Construction and expression of enhanced green fluorescent protein and HSV1-TK co-expression vector

Objective To construct EGFP and HSV1-TK co-expression vector and to detect its expression level in hepatoma carcinoma cell line HepG2, and thus to provide experimented bases for the target gene therapy of liver cancer. Methods HSV1-TK gene was isolated from pORF-HSV1TK plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP.The recombinant plasmid pIRES2-EGFP-TK was identified by restriction endonuclease digestion and DNA sequencing. Then recombinant plasmid was transfected into hepatoma carcinoma cell line HepG2 by liposome reagent,and the expression of EFGP in cell were observed by fluorescence microscopy , TK protein and mRNA was analyzed by Western blotting and RT-PCR respectively. Results The sequence of the cloned DNA fragment was identical to HSV1-TK that was reported on Gene bank.The recombinant expression plasmid was successfully transfected into HepG2 cell line, and green fluorescent was observed under fluorescent microscope, positive clones were picked out by G418 screening. The optimal G418 screening concentration was 600g/L.The mRNA expression level of TK was high and its protein expression was found. Conclusion The recombinant eukaryotic co-expression vector of EGFP and HSV1-TK was successfully constructed and effectively expressed in HepG2 cell line.