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脑膜炎球菌多糖结合疫苗的分子形状与其免疫学性质的相关性
许孟芳
学位类型硕士
导师胡涛
2015-10
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物工程
关键词脑膜炎a群荚膜多糖 结合疫苗 结构特性 免疫特性
其他摘要

脑膜炎奈瑟菌(Neisseria meningitides)是导致婴儿和儿童患严重感染性疾病的重要病菌之一,可引起脑膜炎疾病及其它临床症状。由于该菌的主要致病成分为位于菌体表面的荚膜多糖,所以荚膜多糖常用作疫苗免疫人体。然而,荚膜多糖产生的免疫力持续时间短、几乎没有免疫记忆,并且在婴儿和儿童体内不起作用。结合疫苗的诞生成功解决了这些问题,因为当荚膜多糖与载体蛋白结合后,其结合物的形状和分子量都显著增大,成为T细胞依赖性抗原,因此产生的免疫力持续时间长、有免疫记忆,且可以较好地保护婴幼儿。然而,尽管脑膜炎多糖结合疫苗的应用广泛,至今未有文献全面报道其结构参数、三维形状等结构信息与其免疫学特性。已有多篇文献提出:药物(含疫苗)的分子形状是影响其体内活性(如细胞内化、吞噬作用等)的重要因素。成功建立其结构特性与其免疫学特性的相关性可为今后结合疫苗的研发提供新的思路。本论文的主要成果在于:首先,提出了一个崭新的脑膜炎荚膜多糖与载体蛋白的偶联方法;其次,得到了四种结合程度不同的结合疫苗,并分别测得其结构特性和免疫特性,并初步建立起多糖结合疫苗的结构特性与其免疫学特性的相关性。具体如下:首先,通过溴化氰活化的方法将脑膜炎A群荚膜多糖与鸡卵清白蛋白(OVA)偶联,采用胱胺为间隔臂,随后通过分子筛的方法得到四种结合程度不同的结合疫苗。其次,用小角X射线散射技术(SAXS)、动态光散射技术和分析超离心技术分别对其进行结构性质的测定。结果显示结合疫苗呈杆状结构,结合程度最大的疫苗的水化体积为1,370.0 nm3、最大长度为27.8 nm、回转半径为9.1 nm、分子量约为2,100k Da、沉降系数为20。最后,采用ELISA法分别测定其抗体滴度,发现在本实验研究范围内,结合程度越大的疫苗具有越高的多糖特异性抗体滴度,推测对人体的保护效果也越佳。

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Neisseria meningitides is a leading cause of severe bacterial infections in infants and young children. As a major virulence factor, meningococcal capsular polysaccharide (PS) is poorly immunogenic with little immunological memory. Conjugation of PS with carrier protein can significantly increase the PS-specific immunogenicity and induce immunological memory. Conjugation of PS with a carrier protein can significantly increase the PS-specific immunogenicity and induce immunological memory.Although the rapid development of conjugate vaccine during the past few decades, there are little research focus on its molecular shape and size. In the present study, we offered a brand new conjugating method which taking cystamine dihydrochloride as linker, and, the most important innovation is, we gained four conjugate vaccines with different conjugate levels by using size exclusion chromatography, and each of their structural property and immunological property were investigated. The details were as following:First of all, PS was conjugated with ovalbumin by using CNBr method, and the linker is cystamine dihydrochloride. The conjugate was separated by size exclusion chromatography, and four conjugate vaccines with different conjugate levels were achieved.Secondly, structural characterization, including of each conjugate vaccine, ovalbumin and polysaccharide (depend on specific situation) was carried out using Small Angel X-ray Scattering, dynamic light scattering and analytical ultracentrifugation. The results suggested that the shape of conjugate vaccine was rod-like. The largest volume of conjugate vaccine in present study was 1,370.0 nm3, maximum particle diameter was 27.8 nm, radius of gyration was 9.1nm, molecular mass was 2,100 kDa, and sedimentation coefficient was 20. Besides, circular dichroism spectra and intrinsic fluorescence spectra were carried out to confirm that conjugating to PS did not change the secondary structure of ovalbumin, 1H NMR and FR-IT were also carried out to demonstrate the successfully conjugating process from chemical perspective.Last but not the least, ELISA was carried out to detect conjugate vaccines’ immunogenicity, and it turns out that conjugate vaccine with highest conjugated level also possesses strongest immunogenicity, which means it can protect people better. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/21360
专题研究所(批量导入)
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许孟芳. 脑膜炎球菌多糖结合疫苗的分子形状与其免疫学性质的相关性[D]. 北京. 中国科学院研究生院,2015.
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