.Institute of Animal Breeding and Genetics, Provincial Key Laboratory of Molecular Design/College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
PLoS One. 2013 Aug 16;8(8):e71688. doi: 10.1371/journal.pone.0071688. eCollection 2013.
As an attempt to increase the resistance to Newcastle Disease Virus (NDV) and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA) gene and myxo-virus resistance (Mx) and detect the gene expression in transfected mouse fibroblasts (NIH-3T3) cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF) cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3) cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA). The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P<0. 05), indicating that Mx-NA jointing contributed to delaying the infection of NDV in single-cell level and the co-transfection of the jointed genes was more powerful than single one due to their synergistic effects.
为了提高对新城疫病毒（NDV）的抵抗力，进一步降低其对家禽业的风险，本工作旨在构建神经氨酸酶（NA）基因和黏液病毒抗性（Mx）的真核基因共表达质粒，并检测转染的小鼠成纤维细胞（NIH-3T3）中的基因表达，最重要的是研究重组质粒对鸡胚成纤维细胞（CEF）的影响。NA 和突变 Mx 基因的 cDNA 片段分别从 pcDNA3.0-NA 和 pcDNA3.0-Mx 质粒中通过 PCR 衍生而来，然后将 NA 和 Mx cDNA 片段插入 pVITRO2 的多克隆位点中，生成真核共表达质粒 pVITRO2-Mx-NA。通过限制性内切酶处理和测序确认重组质粒，并将其转染到小鼠成纤维细胞（NIH-3T3）中。通过 RT-PCR 和间接免疫荧光测定（IFA）测量 pVITRO2-Mx-NA 中的基因表达。将重组质粒转染到 CEF 细胞中，然后进行 RT-PCR 和微细胞抑制试验，以测试 NDV 的抗病毒活性。我们的结果表明，成功构建了共表达载体 pVITRO2-Mx-NA；在 NIH-3T3 和 CEF 细胞中均能检测到 Mx 和 NA 的表达。Mx 和 NA 的重组蛋白可保护 CEF 细胞免受 NDV 感染，直至孵育 72 小时后，但单独突变的 Mx 蛋白或 NA 蛋白可保护 CEF 细胞免受 NDV 感染，直至感染后 48 小时，与单独转染组相比，共转染组的 NDV 感染明显减少（P<0.05），表明 Mx-NA 联合作用可延迟 NDV 在单细胞水平上的感染，联合基因的共转染由于其协同作用比单一基因更有效。
