Construction of recombinant lentiviral vector of siRNA for augmenter of liver regeneration gene and its expression in HepG2 cells

Abstract:

Objective：To construct a lentiviral vector for RNA interference（RNAi） for augermenter of liver regeneration（ALR） gene and to detect its interference efficiency in human hepatoma cell line HepG2 and QGY. Methods：Effective sequence of siRNA target－ing ALR gene was confirmed. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed，synthesized and cloned into the HpaⅠ and XhoⅠ resriction sites of GV118 lentiviral vector. The resulting lentivirus vector containing ALR shRNA was named LV-ALR-shRNA. The competent E.coli cells were transformed and positive clone were screened，identified and sequenced. The recombinant lentivirus was packaged into mature lentivirus by 293T cells and the virus titer was measured by hole dilution method. The packaged lentivirus was used to infect HepG2 and QGY cells. The expression of ALR in the cells was detected by real-time PCR and Western blot. Results：PCR and sequencing verified that the recombinant lentiviral RNAi vector of ALR was constructed successfully. The titer of concentrated virus was 8E+8TU/ml.After being transfected by virus，ALR mRNA and protein levels were obviously reduced in both human hepatoma cell line HepG2 and QGY compared with those of nega－tive control and bland control cells（P<0.05）. Conclusion：The lentiviral RNAi expression vector targeting ALR gene is successfully constructed and it could effectively silence the expression of ALR in human hepatoma cell line HepG2 and QGY.