摘要目的 应用基因工程技术表达和制备肺炎链球菌表面黏附素A(pneumococcal surface adhesin A,PsaA),并与细菌荚膜多糖耦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增强结合疫苗中其他细菌多糖抗原的免疫原性的同时,还能获得对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应答的目的 .方法 从肺炎链球菌基因组中扩增psaA基因,将目的 基因插入原核表达载体pET-28a,获得重组质粒pET28a-psaA,通过转化进入大肠杆菌BL21中,经IPTG诱导,采用DEAE-阴离子交换层析法纯化基因重组rPsaA蛋白.将纯化到的rPsaA蛋白与A群脑膜炎荚膜多糖(group A meningococcal polysaccharide,GAMP)耦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 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%,蛋白质相对分子质量约为37×103,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蛋白与荚膜多糖耦联成功,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.结论 利用基因工程技术获得无组氨酸标签的PsaA蛋白,并将它与A群脑膜炎荚膜多糖耦联,可以在增强荚膜多糖抗原免疫原性的同时,提高疫苗的免疫保护效果,探讨了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.

abstractsObjective To express and purify the pneumococcal surface adhesin A(PsaA) protein,discuss its application as a protein carrier in conjugates vaccine. Methods The gene encoding for the PsaA protein was amplified from the genomic DNA of Streptococcus pneumoniae using PCR. The PCR product was then cloned into the prokaryotic expression vector pET-28a and the recombinant was transformed into host cell E. coli BL21 (DE3). The expression of the recombinant protein(rPsaA) was induced by IPTG and purifled 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 × 103 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. Conclusion The recombinant protein PsaA without His-Tag was obtained and conjugated with GAMP. The strong antibody responses against PsaA and CAMP were obtained in the immunized mice at the same time which may provide the protection against pneumonia and meningitis simultaneously.