Construction and detection of lentiviral expression vector of retinoid X receptor alpha gene

Abstract

Objective To construct recombinant lentiviral expression vector carrying rat retinoid X receptor alpha (RXR-α) gene,and to obtain the titer of the lentiviral stock.Methods Rat RAR-α encoding sequence was amplified,and ligated with pGC-FU-3FLAG lentivirol vector to produce the lentiviral expression vector named pGC-fu-3flag-Rxra.It was identified by polymerase chain reaction ( PCR,the fragment length of 411 bp),DNA sequencing and Western blotting.There was a characteristic band of about 90 kD.Virus packaging cells (293T cells) were cotransfected with lentivirol vector pGC-fu-3flag-Rxra,pHelper 1.0 and Helper 2.0 to produce lentivirus.The titers of recombinant viruses were determined by real time qPCR.There was significant difference in the Ct data between 1.00E-04 μl group and control group (2.775).Results Lentiviral expression vector of rat RXR-α gene proved by sequencing and Western blotting was successfully constructed.The titer of recombinant viruses was 2 × 108 TU/ml.Conclusion Rat RAR-α lentiviral expression vector was successfully constructed. Key words: Retinoid X receptor alpha; Lentviral vector; Hepatic fibrosis