本文選題：腦膜炎奈瑟氏菌 + NhhA　； 參考：《中國協(xié)和醫(yī)科大學》2010年碩士論文

【摘要】： 腦膜炎奈瑟氏菌(Neisseria meningitidis, Nm)屬奈瑟氏菌屬,革蘭氏陰性菌。由其引起的流行性腦脊髓膜炎發(fā)病快,致死率高,后遺癥嚴重,尤其對2歲以下嬰幼兒具有很大危害性。在世界范圍內仍是一種具有嚴重危害性的傳染性疾病。從60年代以來,Nm對磺胺的耐藥現(xiàn)象日益普遍,因此以疫苗為主的預防變得尤為重要。莢膜多糖蛋白結合疫苗對于預防A、C、Y、W135群Nm引起的腦脊髓膜炎起到了重要作用。但由于B群腦膜炎奈瑟氏菌(serogroup B N. meningitidis, MenB)莢膜多糖與人N-乙酰神經(jīng)氨酸聚合物結構相似,因而不具有免疫原性,所以多糖菌苗的策略并不適用于MenB。因此對于MenB疫苗研發(fā)來說,外膜蛋白更值得關注。 本研究從腦膜炎奈瑟菌基因組中克隆得到其外膜蛋白NhhA的基因GNA0992,構建pET20b-NhhA重組質粒,在大腸桿菌BL21 (DE3)中實現(xiàn)了可溶性表達,表達量約占菌體蛋白總量的20%。表達蛋白經(jīng)膠回收純化,透析,超濾濃縮后,純度達90%以上,濃度約1.31 ug/ul。Western blot分析結果顯示重組蛋白具有較好的免疫原性和抗原性。用純化的重組蛋白NhhA免疫BALB/c小鼠,對其免疫原性進行了初步分析。結果顯示,經(jīng)過三次腹腔免疫,血清IgG滴度達到23186,同時殺菌力實驗顯不NhhA能誘導針對B群腦膜炎奈瑟氏菌的補體依賴的殺菌反應。證明了NhhA是一種很好的疫苗候選蛋白。 本研究成功構建了Nm外膜蛋白NhhA重組蛋白表達載體,在大腸桿菌中獲得了可溶性表達；對重組蛋白表達、純化等實驗條件進行了初步的探索；通過免疫BALB/c小鼠,對該重組蛋白的免疫學效應作了初步的分析,為進一步研究Nm疫苗提供了很好的理論依據(jù)和實驗資料,也為疫苗開發(fā)的蛋白靶標篩選工作奠定了基礎。
[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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

【共引文獻】

相關碩士學位論文 前1條

1 彭世澤;重組B群腦膜炎球菌fHBP融合蛋白的表達和免疫原性研究[D];北京協(xié)和醫(yī)學院;2011年

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本文編號：2114934