抗甲型流感病毒短发夹状RNA分子的体外生物合成及功能鉴定.docVIP

文章编号： 抗甲型流感病毒短发夹状RNA分子的体外生物合成及功能鉴定 郭述良，罗永艾（重庆医科大学附属第一医院呼吸内科，重庆400016） 摘 要： 目的 制备抗甲型流感病毒短发夹状RNA分子（short hairpin RNA, shRNA），并鉴定其功能。方法 针对甲型流感病毒(influenza A virus, IAV）核壳蛋白（nucleoprotein, NP）和酸性RNA多聚酶（acidic RNA polymerase, PA）编码mRNA高度保守区序列设计并制备编码shRNA的双链DNA转录模板，分别将其插入pGenSil-1 质粒，构建NP-shRNA和PA-shRNA表达载体，测序正确后，采以T7转录试剂盒制备NP-shRNA和PA-shRNA。将所制备的2种shRNAs分别或联合转入人气道上皮细胞（human bronchial epithelium, HBE），然后感染甲型流感病毒IAV） A/PR8株，通过测定HBE细胞病变效应（cytopathogenic effect, CPE）、培养上清液病毒滴度以及NP、PA mRNA与蛋白表达水平，评估所制备的shRNAs对IAV A/PR8在HBE细胞中复制的抑制效果。结果 编码shRNA的2种双链DNA转录模板序列正确。通过体外转录技术制备的NP-shRNA、PA-shRNA每100 μl含量分别为59.4、50.6 nmol，纯度均在2.0以上，浓度和纯度均高。与未转染shRNA的对照组比较，NP-shRNA、PA-shRNA、NP-shRNA＋PA-shRNA转染组细胞内NP mRNA分别减少41.7%、44.1%和68.5%，PA mRNA分别减少43.4%、60.9%和73.7%， NP蛋白表达分别降低92.3%、84.0%和91.7%，CPE显着减轻，培养上清液病毒滴度显着下降。结论 成功构建了2种抗IAV短发夹RNA分子，所制备的NP-shRNA、PA-shRNA对IAV A/PR8株在HBE细胞中复制有显着的抑制效果。 关键词：流感病毒，甲型；短发夹状RNA分子；RNA干扰 中图法分类号： 文献标识码：A Biosynthesizing and Function Verification of Short Hairpin RNA targeting to Influenza A Virus GUO Shu-liang, LUO Yong-ai (Department of Respiratory Medicine, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China) Abstract: Objective This project mainly investigates the inhibitive effects of short hairpin RNA (shRNA) biosynthesized by in vitro transcription on influenza A virus (IAV) duplication in human bronchial epithelium（HBE）cells. Methods Designing and biosynthesizing NP-shRNA and PA-shRNA targeting to highly conservative sequences of IAV NP mRNA and PA mRNA by in vitro transcription. Following the transfection of NP-shRNA and/or PA-shRNA, HBE cells were infected with IAV A/PR/8/34 H1N1（A/PR8）virus. Cytopathogenic effect (CPE) of the cells, hemagglutination assay (HA) and 50% tissue culture infective dose (TCID50) titer of A/PR8 in culture supernatants, the expression quantity of NP mRNA and PA mRNA by RT-PCR, and the expression level of NP protein by Western-blotting were determined and compared in order to evaluate the