HTRA1 shRNA慢病毒表达载体的构建及感染RPE细胞株的鉴定

Construction of a lentiviral vector of HTRA1 shRNA and identification of its RNA interference effect in a RPE cell line

目的构建HTRA1基因的shRNA慢病毒表达载体,并鉴定HTRA1 shRNA慢病毒感染RPE细胞株的效果。设计实验研究。研究对象HTRA1 shRNA慢病毒载体和RPE细胞。方法设计靶向HTRA1 mRNA的寡核苷酸序列,构建HTRA1 shRNA表达质粒,测序鉴定其序列的正确性。通过载体质粒pGC-LV与辅助质粒pHelper 1.0、pHelper 2.0共转染293T细胞包装慢病毒,收集病毒上清,测定滴度。将包装好的HTRA1 shRNA慢病毒感染RPE细胞,设阴性对照和空白对照,采用实时PCR(RT-PCR)和蛋白印迹法(Western Blot)方法检测HTRA1基因的mRNA水平和蛋白质表达水平的变化。主要指标HTRA1基因的mRNA和蛋白质表达量。结果 DNA测序证实HTRA1 shRNA表达质粒包装了正确的RNA干扰序列。慢病毒滴度测定为8×108TU/ml。HTRA1 shRNA慢病毒感染RPE细胞后,HTRA1基因的mRNA水平和蛋白质表达水平较空白对照组和阴性对照组均明显下降,差异均有统计学意义。结论成功构建了HTRA1 shRNA的慢病毒表达载体,该shRNA慢病毒能够有效地抑制RPE细胞株HTRA1基因的表达。

Objective This study was aimed to construct lentivirus-mediated shRNA expression vector targeting HTRA 1 and iden- tify the RNA interference effect in a RPE cell line. Design Experimental study. Participants HTRA 1 shRNA lentivirus vector and RPE cells. Method One pair of oligonucleotide sequences targeted at human HTRA 1 mRNA was designed and synthesized. The annealed oligonucleotide fragments were subcloned into plasmid vector. Virus particles were collected and enveloped into HEK-293T cells.The RPE cells were infected witlh recombinant lentivirns. Real-time PCR and Western Blot were used respectively to detect the expression of HTRA 1 after lentivirus infection. Main Outcome Measures The mRNA and protein expression of HTRA 1. Results DNA sequencing demonstrated that the lentivirus shRNA vector of HTRA 1 was constructed successfully and the virus was packaged in 293 T cells.The titer of virus was 8×10^8TU/ml. The RPE cell line was successfully infected. The mRNA and protein levels of HTRA 1 were reduced sig- nificantly in RPE cells after lentivirns infection, compared with blank control and negative control. Conclusion It is concluded that the lentiviral shRNA vector of HTRA 1 is constructed, and successfully reduced HTRA1 expression in a RPE cell line.