The Alr In The Pichia Pastoris Expression And Biological Activity Study

Objective: To construct yeast expression plasmid of human augmenter of liver regeneration (hALR), to purify it after expression in GS115,to identify its bioactivity in vitro and prepare polyclonal antibody against hALR as a foundation of studying its biological functions.Methods: With PCR and genetic recombination techniques, the gene fragments of hALR were amplified from recombinant plasmid pcDNA3.1-hALR and recombinant plasmid pPIC9-hALR was constructed. The DNA of hALR in recombinant plasmid pPIC9-hALR was subcloned into plasmids pPIC9K. The recombinant plasmid pPIC9-hALR and pPIC9K-hALR were transformed into GS115 by electroporation. The hALR were expressed in GS115 under the induction of methanol. The hALR were purified with ultrafiltration. All purified products were identified by 12% SDS-PAGE. The effects of hALR on the proliferations of QGY and HepG2 in vitro were evaluated by 3H-TdR methods. In order to acquire thepolyclonal antibody against hALR, rabbits were immunized with purified hALR. The serum of rabbit was detected by ELISA methods.Results: Recombinant plasmid pPIC9-hALR and pPIC9K-hALR were successfully constructed and hALR were efficiently expressed as a secretive protein in GS115. Its molecular weight, with 1.5×104 dalton, was in correspondance with theoretic value. Single band of hALR was watched in SDS-PAGE electrophoresis after ultrafiltration. The hALR could indeed stimulate the proliferations of QGY and HepG2 in vitro at a dose-dependent way, but there was different reactivity to hALR between QGY and HepG2.. It was demonstrated by ELISA that the serum from rabbit immunized with hALR not only reacted with hALR, but also with rALR. The polyclonal antibody against hALR could be used to detect rALR because of the existence of cross-immunogenicity between hALR and rALR.Conclusion: The hALR was efficiently expressed in GS115 and successfully purified by ultrafiltration. The proliferations of QGY and HepG2 were stimulated by hALR in vitro at a dose-dependent way, but both reactivities to hALR were different. The successful preparation of polyclonal antibody against hALR made it possible to detect hALR and rALR...