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BioMed Research International
Volume 2014, Article ID 368581, 8 pages
http://dx.doi.org/10.1155/2014/368581
Research Article

Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

1Laboratory of Molecular Virology and Immunology, College of Veterinary Medicine, Agricultural University of Hebei, Hebei Engineering and Technology Research Center of Veterinary Biotechnology, Baoding 071001, China
2Section of Biology, Zhongyi Middle School of Xingtai, Hebei, China
3Department of Biotechnology, College of Environmental and Chemical Engineering, Yanshan University, Qinhuangdao 066004, China

Received 21 February 2014; Accepted 14 April 2014; Published 8 May 2014

Academic Editor: Ramesh Prabhu

Copyright © 2014 Liyue Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid by Spe I and Xho I sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesized in vitro and amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.