麻疯树逆境蛋白(curcin 2)基因的原核表达

Expression of Stress Protein Curcin 2 Gene in E.coli

将编码麻疯树逆境蛋白curcin 2成熟蛋白的M片段和编码前体蛋白的P片段分别定向克隆到质粒载体pQE-30、pQE-31后,获得重组质粒pQE-30-M、pQE-31-P.该重组质粒转化大肠杆菌M15菌株,经IPTG诱导后M片段表达的蛋白质主要以包涵体形式存在,蛋白质的表达量与IPTG浓度及诱导时间有关.重组蛋白可与RGS-His抗体及curcin抗体分别发生专一性抗原抗体反应并被His TrapTMHP柱亲和纯化.体外抗真菌活性实验表明,复性处理后的curcin 2融合蛋白使玉米弯胞杆菌[Curvularia lunata(Walk)Boed]的菌丝生长受到一定程度的抑制.所有检测表明P片段未在M15中表达.以上实验结果为该信号肽的功能研究及curcin 2基因及其蛋白在农业生产上的研究和应用提供实验基础.

The M fragment encoding stress protein curcin 2 mature protein and the P fragment encoding curcin 2 precursor protein were inserted into a prokaryotic expression vector QIA express System pQE-30 and pQE- 31, respectively. The recombination plasmids pQE-30-M and pQE-31-P were obtained. The M fragment was overexpressed in E. coli M15. The recombinant gave rise to a protein in insoluble inclusion bodies in response to the IPTG induction. The protein content was related to IPTG concentration and the induced time. The antibody against RGS-His recognized specific band of 31.9 kD on Western blot, and the antiserum against curcin also recognized the same band. After the inclusion bodies were denatured and renatured, the purified fusion proteins was obtained by His Trap^TM HP chromatogrphy column. All the detection showed that the P fragment was not expressed. These results may provide experiment foundation to study the functions of signal peptide and curcin 2.