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利用酵母双杂交技术研究甲型H1N1流感病毒非结构蛋白NS1和人切割与多聚腺苷酸化特异因子30kDa亚基(CPSF30)的相互作用,构建了一个酵母模型用于筛选NS1和CPSF30相互作用的拮抗剂,从而为筛选抗甲型H1N1流感病毒药物奠定基础。采用连续重叠PCR技术克隆得到H1N1流感病毒NS1基因。提取人HeLa细胞RNA,通过RT-PCR克隆得到人CPSF30基因。将NS1基因克隆到表达载体pGBKT7中获得诱饵载体pGBKNS1,将CPSF30基因克隆到载体pGADT7中获得捕获载体pGADCPSF;将pGBKNS1和pGADCPSF共转入酿酒酵母AH109,获得重组酿酒酵母AH109[pGADCPSF+pGBKNS1]。利用该模型筛选了30余种中成药,发现双黄连口服液等4种中成药能抑制NS1和CPSF30之间的相互作用。
Yeast two-hybrid technique was used to study the interaction between human nonstructural protein H1 (H1N1) and 30 kDa subunit of human polyadenylation specific factor (CPSF30), and a yeast model was constructed to screen the interaction between NS1 and CPSF30 Antagonist, which laid the foundation for the screening of anti-H1N1 influenza virus drugs. The H1N1 influenza virus NS1 gene was cloned by using continuous overlap PCR technique. Human HeLa cell RNA was extracted and the human CPSF30 gene was cloned by RT-PCR. The NS1 gene was cloned into the expression vector pGBKT7 to obtain the bait vector pGBKNS1. The CPSF30 gene was cloned into the vector pGADT7 to obtain the capture vector pGADCPSF. The pGBKNS1 and pGADCPSF were co-transformed into Saccharomyces cerevisiae AH109 to obtain the recombinant Saccharomyces cerevisiae AH109 [pGADCPSF + pGBKNS1]. Using this model, more than 30 kinds of proprietary Chinese medicines were screened and found that 4 kinds of proprietary Chinese medicines, such as Shuanghuanglian Oral Liquid, can inhibit the interaction between NS1 and CPSF30.