文章摘要
许秀华,郑 峰,岳 岩,汪春晖,周婷婷,胡龙华.抗金黄色葡萄球菌富丝氨酸重复蛋白SraPL⁃Lectin抗体在小鼠巨噬细胞抗感染中的功能研究[J].南京医科大学学报,2019,(4):472~477
抗金黄色葡萄球菌富丝氨酸重复蛋白SraPL⁃Lectin抗体在小鼠巨噬细胞抗感染中的功能研究
Function study of the anti⁃infection effects for an anti⁃SraPL⁃Lectin antibody in mouse macrop⁃hages infected by Staphylococcus aureus
投稿时间:2019-01-29  
DOI:10.7655/NYDXBNS20190402
中文关键词: 金黄色葡萄球菌  富丝氨酸重复蛋白  单克隆抗体  巨噬细胞  免疫复合物
英文关键词: Staphylococcus aureus  serine⁃rich repeat protein  monoclonal antibody  macrophages  immune complex
基金项目:国家科技重大专项(2017ZX10303401);国家自然科学基金(81460327);江苏省博士后基金(1701155C);全军应用基础研究项目(BWS14J046);江苏省青年医学人才项目(QNRC2016846)
作者单位
许秀华 南昌大学第二附属医院检验科江西医学检验重点实验室江西 南昌 3300362东部战区疾病预防控制中心江苏 南京 210002 
郑 峰 东部战区疾病预防控制中心江苏 南京 210002 
岳 岩 解放军总医院眼科北京 100853 
汪春晖 东部战区疾病预防控制中心江苏 南京 210002 
周婷婷 东部战区疾病预防控制中心江苏 南京 210002 
胡龙华 南昌大学第二附属医院检验科江西医学检验重点实验室江西 南昌 330036 
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中文摘要:
      目的:阐述单克隆抗体anti?SraPL?Lectin在小鼠巨噬细胞吞噬杀伤金黄色葡萄球菌过程中的功能。方法:PCR扩增sraP相应基因片段,并克隆至pET28a表达载体,用0.1 mmol/L 异丙基硫代半乳糖苷(isopropyl β?D?thiogalactoside,IPTG)诱导表达,镍柱纯化重组蛋白,经Western blot分析anti?SraPL?Lectin的特异性,采用qPCR检测巨噬细胞炎症因子诱导表达水平,CCK8检测金黄色葡萄球菌的增殖抑制率,涂板计数巨噬细胞上清和裂解液中金黄色葡萄球菌的菌落数。结果:anti?SraPL?Lectin不仅能与不同截短类型的SraP重组蛋白结合,而且能与金黄色葡萄球菌细胞壁蛋白发生特异性结合;anti?SraPL?Lectin能下调小鼠巨噬细胞促炎因子[肿瘤坏死因子?α(tumor necrosis factor?α,TNF?α)、白介素(interleukin,IL)?1β、IL?12p40]的表达并上调抑炎因子(IL?10)的表达。庆大霉素和anti?SraPL?Lectin共同作用下,未见金黄色葡萄球菌出现明显的增殖。anti?SraPL?Lectin能降低巨噬细胞上清中金黄色葡萄球菌的载量。结论:单克隆抗体anti?SraPL?Lectin与金黄色葡萄球菌形成免疫复合物能减轻巨噬细胞的炎症反应,促进巨噬细胞对金黄色葡萄球菌的清除。
英文摘要:
      Objective:To determin the functions of an anti?SraPL?Lectin monoclonal antibody in the process of phagocytosis and killing Staphylococcus aureus(S.aureus)in the mouse macrophages. Methods:Differernt gene sequence of sraP was amplied by PCR and specific amplification products were inserted into pET28a plasmid. The rpET28a?SraPL?Lectin plasmid was transferred into E.coli.BL21 and induced by 0.1 mmol/L IPTG at 25 ℃. The recombinant protein was purified by nickel column and the specificity of this antibody was detected by Western blot. The expression level of inflammatory factors in macrophages was detected by qPCR. CCK8 assay was carried out to assess the inhibition rate of S.aureus proliferation. The number of S.aureus colonies in the supernatant and lysate of macrophages was counted on the coated plate. Results:Anti?SraPL?Lectin monoclonal antibody could specifically bind to recombinant SraP truncated proteins and cell wall protein of S.aureus. The co?incubation of monoclonal antibody with S.aureus could induce the down?regulation of pro?inflammatory cytokine(TNF?α,IL?1β,IL?12p40)and up?regulation of anti?inflammatory cytokine(IL?10)in macrophages. The proliferation of S.aureus USA300 LAC was obviously inhibited in the co?effect of gentamicin and antibody. Anti?SraPL?Lectin reduced the amount of S.aureus in macrophage supernatant. Conclusion:The immune complex of anti?SraPL?Lectin antibody and S.aureus can alleviate the immune response of macrophages and promote the clearance of macrophages to S.aureus.
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