Bioinformatics analysis and anti-oxidative stress effect of Tp0100 protein in Treponema pallidum
Aim To analyze the biological characteristics and identify the anti-oxidative stress function of thioredoxin Tp0100 in Treponema pallidum.Methods The basic physicochemical properties,phosphorylation sites,glycosylation sites,sec-ondary and tertiary structures,B cell epitopes,and homology with other bacterial genera of Tp0100 protein in T.pallidum were ana-lyzed by using bioinformatics methods such as NCBI,SignalP5.0,TMHMM,and so on.The prokaryotic expression system of Tp0100 was constructed and induced to express recombinant protein Tp0100(rTp0100).New Zealand rabbit immune serum was pre-pared,and the titers of specific IgG antibody against rTp0100 were detected by ELISA.The levels of reactive oxygen species(ROS)released by macrophages stimulated by rTp0100 were detected by flow cytometry and fluorescence microscopy.Results Tp0100 recombinant protein contains 185 amino acids with a relative molecular weight of 20.5 kDa.It has signal peptides,phosphorylation sites,no transmembrane domains,and no glycosylation sites.The secondary structure of amino acid has 59 α-helix,44 extended chains,21 β turns,and 61 random curls,which exist 2 consecutive B-cell epitopes.Tp0100 has high homology with Neisseria gonor-rhoeae thioredoxin.The recombinant plasmid pET28a-Tp0100 was successfully constructed and the rTp0100 with high purity was ob-tained.Immunization of New Zealand rabbits with rTp0100 induced the production of high titer of specific IgG antibodies.rTp0100 protein stimulation can reduce ROS levels in macrophages.Conclusion Tp0100 is a hydrophilic protein with B-cell epitopes.rTp0100 reveals good antigenicity and can inhibit the production of ROS in macrophages.
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