寡糖修饰对布鲁氏菌抗原蛋白rRS-α免疫原性影响的研究

In classical immune theory, the protein is the main component of immune response, but recently, the preliminary indication of research shows that oligosaccharide modification of proteins can change the biological activity of proteins, especially which is able to enhance the protein immunogenicity. Therefore, the synthesized brucella O-side chain oligosaccharides and natural LPS core oligosaccharides will be prepared, and then the oligosaccharides will conjugate immune-proteins rRS-α, in this program. The effects of oligosaccharide modification on the proteins physical and chemical properties such as isoelectric point, stability and solubility will be evaluated. In vivo, the oligosaccharide conjugate proteins will be investigated on the change of the humoral response, cellular immune response and immune protection. In vitro, the antigen capturing and presenting of oligosaccharide conjugate proteins and non-conjugate proteins in macrophages and dendritic cells will be compared, and the changes of immune regulation pathways of dendritic cells and macrophages could be revealed in order to elaborate the basic principles of oligosaccharide modification on the proteins immune response. The goal of this subject is to prove that oligosaccharide modification can change the immune response level and protective immunity rate of brucella protective antigens, to reveal the primary principles of oligosaccharide modification, and to provide a scientific basis for development of brucellosis oligosaccharide conjugate protein vaccine.

传统的免疫学理论认为，蛋白质是引起免疫应答的主要成分。近年来，国外的研究初步表明，寡糖修饰影响蛋白质的生物学活性，对蛋白质免疫原性有显著增强作用。本课题利用化学方法合成的布鲁氏菌O-侧链寡糖和纯化的LPS核心寡糖，对免疫原性蛋白rRS-α进行修饰，制备寡糖修饰蛋白。研究寡糖修饰对蛋白质理化特性的影响，在体内，研究寡糖修饰对蛋白质的体液免疫、细胞免疫应答水平及保护率的影响；在体外，通过巨噬细胞和树突细胞研究寡糖修饰蛋白和非修饰蛋白捕获和提呈作用，及对细胞表面TLR2和TLR4受体系统调节通路的影响。揭示寡糖修饰影响蛋白质性质的基本原理，阐明寡糖修饰影响布鲁氏菌保护性抗原的免疫应答水平和免疫保护率的信号调节作用的基本机制，为布鲁氏菌病寡糖修饰蛋白疫苗的研制提供科学依据,奠定理论基础。

近年来，国外的研究初步表明，寡糖修饰影响蛋白质的生物学活性，对蛋白质免疫原性有显著增强作用。本项目旨在通过寡糖修饰布鲁氏菌蛋白，希望提高蛋白免疫原性，并对其提高蛋白免疫原性的机制进行研究，揭示寡糖影响蛋白免疫原性的机制。.本课题利用不同类型的寡糖，对免疫原性蛋白rRS-α和Ahcy进行修饰，制备寡糖修饰蛋白。研究寡糖修饰对蛋白质理化特性的影响，在体内，研究寡糖修饰对蛋白质的体液免疫、细胞免疫应答水平及保护率的影响；在体外，通过淋巴细胞研究寡糖修饰蛋白和非修饰蛋白捕获和提呈作用，及对细胞表面TLR2和TLR4受体系统调节通路的影响。.研究表明，几丁质寡糖修饰降低了蛋白免疫原性，而甘露寡糖可以提高蛋白免疫原性，其中甘露六糖和甘露二糖修饰可以显著提高Ahcy蛋白免疫原性，但是降低了RSα的免疫原性，Ahcy的甘露六糖修饰可以提高该蛋白的免疫保护。细胞实验表明，甘露六糖修饰提高了淋巴细胞表面TLR4受体的表达。.项目可以为揭示寡糖修饰影响蛋白质性质的原理提供依据，实验揭示寡糖修饰可以通过TLR4受体途径影响布鲁氏菌保护性抗原的免疫应答水平和免疫保护率的信号调节作用的基本机制，为布鲁氏菌病寡糖修饰蛋白疫苗的研制提供科学依据,奠定理论基础。