甲型H1N1流感病毒非结构蛋白NS1与人CPSF30结合拮抗剂筛选模型的建立及药物筛选  

Development of a yeast two-hybrid screen for selection of A/H1N1 influenza NS1 non-structural protein and human CPSF30 protein interaction inhibitors

作　　者：孔建强[1] 沈君豪[1] 黄勇[1] 阮仁余[1] 项斌[1] 郑晓东[1] 程克棣[1] 王伟[1] 

机构地区：[1]中国医学科学院、北京协和医学院药物研究所,卫生部天然药物生物合成重点实验室、中草药物质基础与资源利用教育部重点实验室,北京100050

出　　处：《药学学报》2010年第3期388-394,共7页Acta Pharmaceutica Sinica

基　　金：科技部甲型H1N1流感联防联控应急科研项目;中央级公益性科研院所基本科研业务费专项(2010ZD04)

摘　　要：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。Influenza A/H1N1 virus-encoded nonstructural, or NS1, protein inhibits the 3′-end processing of cellular pre- mRNAs by binding the cellular protein： the 30-kDa subunit of CPSF （cleavage and polyadenylation specificity factor, CPSF30）. CPSF30 binding site of the NS1 protein is a potential target for the development of drugs against influenza A/H1N1 virus. A yeast two-hybrid screening system was constructed and used for screening Chinese medicines that inhibit the interaction of the A/H1N1 flu NS1 protein and human CPSF30 protein. The NS1 gene of A/H1N1 virus was amplified by consecutive polymerase chain reaction （PCR）, and the human CPSF30 gene of HeLa cell cloned by reverse transcriptase-polymerase chain reaction （RT-PCR）. Then the two gene fragments confirmed by sequencing were subcloned into the yeast expression vectors pGBKT7 and pGADT7, respectively. The two constructs, bait vector pGBKNS1 and prey vector pGADCPSF, were co-transformed into yeast AH109. The eight individual yeast colonies were picked and subjected to verification by PCR/gel electrophoresis. The inhibition of the NS1-CPSF30 interaction was allowed the identification of selective inhibitors. The four of more than thirty identified Chinese medicines, including ‘Shuanghuanglian oral liquid’, showed the strong inhibition of the NS1-CPSF30 interaction.

关 键 词：H1N1流感病毒 非结构蛋白NS1 CPSF30 酵母模型 中药筛选 

分 类 号：R963[医药卫生—微生物与生化药学]