蜡样芽孢杆菌非溶血性肠毒素基因的原核表达及多克隆抗体制备

发布时间：2018-01-25 10:53

本文关键词： 蜡样芽孢杆菌非溶血型肠毒素原核表达多克隆抗体　出处：《中国病原生物学杂志》2017年07期 　论文类型：期刊论文

【摘要】：目的分别对非溶血性肠毒素基因(Nhe)的3个亚基进行原核表达,并对蜡样芽孢杆菌分泌的非溶血性肠毒素蛋白进行免疫学鉴定。方法根据NCBI数据库非溶血性肠毒素NheA、NheB、NheC基因序列设计引物,以蜡样芽孢杆菌的基因组为模板,PCR扩增NheA、NheB、NheC基因。分别将3个基因克隆到原核表达载体pET-30a(+)后转化到大肠埃希菌BL21(DE3),IPTG诱导表达目的蛋白,进行SDS-PAGE分析以及Western blot分析,并免疫新西兰大白兔,制备多克隆抗体。结果经PCR和酶切鉴定,成功构建重组表达载体。重组表达载体分别转化至BL21(DE3),经IPTG诱导表达相应的目的蛋白,分子质量单位分别为NheA 40ku,NheB 44ku,NheC 40ku。经电离飞行时间质谱鉴定,重组蛋白NheA、NheB和NheC表达正确。3种目的蛋白经SDS-PAGE电泳后分别割胶回收,免疫新西兰大白兔,获得重组多克隆抗体。结论成功构建了NheA、NheB、NheC基因重组表达载体,表达的重组蛋白免疫新西兰兔获得相应的克隆抗体,为进一步研究非溶血型肠毒素的结构和功能奠定了基础。
[Abstract]:Objective to express three subunits of non-hemolytic enterotoxin gene (NHE) in prokaryotic expression. The nonhemolytic enterotoxin protein secreted by Bacillus cereus was identified by immunology. Methods Primer was designed according to the sequence of NheAn NheBnheC gene in NCBI database. The genomic DNA of Bacillus cereus was amplified by PCR. Three genes were cloned into the prokaryotic expression vector pET-30a () and transformed into Escherichia coli BL21DE3 / IPTG to induce the expression of the target protein. The polyclonal antibodies were prepared by immunizing New Zealand white rabbits with SDS-PAGE analysis and Western blot analysis. The results were identified by PCR and enzyme digestion. The recombinant expression vector was transformed into BL21DE-3G, and the target protein was induced by IPTG. The molecular weight unit was NheA 40ku. NheB 44kuN NheC40ku. the recombinant protein NheA was identified by ionization time of flight mass spectrometry. NheB and NheC were correctly expressed. 3 kinds of target proteins were extracted by SDS-PAGE electrophoresis and then immunized with New Zealand white rabbits to obtain recombinant polyclonal antibodies. Conclusion NheA was successfully constructed. The recombinant expression vector of NheC gene was obtained by immunizing New Zealand rabbits with the recombinant protein, which laid a foundation for further study on the structure and function of non-hemolytic enterotoxin.
【作者单位】：江苏大学生命科学研究院;
【分类号】：R392
【正文快照】： 蜡样芽孢杆菌(Bacillus cereus)是一种革兰阳性菌,能产生芽孢,广泛存在于人类的周围环境中[1]。蜡样芽孢杆菌被认为是一种条件致病菌[2],可引起眼内炎、败血症等,而其引起的食物中毒最为常见也最为重要[3]。蜡样芽孢杆菌能产生多种毒素,引起腹泻或呕吐。引起腹泻类型的毒素包

【参考文献】