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.
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.
Biomed Res Int. 2014;2014:368581. doi: 10.1155/2014/368581. Epub 2014 May 8.
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.
猪繁殖与呼吸综合征病毒（PRRSV）仍然是威胁养猪业的最重要传染病之一。为构建含有绿色荧光蛋白（GFP）基因的北美型 II 型 PRRSV 感染性克隆，我们采用重叠 PCR 方法，以 pcDNA-EF1-GFP 质粒和含有 PRRSV 感染性基因组的 FL12 质粒为模板，扩增上下游 PRRSV Nsp2 基因片段侧翼的 gfp 基因。将 Nsp2 片段侧翼的 gfp 基因插入 FL12 质粒的 Nsp2 基因中，通过 Spe I 和 Xho I 位点生成含有 gfp 基因的 PRRSV 感染性重组质粒（FL12-GFP）。通过转染体外合成的 PRRSV mRNA 并在 Marc-145 细胞中扩增，在猪肾细胞（BHK-21）中拯救出表达 GFP 的 PRRSV（PRRSV-GFP）。鉴定了 PRRSV-GFP 的感染性和复制能力。结果表明，采用重叠 PCR 策略，成功地将 gfp 基因插入到 PRRSV 感染性克隆质粒 FL-12 的 Nsp2 基因中，并与 PRRSV Nsp2 基因融合，生成 FL12-GFP 质粒。通过转染 BHK-2 细胞中的 PRRSV mRNA，生成重组 PRRSV-GFP。与亲本病毒一样，重组 PRRSV-GFP 保持对 Marc-145 细胞和猪肺泡巨噬细胞（PAMs）的感染力。这项研究为进一步研究 PRRSV 提供了必要的条件。
