| 余翔,孙培原,吴移谋.梅毒螺旋体Tp0100蛋白的生物信息学分析及其抗氧化应激作用.[J].中南医学科学杂志.,2024,(4):501-505. |
| 梅毒螺旋体Tp0100蛋白的生物信息学分析及其抗氧化应激作用 |
| Bioinformatics analysis and anti-oxidative stress effect of Tp0100 protein in Treponema pallidum |
| 投稿时间：2024-03-29 修订日期：2024-05-22 |
| DOI：10.15972/j.cnki.43-1509/r.2024.04.001 |
| 中文关键词: 梅毒螺旋体 Tp0100蛋白 生物信息学分析 免疫原性 氧化应激 活性氧 |
| 英文关键词:Treponema pallidum Tp0100 bioinformatics analysis immunogenicity oxidative stress ROS |
| 基金项目:国家自然科学基金项目(31872643) |
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| 中文摘要: |
| 目的分析梅毒螺旋体硫氧还蛋白Tp0100的生物学特性及其抗氧化应激作用。 方法利用NCBI、SignalP5.0、TMHMM等生物信息学软件分析Tp0100蛋白的基本理化性质、磷酸化位点、糖基化位点、二级结构、三级结构、B细胞表位和与其他菌属的同源性。构建Tp0100原核表达体系,诱导表达重组蛋白Tp0100(rTp0100)。制备相应新西兰兔免疫血清,ELISA法检测特异性IgG抗体滴度。应用流式细胞仪和荧光显微镜检测rTp0100刺激巨噬细胞释放活性氧(ROS)水平。 结果Tp0100蛋白共有185个氨基酸,相对分子质量为20.5 kDa,有信号肽、磷酸化位点,无跨膜域,无糖基化位点。二级结构中氨基酸α螺旋有59个,延伸链44个,β转角21个,无规则卷曲61个；有2个连续的B细胞表位；Tp0100与淋病奈瑟菌硫氧还蛋白具有高度的同源性。成功构建重组质粒pET28a-Tp0100并获得高纯度的rTp0100。该蛋白免疫新西兰兔能够诱导产生高滴度特异性IgG抗体。rTp0100蛋白刺激能降低巨噬细胞内ROS水平。 结论Tp0100蛋白为亲水性蛋白,有B细胞表位；rTp0100具有良好的抗原性,可抑制巨噬细胞内ROS的产生。 |
| 英文摘要: |
| AimTo analyze the biological characteristics and identify the anti-oxidative stress function of thioredoxin Tp0100 in Treponema pallidum. MethodsThe basic physicochemical properties, phosphorylation sites, glycosylation sites, secondary and tertiary structures, B cell epitopes, and homology with other bacterial genera of Tp0100 protein in T.pallidum were analyzed 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 prepared, 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. ResultsTp0100 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 gonorrhoeae thioredoxin. The recombinant plasmid pET28a-Tp0100 was successfully constructed and the rTp0100 with high purity was obtained. 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. ConclusionTp0100 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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