淋病奈瑟菌肽基脯氨酰异构酶的原核表达及其作为候选疫苗的初步评价

Prokaryotic expression and potential as a candidate vaccine of peptidyl-prolyl isomerase from Neisseria gonorrhoeae

摘要:

【背景】淋病是我国主要的性传播疾病之一，感染淋病奈瑟菌可促进人类免疫缺陷病毒(human immunodeficiency virus, HIV)的传播和感染。目前我国淋病发病人数呈上升趋势，随着多重耐药菌株的出现，亟须研发保护性疫苗来防治淋病的传播和感染。【目的】分析淋病奈瑟菌(Neisseria gonorrhoeae, NG)肽基脯氨酰异构酶(peptidyl-prolyl isomerase, PPIase)蛋白的高级结构和表位，探讨其作为疫苗和分子诊断靶点的潜力。【方法】利用生物信息学软件分析PPIase蛋白的极性、亲水性、柔韧性、表面可及性、二级和三级结构，以及T、B细胞表位等；用pET32a(+)质粒构建PPIase蛋白的原核表达系统并纯化蛋白，用纯化的重组蛋白和超声波破碎的NG全菌抗原分别免疫BALB/c小鼠，收获免疫血清；制备NG全细胞抗原，分别以全细胞抗原酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)和间接免疫荧光试验检测重组PPIase蛋白血清抗体与NG全细胞表面抗原的结合情况。【结果】生物信息学分析结果显示，PPIase蛋白主要以α螺旋(39.34%)、无规则卷曲(30.51%)和延伸链(20.59%)为主；经表位分析筛选出5个B细胞优势表位、9个细胞毒性T淋巴细胞优势表位和18个辅助性T淋巴细胞优势表位；通过原核表达系统实现了PPIase重组蛋白的表达和纯化；纯化的重组蛋白可刺激BALB/c小鼠产生高效价的抗体，全细胞抗原的ELISA试验和间接免疫荧光试验的结果表明该蛋白诱导产生的血清抗体能与NG外膜表面抗原结合。【结论】PPIase蛋白作为一种外膜蛋白，具有良好的免疫原性和免疫反应性，其所诱导产生的特异抗体可以结合到NG表面，PPIase蛋白有作为NG候选疫苗的潜力和价值。

Abstract:

[Background] Gonorrhoea is one of the major sexually transmitted diseases in China, and the infection with Neisseria gonorrhoeae (NG) can promote the transmission and infection of HIV. The incidence of gonorrhoea in China is on the rise, and the emergence of multidrug resistant strains makes it an urgent need to develop protective vaccines for preventing the spread and infection of gonorrhea. [Objective] To unveil the advanced structure and epitopes of peptidyl-prolyl isomerase (PPIase) of NG, and explore the potential of PPIase as a vaccine and a molecular diagnostic target. [Methods] Bioinformatics tools were employed to analyze the polarity, hydrophilicity, flexibility, surface accessibility, secondary and tertiary structures, and T and B cell epitopes of PPIase. The prokaryotic expression system of PPIase was constructed with pET32a(+), and the recombinant protein was purified. The BALB/c mice were immunized with the purified recombinant protein and the NG cells disrupted by ultrasound, respectively, and the sera were then harvested. The surface antigens of NG whole cells were prepared. The binding of serum antibody induced by PPIase to the surface antigens of NG whole cells was examined by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay, respectively. [Results] The secondary structure of PPIase was mainly composed of α helix (39.34%), random coil (30.51%), and extended strand (20.59%). The epitope analysis revealed 5 B cell dominant epitopes, 9 cytotoxic T lymphocyte dominant epitopes, and 18 helper T cell dominant epitopes. The recombinant PPIase protein was expressed and purified by the prokaryotic expression system. The purified recombinant protein can stimulate BALB/c mice to produce high-titer antibodies, and the serum antibody induced by the recombinant PPIase protein can bind to the NG surface antigen. [Conclusion] PPIase as an outer membrane protein demonstrates good immunogenicity and immunoreactivity, and the specific antibody induced by this protein can bind to the surface antigen of NG. Therefore, PPIase has the potential as a candidate vaccine against NG.