关键词: HTRA1, RPE细胞, 慢病毒, RNA干扰

Abstract: Objective This study was aimed to construct lentivirus-mediated shRNA expression vector targeting HTRA1 and identify the RNA interference effect in a RPE cell line. Design Experimental study. Participants HTRA1 shRNA lentivirus vector and RPE cells. Method One pair of oligonucleotide sequences targeted at human HTRA1 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 with recombinant lentivirus. Real-time PCR and Western Blot were used respectively to detect the expression of HTRA1 after lentivirus infection. Main Outcome Measures The mRNA and protein expression of HTRA1. Results DNA sequencing demonstrated that the lentivirus shRNA vector of HTRA1 was constructed successfully and the virus was packaged in 293 T cells．The titer of virus was 8×108 TU/ml. The RPE cell line was successfully infected. The mRNA and protein levels of HTRA1 were reduced significantly in RPE cells after lentivirus infection, compared with blank control and negative control． Conclusion It is concluded that the lentiviral shRNA vector of HTRA1 is constructed, and successfully reduced HTRA1 expression in a RPE cell line.

Key words: HTRA1 gene, RPE cell, lentivirus, RNA interference