Expression of human augmenter of liver regeneration in pichia pastoris yeast and its bioactivity in vitro

摘要

AIM: To construct a yeast expression system of human augmenter of liver regeneration (hALR) and to examine its bioactivity in vitro. METHODS: With PCR and gene recombination techniques, cDNA of open reading frame of hALR was obtained from recombinant plasmid pcDNA3.1-hALR and inserted into plasmid pPIC9. The cDNA of hALR from recombinant plasmid pPIC9-hALR demonstrated by sequencing was subcloned into plasmid pPIC9K. The recombinant plasmid pPIC9K-hALR was transformed into GS115 with electroporation. hALR was expressed by GS115 under the induction of 5 mL/L methanol and purified with ultrafiltration after it was analyzed by 15% SDS-PAGE and Western blot. The effects of hALR on in vitro proliferation of QGY and HepG_2 cells were evaluated by ~3H-TdR methods. RESULTS: The correctness and integrity of recombinant plasmids pPIC9-hALR and pPIC9K-hALR were identified by restriction digestion, PCR and sequencing methods, respectively. hALR as a secretive protein was successfully expressed by GS115. Its molecular weight was about 15 ku and the target protein was about 60% of the total protein in the supernatant from GS115 with plasmid pPIC9K-hALR. The results of Western blot of hALR showed the specific band. The high qualitative hALR was obtained through ultrafiltration. hALR could stimulate in vitro proliferation of QGY and HepG_2 cells in a dose-dependent manner, but there was a difference in reactivity to hALR between QGY and HepG_2. CONCLUSION: The hALR as a secretive protein can be successfully expressed by GS115. It may stimulate in vitro proliferation of QGY and HepG_2 cells at a dose-dependent manner. But QGY and HepG_2 cells have different reactivities to hALR.