抗金黄色葡萄球菌富丝氨酸重复蛋白SraP_(L-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-SraP_(L-Lectin) antibody in mouse macrop-hages infected by Staphylococcus aureus

作　　者：许秀华 郑峰 岳岩[3] 汪春晖 周婷婷 胡龙华[1] Xu Xiuhua;Zheng Feng;Yue Yan;Wang Chunhui;Zhou Tingting;Hu Longhua(DepartmentClinical Lciboratory Medicine, the Second Affiliated Hospital of Nanchang University, Key Lciboratoryof Medicine in Jiangxi Province, Nanchang 330006;Disease Control and Prevention of Eastern Theater Command,Nanjing 210002;Department of Ophthalmology, The General Hospital of PLX,Beijing 100853, China)

机构地区：[1]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 [2]东部战区疾病预防控制中心,江苏南京210002 [3]解放军总医院眼科,北京100853

出　　处：《南京医科大学学报（自然科学版）》2019年第4期472-477,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：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)

摘　　要：目的:阐述单克隆抗体anti-SraP_(L-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(isopropylβ-D-thiogalactoside,IPTG)诱导表达,镍柱纯化重组蛋白,经Western blot分析anti-SraP_(L-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-SraP_(L-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-SraP_(L-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-SraP_(L-Lectin)共同作用下,未见金黄色葡萄球菌出现明显的增殖。anti-SraP_(L-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-SraP_(L-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。Objective:To determin the functions of an anti-SraPL-Lectinmonoclonal 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 pET28 a plasmid. The rpET28 a-SraPL - Lectinplasmid 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-Lectinmonoclonal 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-regulationofpro-inflammatorycytokine(TNF-α,IL-1β,IL-12 p40)andup-regulationofanti-inflammatorycytokine(IL-10)inmacrophages.TheproliferationofS.aureus USA300 LACwasobviouslyinhibitedintheco-effectofgentamicinandantibody.Anti-SraPL-Lectinreduced the amount of S.aureus in macrophage supernatant. Conclusion:The immune complex of anti-SraPL-Lectinantibody and S.aureus can alleviate the immune response of macrophages and promote the clearance of macrophages to S.aureus.

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 

分 类 号：R378.1[医药卫生—病原生物学]