Construction and identification of recombinant lentiviral plasmid of receptor for advanced glycation end products / 国际生物医学工程杂志

ABSTRACT

Objective To construct a recombinant lentiviral plasmid of receptor for advanced glycation end products (RAGE) and establish RAGE stable expressing hepatocellular carcinoma cell models in order to explore the pathogenesis of RAGE in the occurrence and development of hepatocellular carcinoma. Methods RAGE gene fragments were amplified by PCR and cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro. Then the recombinant lentiviral plasmid and packaging plasmid were used to package lentivirus in HEK293T cells by calcium phosphate transfection. Lentivirus supernatant was collected to infect hepatoma cells HepG2 and Huh7. After that cells were screened by puromycin. Finally, RAGE expression was detected by real-time PCR and Western blot. Results RAGE gene fragment was successfully amplified and inserted into lentiviral expression vector. After packaging the lentivirus-infected liver cancer cells, real-time quantitative PCR and Western blot results showed that the mRNA (P<0.05) and protein expression of RAGE cells stably expressing RAGE were significantly higher than that of the control cells. Conclusions The RAGE overexpressing hepatocarcinoma cell line was successfully constructed by using lentiviral expression vector, which laid a good foundation for further study on the pathogenesis of RAGE.