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
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全军应用基础研究项目(BWS14J046);江苏省博士后基金(1701155C)


Fabrication and functional andlysis of a monoclonal antibody against binding region of Staphyloc⁃occus aureus serine⁃rich repeat protein(SraPL⁃lectin)
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    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。

    Abstract:

    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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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(自然科学版),2018,(11):1494-1498

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  • 收稿日期:2018-05-14
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  • 在线发布日期: 2018-12-03
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