牛结核分枝杆菌BfrB蛋白生物信息学分析及多克隆抗体制备

Bioinformatic analysis and polyclonal antibody preparation of Mycobacterium bovis BfrB protein

目的旨在预测和分析牛结核分枝杆菌(Mycobacterium bovis)BfrB蛋白的结构和潜在功能,并进行克隆表达及多克隆抗体的制备,为新型疫苗、诊断靶点等的开发提供理论基础。方法利用生物信息学软件预测分析BfrB蛋白的生物信息学特征,克隆其编码基因并与原核表达载体pET32a连接,构建重组表达载体并利用IPTG诱导表达,镍柱亲和层析法纯化重组BfrB蛋白,SDS-PAGE和Western blot验证分析重组BfrB蛋白,以该蛋白免疫新西兰大白兔制备多克隆抗体,并测定其效价。结果BfrB蛋白含有181个氨基酸,分子式为C_(903)H_(1405)N_(255)O_(276)S_(6),理论分子质量为20.442 ku,无信号肽和跨膜结构域,是一种定位于细胞质的亲水性的储铁蛋白。该蛋白有3个固定无序结构域、15个磷酸化位点、16个甲基化位点和2个乙酰化位点,无糖基化位点。其二级结构中α-螺旋占70.01%,延伸链占4.42%,β-折角占3.87%,无规则卷曲占21.55%,三级结构与二级结构预测结果一致,空间结构(四级结构)为24个亚单位(三级结构)组成的聚合物。BfrB蛋白含有9个B细胞抗原表位、7个CD4+T细胞抗原表位和3个CD8+T细胞表位。该蛋白与牛结核分枝杆菌BCG、结核分枝杆菌H37Rv等的BfrB蛋白亲缘关系较近,同源性高达99%以上。与BfrB蛋白存在相互作用的蛋白有Oxidase、rpsL、katG、hemH等,其中BfrB蛋白与Oxidase蛋白之间的相互作用关系最强。成功克隆BfrB蛋白编码基因,大小与预期相符,与表达载体pET32a连接后,经双酶切、测序验证表明重组表达载体pET32a-BfrB构建正确。IPTG诱导表达、纯化后获得条带单一、纯度较高的重组BfrB蛋白,Western blot显示重组BfrB蛋白具有良好的免疫原性,能被相应抗体识别。用该蛋白免疫新西兰大白兔能够诱导产生抗体,且其效价高达1∶512000。结论成功预测和分析了牛结核分枝杆菌BfrB蛋白的结构及功能,并获得BfrB蛋白及多克隆抗体,为后续该蛋白的研究奠定理论基础,也为牛结核病的防控等提供参考依据。

Objective The aim of this study was to predict and analyze the potential structure and function of Mycobacterium bovis BfrB protein,and to cloned and expressed it,as well as prepared polyclonal antibody,provided a theoretical basis for the development of novel vaccines and diagnostic targets.Methods The bioinformatics characteristics of BfrB protein were predicted and analyzed by bioinformatics software,its coding gene was cloned and linked to the prokaryotic expression vector pET32a,the recombinant expression vector was constructed,and the expression was induced by IPTG.The recombinant BfrB protein was purified by Ni-NTA spin columns,and it was analyzed by SDSPAGE and Western blot.Polyclonal antibody was prepared by immunizing New Zealand white rabbit with the recombinant BfrB protein,and its titer was determined.Results The BfrB protein consisted of 181amino acids,with molecular formula C_(903)H_(1405)N_(255)O_(276)S_(6),and theoretical molecular weight was 20.442ku,without signal peptide and transmembrane domain,it was a hydrophilic ferritin located in the cytoplasm.The BfrB protein had three intrinsic disordered domains,fifteen phosphorylation sites,sixteen methylation sites and two acetylation sites,without glycosylation sites.The secondary structure content of a-helix was 70.1%,extended strand was 4.42%,β-turn was 3.87%and random coil was 21.55%,and the tertiary structure was consistent with the predicted results of the secondary structure.The spatial structure(quaternary structure)was a polymer composed of 24subunits(tertiary structure).BfrB protein had nine B cell epitopes,seven CD4+T cell epitopes and three CD8+T cell epitopes.The protein of M.bovis was closely related to BfrB proteins of M.bovis BCGand M.tuberculosis H37Rv,the homology was up to 99%.BfrB protein interacted with multiple proteins in Oxidase,rpsL,katG,hemH and others,in which the interaction between BfrB protein and Oxidase protein was the strongest.The BfrB protein coding gene was successfully cloned,and the size was consistent with the expectation.After connected with the expression vector pET32a,the recombinant expression vector pET32a-BfrB was correctly constructed by double enzyme digestion and sequencing.The recombinant BfrB protein with single band and high purity was obtained after IPTG induced expression and purification.Western blot showed that the recombinant BfrB protein had good immunogenicity and could be recognized by corresponding antibodies.Immunizing New Zealand white rabbit with this protein induced the production of antibodies,and its titer was as high as 1∶512000.Conclusion The structure and biological function of M.bovis BfrB protein were successfully predicted and analyzed,BfrB protein and polyclonal antibody were obtained,which laid a theoretical foundation for the subsequent study of the protein and also provided a reference for the prevention and control of bovine tuberculosis.