CAJ | 학술논문

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.
목적 응용기인공정기술표체화제비폐염련구균표면점부소A(pneumococcal surface adhesin A,PsaA),병여세균협막다당우련제비성다당단백결합역묘,탐토PsaA작위폐염구균단백재체재증강결합역묘중기타세균다당항원적면역원성적동시,환능획득대폐염구균적항체반응,종이체도용일충결합역묘능유도출침대량충세균적항체면역응답적목적 .방법 종폐염련구균기인조중확증psaA기인,장목적 기인삽입원핵표체재체pET-28a,획득중조질립pET28a-psaA,통과전화진입대장간균BL21중,경IPTG유도,채용DEAE-음리자교환층석법순화기인중조rPsaA단백.장순화도적rPsaA단백여A군뇌막염협막다당(group A meningococcal polysaccharide,GAMP)우련성다당단백결합역묘후,용소서동물모형진행면역실험,검측해역묘적면역원성,용ELISA법측정해역묘재소서체내산생적침대폐염구균화뇌막염구균량충병원균특이성항원적항체수평.결과 성공극륭기인중조표체질립,이차표체적PsaA단백재재체상적조안산표첨지전종지표체,불대유조안산표첨,보증역묘적안전성.SDS-PAGE기술분석표명:rPsaA단백고효표체,약위균체단백적60%,단백질상대분자질량약위37×103,이차단백적가용성호,불형성포함체,용DEAE-음리자교환층석법순화기순도가체80%이상.순화도적PsaA단백여협막다당우련성공,응용우소서면역실험,PsaA단백재체능현저증강A군뇌막염협막다당항원적면역원성,병동시산생침대폐염구균단백항원화뇌막염구균다당항원적특이성항체.결론 이용기인공정기술획득무조안산표첨적PsaA단백,병장타여A군뇌막염협막다당우련,가이재증강협막다당항원면역원성적동시,제고역묘적면역보호효과,탐토료급인동접충일충역묘능동시예방폐염화뇌막염량충전염병적가능성.
Objective 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.