摘要
目的:实现成纤维细胞生长因子受体3(FGFR3)胞外配体结合区FGFR3D23的表达与纯化,并鉴定其与A型肉毒毒素重链C端(Bo NT/AHc)的相互作用。方法:全基因合成的FGFR3D23基因片段经酶切连入p TIG(+)质粒,转化大肠杆菌BL21(DE3)后IPTG诱导表达,获得的包含体经Ni2+柱纯化,透析复性后通过pull-down及ELISA检测纯化蛋白与Bo NT/AHc的相互作用。结果与结论:SDS-PAGE分析结果显示得到相对分子质量约27×103的蛋白特异表达条带,表达量为1.39 mg/L,包含体占73.4%;纯化和复性得到的目的蛋白纯度约96%,复性率约3%;pull-down及ELI-SA实验结果证明FGFR3D23可以与Bo NT/AHc特异性地相互作用。
Objective： To express and purify fibroblast growth factor receptor 3 extracellular ligand-binding region （FGFR3D23）, and to investigate its interaction with the C-terminal of botulinum neurotoxin serotype A heavy chain （BoNT/AHc）. Methods： The synthesized FGFR3D23 gene was digested and inserted into pTIG（＋） plasmid. Then the recombinant vector was transformed into host strain E.coli BL21（DE3）. After IPTG induction, the expressed in？clusion bodies were purified by Ni2＋-chelating chromatography. The purified protein was refolded by dialysis, and its interaction with BoNT/AHc was detected by pull-down and ELISA assays. Results ＆amp; Conclusion： The recombi？nant protein with relative molecular weight of 27＆#215;103 was identified by SDS-PAGE analysis. FGFR3D23 was at a final protein concentration of 1.39 mg/L, in which inclusion bodies accounted for 73.4%. After purification and re？folding, the purity of the recombinant protein was about 96%, and the refolding rate was about 3%. Pull-down and ELISA assays showed that FGFR3D23 protein could interact with the BoNT/AHc.
出处
《生物技术通讯》
CAS
2015年第2期203-206,共4页
Letters in Biotechnology
关键词
成纤维细胞生长因子受体3
胞外配体结合区
A型肉毒毒素重链C端
相互作用
fibroblast growth factor receptor 3
extracellular ligand-binding region
C-terminal of the heavychain of botulinum neurotoxin serotype A
interaction
