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首页> 中文期刊> 《畜牧兽医学报》 >神经氨酸酶基因联合抗黏液病毒基因的抗病毒作用

神经氨酸酶基因联合抗黏液病毒基因的抗病毒作用

         
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摘要

This experiment was conducted to build the neuraminidase (NA) gene and anti-mucus virus (Mx) dual eukaryotic gene co-expression plasmid,and detect the gene expression in trans-fected mouse fibroblasts (NIH-3T3) cells, and to investigate the influence of the recombinant plasmid on the chicken fibroblasts (CEF ) cells. The cDNA fragment of NA and mutant Mx gene were derived from pcDNA3. 0-NA, pcDNA3. 0-Mx plasmid by PCR, respectively. NA cDNA fragment and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to construct the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequenced, then the mouse fibroblasts (NIH-3T3) cells were transfected. The expression of genes in pVITRO2-Mx-NA were identified by RT-PCR and indirect immunofluorescence assay (IFA). Then the recombinant plasmid was transfected into CEF cells,RT-PCR and the micro-cell inhibition test were used to test the antiviral activity for NDV (Newcastle disease virus). The restriction endonuclease digestion and sequencing results suggested that co-expression vector pVITR02-Mx-NA was constructed successfully, the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. Recombinant proteins of Mx and NA protected CEF cells from NDV infection during 0-72 hours of incubation, the mutagenesis Mx or NA protein protected CEF cells from NDV infection during 0-48 hours of incubation; Compared with single-gene transfection group,co-transfection group significantly delayed the NDV infection time of CEF cells, the difference was statistically significant (P <0. 05). The recombinant eukaryotic expression vector pVITRO2-Mx-NA was constructed and expressed in CEF cell successfully, which would contribute to delaying the infection of NDV in cell level, it revealed the co-transfection of the combined genes is more powerful than single one, they had synergistic effects.%拟构建神经氨酸酶(NA)基因和抗黏液病毒(Mx)基因双基因真核共表达载体,并检测基因表达以及转染鸡成纤维(CEF)细胞后的抗病毒效果.克隆了NA基因和突变型Mx基因的cDNA；分别定向插入真核细胞双顺反子载体pVITRO2中,酶切和测序鉴定双基因共表达载体pVITRO2-Mx-NA的正确性.用pVITRO2-Mx-NA转染NIH-3T3细胞,通过RT-PCR和间接免疫荧光(IFA)方法检测目的基因的表达.随后用pVITRO2-Mx-NA转染CEF细胞,利用RT-PCR及微量细胞病变抑制法测定重组蛋白抗新城疫病毒(NDV)的效果.结果:酶切和测序分析表明成功构建了双基因真核共表达载体pVITRO2-Mx-NA,在转染后的NIH 3T3和CEF细胞中同时检测到了NA和Mx基因的表达.联合基因表达的重组蛋白可保护CEF细胞在孵育的72 h内免受NDV的感染,而转染了突变型Mx或者NA单基因真核表达载体的CEF细胞在孵育的48 h内亦未受到NDV的侵染；与单基因转染组相比,联合基因转染组明显延迟NDV感染CEF细胞的时间,差异显著(P＜0.05).构建的双基因真核表达载体pVITRO2-Mx-NA转染CEF细胞后,其表达的重组蛋白Mx-NA在细胞水平上具有协同延缓NDV感染的能力,且优于单个基因.

著录项

  • 来源
    《畜牧兽医学报》 |2013年第1期|78-86|共9页
  • 作者

    傅德智; 张亚妮; 陈昊; 李伟; 郑蒙蒙; 王丹; 张振韬; 施青青; 李碧春;

  • 作者单位

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

    苏州大学第一附属医院,苏州215006;

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

    扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类 基因的重组;
  • 关键词

    NA; Mx; 双基因表达载体; 抗病毒活性; 抗NDV;

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