中文摘要：

目的获得鹅源副粘病毒（GPMV）NA-1株M、F和HN基因真核表达蛋白。方法将NA-1株GPMV经SPF鸡胚增殖、纯化,提取病毒基因组RNA。参考GenBank登录的NA-1株GPMV基因组序列,设计3对特异性引物,利用RT-PCR法,分别扩增M、F和HN基因开放阅读框（ORF）,将目的基因克隆至pMD18-T-Simple载体,并进行酶切和序列测定。将目的片段克隆至pCI-neo表达载体,酶切鉴定正确的重组质粒,分别命名为pCI-M、pCI-F和pCI-HN。将重组表达质粒分别转染Vero细胞,用间接免疫荧光试验鉴定目的蛋白的表达。结果RT-PCR法分别扩增出M、F和HN基因,大小与预期一致。克隆载体及表达载体酶切及测序鉴定正确;重组真核表达质粒转染Vero细胞后,荧光显微镜下可见特异性荧光。结论利用pCI-neo真核表达载体在Vero细胞中成功表达了NA-1株GPMV M、F和HN基因,为下一步研究该病毒的出芽机制以及各结构蛋白的功能奠定了基础。

英文摘要：

Objective To express M, F and HN genes of goose paramyxovirus （GPMV） NA-1 strain in eukaryotic cells. Methods The NA-I strain of GPMV was proliferated in SPF chick embryo cells and purified for extraction of genomic RNA. Design 3 pairs of specific primers according to the sequence of genome of GPMV NA- 1 strain reported in GenBank for amplification of open reading frame （ORF） of M, F and HN genes by RT-PCR. Insert the amplified target genes into pMD 18-T Simple vector and identify the constructed recombinant plasmid by restriction analysis and sequencing. Clone the recovered target gene fragment to expression Vector pCl-neo and screen the correctly constructed recombinant plasmids by restriction analysis, which were named as pCl-M, pCl-F and pCl-HN respectively. Transfect Vero cells with the construtted recombinant plasmids and identify the expressed product by indirect IFA.Results The lengths of amplified M,F and HN genes were consistent with those expected. Restiction analysis and sequencing result proved that both cloning and expression vectors were constructed correctly. Indirect IFA of transfected Vero cells showed specific reactions of expressed pnoducts with the corresponding mouse superimmune sere. Conclusion The M, F and HN genes were successfully expressed in Vero cells by using eukaryotic expression vector pCl-neo, which laid a foundation of further study on budding mechanism of GPMV and function of structural protein.