施马伦贝格病毒核衣壳蛋白的原核表达、纯化及多克隆抗体的制备

doi:10.11843/j.issn.0366-6964.2013.10.017

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (10): 1629-1636.doi: 10.11843/j.issn.0366-6964.2013.10.017

施马伦贝格病毒核衣壳蛋白的原核表达、纯化及多克隆抗体的制备

张永宁¹，吴绍强¹，吕继洲¹，冯春燕¹，王彩霞¹，袁向芬¹，邓俊花¹，林祥梅^1*，Wernike Kerstin^2*

（1. 中国检验检疫科学研究院动物检疫研究所，北京 100029；2. 德国弗里德里希洛弗勒研究所，格赖夫斯瓦尔德 17493）

收稿日期:2013-05-03 出版日期:2013-10-23 发布日期:2013-10-23
通讯作者: 林祥梅，E-mail：xiangmeil@gmail.com；Wernike Kerstin，E-mail：kerstin_wernike@fli.bund.de
作者简介:张永宁（1980-），男，山东日照人，助理研究员，博士，主要从事外来动物疫病检疫研究，E-mail: zhangyn@caiq.gov.cn
基金资助:
“十二五”国家科技支撑计划课题（2013BAD12B01）；国家质检总局科技计划项目（2013IK054）；中国检验检疫科学研究院基本科研业务费专项资金（2012JK011）

Prokaryotic Expression and Purification of the Nucleocapsid Protein of Schmallenberg Virus and Preparation of Its Polyclonal Antibodies

ZHANG Yong-ning¹, WU Shao-qiang¹, LYU Ji-zhou¹, FENG Chun-yan¹, WANG Cai-xia¹, YUAN Xiang-fen¹, DENG Jun-hua¹, LIN Xiang-mei^1*, WERNIKE Kerstin^2*

(1. Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing100029, China; 2. Friedrich Loeffler Institute, Greifswald 17493, Germany)

Received:2013-05-03 Online:2013-10-23 Published:2013-10-23

摘要/Abstract

摘要：

本研究旨在表达施马伦贝格病毒（Schmallenberg virus，SBV）的核衣壳蛋白（N），进而制备其多克隆抗体。以SBV (BH80株) 的RNA为模板，RT-PCR扩增N基因序列，并将其克隆至原核表达载体pET-28a-c(+)中，获得pET-28a-SBV-N重组质粒。经酶切和测序鉴定后，将重组质粒转化E. coli BL21（DE3），IPTG诱导表达His-SBV-N融合蛋白。在非变性条件下用Ni-NTA柱纯化His-SBV-N，并以此为抗原免疫新西兰大白兔制备多抗。经免疫亲和层析纯化后，利用Western blot和间接免疫荧光对多抗进行鉴定。结果表明：（1）His-SBV-N融合蛋白在E. coli BL21（DE3）中同时以可溶性和包涵体2种形式表达，相对分子质量约为30 ku；（2）制备的多抗效价高达1∶64 000。该多抗不仅能与His-SBV-N融合蛋白发生特异性反应，而且还能识别SBV的不同毒株，但与布尼亚病毒科内亲缘关系最近的沙门达病毒（Shamonda virus）、道格拉斯病毒（Douglas virus）和赤羽病病毒（Akabane virus）的核衣壳蛋白存在交叉反应。His-SBV-N融合蛋白及其多抗的成功制备，为施马伦贝格病血清学检测方法的建立奠定了物质基础。

Abstract:

The present study was conducted to prokaryotically express the nucleocapsid (N) protein of Schmallenberg virus (SBV) and prepare its polyclonal antibodies. Using the total RNA of SBV (isolate BH80) as a template, RT-PCR was utilized to amplify the full-length coding sequence of the N gene, which was then cloned into the prokaryotic expression vector pET-28a-c(+) to construct the recombinant plasmid pET-28a-SBV-N. After verification using the double-enzyme cleavage method and sequence analysis, the recombinant plasmid pET-28a-SBV-N was transformed into the competent E. coli BL21 (DE3) cells which were then induced by isopropyl-β-thiogalactopyranoside (IPTG). The recombinant His-SBV-N fusion protein was purified under native conditions using a nickel ion metal chelate column. Subsequently, the purified His-SBV-N protein was used as an immunogen to immunize the New Zealand white rabbits to prepare its polyclonal antibodies. After purification of the prepared polyclonal antibodies using the immunoaffinity chromatography method, Western blot analysis and indirect immunofluorescence assay were used to validate the polyclonal antibodies. Our results demonstrated that: (1) The recombinant His-SBV-N fusion protein was efficiently expressed in E. coli BL21(DE3) in the form of both soluble expression and inclusion bodies, and the molecular weight of His-SBV-N protein was about 30 kDa; (2) The titer of the prepared polyclonal antibodies was 1: 64 000. The prepared polyclonal antibodies not only can specifically react with the recombinant His-SBV-N fusion protein, but it also can recognize different isolates of SBV. However, the prepared polyclonal antibodies also have cross-reactivity with the N protein of the most genetically related viruses, such as Shamonda virus, Douglas virus and Akabane virus, within the family Bunyaviridae. Undoubtedly, the successful preparation of both His-SBV-N fusion protein and its polyclonal antibodies lays a material foundation for the establishment of serological methods for SBV detection.

中图分类号:

S852.659.5

张永宁，吴绍强，吕继洲，冯春燕，王彩霞，袁向芬，邓俊花，林祥梅，Wernike Kerstin. 施马伦贝格病毒核衣壳蛋白的原核表达、纯化及多克隆抗体的制备[J]. 畜牧兽医学报, 2013, 44(10): 1629-1636.

ZHANG Yong-ning, WU Shao-qiang, LYU Ji-zhou, FENG Chun-yan, WANG Cai-xia, YUAN Xiang-fen, DENG Jun-hua, LIN Xiang-mei, WERNIKE Kerstin. Prokaryotic Expression and Purification of the Nucleocapsid Protein of Schmallenberg Virus and Preparation of Its Polyclonal Antibodies[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1629-1636.

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参考文献

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施马伦贝格病毒核衣壳蛋白的原核表达、纯化及多克隆抗体的制备

Prokaryotic Expression and Purification of the Nucleocapsid Protein of Schmallenberg Virus and Preparation of Its Polyclonal Antibodies

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