肝细胞生长因子的表达、纯化和基本活性研究  

Expression, purification and preliminary activity study of recombinant hepatocyte growth factor prorein in E. coil

作　　者：郑筱娇[1] 高洲[1] 沈蓉蓉[1] 赵行[1] 岑东 罗建平 吕建新[1] 裴仁治 滑世轩[4] 

机构地区：[1]温州医学院浙江省医学遗传学重点实验室,325027 [2]宁波市鄞州疾病预防控制中心 [3]宁波市鄞州人民医院 [4]河南省人民医院

出　　处：《中华微生物学和免疫学杂志》2012年第11期967-971,共5页Chinese Journal of Microbiology and Immunology

基　　金：浙江省医药卫生科技项目（2007A175）;宁波市科技计划（2007C10065,2010A610031）

摘　　要：目的通过构建肝细胞生长因子（HGF）基因的重组原核表达载体，转化大肠杆菌，制备HGF重组蛋白并初步证实其活性。方法克隆HGF基因插入载体pET-26b（＋），构建重组原核表达载体pET-26b（＋）-HGF，转化E-coliRosseta（DE3）。IPTG诱导转化菌表达重组蛋白，采用Ni—NTA树脂亲和层析法进行纯化，复性后冷冻干燥。结果HGF基因重组原核表达载体pET．26b（＋）．HGF构建成功。转化pET-26b（＋）-HGF的EcoliRosseta（DE3）以包涵体形式大量表达目的蛋白，占菌体总蛋白的38％，并经Westernblot证实。经Ni—NTA树脂亲和层析法纯化后HGF蛋白纯度约为95％，作用于人非小细胞肺癌细胞系A549细胞发现可促进其增殖、迁徙并抑制凋亡。结论成功构建了HGF基因重组原核表达载体pET-26b（＋）-HGF，转化E-coliRosseta（DE3）后成功表达，纯化复性回复重组HGF蛋白结构，经体外实验证实具有生物学活性。Objective To prepare hepatocyte growth factor（HGF） recombinant protein and con- firm its activity preliminarily according to building HGF gene prokaryotic expression vector and transforming into E. coll. Methods Clone HGF inserted into the vector pET-26b （＋） to construct prokaryotic expression vector pET-26b（ ＋ ） -HGF and transform into E. coli Rosseta （ DE3 ）. The transformed bacteria induced by IPTG was purified through Ni-NTA resin affinity chromatography frozen-drying after renaturation. Results HGF gene recombinant prokaryotic expression vector pET-26b（＋）-HGF was constructed success- fully. E. coli Rosseta（ DE3 ） which was transformed into pET-26b（＋）-HGF expresses the target protein as the form of inclusion bodies, accounting for 38% of the total bacterial proteins, and confirmed by Western blot. HGF protein which was purified by Ni-NTA resin affinity chromatography, has a purity of about 95 %, and can promote proliferation, migration, and inhibition of apoptosis for human non-small cell lung cancer cell line A549 cells after interaction. Conclusion HGF gene recombinant prokaryotic expression vector pET-26b （＋） -HGF was constructed and expressed in transformed E. coli Rosseta （ DE3 ） successfully. They resumed their recombinant HGF protein structure after purification and renaturation, and had biological activity con- firmed by in vitro studies.

关 键 词：肝细胞生长因子 蛋白 重组 纯化 活性 

分 类 号：R363[医药卫生—病理学]