鸭瘟病毒VP26基因克隆和分子特性分析  

Cloning and Molecular Characterization of VP26 Gene of Duck Plauge Virus

作　　者：蔡铭升[1] 程安春[1,2] 汪铭书[1,2] 朱德康[1,2] 罗启慧[2] 招丽婵[1] 陈孝跃[1] 

机构地区：[1]四川农业大学动物医学院禽病防治研究中心,雅安625014 [2]动物疫病与人类健康四川省重点实验室,雅安625014

出　　处：《畜牧兽医学报》2009年第7期1112-1119,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30771598);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);现代农业产业技术体系建设专项资金资助(nycytx-45-12);教育部高等学校科技创新工程重大项目培育资金项目(706050)

摘　　要：利用作者实验室已构建的DPV CHv株基因文库重组质粒的DNA测序信息,结合NCBI的ORF Finder和BLAST工具分析得到了编码该病毒VP26蛋白基因(UL35)的ORF,然后采用PCR扩增出了VP26基因并将其克隆到pMD18-T载体上,经PCR和酶切鉴定以及进一步的核酸斑点杂交试验证实该基因即为DPV的VP26基因。分子特性分析表明:该基因大小为354 bp,编码117 aa,包含1个保守结构域,为Herpes_UL35,与小衣壳蛋白家族相关并在Herpes_UL35基因编码蛋白之间高度保守,而且DPVVP26蛋白与GenBank上多种α疱疹病毒同源蛋白的核酸和氨基酸序列具有较高的同源性。系统进化树分析表明,DPV在分类地位上归属于Alphaherpesvir-us亚科,而且有可能属于新的属。另外,亚细胞定位分析表明,VP26主要定位于细胞核中。密码子偏爱性分析结果显示,编码VP26相同氨基酸的不同密码子使用频率有较大的差异,DPV VP26与大肠杆菌、酵母和人的密码子使用偏爱性差异分别有18、19和25个。上述研究结果为进一步研究DPV VP26蛋白的功能和作用机理提供分子生物学依据。A complete open reading frame （ORF） of UL35 gene encoding the protein VP26 of duck plauge virus （DPV） was obtained according to the DNA sequencing information of a recombinant plasmid of the DPV CHv strain gene library constructed in our laboratory combining together with the analysis of ORF Finder and BLAST tool of NCBI. Then the DPV VP26 gene was cloned into pMD18-T,and was strongly confirmed by PCR amplification, restriction digestion and oligonucleotide probe hybridization. Molecular characterization analysis indicated that the DPV VP26 gene was composed of 354 nucleotides, and encoding a polypeptide of 117 amino acid residues, which had a Herpes_UL35 conserved domain related to small nucleocapsid protein family and highly conserved among the Herpes_UL35 proteins. Moreover, the nucleic acid and amino acid sequence of DPV VP26 had higher homology with its homologous protein of Alphaherpesvirus than others. Phylogenetic tree analysis showed that on taxonomic status the DPV could be grouped in Alphaherpesvirus subfamily, and might be a new genus of Alphaherpesvirus. Subcellular location analysis demonstrated that the VP26 mainly located in nucleus. Besides, condon preference analysis demonstrated that the alternative codons for the same amino acid in VP26 had distinctly different frequency and the DPV VP26 had 18, 19 and 25 codons＇ frequency evidently disagreeing with E. coli, yeast and human, respectively. These results provide a molecular biology evidence for the further study on the function and mechanism of DPV VP26.

关 键 词：鸭瘟病毒 VP26基因 克隆 分子特性 

分 类 号：S852.659.1[农业科学—基础兽医学]