China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (9): 3799-3810.doi: 10.16431/j.cnki.1671-7236.2023.09.036

• Basic Veterinary Medicine • Previous Articles     Next Articles

Bioinformatics Analysis and Expression Purification of IspD Protein in Listeria monocytogenes

SHI Chao1,2, ZHANG Wei1,2, LIU Suping1,2, ZHANG Mengqi1, HE Xin1, ZHAO Mingyan1, ZHOU Xia1,2, WANG Zhen1,2, ZHANG Hui1,2   

  1. 1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;
    2. Key Laboratory of Animal Disease Prevention and Control, Xinjiang Production and Construction Corps, Shihezi 832003, China
  • Received:2023-02-10 Online:2023-09-05 Published:2023-08-24

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Abstract: 【Objective】 The aim of this study was to predict and analyze the potential biological function of IspD protein in Listeria monocytogenes,and express and purify IspD protein,which provided a theoretical support for subsequent research of IspD protein.【Method】 The coding sequence (gene ID:986270) and amino acid sequence (accession No.:NP_464611.1) of IspD protein in Listeria monocytogenes were obtained from NCBI.The bioinformatics software was used to analyze the open reading frame (ORF) of coding sequence of IspD protein,and predict and analyze the amino acid for basic characteristics,spatial structure,antigenic epitopes,modification sites,protein interaction and similarity.The coding sequence of IspD protein was codon optimized and synthesized.The recombinant plasmid pET-32a-IspD was constructed by seamless cloning method,then transformed into E.coli BL21(DE3) competent cells.Verified to be the correct positive clonal strain was confirmed by sequencing and double enzyme digestion was induced by IPTG overnight.The IspD fusion protein was purified by Ni-NTA spin columns.SDS-PAGE and Western blotting were used to verify the expression,purification and reactogenicity of IspD fusion protein.【Result】 The coding gene of IspD protein was lmo1086,the full-length was 711 bp,which had 4 ORFs,ORF1 was the longest ORF,which could encode 236 amino acids.Amino acid sequence analysis results showed that IspD was a hydrophobic protein without transmembrane domain and signal peptide,which was subcellular located in cytoplasm and was 2-C-methyl-D-ethrolitol-4-phosphate cytidylytransferase.Alpha helix,extended chain,beta turn and random coil were involved in the composition of the secondary structure,the prediction of the tertiary structure model was consistent with the characteristics of the secondary structure.IspD protein had multiple T cells and B cells epitopes,intrinsically disordered domain,phosphorylation,glycosylation,methylation and acetylation modification sites.There was a lot of protein interaction with IspD,such as DXR,IspF,ipk and so on,and IspD connected with DXR protein which was the strongest interaction through the hydrogen bonds and salt bridges.The expression strain pET-32a-IspD was successfully constructed.SDS-PAGE results showed that IspD fusion protein was highly expressed in the supernatant.Western blotting analysis indicated the purified IspD fusion protein had reactogenicity.【Conclusion】 IspD protein protein had potential value as a candidate diagnosis,vaccine development and drug target.IspD fusion protein was obtained by expression and purification,which provided a theoretical basis for subsequent study of the specific function of IspD protein.

Key words: Listeria monocytogenes; IspD protein; bioinformatics; expression; purification

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