绵羊痘病毒新疆株的分离及结构蛋白P32基因序列分析  

Isolation and Identification of Sheep Pox Virus Isolated from Xinjiang and Analysis of the Sequence of Structural Protein P32

作　　者：魏婕[1] 薛英[1] 苗书魁[1] 窦小龙[2] 任晓冰[2] 马文戈[1] 黄炯[1] 

机构地区：[1]新疆畜牧科学院兽医研究所,乌鲁木齐830000 [2]新疆农业大学,乌鲁木齐830052

出　　处：《新疆农业科学》2015年第4期732-739,共8页Xinjiang Agricultural Sciences

基　　金：国家科技支撑计划"野生动物跨境传播重要动物疫病预防及鉴别诊断研究"(2013BAD12B04);农业部重点科研项目"边境地区动物疫病防控技术体系研究"(201103008)

摘　　要：【目的】对绵羊痘病毒当地流行毒株的分离和其结构蛋白P32基因的克隆和序列进行分析,明确引起新疆本地绵羊流行痘病的病毒种类,掌握当前流行毒株的变异情况,为绵羊痘的预防控制及将来研制高效安全疫苗提供科学依据。【方法】将两例疑似患有羊痘的绵羊肺脏组织病料接种于MDBK细胞进行病毒分离,细胞病变组织经处理后通过电子显微镜观察分离病毒。以分离病毒基因组DNA为模板,通过PCR扩增绵羊痘病毒结构蛋白P32基因,并对目的基因进行克隆和基因序列和蛋白生物信息学分析。【结果】两例病料组织接种MDBK细胞后均产生明显的细胞病变,电子显微镜观察可见到约200 nm大小、椭圆形有囊膜的典型痘病毒粒子。对两株病毒表面膜结构蛋白P32基因PCR扩增均可获得与预期大小一致的约972 bp片段,氨基酸序列分析表明分离毒株SPV/Miquang/2013和SPV/Kuche/2013病毒P32基因与绵羊痘病毒参考株同源性分别达96.9%-99.4%和97.5%-100%,生物信息学分析发现SPV/Miquang/2013株P32蛋白有两个关键氨基酸位点发生变异,即第78位谷氨酸突变为赖氨酸,第293位异亮氨酸突变为苏氨酸。【结论】从疑似病例中分离到两株病毒,通过电镜观察及P32基因扩增测序,与其他羊痘病毒比对后鉴定分离毒株为绵羊痘病毒,分别命名为SPV/Miquan/2013和SPV/Kuche/2013。SPV/Miquan/2013的P32蛋白同其他绵羊痘病毒的两个氨基酸位点发生明显突变。【Objective】Two viruses were isolated from lung of sheep suspected of carrying sheep pox viruses,respectively,and then the related gene homologous evolution was identified and analyzed in order to understand the epidemiological characteristics of sheep pox virus in Xinjiang and lay the foundation of prevention and efficient vaccine for the future. 【Method】Isolating the virus from MDBK cells with CPE by tissue extrac-ting solution inoculation,virus＇ morphology was observed by electron microscopy. The surface membrane protein P32 gene of the viruse was amplified by PCR and then the homologous evolution and the bioinformatics of the protein was analyzed. 【Result】The tissue extracting solution inoculated with the MDBK cells could found the CPE; The virus observed by electron microscopy was found to be a 200 nm and enveloped oval viral particle;. The results showed that fragments about 972 bp were amplified from viral DNAs,which had most highly identity with other sheep poxviruses published in Gen Bank. The homology analysis of P32 protein amino acid showed that the two viruses had 96. 9%-99. 4% and 97. 5%-100% identity with other sheep poxviruses,respectively. It was found that the 78 th and 293 rd amino acid had mutation.【Conclusion】Two viruses were isolated from the suspected tissue,these viruses were identified to be sheep pox virus through the electron microscopy observation and amplification of the P32 gene with other pox viruses in Gen Bank. They were named SPV/Miquan /2013 and SPV/Kuche /2013,respectively. SPV/Miquan /2013 amino acids had significant mutation with other sheep pox viruses.

关 键 词：绵羊痘病毒 分离 鉴定 序列分析 

分 类 号：S852.654[农业科学—基础兽医学]