肺炎衣原体外膜蛋白Omp85的重组表达及其复性条件研究

发布时间：2018-01-08 06:33

本文关键词：肺炎衣原体外膜蛋白Omp85的重组表达及其复性条件研究　出处：《药物生物技术》2015年05期 　论文类型：期刊论文

【摘要】：肺炎衣原体(Chlamydia pneumoniae,CPn)可致人急性呼吸道疾病,是引起社区获得性肺炎(Community acquired pneumonia,CAP)的主要病原体之一。Omp85蛋白是CPn上一个重要的、与其粘附和入侵宿主相关的表面蛋白,也是肺炎衣原体肺炎病的候选诊断分子和候选亚单位疫苗,因此成功表达与纯化复性的重组Omp85蛋白对揭示CPn致病机制,研发肺炎衣原体肺炎诊断试剂及疫苗具有重要的意义。该研究通过生物信息学方法分析并选取Omp85表位富集区,优化并化学合成其核苷酸序列,构建重组载体并转化大肠杆菌,使用SDS-PAGE方法筛选高表达工程菌并分析其表达形式,结果发现目的蛋白主要以包涵体形式表达。使用镍柱亲和层析复性、葡聚糖凝胶层析复性、稀释复性和透析复性4种方法对目的蛋白进行复性,均获得复性蛋白,葡聚糖凝胶层析复性方法对Omp85的复性效果优于其它3种。该研究为进一步揭示CPn致病机制以及研发肺炎衣原体肺炎的诊断试剂和疫苗奠定了基础。
[Abstract]:Chlamydia pneumoniae (Chlamydia pneumoniae, CPn) can cause acute respiratory disease, is caused by community-acquired pneumonia (Community acquired, pneumonia, CAP) of the main pathogens of.Omp85 CPn protein is an important protein and its surface, adhesion and invasion of host related, and chlamydia pneumonia disease candidate molecular diagnosis and candidate subunit the vaccine, therefore successful expression and purification of refolded recombinant Omp85 protein to reveal the pathogenic mechanism of CPn, has important significance to the development of Chlamydia pneumoniae pneumonia diagnosis reagent and vaccine. The study by bioinformatic analysis and selection of Omp85 epitope rich region, and optimization of chemical synthesis of the nucleotide sequence, the recombinant plasmid was transformed into Escherichia coli and use the SDS-PAGE method to screen high expression engineering bacteria and analysis of their expression, results showed that target protein mainly expressed in the form of inclusion bodies. Refolding by nickel affinity chromatography, Sephadex gel chromatography renaturation, 4 methods of dilution refolding and dialysis refolding renaturation of proteins, were refolded protein, Sephadex gel chromatography renaturation method for the refolding of Omp85 is better than the other 3. This study lays a foundation for further revealing the pathogenesis of CPn and the mechanism of Chlamydia pneumonia in research and development of diagnostic reagent and vaccine.

【作者单位】：南京军区军事医学研究所;中国药科大学生命科学与技术学院;南京医科大学基础医学院;
【基金】：国家传染病重大专项(No.2013ZX10004804-003) 全军十二五重大项目(No.AWS11C001)的资助
【分类号】：R375.2;R392
【正文快照】： 肺炎衣原体(Chlamydia pneumoniae,CPn)是一类致人急性呼吸道疾病的细胞内寄生的病原体[1],是继肺炎链球菌和流感嗜血杆菌之后引起社区获得性肺炎(Communityacquired pneumonia,CAP)的主要病原体,大约7%~10%的CAP是由CPn引起的,此外CPn还与动脉粥样硬化和心血管疾病相关[2-6],

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