Cloning and Expression of VP1 Gene of Swine Vesicular Disease Virus

ZHONG Jin-dong; HUA Qun-yi; XIAO Rong-hai; XIA Xue-shan; YANG Yun-qing; ZHOU Xiao-li; DEN Zu-hong

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August 2006

ZHONG Jin-dong, HUA Qun-yi, XIAO Rong-hai, XIA Xue-shan, YANG Yun-qing, ZHOU Xiao-li, DEN Zu-hong and Cloning and Expression of VP1 Gene of Swine Vesicular Disease Virus[J]. Virologica Sinica, 2006, 21(4): 385-389.

Citation: ZHONG Jin-dong, HUA Qun-yi, XIAO Rong-hai, XIA Xue-shan, YANG Yun-qing, ZHOU Xiao-li, DEN Zu-hong, . Cloning and Expression of VP1 Gene of Swine Vesicular Disease Virus .VIROLOGICA SINICA, 2006, 21(4) : 385-389.

猪水泡病病毒VP1基因的克隆和表达

钟金栋 ¹ ,
花群义 ^2*,, ,
肖荣海 ² ,
夏雪山 ¹ ,
杨云庆 ² ,
周晓黎 ² ,
董俊 ² ,
邓祖洪 ³

1.
1.昆明理工大学生化学院,云南昆明650224
2.
云南出入境检验检疫局技术中心，云南昆明650228
3.
西双版纳兽医站,云南景洪666100

通讯作者： 花群义, ;

摘要

以猪水泡病病毒RNA为模板，应用反转录聚合酶链式反应(RT-PCR)技术，扩增了849bp的VP1基因，通过T-A克隆技术，将VP1基因片段克隆至pMD18-T克隆载体质粒中，构建SVDV VP1基因克隆重组质粒，进行核苷酸序列分析。然后亚克隆插入pBAD/Thio TOPO表达载体，经测序鉴定，筛选获得VP1基因正向插入、有正确读码框的阳性克隆，成功构建了猪水泡病病毒VP1基因重组表达载体。经L-Arabinose诱导表达，可稳定、高效地表达VP1蛋白抗原。SDS-PAGE结果表明，以终浓度为0.002%的L－阿拉伯醛糖进行诱导，5h后表达可达到高峰，表达蛋白为融合蛋白，质量约47.13 kDa，表达产量约占菌体总蛋白的16％。Western blotting检测表明，诱导的蛋白能与猪水泡病阳性血清发生特异性反应。融合蛋白中含有猪水泡病病毒VP1蛋白抗原，为应用该表达蛋白抗原制备SVD免疫血清学诊断试剂和新型疫苗构建奠定基础。
- 猪水泡病病毒
- , VP1
- , 克隆
- , 表达

Cloning and Expression of VP1 Gene of Swine Vesicular Disease Virus

1.
1. Faculty of Bioengineering and Chemical Engineering, Kunming University of Science and Technology, Kunming 650224, China
2.
Technology Center of Yunnan Entry-Exit Inspection and Quarantine Bureau, Kunming 650228, China
3.
Xishuangbanna Veterinary Station, Jinghong 666100, China

Corresponding author: HUA Qun-yi,

Abstract

The VP1 gene of Swine vesicular disease virus (SVDV) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) yielding a product of 849bp cDNA fragment. Using T-A cloning technique, the PCR product was cloned into pMD18-T vector. The purified VP1 gene was subcloned into the pBAD/Thio TOPO vector and the plasmid was identified by PCR. It was sequenced to confirm the authenticity of the sequence and orientations. SDS-PAGE and Western blotting revealed that the VP1 protein was expressed in Escherichia coli LGM194 at a high level and the recombinant fusion protein contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. The optimal amount of the expressed fusion protein was 16% of total bacterial protein after being induced with L-arabinose at 0.002%concentration for 5 hours. It had a molecular mass of approximately 47.13 kDa and was immunologically reactive. The recombinant protein will be characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of swine vesicular disease.
- Swine vesicular disease virus（SVDV）
- , VP1 gene
- , 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 Expression of VP1 Gene of Swine Vesicular Disease Virus

Corresponding author: HUA Qun-yi,

1. 1. Faculty of Bioengineering and Chemical Engineering, Kunming University of Science and Technology, Kunming 650224, China
2. Technology Center of Yunnan Entry-Exit Inspection and Quarantine Bureau, Kunming 650228, China
3. Xishuangbanna Veterinary Station, Jinghong 666100, China

Abstract: The VP1 gene of Swine vesicular disease virus (SVDV) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) yielding a product of 849bp cDNA fragment. Using T-A cloning technique, the PCR product was cloned into pMD18-T vector. The purified VP1 gene was subcloned into the pBAD/Thio TOPO vector and the plasmid was identified by PCR. It was sequenced to confirm the authenticity of the sequence and orientations. SDS-PAGE and Western blotting revealed that the VP1 protein was expressed in Escherichia coli LGM194 at a high level and the recombinant fusion protein contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. The optimal amount of the expressed fusion protein was 16% of total bacterial protein after being induced with L-arabinose at 0.002%concentration for 5 hours. It had a molecular mass of approximately 47.13 kDa and was immunologically reactive. The recombinant protein will be characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of swine vesicular disease.