羊口疮病毒生物学特性研究

发布时间：2018-04-30 09:59

本文选题：羊口疮病毒 + ORFV-Q分离株　；参考：《内蒙古农业大学》2016年硕士论文

【摘要】：本研究从山东省青岛市某羊场采集疑似羊口疮病料痂皮,经剪碎、研磨、离心等处理后在羊睾丸细胞上进行接种培养传代,分离到一株病毒,通过对其形态特征观察、理化特性研究、动物回归实验、病毒滴度测定及分子生物学鉴定,证实分离到羊口疮病毒。根据GenBank中已公布的Orfv的全基因组序列和B2L、F1L、VIR的基因序列,设计3对特异性引物,应用PCR扩增、胶回收、重组质粒转化、阳性菌筛选、质粒测序等方法对分离株的B2L、F1L和VIR基因进行扩增、克隆和测序,并将其基因序列与其他已公布的不同来源的Orfv分离株进行比较分析。结果表明,本研究利用羊睾丸细胞成功分离出一株羊口疮病毒,并命名为ORFV-Q。本分离株能够引起羊睾丸细胞产生明显细胞病变,通过传代至11代,细胞开始出现病变和出现80%CPE的时间分别稳定在接种后24h和72h左右。病毒粒子为椭圆形,外周有囊膜包被,在细胞质内增殖,病毒对温度、氯仿、乙醚等均敏感。接种毒株的羊只出现丘疹、脓疱等典型的羊口疮症状,毒株TCID50为10-5.5。相关基因序列分析表明, ORFV-Q分离株的B2L基因、F1L基因和VIR基因与GenBank已公布毒株的该序列之间核苷酸同源性分别为96.8%-98.9%、96.2%～99.9%、94.9%～99.8%,且该分离株与中国陕西株和湖北株同源关系最近。
[Abstract]:In this study, a virus was isolated from a sheep farm in Qingdao City, Shandong Province, and a virus was isolated from the sheep testicular cells after shearing, grinding, centrifugation and other treatments. Studies on physical and chemical properties, animal regression test, virus titer determination and molecular biological identification confirmed that sheep mouth sore virus was isolated. According to the whole genome sequence of Orfv and the sequence of VIR gene of B2LX F1LN, three pairs of specific primers were designed, which were amplified by PCR, recovered by gel, transformed into recombinant plasmid, screened by positive bacteria. Plasmid sequencing methods were used to amplify, clone and sequence the B2LF1L and VIR genes, and to compare and analyze the sequence of B2LF1L and VIR genes with other published Orfv isolates from different sources. The results showed that a sheep mouth sore virus was successfully isolated from sheep testicular cells and named ORFV-Q. The isolated strain could induce obvious cytopathic changes in sheep testicular cells. After passage to the 11th passage, the time between the onset of pathological changes and the occurrence of 80%CPE was stable at 24 h and 72 h after inoculation, respectively. The virus particles were oval and enveloped with envelope, which proliferated in the cytoplasm. The virus was sensitive to temperature, chloroform, ether and so on. The sheep inoculated with the virus strain only showed papules, pustules and other typical symptoms of aphthous sores, the TCID50 of the virus strain was 10-5.5. The sequence analysis of related genes showed that the nucleotide homology between B2L gene F1L gene and VIR gene of ORFV-Q isolate and that of GenBank strain was 96.8- 98.9, respectively. The nucleotide homology of the isolated strain was 99.8% with Shaanxi strain and Hubei strain respectively.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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