摘要
目的:根据外膜蛋白FopA的序列信息,建立土拉弗朗西斯菌FopA蛋白全长(FopA-L)和部分(FopA-S)的特异性抗原的BL21表达系统,获得高活性的重组FopA-L、FopA-S蛋白并制备相应的多克隆抗体,为土拉菌的监测、诊断和治疗提供依据。方法:通过pET100质粒构建FopA-L及FopA-S的表达载体,转化大肠杆菌BL21细胞并诱导表达FopA-L及FopA-S蛋白,螯合镍离子次氨基三乙酸(Ni-NTA)亲合层析纯化FopA蛋白,用重组蛋白免疫大耳白兔制备多克隆抗体,通过ELISA、Western印迹、胶体金免疫层析技术等方法进行检测。结果:构建了FopA-L及FopA-S的表达载体,获得相应的高表达目的蛋白BL21细胞株,用表达的蛋白为抗原成功制备了FopA特异性的抗体,效价皆在1∶100000以上且特异性良好。结论:FopA-S与FopA-L两种抗原和相应抗体的制备为建立土拉菌快速检测方法奠定了基础。
Objective：To establish an BL21 expression system of Francisella tularemia-specific antigen（FopA-L and FopA-S）,then obtain their highly active recombinant protein and the polyclonal antibodies which can be applied for Francisella monitoring,diagnosis and treatment.Methods：Constructing a plasmid expression vector pET100 FopA-L and FopA-S,then transforming FopA plasmid into E.coli BL21 cells,induced by IPTG,purifing protein by chelate nickel ion-NTA（Ni-NTA）affinity chromatography,making polyclonal antibody by recombinant FopA-L and FopA-S proteins immune rabbits and antibody were determined by Western blot,ELISA and GICA.Results：Constructed expression vectors pET100 of FopA-L and FopA-S,the BL21 cell lines obtained can highly express target proteins,antibody specifically combined with them which can be used in Francisella detecting were prepared successfully,with a titer of more than 1∶100000 and highly specific.Conclusion：Preparation the FopA-L and FopA-S antigen and antibody laid the foundation for the establishment of rapid detection method of Francisella.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第12期76-81,共6页
China Biotechnology
基金
财政部质检公益项目(2007GYJ023
2007GYJ024)资助项目
关键词
土拉菌
FopA
大肠杆菌
表达
多克隆抗体
Francisella tularemia FopA E.coli Expression Polyclonal antibody
