猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)是造成猪肺炎和胸膜炎的重要病原菌之一,其病死率高达80％～100％.为了寻找APP转铁结合蛋白B(transferrin binding protein B,TbpB)与猪转铁蛋白(trans-ferrin,Tf)相互作用的关键氨基酸,以达到为APP铁摄取机制研究及相关疫苗开发奠定基础的 目的,本研究在对野生型TbpB进行序列分析、三维结构建模、克隆的基础上,通过GRAMM-X分子对接软件在线预测APP TbpB与猪Tf结合的关键氨基酸,利用重叠延伸PCR(SOE PCR)技术构建突变质粒,ZYM-5052半乳糖自诱导培养基诱导大肠杆菌表达该突变蛋白后与猪Tf进行斑点结合试验,野生型TbpB、突变TbpB以及PBS分别免疫小鼠,采用间接ELISA法检测不同阶段小鼠血清中特异性抗体的水平.分子对接结果显示,预测的关键氨基酸为第190位苯丙氨酸,质粒测序结果显示,已成功将编码190位苯丙氨酸的密码子突变为编码丙氨酸的密码子;斑点结合试验结果显示,突变TbpB与猪Tf不再发生结合反应;小鼠免疫试验结果表明与野生型TbpB相比,突变TbpB可诱导产生更高水平的抗体;APP TbpB 190位苯丙氨酸在与猪Tf反应中起着关键作用,突变TbpB具有良好的免疫原性,为进一步研究TbpB的功能、结构以及开发相关疫苗奠定了理论基础.
Study on mutation and immunogenicity of Actinobacterium pleuropneumoniae transferrin binding protein B F190A
Actinobacillus pleuropneumoniae(APP)is one of the important pathogens causing swine pneumonia and pleurisy.The mortality rate is as high as 80％-100％.This study aims to find the key amino acids for the interaction between transferrin binding protein B(TbpB)and por-cine transferrin(Tf),and to provide a foundation for the study of APP iron uptake mechanism and the development of related vaccines.Based on sequence analysis,three-dimensional structure mod-eling and cloning of wild-type TbpB,the key amino acids of APP TbpB binding to pig Tf were pre-dicted online by GRAMM-X molecular docking software.The mutant plasmid was constructed by overlapping extended PCR(SOE PCR)technology.The mutant protein was expressed by ZYM-5052 galactose self-induced medium in Escherichia coli.Dot blot assay was performed to identify its functions.The level of specific antibodies in serum of mice at different stages was detected by indirect ELISA method followed by mutant TbpB and PBS immunized to mice respectively.The molecular docking results showed that the predicted key amino acid was phenylalanine at position 190,The plasmid sequencing showed that the codon encoding phenylalanine at position 190 had been successfully mutated into a codon encoding alanine,Dot blot assay results showed that the mutant TbpB could not binding to pig Tf anymore.The results of mouse immunoassay showed that mutant TbpB could induce higher levels of antibodies compared with wild-type TbpB;APP TbpB 190-position phenylalanine plays a key role in the reaction with pig Tf and the mutant TbpB has good immunogenicity,which provide a theoretical foundation for further study of the function and structure of TbpB and the development of related vaccines.
Actinobacillus pleuropneumoniaetransferrin binding protein Bsite-directed muta-tionsdot blot assayimmunogenicity
NETL
NSTL
万方数据
