猪伪狂犬病病毒山东分离株gE和gD基因的克隆及序列分析

发布时间：2018-02-01 23:13

本文关键词： 猪伪狂犬病毒 gE基因 gD基因克隆序列分析　出处：《中国兽医学报》2017年11期 　论文类型：期刊论文

【摘要】：近年来,我国猪场内很多规模化猪场的伪狂犬病病毒野毒呈阳性,大部分中小规模猪场和小养殖场感染情况更为糟糕,使得伪狂犬病病毒的变异率增加,给该病的防控带来了更大的压力。为了深入了解本病的流行情况及研制新型疫苗用于该病的防治,本研究在流行病学调查的基础上从发生疑似伪狂犬病的2个猪场仔猪组织内各分离到1株PRV野毒株,分别命名为LY株和YSH株。参照已发表的PRV gE和gD基因序列,设计合成2对分别扩其全长的引物,使用高保真酶进行PCR扩增,将扩增得到的全长序列克隆到pEASY-Blunt载体中,并转化Trans1-T1感受态细胞,挑取阳性菌落的质粒进行酶切、测序鉴定,并与国内外其他毒株进行比较分析,构建遗传进化关系图。结果表明,2毒株与国内外其他毒株比较,gE、gD基因核苷酸序列的同源性分别为97.7%~100.0%和99.0%~100.0%;氨基酸序列的同源性分别为95.5%~100.0%和98.9%~100.0%,以gE、gD基因氨基酸序列建立的进化树分析结果显示,近3~5年分离的PRVs形成一个相对独立的新分支。经过同源性及进化分析,可知新分离的2株伪狂犬病病毒株与欧美株的亲缘关系远,与亚洲株内中国株的亲缘关系近,初步判定属于国内流行株,并可能存在一定的抗原变异,这为进一步研究山东省猪群伪狂犬病病毒分子流行病学奠定了基础。
[Abstract]:In recent years, China's wild strains of pseudorabies virus of swine in many large-scale pig farms were positive, the majority of small and medium-sized and small scale pig farms infection situation is worse, the variation of pseudorabies virus increased, has brought greater pressure to the prevention and control of the disease. In order to deeply understand the epidemic situation of the disease and the development of new vaccines for the prevention of the disease, 2 pig tissue in this study on the basis of the epidemiological survey from the suspected case of pseudorabies in piglets in the isolated 1 PRV field strains, which were named LY and YSH strains. PRV gE and gD gene sequence has been published in the design and synthesis of 2 respectively. Expanding the full-length primers for PCR amplification using high fidelity enzyme, the PCR sequences were cloned into pEASY-Blunt vector, and transformed into Trans1-T1 competent cells, the plasmid selected positive clones were identified by enzyme digestion, sequencing and identification. A comparative analysis of other strains at home and abroad, to construct the genetic evolutionary relationship diagram. The results showed that 2 strains and other strains at home and abroad, gE, gD gene nucleotide sequence homology were 97.7%~100.0% and 99.0%~100.0%; the homology of amino acid sequences were 95.5%~100.0% and 98.9%~100.0%, to gE, the result of phylogenetic analysis of gD gene amino acid sequence set that separation in the last 3~5 years PRVs formed a new branch independently. Through phylogenetic analysis, genetic relationship of the 2 isolated strains of pseudorabies virus strains and strains and far, and the genetic relationship among Chinese strains Asia strains near, tentatively identified domestic strains, and there may be antigenic variation of certain, which laid the foundation for further study on molecular epidemiology of pseudorabies virus in pigs in Shandong province.

【作者单位】：山东省农业科学院畜牧兽医研究所山东省畜禽疫病防治与繁育重点实验室;
【基金】：山东省现代农业产业技术体系疫病控制岗位项目(SDAIT-08-07) 山东省农业重大应用技术创新课题山东省科技发展计划(2014GNC111011) 山东省农业科学院青年英才培养工程;山东省农业科学院青年科研基金项目(2014QNM40);山东省农业科学院青年科研基金项目(2016YQN53)
【分类号】：S852.651
【正文快照】： *Corresponding author,E-mail:junli79@163.com猪伪狂犬病(pseudorabies,PR)也称奥叶基氏病(Aujeszky disease)、传染性延髓麻痹、奇痒症,是由猪伪狂犬病病毒(pseudorabies virus,PRV)引起的以发热、奇痒、脑脊髓炎等为主要特征的一种烈性传染病。PRV属于疱疹病毒科(herpesvi

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