Construction of a recombinant adenovirus vector containing Apoptin gene

Objective To construct a recombinant adenovirus carrying Apoptin gene so as to provide basis for further studying Apoptin gene functions and ascertaining corresponding mechanism. Methods The shuttle vector pAdTrack-CMV-VP3 containing Apoptin gene was linearized with Pmel and subsequently electrotransformed into BJ5183-AD-1 cells for homologous recombination. The DNA containing Apoptin gene of the recombinant adenovirus was obtained by digesting with PacI and then transfected into 293 cells, where the recombinant adenovirus containing Apoptin gene was identified by RT-PCR and purified by cesium choride density centrifugation. Finally, the titre of the recombinant adenovirus was determined by biological and physical assays. Results Digestion with PacI proved successful homologous recombination. Green fluorescence showed successful recombinant adenovirus with infectiveness. RT-PCR confirmed that the recombinant adenovirus had coding of Apoptin gene proteins. The titre of the recombinant adenovirus was 84.52×109 VP/ml and 6.561×109 PFU/ml. Conclusion The recombinant adenovirus vector containing Apoptin gene is successfully constructed, with its titre sufficient for in vivo and in vitro experiment.