The Expression Of Human Augmenter Of Liver Regeneration In Pichia Pastoris、Purification And Activity Detection

Object:To lay the foundation for subsequent further study the biological functions of 15kD human augmenter of liver regeneration(ALR), construct the eukaryotic expression system which convenient for purification of pPICZaA-rhALR GS115.Methods:1.With Polymerase chain reaction (PCR), and gene recombination techniques, DNA of rhALR was obtained from recombinant plasmid pPIC9K-rhALR. The purified cDNA of hALR and empty plasmid pPICZaA were digested by double enzymes(Xho I and Xba I) respectively. Both enzyme product after purification were linked by DNA ligase, constructed recombinant plasmid pPICZaA-rhALR.2.The recombinant plasmid pPICZaA-rhALR demonstrated by sequencing, PCR and double digestion was linearized by digestion with SaCI, and transformed into GS115 with electroporation. YPDs containing Zeocin to select positive transformations, then select putative multicopy recombinants on the YPDs containing increasing concentations of Zeocin.The multicopy recombinants were identified by colony PCR. The rhALR was secreted expression by GS115 under the induction of 10 mL/L methanol. The expression supernatant was analyzed by Western Blot(ALR polyclonal antibody and His-tag mouse monoclonal antibody) and SDS-PAGE gel electrophoresis.3.The expression supernatant was purified through Nickel column affinity chromatography method, then dialyzed with intercept molecular weight of 7kD dialysis bag. The purification effect was analyzed by SDS-PAGE gel electrophoresis. The concentration of purified protein was measured by BCA reagent.4. The effects of rhALR on in vitro proliferation of human hepatoma cell lines (QGY and HepG2 cells), as well as the effects of it on reducing QGY cellular proliferation inhibiton rate induced by DDP, were evaluated by MTS reagent. The anti-apoptosis effects of rhALR on QGY cells apoptosis induced by DDP were detected by Flow Cytometer.Results:l.The recombinant plasmid pPICZaA-rhALR of 15kD rhALR for expression in pichia pastoris GS115 was constructed successfully. And it was identified by Polymerase chain reaction(PCR), direct sequencing and restriction enzyme reaction methods respectively.2.Several multicopy recombinants were selected on the YPDs containing 2000μg/ml Zeocin. The recombinant pichia pastoris was proved to be Mut+ type by colony PCR, namely methanol using normal type. The rhALR as a secretory protein was expressed in GS115 efficiently, which with molecular weight about 17.5 kD accounts for 70% of the total protein in the supernatant from pPICZaA-rhALR GS115. The results of Western Blot all showed the specific single band.3.The results of purified product through SDS-PAGE gel electrophoresis analysis all showed the spicific single and concentrated band. The concentration of rhALR protein after purification was (800-1600) μg/ml measured by BCA reagent.4.The rhALR could stimulate in vitro proliferation of QGY and HepG2 cells in a dose-dependent manner, also could reduce cellular proliferation inhibition rate and play an anti-apoptosis effect when QGY cells treated with DDP in a dose-dependent manner.Conclusion:The rhALR as a secretory protein is expressed in pPICZaA-rhALR GS115 efficiently and purified through Nickel column affinity chromatography successfully and in vitro experiment confirm that it owns well bioactivity.