title: Preparation of North American type II PRRSV infectious clone expressing green fluorescent protein.
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.
authors.affiliation: Laboratory 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.
authors.forename: Liyue
authors.initials: L
authors.lastname: Wang
authors.affiliation: Laboratory 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 ; Section of Biology, Zhongyi Middle School of Xingtai, Hebei, China.
authors.forename: Kao
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Biotechnology, College of Environmental and Chemical Engineering, Yanshan University, Qinhuangdao 066004, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Lin
authors.affiliation: Laboratory 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.
authors.forename: Wenyan
authors.initials: W
authors.lastname: Li
authors.affiliation: Laboratory 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.
authors.forename: Jiexia
authors.initials: J
authors.lastname: Wen
authors.affiliation: Laboratory 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.
authors.forename: Jianlou
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Laboratory 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.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biotechnology, College of Environmental and Chemical Engineering, Yanshan University, Qinhuangdao 066004, China.
authors.forename: Xiujin
authors.identifier: 0000-0002-8587-8220
authors.initials: X
authors.lastname: Li
authors.affiliation: Laboratory 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.
authors.forename: Fei
authors.initials: F
authors.lastname: Zhong
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D019976:Cloning, Organism; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000235:genetics*; D007668:Kidney / Q000378:metabolism* / Q000473:pathology / Q000821:virology*; D008856:Microscopy, Fluorescence / Q000379:methods; D009656:North America; D019316:Porcine respiratory and reproductive syndrome virus / Q000502:physiology*; D016662:Virus Integration / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins