肺炎衣原体外膜蛋白Omp85的重组表达及其复性条件研究

Expression,Purification and Renaturation of Recombinant Surface Protein Omp85 of Chlamydia Pneumoniae

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。

Chlamydia pneumoniae（CPn） causes acute respiratory disease and is one of the main pathogens that cause community acquired pneumonia（CAP）. Omp85 protein is an important surface protein of CPn and is associated with its adherence to and entry into host cells. Also Omp85 is a candidate antigen for development of diagnostic agent and subunit vaccine. So expression and purification of renatured Omp85 are important for revealing pathogenic mechanism of CPn as well as developing diagnostic agent and vaccine against CPn infection. In this study, amino acid sequence of Omp85 was analyzed and epitope-enriched region was selected. The nucleotide sequence was optimized and chemically synthesized. Then recombinant vector was constructed and transformed to E. coli BL21 （DE3） , and recombinant E. coli which expressed Omp85 highly was selected. The recombinant Omp85 was found to be expressed in inclusion body. To renature it, Ni-NTA affinity renaturation, sephadex chromatography renaturation, dilution renaturation, and dialysis renaturation were used, in which sephadex chromatography renaturation gave the best result. This research has laid a foundation for further revealing pathogenic mechnism of CPn as well as developing diagnostic agent and vaccine against CPn infection.