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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。
Yeast two-hybrid system was used to construct yeast two-hybrid bait vector with three open reading frames of IHHNV of Litopenaeus vannamei, and the toxicity of the expressed product to yeast cells and the self-activation of reporter gene were tested. Yeast two hybrid library screening and protein interaction with the virus provide a reference. The gene fragment of three open reading frames (IHPH) of Litopenaeus vannamei was amplified by PCR, and the gene fragment was ligated with the double-digested yeast two-hybrid bait plasmid pGBKT7 using In-Fusion technology to obtain the recombinant plasmid pGBKT7 -CP, pGBKT7-NS1 and pGBKT7-NS2, and self-activated and tested for toxicity. The results showed that three recombinant plasmids, pGBKT7-CP, pGBKT7-NS1 and pGBKT7-NS2, had no self-activation activity on MEL1 reporter gene, no self-activation on AUR1-C reporter gene, pGBKT7-CP, pGBKT7-NS1, pGBKT7-NS2 OD600 of the broth was greater than 0.8 after cultured in SD / -Trp liquid medium for 16 h, indicating that the bait vector is non-toxic and does not have the function of autonomous activation of the reporter gene. Activating bait vectors can be used in yeast two-hybrid assays.