论文部分内容阅读
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IAV A/PR8株,通过测定HBE细胞病变效应(cyto-pathogenic 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100μl含量分别为59.4、50.6nmol,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%,PAmRNA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显著减轻,培养上清液病毒滴度显著下降。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细胞中复制有显著的抑制效果。
Objective To prepare short hairpin RNA (shRNA) of anti-influenza A virus and identify its function. Methods A double-stranded DNA transcription template encoding shRNA was designed and synthesized based on the highly conserved region of nucleoprotein (NP) and acidic RNA polymerase (PA) of influenza A virus (IAV) The recombinant plasmids were inserted into pGenSil-1 plasmids respectively to construct NP-shRNA and PA-shRNA expression vectors. After sequencing, NP-shRNA and PA-shRNA were prepared by T7 transcription kit. The two kinds of shRNAs were transfected into human bronchial epithelium (HBE) separately or in combination, and then infected with IAV A / PR8 strain. By measuring the cyto-pathogenic effect (HBE) Liquid virus titer and NP, PA mRNA and protein expression levels were evaluated to evaluate the inhibitory effect of the prepared shRNAs on the replication of IAV A / PR8 in HBE cells. Results The sequence of the two double stranded DNA transcription templates encoding shRNAs was correct. The NP-shRNA and PA-shRNA prepared by in vitro transcription technology were 59.4 and 50.6 nmol respectively, with the purity higher than 2.0 and high concentration and purity. Compared with the untransfected shRNA group, the NP mRNA expression decreased by 41.7%, 44.1% and 68.5% in NP-shRNA, PA-shRNA and NP-shRNA + PA-shRNA transfected groups respectively, while PA mRNA decreased by 43.4% and 60.9 % And 73.7% respectively. The NP protein expression decreased by 92.3%, 84.0% and 91.7%, respectively. CPE was significantly reduced and the virus titer in culture supernatant was significantly decreased. Conclusion Two kinds of anti-IAV short hairpin RNA molecules were constructed successfully. The NP-shRNA and PA-shRNA prepared by this method can significantly inhibit the replication of IAV A / PR8 strain in HBE cells.