论文部分内容阅读
目的鉴定基于聚合酶亚基间相互作用的靶点特异性抗流感病毒药物筛选酵母双杂交体系。方法挑取筛选平板SD/-Ade/-His/-Leu/-Trp/X-α-Gal阳性单菌落(共转化质粒PB1-pGBKT7与PA-pGADT7的AH109酵母细胞)接种培养,PCR验证重组质粒共转是否成功;glass-beads法提取酵母总蛋白,Western-blot验证融合蛋白在酵母细胞中是否表达;将PB1-pGBKT7质粒PB1基因克隆至载体pAcGFP1-C,PA-pGADT7质粒PA基因克隆至载体pProLabel-C,将构建的重组质粒PB1-pAcGFP1、PA-pProLabel共转染人胚肾GP2-293细胞,Co-IP验证在哺乳动物细胞中流感病毒聚合酶亚基PB1、PA具有相互作用。结果 PB1、PA基因PCR结果均为阳性;PB1-Myc融合蛋白Western-blot结果为阴性,PA-HA融合蛋白Western-blot结果为阳性;PB1-pAcGFP1、PA-pProLabel重组质粒PCR验证结果 PB1、PA基因阳性;Western-blot检测PB1-GFP融合蛋白阳性;Co-IP结果表明流感病毒RNA聚合酶亚基PB1与PA在哺乳动物细胞内具有相互作用。结论基于RNA聚合酶亚基间相互作用的抗流感病毒药物筛选系统构建成功,可以用于抗流感病毒靶点特异性的高通量药物的筛选。
OBJECTIVE: To screen yeast two-hybrid system based on target-specific anti-influenza virus drugs based on the interaction between polymerase subunits. Methods Single colonies positive for SD / -Ade / -His / -Leu / -Trp / X-α-Gal were selected and cultured in AH109 yeast cells co-transformed with PB1-pGBKT7 and PA-pGADT7. The recombinant plasmid The total protein of yeast was extracted by glass-beads method and the expression of fusion protein was verified by Western-blot in yeast cells. The PB1 gene of PB1-pGBKT7 plasmid was cloned into vector pAcGFP1-C and the PA gene of PA-pGADT7 plasmid was cloned into vector pProLabel-C. The constructed recombinant plasmids PB1-pAcGFP1 and PA-pProLabel were co-transfected into human embryonic kidney GP2-293 cells. Co-IP verified the interaction of influenza virus polymerase subunits PB1 and PA in mammalian cells. Results PB1-PAc gene PCR results were positive; PB1-Myc fusion protein Western-blot results were negative, PA-HA fusion protein Western-blot results were positive; PB1-pAcGFP1, PA-pProLabel recombinant plasmid PCR validation PB1, PA The results of Co-IP showed that influenza virus RNA polymerase subunit PB1 interacts with PA in mammalian cells. Conclusion The anti-influenza virus drug screening system based on RNA polymerase subunit interaction was successfully constructed and could be used to screen anti-influenza virus target-specific high-throughput drugs.