Cloning Of Recombinate Human Augmenter Of Liver Regeneration And Initial Expression In Pichia Pastoris

Augmenter of liver regeneration(ALR) plays a part in advancing hepatocytes'regeneration,anti-fibrosis and so on,it can promote the hepatic cell to regenerate when liver function failure or severe hepatitis caused by many reasons happen.We have made the expression of human-ALR in the E.coli come true,but the production is an inactive monomer,about 15KD.Considering the eukaryon expression system has the functions of phosphorylation,glycosylation,formation of disulfide bond and other protein processing,and many proteins have implement their expressions in active forms in the eukaryon expression system.So we carried out this research,hoping to realize the expression of hALR in eukaryon expression system in an active form.The research was carried out in two parts.The first part was to constructed the recombinant in E.coli DH5a successfully.We used the strain preserved in our laboratory as the template,PCR(polymerase chain reaction) was carried out,and we got the PCR fragment of about 424bp,which matched our expectation.PCR production was cut by BamHâ… and EcoRâ… ,and ligated to the vector pPIC3.5K that also cut by the same BamHâ… and EcoRâ… ,then transformed competent E.coli DH5a,extracted the plasmid in the positive E.coli DH5a.Cut the recombinant plasmid with BamHâ… and EcoRâ… ,PCR amplification was prepared for the initial identification,then sent the positive plasmid to sequence,finally we acquired the correct recombinant pPIC3.5K - ALR.The second part,the recombinant pPIC3.5K - ALR was linearized by Salâ… ,then electrotransformed competent Pichia pastoris GS115,the Mut~+ trasformants were differentiated from Mut~s via comparison of patches growth rate on MM and MD plates, by means of PCR analysis,the positive recombinant Pichia pastoris strains were selected.Then the positive strains was induced by BMMY,sampled at 0h,12h,24h,48h,72h and 96h after induction.SDS-PAGE was carried out on the supernant and cell sediment of different induction time,finally we got the protein of 40KD in the cell sediment,which meted our expectation,so we initially realized the expression of hALR in Pichia pastoris.