Comparison of Conventional Plasmid Vector and Semliki forest virus-derived Vectors in Expressing Growth Hormone Releasing Hormone (GHRH)

The elements for Semliki forest virus (SFV) RNA replicon, a kind of new generation vector, comes from the Alphavirus genus. It was designed to overcome the poor efficacy of some current plasmid vector. Genes coding for viral replicases are reserved while genes coding for structure proteins are replaced by foreign gene in RNA replicon. High level replication of RNA and expression of foreign gene in cytoplasm are regulated by the replicases. To evaluate the effects of the SFV RNA replicon on the efficiency of gene expression, LacZ gene was inserted into pIRES-neo which digested by BamHⅠand dephosphorylated by shrimp alkaline phosphatase, and pIRES-neo-LacZ vector was constructed. RNA replicon vector pCMV-rep-LacZ and two conventional CMV promoter-based vector (pLNCX-LacZ and pIRES-neo-LacZ) were transfected by Lipofectin to prepared 293 cells, respectively. RNA replicon vector pCMV-Rep-GHRH(Growth hormone releasing hormone) and two conventional CMV promoter-based vector(pCDNA3.1(+)-GHRH and pIRES-neo-GHRH) were transfected by Lipofectin to prepared 293 cells, respectively, too. Results of quantitating for β-galactosidase expressed from transfected cells and quantitating for GHRH expressed from transfected cells by RIA and RT-PCR showed that the express level of RNA replicon vector was superior to ordinary vector and 2~3 times higher than normal plasmid vector. This will provide a beneficial exploring to improve the efficiency of gene expression in eukaryotic cell.