P253R突变型FGFR2Ⅲc胞外段的表达、复性及活性研究(英文)  

Expression,Refolding,and Activity Studies of the FGFR2Ⅲc Soluble Ectodomain Mutant P253R

作　　者：刘雪婷[1] 喻志红[1] 何水连[1] 陈安安[1] 王丁丁[2] 何颖[1] 张志成[1] 汪炬[1] 

机构地区：[1]暨南大学生物工程研究所,广州510632 [2]广东药学院生命科学与生物制药学院,广州510006

出　　处：《药物生物技术》2011年第6期475-480,共6页Pharmaceutical Biotechnology

基　　金：The ministry of science and technology of china(2009ZX09103-632);The national natural science foundation of china(91029742)

摘　　要：成纤维细胞生长因子(Fibroblast growth factor,FGF)具有促进细胞增殖,诱导新生血管形成等重要生理作用,与肿瘤的发生及发展也有十分密切的关系。P253R是Apert综合症中发现的一种突变,可使FGFR2IIIc与FGF2亲和力大大增加,从而导致过度自分泌和旁分泌活性,引起骨骼发育异常。从人胎盘组织提取总RNA,通过RT-PCR扩增得到野生型FGFR2IIIc胞外段基因片段(wsFGFR2);再利用重叠延伸法以wsFGFR2基因片段为模板扩增得到P253R突变型FGFR2IIIc胞外段基因片段(msFGFR2,P253R是Apert综合征中发现的一种突变,可使FGFR2与FGF2亲和力大大增加)。msFGFR2基因克隆到pET3c载体,并在大肠杆菌BL21(DE3)中以包涵体的形式表达。经凝胶层析复性和肝素亲和层析纯化得到复性成功的活性蛋白,复性率为12%,纯度大于95%。MTT结果表明,msFGFR2能显著抑制FGF2促NIH3T3细胞增殖能力,并抑制前列腺癌DU145细胞的增殖,提示msFGFR2能有效阻断FGFs的细胞内信号通路,具有很好的产业化和临床应用前景。Fibroblast growth factor （FGF） can promote cell proliferation and induce angiogenesis. It has been correlated with tumorigenesis. A mutant form of the FGF receptor 2IIIc （FGFR2） , P253R, which is found in Apert syndrome, has a higher affinity for FGF2 and causes excessive autoerine and paracrine signaling,leading to skeletal dysplasia. Total RNA was extracted from human placental tissue and the ectodomain of FGFR2IIIc （wsFGFR2） was amplified using reverse- transcriptase polymerase chain reaction （RT-PCR）. The P253R mutant （msFGFR2, P253R mutant found in Apert syn- drome which has a higher affinity for FGF ligands ） was obtained through overlap-extension PCR by using wsFGFR2 as template, msFGFR2 was cloned into a pET3c vector and expressed them in the form of inclusion bodies in Escherichia coli strain BL21 （ DE3 ）. Successfully refolded proteins were isolated by gel chromatography and purified by heparin affinity chromatography. The refolding yield of msFGFR2 was 12%, with purity higher than 95%. MTT cell proliferation assay showed that msFGFR2 significantly inhibited tHe stimulatory effect of FGF2 on NIH3T3 cell proliferation, msFGFR2 also effectively inhibited the proliferation of the prostate cancer cell line DU145, indicating that msFGFR2 could block the FGF signaling pathway and had a potential clinical application in cancer therapy.

关 键 词：可溶性成纤维细胞生长因子受体2 包涵体 复性 转化生长因子-2 

分 类 号：Q812[生物学—生物工程]