Construction of Eukaryotic Expression Plasmid of Human Full-length Adiponectin and Its Expression in LX-2 Cells

Objective To construct a full-length adiponectin eukaryotic expression plasmid,and investigate its transfection and expression in human hepatic stellate(LX-2) cells. Methods Human adipose tissue mRNA was extracted and the full-length adiponectin gene fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR). Both amplified target gene fragment and empty vector plasmid P7 were double-digested,then the digested fragments were ligated and transformed into E. coli. Successful constructed plasmid was transfected to LX-2 cells by FuGENE HD reagents. Immunofluorescence staining was explored to detect the transfection efficiency and cellular localization,and Western-blot was used to detect the adiponectin protein expression. Results The sequence of DNA fragment from constructed P7-adiponectin plasmid was identified to that published in GenBank. The transfection efficiency detected by immunofluorescence staining was about 40%,A 30 000 protein was detected by western-blot which was consistent with the expected molecular weight. Conclusion Human full-length adiponectin eukaryotic expression vector was successfully constructed,it could be effectively transfected into LX-2 cells and could express the adiponectin protein.