文章摘要
岳 岩,郑 峰,曹清心,王怡雯,周婷婷,王长军.金黄色葡萄球菌富丝氨酸重复蛋白SraPL⁃Lectin特异性抗体的制备及其功能分析[J].南京医科大学学报,2018,(11):1494~1498
金黄色葡萄球菌富丝氨酸重复蛋白SraPL⁃Lectin特异性抗体的制备及其功能分析
Fabrication and functional andlysis of a monoclonal antibody against binding region of Staphyloc⁃occus aureus serine⁃rich repeat protein(SraPL⁃lectin)
投稿时间:2018-05-14  
DOI:10.7655/NYDXBNS20181102
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  感染
英文关键词: Staphylococcus aureus  serine⁃rich repeat protein  monoclonal antibody  infection
基金项目:全军应用基础研究项目(BWS14J046);江苏省博士后基金(1701155C)
作者单位
岳 岩 中国人民解放军总医院医务科北京 100853 
郑 峰 南京军区军事医学研究所江苏 南京 210002 
曹清心 南京军区军事医学研究所江苏 南京 210002 
王怡雯 南京军区军事医学研究所江苏 南京 210002 
周婷婷 南京军区军事医学研究所江苏 南京 210002 
王长军 南京军区军事医学研究所江苏 南京 210002 
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中文摘要:
      目的:制备金黄色葡萄球菌富丝氨酸重复蛋白SraPL?Lectin单克隆抗体,并分析其体外功能。方法:以USA300基因组为模板,PCR扩增SraPL?Lectin基因序列,克隆到pET28a表达载体中,0.1 mmol/L IPTG 25 ℃诱导6 h,镍柱亲和层析纯化SraPL?Lectin?His蛋白。以纯化的SraPL?Lectin?His重组蛋白为抗原免疫Balb/c小鼠,采用常规融合技术制备杂交瘤细胞,间接ELISA法、Western blot法筛选和鉴定阳性杂交瘤细胞株。扩大培养阳性细胞株后注射小鼠腹腔,收集腹水,经Protein G柱纯化anti?SraPL?Lectin单克隆抗体。以不同终浓度的单克隆抗体预先孵育A549细胞和注射小鼠腹腔,涂板计数单克隆抗体阻断细菌黏附、侵入和感染的能力。结果:成功构建了SraPL?Lectin?pET28a重组表达载体,经镍柱纯化后获得高纯度的重组蛋白,获得稳定分泌单克隆抗体的细胞株,小鼠腹水经Protein G柱纯化后获得纯度高达95%以上的特异性抗体,效价1∶32万。终浓度100 ng/mL单克隆抗体预先孵育A549细胞2 h,能够显著降低金黄色葡萄球菌对A549细胞的黏附和侵入。提前1 d向小鼠腹腔注射1 μg anti?SraPL?Lectin单克隆抗体,能够显著降低小鼠血液中的金黄色葡萄球菌数量。结论:本研究制备的anti?SraPL?Lectin单克隆抗体能够阻断金黄色葡萄球菌对宿主细胞的黏附和侵入,为将SraPL?Lectin作为防控金黄色葡萄球菌感染药物靶点的研发奠定了基础。
英文摘要:
      Objective:To prepare a monoclonal antibody against SraPL?lectin,a serine?rich repeat protein of Staphylococcus aureus,and analyze its function. Methods:The SraPL?lectin gene was amplified by PCR from USA 300 DNA,and inserted into pET28a plasmid. The pET28a? SraPL?lectin was transferred into E.coli. BL21(DE3)and induced with 0.1 mmol/L isopropyl?β?d?thiogalactoside(IPTG)for 6 h at 25 ℃. The recombinant protein was purified by His label affinity chromatography. The purified protein was used as an antigen to generate an anti?SraPL?lectin monoclonal antibody. The mAb was identified by ELISA and Western blot. A549 cells were pre?incubated monoclonal antibodies with different final concentrations. Mice were injected with 1 μg anti?SraPL?lectin monoclonal antibodies one day before challenge. The effect of adhesion and invasion was counted by plating bacteria on TSA. Results:We successfully constructed a prokaryotic expression pET28a?SraPL?lectin vector,and the SraPL?lectin recombinant protein was expressed and purified. We also generated a clone of hybridoma with SraPL?lectin binding activity,and obtained anti?SraPL?lectin monoclonal antibody purified from mouse ascites by protein G column. The titer of mAb was 1∶320 000. Pre?incubation of A549 cells with a final concentration of 100 ng/mL for 2 h significantly reduced the adherence and invasion of S. aureus on A549 cells. The number of bacteria in blood was significantly decreased when mice were administered 1 μg anti?SraPL?Lectin monoclonal antibodies. Conclusion:The anti?SraPL?Lectin monoclonal antibody prepared in this study significantly reduced the adherence and invasion of S. aureus on host cells. This study lays the foundation for the development of SraPL?Lectin as a target for the prevention and treatment of S. aureus infection in future.
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