Construction and Expression of the Eukaryotic Expression Vector Containing AIF Gene

DENG Wen-wen, LIU Shu-kun, ZHANG Zun-zhen

Abstract

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.

 

Keywords: Apoptosis-inducing factor, Eukaryotic expression vector, TA cloning, Transfection

 

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References


Susin SA, Leornzo HK. Zamzami N, et ul. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature,1999;397(6718):441-446.

Cande C, Cecconi F. Dessen P, et al. Apoptosis-inducing factor ( AIF ): key to the conserved caspase-independent pathways of cell death. J Cell Sci,2002; 115(24):4727-4734.

Van Loo G, Saelens X. Van Gurp M, et al. The role of mitochondrial factors in apoptosis;a Russian roulette with more than one bullet. Cell Death Difference, 2002; 9 ( 10 ): 1031- 1042.

Otera H. Ohsakaya S, Nagaura Z, et al. Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space. EMBO J. 2005; 24 (7):1375-1386.

Van Gurp M, Festjens N, Van Loo G, et al. Mitochondrial intermembrane proteins in cell death. Biochem Biophys Res Commun, 2003;04 (3); 487-497.

Klein JA, Longo-Guess CM, Rossmann MP, et al. The harlequin mouse mutation down regulates apoptosis-inducing factor. Nature,2002;419(6905) :367-374.

Reix S, Mechawar N, Susin SA, et al. Expression of cortical and hippocampal apoptosis-inducing factor (AIF) in aging and Alzheimer's disease. Neurobiol Aging,2007;28(3) ;351-356.

Anne-Sophie A. Iman L, Dounia D, et al. Apoptosis-inducing factor regulates skeletal muscle progenitor cell number and muscle phenotype. PLoS ONE.2011 ;6( 11): 1-15.

Delavallee L. Gabon L, Galan-Malo P. et al. AIF-mediated caspase-independent necroptosis: a new chance for targeted therapeutics. IUBMB Life,2011 ;63(4):221-232.


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