| Human augmenter of liver regeneration (hALR) is a new hepatic growth factor cloned from liver tissues of human fetal liver. It has the biological functions of stimulating the liver cell DNA synthesization to promote the liver regeneration. The further studies indicated that it also could increase the survival rate of acute hepatic failure and has the preventive effects on liver fibrosis. In this study, we expressed recombinant hALR (rhALR) in Pichia Pastoris GS115 with secretion, obtained high purity of rhALR by chromatography, and examine its bioactivity in vitro and in vivo.With PCR and genetic recombinant techniques, the coding region of hALR gene was amplified from recombinant plasmid pBV220- hALR, and the recombinant expression vector pPICZαA-hALR was constructed. After identified by sequencing, the vector pPICZαA-hALR was linearized by SacI and concentrated by isopropanol method and then transformed into P. pastoris GS115 by the methods of electroporation and spheroplast transformation. After identified by PCR and selected in the ZeocinTM, the recombinant transformants were induced with 0.5% methanol and rhALR-like could be found in the supernatant of culture. The 30kD protein was confirmed to be dimmer of rhALR by Western-blot. Quantitative analysis showed that rhALR produced by yeast was in a level of 66% of the total protein of the culture supernatant, with a yield of 100mg/L.Under the processing by 70% saturation of ammonium sulfate precipitation, Sephadex G25 desalination and low salt dialysis, the product was purified by Sepharose DEAE Fast Flow and Sephadex G75, resulting rhALR with 90% purity and 26% recovery. The bioactivity assay of the purified products showed that rhALR could stimulate the proliferation of HepG2, SMMC-7721 and NIH-3T3 in vitro. In 1/3 hepatectomy model of rats, the MI (mitotic index) and PCNA (proliferative cell nuclear antigen) in rhALR group was significant higher than in control. In acute liver injured rat induced by D-galactose, 100μg/L rhALR could increase the survival rate of liver injured rats.In conclusion, we successfully constructed the recombinant P. pastoris and obtained rhALR with high purity and strong bioactivity. |
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