Molecular Cloning and Expression of the fp25k Gene of HaSNPV in E. coli and Preparation of Its Antiserum

WU Dong; DENG Fei; HU Zhi-hong; YUAN Li; SUN Xiu-lian

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August 2004

WU Dong, DENG Fei, HU Zhi-hong, YUAN Li and SUN Xiu-lian. Molecular Cloning and Expression of the fp25k Gene of HaSNPV in E. coli and Preparation of Its Antiserum[J]. Virologica Sinica, 2004, 19(4): 380-384.

Citation: WU Dong, DENG Fei, HU Zhi-hong, YUAN Li, SUN Xiu-lian. Molecular Cloning and Expression of the fp25k Gene of HaSNPV in E. coli and Preparation of Its Antiserum .VIROLOGICA SINICA, 2004, 19(4) : 380-384.

棉铃虫病毒HaSNPV fp25k基因的克隆表达及抗体制备*

胡志红 ^1,2 ,
孙修炼 ¹ ,

1.
1. 中国科学院武汉病毒研究所分子病毒学实验室, 湖北武汉430071
2.
中国科学院水生生物研究所, 湖北武汉 430072
3.
中国科学院研究生院，北京100039

摘要

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus, HaSNPV) fp25k基因的序列，设计引物，引入适当的酶切位点，利用PCR扩增出基因片段。将该基因片段克隆至原核表达载体pProEXHTb，经IPTG诱导，在大肠杆菌DH5中获得了高效表达，表达产物的大小为32kDa。纯化蛋白产物免疫家兔制备抗血清。该抗血清可与原核表达的GST-FP25K融合蛋白及在感染的昆虫细胞中表达的FP25K蛋白发生特异性免疫反应。该抗体的获得为深入研究FP25K蛋白的功能提供了基础。
- 棉铃虫单核衣壳核多角体病毒（HaSNPV）
- , FP25K
- , 克隆
- , 表达
- , 抗体

Molecular Cloning and Expression of the fp25k Gene of HaSNPV in E. coli and Preparation of Its Antiserum

WU Dong ^1,2 ,
DENG Fei ¹ ,
HU Zhi-hong ¹ ,
YUAN Li ¹ ,
SUN Xiu-lian ^1*,,

1.
1. Laboratory of Molecular Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
2.
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
3.
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China

Corresponding author: SUN Xiu-lian,

Abstract

The PCR product of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus HaSNPV fp25k gene was cloned into a prokaryotic expression vector pProEXHTb. After inducing with IPTG, the fp25k gene was successfully expressed in E.coli DH5α. The expression product was 32 kDa. This protein was purified and injected into rabbit to raise polyclonal antiserum. Western blot analysis using this antiserum showed the specific reaction to FP25K protein expressed in both prokaryotic with GST fusion and viral infected insect cells. The antibody made it possible to analysis the function this protein in infected insect cells.
- HaSNPV
- , FP25K
- , Cloning
- , Expression
- , polyclonal antiserum.

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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

Molecular Cloning and Expression of the fp25k Gene of HaSNPV in E. coli and Preparation of Its Antiserum

Corresponding author: SUN Xiu-lian,

1. 1. Laboratory of Molecular Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
2. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
3. Graduate School of the Chinese Academy of Sciences, Beijing 100039, China

Abstract: The PCR product of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus HaSNPV fp25k gene was cloned into a prokaryotic expression vector pProEXHTb. After inducing with IPTG, the fp25k gene was successfully expressed in E.coli DH5α. The expression product was 32 kDa. This protein was purified and injected into rabbit to raise polyclonal antiserum. Western blot analysis using this antiserum showed the specific reaction to FP25K protein expressed in both prokaryotic with GST fusion and viral infected insect cells. The antibody made it possible to analysis the function this protein in infected insect cells.