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目的构建基于聚合酶亚基间相互作用的双靶点特异性抗流感病毒药物筛选酵母双杂交体系。方法将流感病毒A/PR8/34株PB2、PA基因克隆至载体pGADT7,PB1基因克隆至载体pGBKT7,将构建的酵母双杂交重组质粒PB2-pGADT7、PB1-pGBKT7和PA-pGADT7、PB1-pGBKT7分别共转化酵母AH109,利用SD/-Ade/-His/-Leu/-Trp/X-α-Gal培养基筛选阳性菌落。结果 PB2-PB1共转化效率为1.4×104cfu/μg,PA-PB1共转化效率为1.6×104cfu/μg;双靶点特异性的酵母双杂交系统在SD/-Ade/-His/-Leu/-Trp/X-α-Gal上均有兰色菌落生长;PB2、PB1、PA基因PCR结果均为阳性。结论初步构建了流感病毒聚合酶亚基间相互作用的酵母双杂交系统,可用于抗流感病毒靶点特异性的药物高通量筛选。
Objective To construct a two-target specific anti-influenza virus drug based on the interaction between polymerase subunits to screen yeast two-hybrid system. Methods PB2 and PA genes of influenza A / PR8 / 34 strains were cloned into vector pGADT7, and PB1 gene was cloned into vector pGBKT7. The constructed yeast two-hybrid recombinant plasmids PB2-pGADT7, PB1-pGBKT7 and PA-pGADT7 and PB1-pGBKT7 Yeast AH109 was co-transformed and positive colonies were screened using SD / -Ade / -His / -Leu / -Trp / X-α-Gal medium. Results The co-transformation efficiency of PB2-PB1 was 1.4 × 104 cfu / μg and PA-PB1 co-transformation efficiency was 1.6 × 104 cfu / μg. The double target-specific yeast two-hybrid system was expressed in SD / -Ade / -His / -Leu / Trp / X-α-Gal on both blue colonies; PB2, PB1, PA gene PCR results were positive. Conclusion The yeast two-hybrid system of influenza virus polymerase subunit interaction was preliminarily constructed and could be used for high-throughput screening of anti-influenza virus target-specific drugs.