猪种布鲁氏菌TIR结构域蛋白的生物信息学分析、重组表达及蛋白互作研究

Bioinformatics Analysis,Recombinant Expression and Protein Interactions Research of Brucella suis TIR Domain Containing Protein

【目的】本研究以猪种布鲁氏菌Toll/白介素-1受体(TIR)结构域蛋白(Brucella suis TIR domain containing protein,Bs-TDCP)为研究对象,进行基因克隆、生物信息学分析及蛋白互作研究,为后续开发适合动物或人类使用的有效布鲁氏菌疫苗奠定基础。【方法】利用GenBank数据库查找黑腹果蝇髓样分化因子88(Dm-MyD88)和人MyD88(Hs-MyD88)基因序列,设计特异性引物进行基因序列扩增。根据猪种布鲁氏菌测序结果,获得Bs-TDCP基因序列。通过ExPASy、SWISS-MODEL、TMHMM、SMART等在线工具分析Bs-TDCP蛋白理化性质、二级和三级结构、跨膜及功能结构域,并进一步研究Bs-TDCP蛋白与Toll样受体(TLRs)信号通路的关键胞内配体MyD88分子之间的蛋白串扰。【结果】猪种布鲁氏菌TIR结构域蛋白Bs-TDCP基因的开放阅读框(ORF)大小为828 bp,编码275个氨基酸,其中丙氨酸占12.7%,分子式为C_(1345)H_(2196)N_(390)O_(424)S_(6),Bs-TDCP蛋白分子质量为35.85 ku,理论等电点为9.37,不稳定指数为50.44,属于不稳定疏水性蛋白,不包含跨膜结构域和信号肽序列(SPS),但其具有典型的TIR结构域,Bs-TDCP蛋白二级结构由α-螺旋、延伸链、β-转角和无规则卷曲组成,占比分别为77.45%、8.36%、2.91%和11.27%。重组蛋白Bs-TDCP(rBs-TDCP)、重组蛋白Dm-MyD88(rDm-MyD88)和重组蛋白Hs-MyD88(rHs-MyD88)亲和纯化效果良好。蛋白体外结合试验结果表明,不含His标签的Bs-TDCP(rBs-TDCP^(△))与MyD88分子之间存在蛋白互作。【结论】rBs-TDCP^(△)分别与rDm-MyD88、rHs-MyD88配体存在一定程度的蛋白互作,本研究结果为开发供人类使用的新型布鲁氏菌疫苗提供理论参考。

【Objective】In this study,Toll/interleukin-1 receptor(TIR)domain protein(Bs-TDCP)of Brucella suis was used as the object of study,and gene cloning,bioinformatics analysis and protein interaction were performed.This will lay the foundation for the subsequent development of effective Brucella vaccines suitable for animal or human.【Method】The GenBank database was used to find the gene sequences of Drosophila melanogaster myeloid differentiation factor 88(Dm-MyD88)and Homo sapiens MyD88(Hs-MyD88),and sequence-specific primers were designed for gene sequence amplification.Based on the sequencing results of Brucella suis,the Bs-TDCP gene sequence of TIR domain protein was obtained.ExPASy,SWISS-MODEL,TMHMM,SMART and other online tools were used to analyze the physical and chemical properties,secondary and tertiary structures,transmembrane and functional domains of Bs-TDCP protein,and further study the protein cross-talk between Bs-TDCP proteins and MyD88 molecules,a key intracellular ligand of Toll-like receptors(TLRs)signaling pathway.【Result】The open reading frame(ORF)of Bs-TDCP gene of the TIR domain protein of Brucella suis was 828 bp,encoding 275 amino acids,of which 12.7%was alanine.The molecular formula was C_(1345)H_(2196)N_(390)O_(424)S_(6),the molecular weight of the protein was 35.85 ku,and the theoretical isoelectric point was 9.37.The instability index of Bs-TDCP was 50.44,which was an unstable hydrophobic protein.It did not contain transmembrane domains and signal peptide sequences(SPS),but it had a typical TIR domain.The secondary structure of Bs-TDCP protein was composed of alpha helix,extended strand,beta turn and random coil,the percentages were 77.45%,8.36%,2.91%and 11.27%,respectively.The affinity purification effect of recombinant protein Bs-TDCP(rBs-TDCP),recombinant protein Dm-MyD88(rDm-MyD88),and recombinant protein Hs-MyD88(rHs-MyD88)was good.The results of the protein binding assay in vitro showed a protein interaction between Bs-TDCP without His tag(rBs-TDCP^(△))and MyD88 molecules.【Conclusion】rBs-TDCP^(△)had some protein interaction with rDm-MyD88 and rBs-MyD88 ligands,respectively.The results provided theoretical reference for the development of novel brucellosis vaccines for human use.