The Study And Evaluation Of Various Expression Engineering Systems Expressing Human Hepatocyte Growth Factor And A Preliminary Experimental Study For Gene Therapy

Hepatocyte growth factor(HGF)is composed of a disulfide linked 69 kDa α chain and a 34 kDa βchain. HGF is the most potent stimulator of DNA synthesization mature hepatocyte. It regulates cell growth, development, motility and morphogenesis. HGF plays a major role in the regenreration of the liver, may well be a key factor in kidney regeneration, and may regulate wound healing. But native HGF is not easily obtained by purification from plasma. In ordre to acquire a great quantity of bioactive HGF, we have design to set up various HGF expression engineering system, including prokaryotic expression engineering system, mammalian expression engineering system and Pichia pastoris expression engineering system,etc. In the first part of this study, the HGF nucleic acid sequence and RE map site were analyzed using bio-software. Aware of the sequence by analysis of multiple RE mapping could help us construct expression vector successfully. In the second part of this study, two prokaryotic expression vectors :PBV220-HGFα and PBV220-HGFβ were constructed, and the recombinaint plasmid were transformed into host strains,such as DH5α,JM109,BL21(DE3). Thus we obtained high expressing engineering bacteria strain, BL21(DE3)-HGFαand BL21(DE3)HGF-β. The molecular weights of the recombinant HGF was analyzed by SDS-PAGE. The expressed product identified by Western-Blot. The condition in expressing period, such as the composing of culture medium, culturing temperature, and induction time et al were experimented and a perfect culturing condition was confirmed to express HGF. Recombinant HGF produce as inclusion body in Escherichia coli was purified via several steps including solubilization of the inclusion bodise in 8 M urea and refolding by dilution of denaturant. Using primary rat hepatocyte bioassay, we have tested the biological activity of recombinant HGF stimuldted DNA sythesization in primary adult rat hepatocytes. In the third part of this study, we constructed a mammalian expression vector pRC/CMV-HGF. The analysis of multiple RE mapping indicated that the recombinant plasmid has correct cloned. The recombinaint plasmid were transfected into CHO cells. Screening with G418 at 800μg/ml, we obtained the transfected cell clones. The medium of stable expression cell lines stimulate the primary adult rat hepatocytes by 3H-TdR incorperation analysis. All these demonstrated that the mammalian expression system product bioactive HGF.In the fourth part of this study, the expression of HGF gene in Pichia pastoris was investigated .The HGF cDNA without coding signal peptide sequences was inserted into clone vector pMD18－T－HGF, after the sequence analysis, the target sequence was insert into expression vector pPIC9k containing AOXl promoter and the sequences of alpha secreting signal peptides. The analysis of multiple RE mapping indicated that the recombinant plasmid has correct constructed. Then the recombinant plasmid were transformed into Pichia pastoris host strain KM71. Then transformant was screened out and cultured in flasks, and HGF was expressed under the induction of 0.5% methanol. Biological assay proved that the expressed product could stimulate the proliferation of primary adult rat hepatocytes cells. Thus a highly recombinant Pichia Pastoris efficient expression was constructed. The availability of a recombinant HGF opens the way for on vivo experimental studies and to the possibility of using HGF as a clinical therapeutic agent.In the fifth part of the study, we investigate HGF gene transfection and expression in human umbilical vein endothelial cells( ECV304) with liposome in vitro and study its effect on the bioactivity of these cells. The recombinaint plasmid was constructed and transfected into human umbilical vein endothelial cells with liposome:GeneSHUTTLE-20, and positive clones were selected by G418. The transfection and the expression of HGF in ECV304 were tested by RT-PCR and immunohistochemistry. The activity of HGF was estimated by MTT assay. After G418 selection, the cell clones...