IHHNV编码蛋白酵母双杂交诱饵载体的构建及自激活研究

Construction and Identification of Yeast Two Hybrid Bait Vector for Screening of IHHNV Encoding Protein

应用酵母双杂交体系,构建凡纳滨对虾IHHNV 3个开放阅读框的酵母双杂交诱饵载体,并对该载体的表达产物对酵母细胞的毒性及其对报告基因的自激活作用进行检测,为运用酵母双杂交文库筛选与病毒互相作用的蛋白提供参考。对凡纳滨对虾IHHNV病毒3个开放阅读框的基因片段进行PCR特异性扩增,并运用In-Fusion技术将该基因片段与双酶切的酵母双杂交诱饵质粒pGBKT7进行连接,获得重组质粒pGBKT7-CP、pGBKT7-NS1和pGBKT7-NS2,并进行自激活和毒性检测。结果表明,pGBKT7-CP、pGBKT7-NS1和pGBKT7-NS2等3个重组质粒对MEL1报告基因无自激活性;对AUR1-C报告基因无自激活作用,pGBKT7-CP,pGBKT7-NS1,pGBKT7-NS2和空载体的酵母菌在SD/-Trp液体培养基中培养16 h后,菌液的OD_(600)均大于0.8,说明诱饵载体无毒性且不具自主激活报告基因的功能,所获得的无自激活作用的诱饵载体可用于酵母双杂交试验。

A yeast two hybrid system bait vector for screening of Litopenaeusvannamei IHHNV encoding 3 protein was constructed and identified, and its effects on the growth of yeast cells and the activation of reporter genes were studied, which could provide references for screen- ing vires-related protein using yeast two hybrid system. Full fragment of ORF of 3 IHHNV reeds was amplified using PCR and directly ligated to the pGBKT7 vector using In-Fusion. The recombinant plasmids pGBKT7-CP, pGBKT7-NS1 and pGBKT7-NS2 were obtained and their toxicity and transcriptional activation were tested. The results showed that the above plasmids had no serf-activation to the MEL1 reporter gene or AUR1-C reporter gene. After being cultured 16h in SD/-Trp liquid medium, the OD600 of the yeast was greater than 0.8, which in- dicated that bait vector was not toxic and had no self-activation to reporter genes, hence the obtained bait vector could be used for relevant experiments concerning yeast two hybrid system.