利用酵母双杂交技术筛选与wsv112互作的宿主蛋白

Identification of the Host Interactors of wsv112 of WSSV by Yeast Two-Hybrid

中文关键词: 对虾白斑综合征病毒  wsv112  dUTPase  酵母双杂交

英文关键词: WSSV  wsv112  dUTPase  Yeast two-hybird

中文摘要:

      对虾白斑综合征病毒(WSSV)开放阅读框wsv112编码脱氧尿苷焦磷酸酶(dUTP pyrophosphatase, dUTPase)。为研究wsv112与宿主的互作关系，本研究采用酵母双杂交Gal4系统从凡纳滨对虾(Litopenaeus vannamei)肠道cDNA文库中筛选与wsv112互作的候选蛋白。以WSSV为模板，构建pGBKT7-112诱饵载体，转化到Y2H Gold酵母菌感受态细胞中，转化菌液涂布到不同缺陷型培养基上，检测诱饵载体的自激性和毒性。将凡纳滨对虾肠道cDNA文库与诱饵菌株pGBKT7-112接合，通过筛选力度不同的缺陷型培养基、颜色反应、PCR、测序鉴定等步骤筛选阳性克隆，将阳性菌株提取质粒，再经过回复杂交实验验证筛选出的阳性质粒与诱饵载体的作用。研究表明，诱饵载体pGBKT7-112无自激性和毒性，可用于酵母双杂交实验；经初筛和回复杂交实验最终得到2个阳性质粒，经过NCBI数据库对比，其编码的蛋白分别与日本囊对虾(Penaeus japonicus) C型凝集素(AGW27416.1)和克氏原螯虾(Procambarus clarkii) 40S核糖体蛋白S20小亚基蛋白(ALE99171.1)具有37%和98%同源性。本研究为wsv112的调控机制提供新的线索。

英文摘要:

      The ORF wsv112 of white spot syndrome virus (WSSV) encodes a dUTPases. It plays an essential role in nucleotide biosynthesis. Hydrolysis of dUTP by UTPase produces dUMP, required for the de novo synthesis of dTTP, and maintains low cellular ratios of dUTP/dTTP, thus preventing the mis-incorporation of uracil into chromosomal DNA. In order to identify the host interactors of wsv112，wsv112 was cloned into the bait vector pGBKT7 and used to screen an intestine cDNA library of Litopenaeus vannamei, which had previously been constructed by yeast two-hybrid sequencing transformation. The positive clone was identified through different culture media, color change, polymerase chain reaction (PCR), and sequencing. The result showed that the bait plasmid pGBKT7-112 showed no virulence or self-activating effect on yeast strain Y2H Gold. A total of 526 blue clones were screened, which were analyzed by PCR and homology analysis using the BLAST in NCBI, and 6 possible interaction proteins of Litopenaeus vannamei were obtained. Then through the Yeast two-hybrid reply hybridization experiment, only two gene interactions were confirmed with the wsv112. They were identified as lectin C gene of Marsupenaeus japonicas (AGW27416.1) and 40S ribosomal protein S20 gene of Procambarus clarkia (ALE99171.1) with 37% and 98% identity. This study may provide a theoretical basis for further study of the wsv112 interaction mechanisms.