Construction and Expression of the Eukaryotic Expression Vector Containing AIF Gene

Objective To construct the eukaryotic express vector containing apoptosis-inducing factor (AIF) gene and to study its expression in A549 cells. Methods According to the GenBank AIF mRNA sequence, specific primers to amplify AIF gene from lung carcinoma cell line A549 by RT-PCR was designed. The amplified AIF gene fragment was cloned into plasmid pUC-T by TA cloning, then double enzyme digestion and DNA sequencing were used to identifying the positive recombinant AIF-pUC-T. The target fragment was retrieved and cloned into the eukaryotic express vector pcDNA3.1 (+). The positive recombinant AIF-pcDNA3.1 (+) was transfected into A549 cells, and expression of AIF gene was verified by RT-PCR and Western blot. Results AIF target gene was successfully amplified and cloned into the pUC-T. The target fragment was retrieved and cloned into the eukaryotic express vector pcDNA3.1 (+), and it was completely coincided with the AIF sequence in GenBank suggested by the result of DNA sequencing. Showed by the results of RT-PCR and Western blot, AIF gene expression in A549 cells transfected with AIF-pcDNA3.1(+) was much higher than that of control cells which was not transfected with AIF-pcDNA3.1(+). Conclusion The AIF eukaryotic expression vector AIF-pcDNA3.1 (+) is successfully constructed in A549 cells and it could be experimental foundations for further study of AIF gene.