Cloning,Expression Of Mouse Scya1,Scya2,Scya12 Gene And Studies On Function Of Their Expression Products

Object:To clone the cDNA sequence of mouse scyal, scya2 and scya12 gene in procaryotic host based system and construct their recombination pithia expression vectors, and express them in pithia pastoris and identify the function of expression proteins.Methods:Mouse scyal, scya2 and scya12 cDNA were amplified with total RNA from mouse liver Tissue by RT-PCR then cloned into cloning vector pGEM-T and sequenced.The recombination cloning vector of mouse scyal, scya2 and scya12 cDNA was respectively double restriction enzyme digested by EcoRI and Notâ… EcoRâ… and Xbaâ… , Xhoâ… å'ŒXbaâ… then cloned into pithia expression vector pPICZαA to construct secreting recombinant expressing plasmids of pPICZaA/scyal, pPICZaA/scya2 and pPICZaA/scyal2.The recombinant expressing plasmids of pPICZaA/scyal, pPICZaA/scya2 and pPICZaA/scyal2 were respectively cloned,sequenced and linearizated then integrated into genome of pichia pastoris X-33, The transformants were selected and identified by PCR.The selected strain was induced by 0.5% methanol to secrete expression,then detect the recombinant proteins by SDS-PAGE.Finaly, the recombinant proteins were roughly extracted to carry out the study of simple bacteriostasis.Results:1-Mouse scya1,scya2 and scya12 cDNA were cloned,identified and sequenced with pGE M-T vector, and their sequencing results are identical to the published sequences in Genbank which enter mumber are respective NM₀11329-2, NM₀11333-3 and NM₀11331-2.The result is to establish foundation for further study to mouse scya1,scya2 and scya12 cDNA2,The cloned mouse scyal, scya2 cDNA were double restriction enzyme digested and cloned into pP ICZαA vector then transformed into bacterium coli, however, the constructed recombinant expression vector could not be enriched by cloning. For this reason, the recombinant expression vector of mouse scyal, scya2 cDNA was not integrated into genome of pichia pastoris X-33 to induce expression.3,The recombinant pichia expression vector of pPICZaA/scya2 was successfully constructed and inte-grated into genome of pichia pastoris X-33 to express.The recombinant protein was discovered in yeast culture supernatant which relative molecular mass is approximately 16600. The bacteriostasis of the rou-ghly extracted recombinant proteins is not discovered.Conclusion:Mouse scyal and scyal2 cDNA could be stably existed and cloned in pGEM-T vector, but their recombinant pichia expression vectors could not be abundantly cloned.The recombinant proteins of scya2 cDNA could be expressed in X-33 strain,this is to establish foundation for abundantly gaining recombinant ptoteins of scya2 cDNA and study of their more function. In this study recombinant pichia expression vectors of Mouse scyal, scya2 and scya12 cDNA firstly were consruct-ed and the recombinant ptoteins of scya2 cDNA firstly were expressed in pichia pastoris X-33.