【作者】 樊小英；

【导师】 朱越雄； 李忠明；

【作者基本信息】 苏州大学 ， 微生物学， 2011， 硕士

【摘要】 作为一种自然界广泛存在的致病菌,肺炎链球菌可引起肺炎、脑膜炎、中耳炎、鼻窦炎和败血症等疾病。过去认为肺炎链球菌荚膜多糖抗原为肺炎链球菌主要致病因子,近年来,随着对肺炎链球菌致病机制的深入研究,发现肺炎链球菌一些自身蛋白可能为肺炎链球菌致病因子,这些蛋白在所有临床分离的肺炎链球菌菌株中均存在,可产生交叉免疫保护作用,而且蛋白疫苗产生的是T细胞依赖性免疫反应,对成人和儿童均有很好的保护作用。将肺炎球菌自身蛋白作为载体与多糖偶联制备蛋白多糖结合疫苗成为现在肺炎球菌疫苗的主要方向。目的:应用基因工程技术在原核E. coli和真核毕赤酵母GS115两种体系中表达和制备肺炎链球菌表面黏附素A(pneumococcal surface adhesin A,PsaA),并与细菌荚膜多糖偶联制备成多糖蛋白结合疫苗,探讨PsaA作为肺炎球菌蛋白载体在结合疫苗中起到增强其它细菌多糖抗原免疫原性的同时,还能获得抗肺炎球菌的抗体反应,从而达到用一种结合疫苗能诱导出针对两种细菌的抗体免疫反应的目的。方法:从肺炎链球菌基因组中扩增psaA基因,将目的基因分别插入原核表达载体pET-28a(+)和真核表达载体pPIC9K,获得非融合表达的重组质粒pET28a-psaA和pPIC9K-psaA,通过转化分别进入大肠杆菌BL21和毕赤酵母GS115中,分别经IPTG和甲醇诱导,同采用DEAE-阴离子交换层析法纯化基因重组rPsaA蛋白。经过比较,将表达纯化较好的rPsaA蛋白与A群脑膜炎荚膜多糖(Group A Meningococcal Polysaccharide, GAMP)偶联成多糖蛋白结合疫苗后,用小鼠动物模型进行免疫实验,检测该疫苗的免疫原性,用ELISA法测定该疫苗在小鼠体内产生的针对肺炎球菌和脑膜炎球菌两种病原菌特异性抗原的抗体水平。结果:在原核E. coli中成功克隆基因重组表达质粒,而且表达的PsaA蛋白在载体上的组氨酸标签之前终止表达,不带有组氨酸标签,保证疫苗的安全性。SDS-PAGE技术分析表明:rPsaA蛋白高效表达,约为菌体蛋白的60%,蛋白分子量约为37 kD,而且蛋白的可溶性好,不形成包涵体,用DEAE-阴离子交换层析法纯化其纯度可达80%以上。纯化到的PsaA蛋白与GAMP多糖偶联成功,应用于小鼠免疫实验,PsaA蛋白载体能显著增强A群脑膜炎荚膜多糖抗原的免疫原性,并同时产生针对肺炎球菌蛋白抗原和脑膜炎球菌多糖抗原的特异性抗体。另外在真核毕赤酵母中也成功表达了rPsaA蛋白,目的蛋白表达量高达90%,纯度约70%。结论:利用基因工程技术在原核表达体系和真核毕赤酵母体系中均成功获得无组氨酸标签的rPsaA蛋白,并将它与A群脑膜炎荚膜多糖成功偶联,应用于小鼠免疫实验,在小鼠体内PsaA蛋白载体能显著增强A群脑膜炎荚膜多糖抗原的免疫原性,并同时产生针对肺炎球菌蛋白抗原和脑膜炎球菌多糖抗原的特异性抗体,提高疫苗的免疫保护效果。为探讨给儿童接种一种疫苗能同时预防肺炎和脑膜炎两种传染病的可能性提供研究基础。更多还原

【Abstract】 Streptococcus pneumoniae carriage can lead to a wide range of localized and systemic diseases such as otitis media, sinusitis, conjunctivitis, pneumonia, septicemia and meningitis. In recent years, with the development of study on Streptococcus pneumoniae, Pneumococcal surface adhesinA (PsaA), pneumococcal surface protein A (PspA) and pneumolysin (Ply) have all been shown to contribute to pneumococcal virulence, use of these common proteins in a vaccine is a logical alternative to the use of polysaccharide vaccines to ensure not only complete serotype coverage for S.pneumoniae, but also a more vigorous immune response since these proteins generate a T-dependent response with immunologic memory. The conjugates vaccine (rPsaA as a carrier protein conjugated with meningococcal polysaccharide) becomes the main direction at present. Objective: The gene expression and the purification of the pneumococcal surface adhesin A (PsaA) protein and its application as a protein carrier in conjugates vaccine. Method: The gene encoding for the PsaA protein was amplified from the genomic DNA of Streptococcus pneumoniae using PCR. Then the PCR product was respectively cloned into the prokaryotic expression vector pET28a and the eukaryotic expression vector pPIC9K. After that, the recombinant was respectively transformed into host cell E. coli BL21 (DE3) and host cell Pichiapastoris GS115. The expression of the recombinant protein (rPsaA) was respectively induced by IPTG and methanol and purified by using DEAE anion-exchange chromatography. The rPsaA was successfully conjugated with Group A Meningococcal Polysaccharide (GAMP). The mice were immunized subcutaneously with the conjugate and the immune responses against GAMP and PsaA were detected by ELISA. Results: The recombinant PsaA was expressed as a 37-kD soluble protein without His-Tag. The rPsaA was successfully conjugated with GAMP. In addition to the immune response against PsaA, The antibody response against GAMP was significant improved in the mice immunized with conjugate vaccine in comparison with those immunized with GAMP alone. Conclusions: The recombinant protein PsaA without His-Tag was obtained and conjugated with GAMP. The strong antibody responses against PsaA and GAMP were obtained in the immunized mice at the same time which may provide the protection against pneumonia and meningitis simultaneously.更多还原