类鼻疽杆菌鞭毛蛋白FliC与EHEC O157∶H7 EspA蛋白的融合表达及其免疫学性质研究  

The expression and immunological properties of the flagella protein of Burkholderia pseudomalle fused with EspA from enterohemorrhagic Escherichia coli O157

作　　者：于波[1] 方瑶[1] 顾江[1] 王海光[1] 程琰[1] 李倩[1] 唐彬[1] 李娜[1] 张小玲[1] 邹全明[1] 毛旭虎[1] 

机构地区：[1]第三军医大学医学检验系临床微生物及免疫学教研室国家免疫生物制品工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2013年第3期251-255,共5页Immunological Journal

基　　金：全军"十二五"重大项目(AWS11J011-04)

摘　　要：目的融合表达类鼻疽杆菌鞭毛蛋白FilC与肠出血性大肠杆菌(EHEC)O157 EspA蛋白,并评价其免疫学性质。方法 PCR扩增类鼻疽杆菌FilC编码基因bpsl3319,连接到基于EHEC O157 EspA的表达载体pEspA上,表达并纯化EspA-FliC融合蛋白,Western blot分析其抗原性;EspA-FliC免疫Balb/c小鼠评价其免疫原性。结果获得了bpsl3319目的基因,构建了重组表达质粒pEspA-bpsl3319;实现了EspA-FilC的高效表达,其表达量约占菌体总蛋白的25%,纯化后蛋白纯度超过85%。Westernblot结果显示EspA-FliC能分别与抗类鼻疽杆菌多克隆抗体、抗EHEC O157∶H7 EspA的单抗和多抗血清发生特异性抗原抗体反应。EspA-FilC免疫Balb/c小鼠可刺激其产生高效价特异的IgG,且此抗体能识别EspA-FilC、EspA以及天然类鼻疽杆菌的FliC。结论重组表达了融合蛋白EspA-FilC,该蛋白具有较好的免疫反应性和良好的免疫原性。此研究为下一步研制类鼻疽杆菌和EHEC O157的二联疫苗奠定了基础。In this study, we aimed to express flagella protein （FilC） of Burkholderia pseudomalle （B.pseudomallei） fused with EspA of enterohemorrhagic Escherichia coli （EHEC） O157：H7, and evaluate its immunological properties. Firstly, gene encoding FilC （bps13319） was amplified and cloned into pEspA. Then,Western blot was used to analyze the reactivity of the fusion protein to anti-B, pseudomallei and anti-EspA antibodies. In addition, purified FilC was used to immunize Balb/c mice, and the antiserum was reacted with EspA,EspA-FilC and whole B. pseudomallei cells. FilC was successfully expressed in inclusion body on pEspA vector, which consisted of about 25% of total proteins. Western blot results showed that EspA-FliC was able to react withanti-EspA monoclonal antibody and anti-B, pseudomallei polyelonal antibodies. Purified EspA-FliC was able to elicite significant EspA-FliC specific IgG in mice. This anti-serum could recognize EspA, EspA-FliC and nativeFliC from B. pseudomaUei. From these results, we can tell that the recombinant EspA-FilC shows good immunoreactivity and immunogenicity, which lays a solid foundation for the future development of combinedvaccines against B. pseudomallei and EHEC O157.

关 键 词：类鼻疽杆菌 鞭毛蛋白FliC 肠出血性大肠杆菌O157 EspA 疫苗 

分 类 号：R392[医药卫生—免疫学]