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

doi:10.16431/j.cnki.1671-7236.2023.08.028

Abstract

Abstract: 【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 sapiensMyD88 (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 C₁₃₄₅H₂₁₉₆N₃₉₀O₄₂₄S₆,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.

Key words: Brucella suis; TIR domain; myeloid differentiation factor 88 (MyD88); protein function

CLC Number:

S852.61

LI Qianqian, LIN Sihan, WANG Liyuan, YANG Bingbing, ZHANG Mingda, SHENG Xiuli, DU Zhiqiang. Bioinformatics Analysis,Recombinant Expression and Protein Interactions Research of Brucella suis TIR Domain Containing Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3303-3312.

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

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