. In result, the construction of yeast one-hybrid library was successful,1.01 X 106clones was screened and 196 positive clones was obtained. The colony PCR products were sequenced and putative proteins encoded by them were inferred by NCBI Blast analysis and four interacted with the stem-loop were identified by library screening. This study provides the basis for further clarification of a regulating role of host cell proteins in the replication of the virus.