Fusion expression and identification of porcine cholecystokinin and somatostatin
The objective of this study was to prepare recombinant porcine cholecystokinin and somatostatin fusion protein(rpCCK-SS)using a prokaryotic expression system.The synthetic porcine cholecystokinin and somatostatin fusion gene(pCCK-SS)was cloned into pET28a and pCold GST vectors,transformed into BL21(DE3)or Transetta(DE3),and the recombinant expression strains were constructed,and identified by double enzyme digestion and sequencing.The concentration and induction time of the inducer were optimized by SDS-PAGE and Western blot.The results showed that the total length of pCCK-pSS gene was 693 bp,encoding 231 aa.BL21(DE3)-pET28a-pCCK-SS expresses the target protein with a molecular weight of 25 kDa and exists in the form of inclusion bodies.Transetta(DE3)-pCold-pCCK-SS was expressed with a molecular weight of 50 kDa in both supernatant and precipitate,mainly in the form of inclusion bodies in precipitate.The optimal expression conditions were 15℃and induced by 0.25 mmol/L IPTG concentration for 8 h.The results showed that rpCCK-SS was successfully expressed by two expression vectors,and the expression effect of pCold GST was better than pET28a,which laid a foundation for further development of new growth promotion agents.
Porcine cholecystokininSwine somatostatinLow temperature inductionFusion expression
万方数据
维普
