文章摘要
邓凯丽,陈思遐,杜兴冉,冯旰珠.鲍曼不动杆菌TonB蛋白多表位的构建与筛选[J].南京医科大学学报,2018,(10):1345~1349
鲍曼不动杆菌TonB蛋白多表位的构建与筛选
Construction and selection of antigen epitopes of Acinetobacter baumannii protein TonB
投稿时间:2018-02-26  
DOI:10.7655/NYDXBNS20181003
中文关键词: 鲍曼不动杆菌  TonB蛋白  抗原表位
英文关键词: Acinetobacter baumannii  TonB antigen  epitopes
基金项目:国家自然科学基金(81670013)
作者单位
邓凯丽 南京医科大学第二附属医院呼吸科江苏 南京 210011 
陈思遐 南京医科大学第二附属医院呼吸科江苏 南京 210011 
杜兴冉 南京医科大学第二附属医院感染科江苏 南京 210011 
冯旰珠 南京医科大学第二附属医院呼吸科江苏 南京 210011 
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中文摘要:
      目的:构建和筛选鲍曼不动杆菌TonB蛋白抗原的B细胞、T细胞表位。方法:运用生物信息学软件预测TonB蛋白的小鼠B细胞及T细胞表位并人工合成相应肽段;构建重组质粒pET28a?tonB并原核表达、纯化获得TonB蛋白;分3次以TonB蛋白在皮下免疫BALB/c小鼠,5周后收集小鼠血清并分离小鼠脾脏细胞;间接ELISA法检测小鼠血清中B细胞表位抗体水平,双抗体夹心ELISA法检测经候选T细胞表位体外刺激后脾细胞分泌γ?干扰素(IFN?γ)量。结果:构建候选B细胞和T细胞表位各4条,分别为PB1、PB2、PB3、PB4和PT1、PT2、PT3、PT4;间接ELISA结果显示,PB3及PB4与TonB蛋白免疫组小鼠血清发生反应,其吸光度值与对照组相比显著升高(P<0.05);双抗体夹心ELISA结果显示,PT2、PT3及PT4刺激免疫组小鼠脾脏细胞产生的IFN?γ量与对照组相比显著升高(P<0.05)。结论:成功构建并筛选出鲍曼不动杆菌TonB蛋白的2个B细胞表位PB3及PB4,3个T细胞表位PT2、PT3及PT4。
英文摘要:
      Objective:To screen B cell and T cell antigen epitopes of Acinetobacter baumannii protein TonB. Methods:B cell epitopes and T cell epitopes were predicted by bioinformatics methods and synthesized. Recombinant pET28a?tonB was cloned and TonB was purified to immunize mice. BALB/c mice were immunized by subcutaneous for 3 times. Five weeks later,serum was collected from each of immunized mice and then negative control were mice used to perform indirect ELISA with synthesized peptides to identify B cell epitopes. Cells of the spleen were separated from every mouse and cultured with each of synthesized peptides respectively,and then cell supernate was collected to detect IFN?γ secretion by double sandwich ELISA for T cell epitopes selection. Results:Construction of 4 candidate B cell and T cell epitopes was performed,including PB1,PB2,PB3,PB4,PT1,PT2,PT3,and PT4,respectively. Indirect ELISA showed that peptide PB3,PB4 reacted with immunized mice serum and the A450 nm value was significant higher than that of the negative mice(P<0.05). Double sandwich ELISA showed that IFN?γ concentration that stimulated by peptide PT2,PT3,and PT4 was significant higher than its negative control group(P<0.05). Conclusion:Two B cell epitopes PB3 and PB4,3 T cell epitopes PT2,PT3,PT4 of TonB were successfully constructed and screened.
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