Molecular Cloning And In Vitro Expression Of Porcine Sirtuin3 Gene

Members of Sirtuin gene family are regulatory silence factors in mammal cells, which influence transcription factors of the cell cycle and the regulation of cellular catobolism, and play a role in cell functioning in aging process. We report here the whole suquence of swine Sirtuin 3 gene has been cloned and further expressed by inserted into prokaryotic genome, and this will lead to studies on biological functions of swine Sirtuin 3 and its application.EST tags were aquired by searching in swine ESTs library with human Sirtuin gene sequence (NM-001017524), then were spliced into one fragment, based on which PCR primers were disigned for the replication of whole gene of swine Sirtuin 3. This gene was transferred into plasmid T carrier, and was sequenced and analysed. The results showed that the 999nt gene contained a intact OFR, encoding 332 amino acids out of which the forgoing 22 residues were signal peptides. This gene sequence has already included in GenBank (EU187292).The intact gene for mature protein of swine Sirtuin 3 was proliferated by PCR and then integrated into pGEX-4T-l carrier, through which a recombinant plasmid pGEX-Sirtuin 3 was aquired. The plasmid was transferred into E. coli BL21 (DE 3) genome, and fused protein was expressed under the IPTG induction. The results of SDS-PAGE showed that this fused protein pGEX-Sirtuin 3 was about 58kDa, conforming its expected molecular size.In conclusion, the whole sequence of swine Sirtuin 3 gene was obtained, a rembinant plasmid expressing swine Sirtuin 3 protein was constructed and the accoring fused protein was verified. Therefore, further studies on swine Sirtuin 3 will be feasible.