Construction of TIP30 expression vector and its expression in HepG2 cells

Objective:To investigate the construction of eukaryotic expressing vector containing human full-length Tip30 gene(pAAV-TIP30) and observe its expression in the transfected hepatocellular carcinoma(HCC) cell line HepG2.Methods:Using the mRNA extracted from normal human liver tissue as a template,Tip30 gene was amplified through reverse transcription polymerase chain reaction(RT-PCR) method.The PCR products were digested with BamH I and Xba I,and cloned into eukaryotic expressing vector pAAV-MCS.The recombinant plasmids were then transfected into the HepG2 cells and the expression of TIP30 protein in the host cells was detected by Western blot analysis.Results:Full-length TIP30 gene was correctly ampli ed by RT-PCR method.The identity of the cloning of TIP30 gene in pAAV-MCS was confirmed by restrictive enzyme digestion and sequencing.Western blot showed that the protein expression of TIP30 increased in the HepG2 cells,after they were transfected with the recombinant plasmids.Conclusion:Recombinant plasmid pAAV-Tip30 has been cloned and transfected into HepG2 cells successfully,which can provide a basis for the further investigation of the effects of TIP30 gene on HCC and the underlying mechanisms as well.