布氏杆菌周质蛋白 EipB优势T-B联合抗原表位预测及其免疫应答特点

Bioinformatics analysis of Brucella periplasmic protein EipB andits immune response characteristics of dominant T-B combined epitope

目的 利用生物信息学方法预测布氏杆菌周质蛋白(Brucella periplasmic protein, EipB)的理化性质、空间结构以及T、B细胞表位,探讨T、B优势表位肽的免疫效应,为布氏杆菌分子肽疫苗的筛选奠定基础。方法 利用在线网站以及生物信息学相关数据库分析EipB蛋白的理化性质、跨膜结构域,信号肽序列,二级和三级结构、亲/疏水性以及该蛋白的T、B抗原表位。选取抗原性最高的B和T表位(分别位于121-137和95-110)通过血蓝蛋白进行连接,免疫小鼠后,通过ELISA检测IgG、IgM、IgA以及IgE等抗体的产生情况;通过流式细胞术检测T细胞分泌细胞因子的情况。结果 布氏杆菌EipB蛋白是由279个氨基酸组成,呈弱碱性,分子质量为31 kDa,性质较为稳定;该蛋白在26-27氨基酸之间含有一个信号肽序列,在1-30氨基酸之间含有一个跨膜结构域;二级结构中α-螺旋,β-转角,延伸连和无规则卷曲等结构;亲水区域明显;该蛋白含有21条优势B细胞表位肽,这些优势B细胞表位所在氨基酸区段与亲水性较高区域相对应。含有CD_(4)^(+)T细胞表位56条;含有CD_(8)^(+)T细胞表位39条。T-B优势细胞表位联接后免疫小鼠,结果显示:血清中的IgG、IgM、IgA以及IgE等抗体显著增加;T细胞产生IFN-γ等细胞因子显著上升。结论 利用生物信息学的方法预测出EipB蛋白含有优势T、B细胞表位。优势T-B表位肽联合免疫小鼠能够诱导小鼠产生较高水平的免疫应答,为布氏杆菌分子肽疫苗的筛选提供参考。

Using bioinformatics methods to predict the physical and chemical properties,spatial structure,and T and B cell epitopes of Brucella Periplasmic Protein(EipB),and to explore the immune effects of T and B dominant epitope peptides,for Brucella Periplasmic Protein(EipB)Lay the foundation for the screening of molecular peptide vaccine against bacillosis.Online websites and bioinformatics-related databases were used to analyse the physicochemical properties,transmembrane structural domains,signal peptide sequences,secondary and tertiary structures,hydrophilicity/hydrophobicity and T and B antigenic epitopes of the EipB protein.And select the B and T epitopes with the highest antigenicity(located at 121-137 and 95-110 respectively)to connect with hemocyanin,and after immunizing the mice,detect the production of IgG,IgM,IgA and IgE antibodies by ELISA;Flow cytometry was used to detect the secretion of cytokines in T cells;The brucellosis EipB protein is composed of 279 amino acids,is weakly basic,has a molecular mass of 31 kDa and is relatively stable;contains a signal peptide sequence and a transmembrane structural domain;has a secondary structure withα-helix,β-turn,extended linkage and irregular coiling;has a distinct hydrophilic region,and the protein contains 21 dominant B-cell epitope peptides in amino acid regions 54-60,243-259,77-93,121-137,174-190,158-174,64-80,230-246,202-218,148-164,91-107,133-149,13-29,35-51,168-184,98-114,194-210,257-273,23-39,250-266,112-128,185-201,224-240,83-99,263-279,105-121,the amino acid regions in which these dominant B-cell epitopes are located correspond to regions of higher hydrophilicity.Contains 56 CD _(4)^(+)T cell epitopes and 39 CD _(8)^(+)T cell epitopes.After immunizing mice with T-B dominant cell epitope combination,antibodies such as IgG,IgM,IgA and IgE in serum increased significantly;T cells produced IFN-γand other cytokines significantly increased.1)Bioinformatics method was used to predict that EipB protein contains dominant B and T cell epitopes.2)Combined immunization of mice with dominant B-T dominant epitope peptides can induce a higher level of immune response in mice,providing a reference for the screening of brucellosis molecular peptide vaccines.