Cloning and Expressing FM DV Non-Structural Protein 3abc Gene in E．cofi

YOU Yong-jin; ZHU Caizhu; GE Yan; CHEN Bo; ZHANG Qiang; RAO Zhong; XU Quan-xing; LU Yong-gan

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April 2003

YOU Yong-jin, ZHU Caizhu, GE Yan, CHEN Bo, ZHANG Qiang, RAO Zhong, XU Quan-xing and LU Yong-gan. Cloning and Expressing FM DV Non-Structural Protein 3abc Gene in E．cofi[J]. Virologica Sinica, 2003, 18(2): 155-158.

Citation: YOU Yong-jin, ZHU Caizhu, GE Yan, CHEN Bo, ZHANG Qiang, RAO Zhong, XU Quan-xing, LU Yong-gan. Cloning and Expressing FM DV Non-Structural Protein 3abc Gene in E．cofi .VIROLOGICA SINICA, 2003, 18(2) : 155-158.

口蹄疫病毒非结构蛋白3abc基因的克隆与表达

尤永进 ¹ ,
朱彩珠 ² ,
葛艳 ¹ ,
陈波 ¹ ,
张强 ² ,
饶忠 ¹ ,
徐泉兴 ¹ ,
卢永干 ²

1.
1．上海市农业科学院畜牧兽医研究所，上海市农业遗传育种重点实验室动物遗传工程研究分室，上海，201106
2.
中国农业科学院兰州兽医研究所，甘肃兰州，730046

摘要

摘要：口蹄疫病毒(Foot．and．mouth disease virus，FMDv)非结构蛋白(NSP)．3ABC可用于注苗与感染动物的鉴别诊断，合成该基因的PCR引物，并在引物5’端和3’端分别加入含BamH I和Hind III限制性酶切位点序列。以FMDV毒株基因组RNA为模板，利用RT．PCR技术扩增3abc基因，得到的基因片段与T载体连接，转化DH5 Q。提取重组载体pT．3ABC，经BamH I，胁d III双酶切后与载体pET32a连接，转化宿主菌BL21(DE3)plysS， IPTG 诱导表达目的蛋白。SDS．PAGE 及Western Blotting检测和鉴定结果表明，在大肠杆菌中成功表达了 NSP-3ABC蛋白，分子量约56kDa，且该表达产物可与FMDV感染的动物血清产生免疫反应。ELISA试验结果显示，表达蛋白可用于FMDV注苗与感染动物的鉴别诊断。
- 口蹄疫病毒
- , 非结构蛋白
- , 克隆
- , 表达

Cloning and Expressing FM DV Non-Structural Protein 3abc Gene in E．cofi

YOU Yong-jin ¹ ,
ZHU Caizhu ² ,
GE Yan ¹ ,
CHEN Bo ¹ ,
ZHANG Qiang ² ,
RAO Zhong ¹ ,
XU Quan-xing ¹ ,
LU Yong-gan ²

1.
Animal Genetic Engineering Research Section，Shanghai Municipal Key Laboratory ofAgricultural Genetic and Breeding， Institute ofAnima l Husbandry and Veterinary Medicine，Shanghai Academy of Agricultural Sciences，Shanghai 201106,China
2.
Lanzhou Veterinary Research Institute，China Academy of Agricultural Sciences，Lanzhou 730046,China

Abstract

Abstract：The Foot and mouth disease virus(FMDV、non．structural protein 3ABC can be used for diferentiation of infection from vaccination．The primers of 3ABC gene were designed an d synthesized， an d the 5’end an d 3’end of primers were adding the sequence of restriction endonuclease BamH I an d Hind III respectively．The 3ABC gene coding region was obtained from the FMDV genome RNA by RT．PCR．Th e amplified fragment was cloned into T．vector．The recombinan t plasmid pT．3ABC was digested with BamH I and ，ld III an d then cloned into pET32a．Th e recombinan t plasmid pET3ABC was transformed into BL21(DE3)plysS and the target protein was induced by IP1℃ ．Expression of NSP．3ABC protein was examined and identificated bV SDS．PAGE，W_estem blotting an d EU SA．Th e results showed that recombinan t plasmid pET3ABC was constructed an d the NSP．3ABC was expressed in Ecoli successfullv．A special electrophoretic ban d in SDS．PAGE (56kDa)Was noted，Westem bloting showed it Can react witIl FM DV infected an imal serum ，and ELISA result showed the expressed protein Can be used to diferentiate the infection from vaccination．
- Foot-and-mouth disease virus
- , Non-stI1lcmral protein
- , Cloning
- , Expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning and Expressing FM DV Non-Structural Protein 3abc Gene in E．cofi

1. Animal Genetic Engineering Research Section，Shanghai Municipal Key Laboratory ofAgricultural Genetic and Breeding， Institute ofAnima l Husbandry and Veterinary Medicine，Shanghai Academy of Agricultural Sciences，Shanghai 201106,China
2. Lanzhou Veterinary Research Institute，China Academy of Agricultural Sciences，Lanzhou 730046,China

Abstract: Abstract：The Foot and mouth disease virus(FMDV、non．structural protein 3ABC can be used for diferentiation of infection from vaccination．The primers of 3ABC gene were designed an d synthesized， an d the 5’end an d 3’end of primers were adding the sequence of restriction endonuclease BamH I an d Hind III respectively．The 3ABC gene coding region was obtained from the FMDV genome RNA by RT．PCR．Th e amplified fragment was cloned into T．vector．The recombinan t plasmid pT．3ABC was digested with BamH I and ，ld III an d then cloned into pET32a．Th e recombinan t plasmid pET3ABC was transformed into BL21(DE3)plysS and the target protein was induced by IP1℃ ．Expression of NSP．3ABC protein was examined and identificated bV SDS．PAGE，W_estem blotting an d EU SA．Th e results showed that recombinan t plasmid pET3ABC was constructed an d the NSP．3ABC was expressed in Ecoli successfullv．A special electrophoretic ban d in SDS．PAGE (56kDa)Was noted，Westem bloting showed it Can react witIl FM DV infected an imal serum ，and ELISA result showed the expressed protein Can be used to diferentiate the infection from vaccination．