摘要
目的在毕赤酵母中分泌表达人肝细胞生长因子(Hepatocyte growth factor,HGF),并检测其活性。方法从真核表达质粒pRC/CMV-HGF中扩增HGF的编码基因,将其克隆入表达载体pPIC9K,构建重组表达质粒pPIC9K-HGF,电穿孔法转化毕赤酵母菌株KM71,PCR筛选阳性菌株,经甲醇诱导表达重组人HGF(rhHGF)。表达产物经SDS-PAGE和Western blot鉴定后,检测其对原代培养的大鼠肝细胞增殖的影响。结果重组表达质粒pPIC9K-HGF经酶切和测序证实构建正确;表达的重组蛋白相对分子质量约80 000,表达量占菌体总蛋白的18%,可与兔抗人HGF多抗特异性结合,并可刺激大鼠肝细胞增殖。结论在毕赤酵母菌中成功分泌表达了rhHGF,表达产物具有促进肝细胞增殖的活性。
Objective To express recombinant human hepatocyte growth factor(rhHGF) in secretory form in Pichia pastoris and determine its activity.Methods HGF gene was amplified from plasmid pRC / CMV-HGF and cloned into expression vector pPIC9K.The constructed recombinant plasmid pPIC9K-HGF was transformed to P.pastoris KM71 strain by electroporation for expression under induction of methanol.The expressed product was identified by SDS-PAGE and Western blot and determined for the effect on proliferation of rat hepatocytes in primary culture.Results Both restriction analysis and sequencing proved that recombinant plasmid pPIC9K-HGF was constructed correctly.The expressed rhHGF,with a relative molecular mass of about 80 000,contained about 18% of total somatic protein,showed specific binding to rabbit anti-human HGF polyclonal antibody and stimulated the proliferation of rat hepatocytes.Conclusion Recombinant human HGF was successfully expressed in P.pastoris in secretory form,which showed activity in promoting the proliferation of hepatocytes.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第8期903-906,910,共5页
Chinese Journal of Biologicals
基金
山西省科技攻关项目(20080311059-6)
细胞生理学省部共建教育部重点实验室主任基金(2010-01)
山西医科大学科技创新项目(01200713)
关键词
肝细胞生长因子
毕赤酵母
肝细胞
细胞增殖
Hepatocyte growth factor(HGF)
Pichia pastoris
Hepatocyte
Cell proliferation