文章摘要
吕洪臻,唐小军,熊四平,徐 鑫,郑 峰,冯振卿,朱 进.抗炭疽毒素保护性抗原单克隆抗体的制备及功能分析[J].南京医科大学学报,2013,(11):1497~1501
抗炭疽毒素保护性抗原单克隆抗体的制备及功能分析
Preparation and function identification of monoclonal antibodies against anthrax protective antigen
投稿时间:2013-06-03  
DOI:10.7655/NYDXBNS20131103
中文关键词: 炭疽杆菌  保护性抗原  单克隆抗体  中和抗体
英文关键词: bacillus anthracis  protective antigen  monoclonal antibody  neutralizing antibody
基金项目:国家自然科学基金(31170884);江苏省自然科学基金(BK2012527)
作者单位
吕洪臻 南京医科大学病理学系,江苏 南京 210029 
唐小军 南京医科大学病理学系,江苏 南京 210029 
熊四平 南京医科大学病理学系,江苏 南京 210029 
徐 鑫 南京医科大学病理学系,江苏 南京 210029 
郑 峰 南京军区军事医学研究所,江苏 南京 210002 
冯振卿 南京医科大学病理学系,江苏 南京 210029 
朱 进 南京医科大学病理学系,江苏 南京 210029
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:原核表达炭疽杆菌保护性抗原PA10蛋白,制备单克隆抗体,并进行功能分析。方法:人工合成炭疽杆菌保护性抗原PA10编码基因并克隆入pUC57载体,酶切鉴定后,将PA10编码基因插入原核表达载体pColdⅡ中,测序验证正确后转化至大肠杆菌BL21(DE3),在IPTG和低温条件下诱导蛋白表达,SDS-PAGE验证产物;用HiTrap IMAC HP柱纯化重组蛋白,Western blot进一步验证。以纯化获得PA10重组蛋白为抗原,常规程序免疫BALB/c小鼠,制备单克隆抗体,并检测所制备抗体的中和活性。结果:获得的PA10重组蛋白免疫小鼠制备单克隆抗体,最终获得了4株特异性的单克隆抗体(分别命名为2G8?5A8?7B3? 9C9)。经体外炭疽毒素保护试验证实,其中1株单抗(7B3)具有较强的中和活性,其保护率可高达96%。结论:本文成功构建并表达炭疽杆菌保护性抗原PA10,制备了具有中和活性的抗PA单克隆抗体,为构建人源化的抗PA中和抗体奠定基础,从而有望用于炭疽病的治疗。
英文摘要:
      Objective:To obtain the anthrax protective antigen in E. coli,prepare its monoclonal antibodies,and analyze the function. Methods:The gene coding for anthrax protective antigen PA10 was synthesized and cloned into pUC57 plasmids. After being identified by enzyme analysis,the target gene was inserted into pColdⅡ plasmid. By sequencing confirmation,the plasmid pCold II-PA10 was transformed into E. coli BL 21(DE3) and induced by IPTG and low temperature for expression,and identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography and identified by Western blotting. After the mice BALB/c were immunized by the recombinant protein of PA10 as antigen,the monoclonal antibodies were prepared. Results:The recombinant protein of PA10 was used to immunize mouse,and obtain 4 strains of specific monoclonal antibodies(respectively named as 2G8, 5A8, 7B3,9C9). The protection of anthrax toxin test in vitro,proved that one of four monoclonal antibody strains(7B3) has a comparatively large activity to neutralize anthrax toxin with protection rate up to 96%. Conclusion:This research successfully constructed the anthrax Protective antigen PA10 in E. coli and prepared its monoclonal antibodies,which laid the foundation for further development of chimeric neutralizing antibodies used to treat anthrax.
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