酵母双杂交筛选与呼肠孤病毒σ1、σ1S互作宿主蛋白

Screening of proteins interacted with orthoreovirus σ1 and σ1S protein by the yeast two-hybrid system

提取猪肠上皮细胞IPEC-1的总RNA,构建了IPEC-1的靶标cDNA文库。随机挑取文库克隆子进行PCR扩增,发现插入片段长度为200～1 500bp,文库的重组率为100%。以猪源呼肠孤病毒GD-1株S1基因节段为模板,通过PCR扩增,定向克隆构建诱饵载体pGBKT7-S1、pGBKT7-S1s。通过菌落PCR和测序验证的质粒被转化至酵母菌株Y2H Gold,诱饵载体pGBKT7-S1、pGBKT7-S1s均能在酵母细胞中正确表达出对应的σ1和σ1S蛋白,且无自激活活性,对酵母细胞无毒性,满足文库筛选实验的要求。将含有pGBKT7-S1、pGBKT7-S1s的酵母菌株Y2H Gold分别与靶标cDNA文库菌株Y187进行杂交,筛选、验证、测序后得到13个与σ1、6个与σ1S相互作用的宿主蛋白,其中的1个蛋白与σ1、σ1S同时存在相互作用。本试验成功构建了适用于研究肠道细菌、病毒作用机制的猪肠上皮细胞IPEC-1cDNA文库,筛选出18个与猪呼肠孤病毒σ1或σ1S的互作蛋白,为研究呼肠孤病毒与宿主相互作用,以及鉴定疾病控制的新靶标提供了线索。

To seek out proteins interact with the σ1 structural protein and σ1S nonstructural protein of porcine orthoreovirus in porcine epithelial cell, the IPEC-1 cells were harvested for total RNA preparation and the cDNA library was further constructed with the Mate ＆ PlateTM Library con- struction system. Clones randomly selected were amplified by PCR and the inserts varied from 200 hp to 1 500 bp and the recombination rate was 100%. The S1 gene segment of GD-1 strain was used as a template and amplified by PCR and bait plasmids pGBKT7-S1 and pGBKT7-SIs were constructed by directed cloning. Plasmids verified by colony PCR and sequencing were transformed into yeast strain Y2H Gold. The well expression of 61 and σ1S were confirmed by Western blot, and showed no self-activation and cytotoxicity,and suited for this screen. The yeast strains Y2H Gold which containing bait vector pGBKT7-S1 or pGBKT7-Sls were mated with target cDNA li- brary strain Y187,respectively. 13 candidate interacting proteins with σ1 and 6 candidate interac- ting proteins with σ1S were verified. In brief,A high quality IPEC-1 cDNA library was constructed and 18 proteins interacting with σ1 or σ1S protein were identified. These results provided good clues for interrogating orthoreovirus host-pathogen interactions and identifying novel targets which may he exploited in disease control.