应用酵母双杂交方法筛选与马传染性贫血病毒Rev蛋白相互作用的蛋白

Identification of equine infectious anemia Rev-interacting proteins using yeast two hybrid system

为研究马传染性贫血病毒(EIAV)蛋白与马巨噬细胞蛋白之间的相互作用,本研究利用SMART技术构建了马巨噬细胞的酵母双杂交c DNA文库,并利用该文库调取与EIAV Rev蛋白相互作用的细胞蛋白,构建诱饵质粒p GBKT7-rev,通过表型检测Rev的自激活和细胞毒性作用,并与构建的文库进行杂交筛选。对获得阳性克隆进行序列测定和比对分析,根据分析结果进行共转化验证。结果显示,文库的容量为3×108 cfu/m L。Rev蛋白在酵母双杂交系统中无自激活现象和细胞毒性作用。此外,本实验共筛选得到5个与EIAV Rev相互作用的宿主细胞蛋白。本研究为进一步研究Rev蛋白的生物学新功能奠定了基础。

To identify the host protein interacted with equine infectious anemia （EIAV） Rev protein, the bait vector of pGBKT7-rev was constructed and tested for its toxicity and self-activation by the phenotype assay. Then, the Rev was used as bait to scan the yeast two hybrid cDNA library of equine macrophage construct using SMART technology. The result indicated that the library titer was up to 3×10^8cfu/mL and that Rev protein was unable to induce self-activity and had no toxicity to the yeast cells. In addition, five host cell proteins interacting with EIAV Rev protein were screened fi＇om the library. The primary identifications of the cell proteins would facilitate future study on the fimction of EIAV Rev protein.