摘要
目的:克隆sFGFR1Ⅲc胞外段进行原核表达和纯化。方法:将sFGFR1Ⅲc胞外段基因转入pET3c载体后转化BL21工程菌,以包涵体形式表达胞外段。经凝胶层析法复性(复性率为20%)和肝素亲和层析纯化后得到纯度95%以上的目的蛋白。通过电泳鉴定其纯度以及MTT法检测其生物学活性。结果:成功获得大小为660bp的人sFGFR1Ⅲc基因片段,转化菌诱导表达了24kDa的sFGFR1Ⅲc胞外段并通过MTT证实它对小鼠胚胎成纤维细胞(NIH3T3)的增殖具有明显的抑制作用,其抑制FGF2引起的细胞增殖呈剂量依赖性,其中作用最为明显的是80ng/ml。结论:成功获得了高纯度的sFGFR1Ⅲc胞外段,该胞外段具备较好的生物学活性,为下一步的研究工作奠定了良好的物质基础。
Objective ：Cloning,expression and purification of human recombinant sFGFR1Ⅲc.Method：The gene of soluble ectodomain of FGFR1Ⅲc（sFGFR1Ⅲc） was cloned into pET3c vector,and then expressed as inclusion body in Escherichia coli strain BL21.Successfully refolded protein was isolated by gel chromatography and purified by heparin affinity chromatography.The refolded yield of sFGFR1Ⅲc was 20%,with purity higher than 95%.Its purity was identified by electrophoreses analysis as well as biological activity detected by MTT cell proliferation assay.Result：It＇s successful to clone a sFGFR1Ⅲc cDNA of 660bp into pET3c vector,and express recombinant sFGFR1Ⅲc of 24kDa induced in E.coli.Also,it has been identified by MTT that sFGFR1Ⅲc was effective in inhibiting the proliferation of the mouse embryonic fibroblast cell line（NIH3T3） in a concentration-dependence manner,and the most efficacious concentration is 80 ng/ml.Conclusion： The Results indicate that a high purity sFGFR1Ⅲc with good bioactivity was obtained and had a potential biological function as the standard material for the further study.
出处
《生物技术》
CAS
CSCD
北大核心
2012年第1期22-26,共5页
Biotechnology
基金
国家科技部"十一五"新药创制重点项目("广谱抗肿瘤新药--tsFGFR2的研究与开发"
2009ZX09103-632)资助~~
