论文部分内容阅读
目的构建分别靶向ezrin(E)、radixin(R)、moesin(M)的siRNA慢病毒表达载体,转染原代大鼠肺微血管内皮细胞,观察其对E、R、M基因表达及流感病毒复制的影响。方法根据siRNA设计原理,化学合成靶向大鼠E、R、M基因的siRNA序列,克隆入慢病毒表达质粒GV248,构建分别靶向E、R、M基因的慢病毒siRNA载体Lenti-E-siRNA、Lenti-R-siRNA、Lenti-M-siRNA,同时构建靶向无关序列的Lenti-control-siRNA;组织块法分离培养大鼠原代肺微血管内皮细胞(PMVEC);Lenti-E-siRNA、Lenti-R-siRNA、Lenti-M-siRNA转染原代大鼠PMVEC后,荧光定量PCR检测E、R、M的mRNA的表达,Western blot法检测ERM蛋白的表达,激光共聚焦显微镜技术检测ERM蛋白的分布;FM1流感病毒鼠肺适应株感染慢病毒转染的PMVEC后,荧光定量PCR检测慢病毒载体对流感病毒复制的影响。结果重组质粒经测序鉴定,Lenti-E-siRNA、Lenti-R-siRNA、Lenti-MsiRNA、Lenti-control-siRNA构建成功。慢病毒载体转染大鼠PMVEC,感染复数(MOI)为10时感染率达80%以上。Lenti-E-siRNA、Lenti-R-siRNA、Lenti-M-siRNA明显抑制PMVEC中E、R、M的转录及表达。流感病毒感染PMVEC中,慢病毒空载体及Lenti-control-siRNA抑制流感病毒的复制,而Lenti-E-siRNA、Lenti-R-siRNA、Lenti-M-siRNA增加PMVEC中流感病毒的mRNA水平。结论成功构建靶向ERM基因的慢病毒载体,证实其可稳定转染PMVEC特异高效地抑制E、R、M基因表达;慢病毒载体本身能抑制PMVEC中流感病毒复制,而靶向E、R、M的慢病毒siRNA却促进流感病毒的复制。
Objective To construct siRNA lentiviral vector targeting ezrin (E), radixin (R) and moesin (M) respectively and transfect the primary rat pulmonary microvascular endothelial cells to observe the expression of E, R and M genes and the expression of influenza virus The effect of copy. Methods According to the siRNA design principle, siRNA sequences targeting E, R and M genes of rat were chemically synthesized and cloned into lentiviral expression plasmid GV248 to construct lentiviral siRNA vector Lenti-E-siRNA targeting E, R and M genes respectively Lenti-R-siRNA, Lenti-M-siRNA and Lenti-control-siRNA targeting irrelevant sequences. Lenti-E-siRNA, Lenti R-siRNA and Lenti-M-siRNA were used to detect the expression of E, R and M mRNA in primary rat PMVEC. Western blot was used to detect the expression of ERM protein. Laser confocal microscopy was used to detect ERM protein . The FM1 influenza virus murine lung-adapted strain was infected with lentivirus-transfected PMVEC and the effect of lentiviral vector on influenza virus replication was detected by real-time PCR. Results The recombinant plasmid was identified by sequencing. Lenti-E-siRNA, Lenti-R-siRNA, Lenti-MsiRNA and Lenti-control-siRNA were successfully constructed. The lentiviral vector was transfected into rat PMVEC and the infection rate was more than 80% at the MOI of 10. Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA significantly inhibited the transcription and expression of E, R and M in PMVEC. In PMVEC infected with lentivirus, lentiviral empty vector and Lenti-control-siRNA inhibited the replication of influenza virus, while Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA increased the mRNA level of influenza virus in PMVEC. Conclusion The lentiviral vector targeting ERM gene was successfully constructed and proved to stably transfect PMVEC specifically and efficiently to inhibit the gene expression of E, R and M. The lentiviral vector itself can inhibit the replication of influenza virus in PMVEC, while targeting E, R, M lentiviral siRNA promotes the replication of influenza virus.