关键词: PNPLA3, 过表达, 干扰, 腺病毒

Abstract: Objective To construct a adenovirus vector of PNPLA3 expression and interference, a gene related to metabolism of glucose and lipid and liver fatty disease. Methods The expressional and interferce primers were designed according to PNPLA3 gene sequence，the sequences were cloned into pAdTrack-CMV or pAdTrack-U6 vector with BglII and xhoI restriction site. The E.coli BJ5183 sensitive bacterias were cotransfected with lined vector cutted by PmeI enzyme and adenovirus vector pAdEasy-1. The obtained recombinant adenovirus vector was cutted with PacI enzyme, then adenovirus was obtained in 293A cells transfected with lined recombinant adenovirus plasmids. The titer of virus was measured based on the expression level of green fluorescent protein. The transfection efficiency of green fluorescent protein into mouse primary hepatocytes was calculated. Results DNA sequencing and digestion identification demonstrated that the expression and interference adenovirus vectors of PNPLA3 gene were constructed. The titer of concentrated virus was 1.5×1011VP/ml.The infection efficiency reached above 90% and interference efficiency decreased endogenous PNPLA3 mRNA in hepatocytes by more than 80% when mouse primary hepatocytes were stabled transfected with 120 infection multiplicity. Conclusion The expressional and interference adenovirus vector of PNPLA3 gene is successfully constructed.

Key words: PNPLA3, Overexpression, Interference, Adenovirus