微生物學(xué)報Acta Microbiologica Sinica

48(6)：790~795; 4 June 2008

基金項目: 國家支撐計劃(2006BAD06A12); 國家“973 項目”(2005CB523201)

*通訊作者,。Tel: +86-931-8342587; E-mail: liukey@public.lz.gs.cn

作者簡介: 付元芳(1981. ), 女, 藏族, 甘肅天祝人, 碩士研究生, 主要從事病毒分子生物學(xué)研究。E-mail: wst258@163.com

付元芳1, 2,，盧曾軍1,，曹軼梅1,，郭建宏1,，張小麗2,，田美娜1,，劉在新1*,，才學(xué)鵬1*

（1 中國農(nóng)業(yè)科學(xué)院蘭州獸醫(yī)研究所，家畜疫病病原生物學(xué)國家重點實驗室,，農(nóng)業(yè)部畜禽病毒學(xué)重點實驗室,，國家口蹄疫參考實驗室，蘭州 730046）

（2 甘肅農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院,，蘭州 730070）

摘要：【目的】口蹄疫病毒（FMDV）非結(jié)構(gòu)蛋白（NSP）3A,、3B 和2C 基因的表達及產(chǎn)物純化與活性檢測?！痉椒ā坷迷吮磉_系統(tǒng)表達了FMDV NSP 3A,、3B 和富含B 細胞抗原位點序列的2C 蛋白。利用高濃度尿素裂解包涵體,，采用稀釋法和氧化型,、還原型谷胱甘肽系統(tǒng)相結(jié)合方法對2C 蛋白進行復(fù)性。用金屬鰲合親合層析的方法對表達的FMDV NSP 3A,、3B 和2C 進行純化,。采用ELISA方法對比檢測了3 種純化蛋白在檢測羊血清NSP 抗體的效果?！窘Y(jié)果】檢測得知3A 和3B 為可溶性表達蛋白,，2C 以包涵體形式表達。通過Western-blot 分析,，表明純化后蛋白能與FMDV 感染動物血清發(fā)生特異性反應(yīng),。純化的3A、3B 和復(fù)性后的2C 融合蛋白與3ABC 抗原的檢測結(jié)果具有很高的符合性,?！窘Y(jié)論】該研究為建立鑒別FMDV 自然感染動物和滅活疫苗免疫動物的酶聯(lián)免疫電轉(zhuǎn)移印跡技術(shù)（EITB）提供了所需的材料。

關(guān)鍵詞：口蹄疫病毒,；非結(jié)構(gòu)蛋白,；酶聯(lián)免疫電轉(zhuǎn)移印跡技術(shù)（EITB）

中圖分類號：Q939 文獻標識碼：A 文章編號：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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口蹄疫病毒非結(jié)構(gòu)蛋白3A,、3B 和2C 基因的表達及產(chǎn)物純化與活性檢測

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