微生物学报Acta Microbiologica Sinica
48(6)：790~795; 4 June 2008
基金项目: 国家支撑计划(2006BAD06A12); 国家“973 项目”(2005CB523201)
*通讯作者。Tel: +86-931-8342587; E-mail: liukey@public.lz.gs.cn
作者简介: 付元芳(1981. ), 女, 藏族, 甘肃天祝人, 硕士研究生, 主要从事病毒分子生物学研究。E-mail: wst258@163.com
付元芳1, 2，卢曾军1，曹轶梅1，郭建宏1，张小丽2，田美娜1，刘在新1*，才学鹏1*
（1 中国农业科学院兰州兽医研究所，家畜疫病病原生物学国家重点实验室，农业部畜禽病毒学重点实验室，国家口蹄疫参考实验室，兰州 730046）
（2 甘肃农业大学动物医学院，兰州 730070）
摘要：【目的】口蹄疫病毒（FMDV）非结构蛋白（NSP）3A、3B 和2C 基因的表达及产物纯化与活性检测。【方法】利用原核表达系统表达了FMDV NSP 3A、3B 和富含B 细胞抗原位点序列的2C 蛋白。利用高浓度尿素裂解包涵体，采用稀释法和氧化型、还原型谷胱甘肽系统相结合方法对2C 蛋白进行复性。用金属鳌合亲合层析的方法对表达的FMDV NSP 3A、3B 和2C 进行纯化。采用ELISA方法对比检测了3 种纯化蛋白在检测羊血清NSP 抗体的效果。【结果】检测得知3A 和3B 为可溶性表达蛋白，2C 以包涵体形式表达。通过Western-blot 分析，表明纯化后蛋白能与FMDV 感染动物血清发生特异性反应。纯化的3A、3B 和复性后的2C 融合蛋白与3ABC 抗原的检测结果具有很高的符合性。【结论】该研究为建立鉴别FMDV 自然感染动物和灭活疫苗免疫动物的酶联免疫电转移印迹技术（EITB）提供了所需的材料。
关键词：口蹄疫病毒；非结构蛋白；酶联免疫电转移印迹技术（EITB）
中图分类号：Q939 文献标识码：A 文章编号：0001-6209 (2008) 06-0790-06
Purification and reactivity of Foot-and-Mouth Disease Virus non-structural protein 3A, 3B and 2C expressed in E. coli
Yuanfang Fu1,2, Zengjun Lu1, Yimei Cao1, Jianhong Guo1, Xiaoli Zhang2,
Meina Tian1, Zaixin Liu1*, Xuepeng Cai1*
(1Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, National Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute of Chinese Academy of Agriculture Science, Lanzhou 730046, China)
(2Veterinary College of Gansu Agriculture University, Lanzhou 730070, China)
Abstract: [Objective] To purify and to detect reactivity of non-structural proteins 3A, 3B and 2C expressed in the Escherichia coli. [Methods] FMDV NSP 3A, 3B and 2C containing the major B-cell antigenic sites were expressed in E. coli. We got renatured 2C protein by lysing of isolated inclusion body using high concentration of urea, and then diluted in a buffer system containing oxidized/reduced glutathione. Purified 3A, 3B and 2C were obtained by Ni-NTA His Bind Resin affinity chromatography. The reactivity of three NSPs with sera of different origin was measured using an indirect ELISA and Western-blot. The reactivity of three proteins was compared with 3ABC and 3D by detecting sera of clinically healthy sheep that were collected from epidemic region of AsiaⅠFMD. [Results] Proteins 3A and 3B were solubly expressed in bacteria, and 2C was expressed to form inclusion body. All three products could react specifically with sera from FMDV infected animal by western-blot and ELISA. The high coincident rates were observed between 3A, 3B, 2C and 3ABC. [Conclusion] The results would provide useful materials for establishment of immunoelectro-transfer blot (EITB) diagnostic method, which could be used for differentiation of the FMDV infected animals from the vaccinated animals．
Keywords: Foot-and-Mouth Disease Virus (FMDV);non-structural protein (NSP); immunoelectro-transfer blot (EITB)
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