摘要
背景：肝细胞生长因子激活因子（HGFA）是活化单链前体HGF（pro—HGF）成为有生物学功能的HGF的关键酶．在受损组织器官的修复和再生中发挥重要作用。目的：制备rhHGFA—Fc融合蛋白并证实其具有肝细胞保护功能。方法：采用基因重组技术构建表达人源化HGFA成熟肽段与人IgGFc段融合蛋白的载体，以脂质体介导法转染人胚肾293细胞。纯化293细胞表达产物，以蛋白质印迹法鉴定纯化蛋白（rhHGFA—Fc融合蛋白）及其对pr0．HGF的酶切活性，以流式细胞术检测融合蛋白介导的肝细胞凋亡抑制作用。结果：SDS—PAGE显示转染重组质粒的293细胞总蛋白纯化产物在62kDa处显示单一蛋白条带。蛋白质印迹法结果显示纯化的rhHGFA-Fc融合蛋白能与抗hHGFA单克隆抗体发生特异性反应，并将pro-HGF酶切为仅链和p链，酶切活性呈剂量依赖性，半效酶切活性（IC50）为8．22nmol／L；流式细胞术结果显示融合蛋白通过激活pro．HGF发挥其抑制肝细胞凋亡的作用。结论：293细胞表达的rhHGFA—Fc融合蛋白纯度高、酶切活性强，可高效激活pro—HGF，对于肝组织严重损伤，特别是肝硬化基础上肝损伤的治疗具有潜在价值。
Background： Hepatocyte growth factor activator （HGFA） is the major protease that cleaves inactive single-chain HGF （pro-HGF） to its active form, and plays an important role in reparation and regeneration of injured tissue or organ. Aims To construct the rhHGFA-Fc fusion protein and to confirm its hepatocyte protective effect. Methods： The vector expressing fusion protein of humanized HGFA active fragment and IgG Fc fragment was constructed by gene recombination, and then transfected into human embryonic kidney 293 ceils by Lipofectamine 2000. The expression product of 293 cells was purified. The purified product （rhHGFA-Fc fusion protein） and its enzyme activity on pro-HGF was identified by Western blotting, its effect on mediating the inhibition of hepatocyte apoptosis was determined by flow cytometry. Results： The purified product of 293 cells transfected with the recombinant plasmid formed a lane at the position of 62 kDa with SDS- PAGE and had a specific reaction with monoclonal antibody against hHGFA by Western blotting, rhHGFA-Fc fusion protein could cleave pro-HGF into α chain β and chain, the enzyme activity was dose-dependent and the ICs0 was 8.22 nmol/L. The flow cytometry results indicated that the fusion protein could inhibit the apoptosis of hepatocyte by activating the pro-HGF. Conclusions： The rhHGFA-Fc fusion protein expressed by 293 ceils has high purity and enzyme activity, and can convert pro-HGF to its active form adequately. It might be a potential drug for severe liver injury especially the cirrhosis.
出处
《胃肠病学》
2010年第1期8-11,共4页
Chinese Journal of Gastroenterology
基金
上海市浦江人才计划资助(No.07pj14069)
