脑膜炎奈瑟氏菌外膜蛋白NhhA的克隆表达及免疫原性分析

发布时间：2018-07-11 10:58

本文选题：脑膜炎奈瑟氏菌 + NhhA　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： 脑膜炎奈瑟氏菌(Neisseria meningitidis, Nm)属奈瑟氏菌属,革兰氏阴性菌。由其引起的流行性脑脊髓膜炎发病快,致死率高,后遗症严重,尤其对2岁以下婴幼儿具有很大危害性。在世界范围内仍是一种具有严重危害性的传染性疾病。从60年代以来,Nm对磺胺的耐药现象日益普遍,因此以疫苗为主的预防变得尤为重要。荚膜多糖蛋白结合疫苗对于预防A、C、Y、W135群Nm引起的脑脊髓膜炎起到了重要作用。但由于B群脑膜炎奈瑟氏菌(serogroup B N. meningitidis, MenB)荚膜多糖与人N-乙酰神经氨酸聚合物结构相似,因而不具有免疫原性,所以多糖菌苗的策略并不适用于MenB。因此对于MenB疫苗研发来说,外膜蛋白更值得关注。本研究从脑膜炎奈瑟菌基因组中克隆得到其外膜蛋白NhhA的基因GNA0992,构建pET20b-NhhA重组质粒,在大肠杆菌BL21 (DE3)中实现了可溶性表达,表达量约占菌体蛋白总量的20%。表达蛋白经胶回收纯化,透析,超滤浓缩后,纯度达90%以上,浓度约1.31 ug/ul。Western blot分析结果显示重组蛋白具有较好的免疫原性和抗原性。用纯化的重组蛋白NhhA免疫BALB/c小鼠,对其免疫原性进行了初步分析。结果显示,经过三次腹腔免疫,血清IgG滴度达到23186,同时杀菌力实验显不NhhA能诱导针对B群脑膜炎奈瑟氏菌的补体依赖的杀菌反应。证明了NhhA是一种很好的疫苗候选蛋白。本研究成功构建了Nm外膜蛋白NhhA重组蛋白表达载体,在大肠杆菌中获得了可溶性表达；对重组蛋白表达、纯化等实验条件进行了初步的探索；通过免疫BALB/c小鼠,对该重组蛋白的免疫学效应作了初步的分析,为进一步研究Nm疫苗提供了很好的理论依据和实验资料,也为疫苗开发的蛋白靶标筛选工作奠定了基础。
[Abstract]:Neisseria meningitis (Nm) belongs to the genus Neisseria and Gram-negative bacteria. The epidemic cerebrospinal meningitis caused by it has the advantages of rapid onset, high mortality and serious sequelae, especially for infants under 2 years of age. It is still a serious infectious disease worldwide. Since 1960's, the drug resistance of Nm to sulfanilamide has become more and more common, so vaccine-based prevention has become more and more important. Capsule polysaccharide protein-binding vaccine plays an important role in the prevention of meningitis caused by Nm of Acanthopycloviridine W135 group. But because the capsule polysaccharide of serogroup B meningitis (Menb) is similar to the structure of human N-acetylneuraminic acid polymer, it has no immunogenicity, so the strategy of polysaccharide vaccine is not suitable for Menb. Therefore, for Menb vaccine research and development, the outer membrane protein is more worthy of attention. In this study, the outer membrane protein gene GNA0992 was cloned from the genome of Neisseria meningitidis, and the recombinant plasmid pET20b-NhA was constructed. The recombinant plasmid was soluble expressed in E. coli BL21 (DE3) and accounted for about 20% of the total bacterial protein. The expressed protein was purified by gel recovery, dialysis and ultrafiltration, and the purity was over 90%. The results of Western blot analysis showed that the recombinant protein had good immunogenicity and antigenicity. The immunogenicity of BALB / c mice was preliminarily analyzed by immunizing BALB / c mice with the purified recombinant protein NhhA. The results showed that the titer of serum IgG reached 23186.The bactericidal activity test showed that Nha could induce complement dependent bactericidal reaction against Neisseria meningitidis. It is proved that NhhA is a good vaccine candidate protein. In this study, we successfully constructed the recombinant protein expression vector of Nm outer membrane protein, obtained soluble expression in Escherichia coli, preliminarily explored the expression and purification of recombinant protein, and immunized BALB / c mice. The immunological effects of the recombinant protein were preliminarily analyzed, which provided a good theoretical basis and experimental data for the further study of Nm vaccine, and laid a foundation for the screening of protein target for vaccine development.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【共引文献】