Construction and expression of eukaryotic expression vector of PEA-15 / 肿瘤研究与临床

ABSTRACT

Objective To construct the eukaryotic expression vector of pcDNA3.1-PEA-15 and express it in the human esophageal cancer (EC-109) cells,and to explore the effect of PEA-15 on EC-109 cells.Methods The PEA-15 gene was amplified from EC-109 by reverse transcriptase polymerase chain reaction (RT-PCR) and ligated to eukaryotic expression vector pcDNA3.1.After confirmation of recombinant plasmid was correctly by endonuc]eases digestion and DNA sequencing,the construct was transfected it into EC-109 through liposome inducing.The expression of PEA-15 in transfected EC-109 was detected by RT-PCR and Western blot.The cell growth inhibition ratio was evaluated by MTT assay.Results RT-PCR indicated that PEA-15 was highly expressed in EC-109 cells.The amplified fragment by RT-PCR was coincident with hypothesis enzyme digestion analysis and DNA sequencing confirmed that the pcDNA3.1-PEA-15 was constructed successfully.The expression of PEA-15 gene was increased obviously in the transfected EC-109 detected by RT-PCR and Western blot respectively (t =4.078,5.269,P ＜ 0.05).The cell growth inhibition ratio in the group which transfected pcDNA3.1-PEA-15 was significantly lower compared with the pcDNA3.1 transfect group after 72 hours (t =6.163,P ＜ 0.05).Conclusions The recombinant eukaryotic expression vector pcDNA3.1-PEA-15 is constructed successfully,and it can be expressed in EC-109.It also shows that PEA-15 has the function on cell growth which suggests that PEA-15 can inhibit the apoptosis of EC-109 cells and its expression involved in esophageal cancer development.