ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2013, Vol. 44 ›› Issue (10): 1629-1636.doi: 10.11843/j.issn.0366-6964.2013.10.017

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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-mei1*, WERNIKE Kerstin2*   

  1. (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

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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.

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