不同地区羊口疮病毒三个主要结构蛋白基因的遗传进化分析

发布时间：2018-03-13 01:16

本文选题：Orfv　切入点：B2L基因　出处：《内蒙古农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：本研究从内蒙古赤峰、武川和山东青岛地区疑似羊口疮的发病羊群采集病料,通过剪碎、研磨、冻融三次离心取上清液加双抗进行无菌处理后在犊牛睾丸细胞、MDBK细胞上接种传代,同时根据GenBank中发表的的羊口疮病毒B2L蛋白基因设计1对引物,对细胞培养物、病料中疑似羊口疮病毒B2L基因的PCR扩增,病料处理差速离心后经负染透射电子显微镜观察。在此基础上根据GeneBank中羊口疮病毒的B2L、F1L、VIR基因设计3对引物,对三个地方的羊口疮病毒进行DNA提取、PCR扩增、连接到pMD19-T载体、转化到DH5a感受态细胞、通过蓝白斑筛选阳性质粒和重组质粒的鉴定后测序。用DNAstar软件进行三个不同地区分离株之间核苷酸同源性比较分析,再将测序的结果利用NCBI上的BLAST的方法与GenBank中公布的Orfv的B2L、F1L、VIR基因核苷酸序列进行同源性比较分析。最后应用DNA Star程序中的Meg Align软件包进行分析,通过Clustal-V Method构建系统进化树。结果表明：从三个不同地区采集的病料在犊牛睾丸细胞和MDBK细胞上接种传代时第一、二代出现明显的细胞病变。从第三代开始细胞病变不明显,但细胞培养物仍能用PCR扩增出明显的条带；透射电镜观察到椭圆形、8字形的毛线团样典型的羊口疮病毒粒子；这些结果表明本次成功分离到Orfv,分别命名为Orfv-W、rfv-C和Orfv-Q。三个分离株之间B2L、F1L和VIR基因核苷酸同源性分别为94.8%-97.4%、93.2%～98%和95.1%-100%。三个分离株与国内外不同来源的Orfv病毒株B2L、F1L和VIR基因核苷酸同源性分别为82.1%～98.0%、93.4%～98.0%和93.8%～99.1%。对B2L、F1L和VIR基因进行系统进化树分析后显示,Orfv-W毒株与山羊分离株亲缘关系近；Orfv-Q毒株与绵羊分离株亲缘关系近；Orfv-C毒株既与绵羊分离株亲缘关系近,也与山羊分离株亲缘关系近,且三个基因分支均与国内西北部地区分离株的亲缘关系接近。
[Abstract]:In this study, sheep diseases were collected from suspected sheep and mouth ulcers in Chifeng, Wuchuan and Qingdao, Shandong Province, and were shredded and ground. The supernatant of frozen thawed three times centrifugation and double antibody were inoculated on the MDBK cells of calf testicular cells after sterile treatment. At the same time, a pair of primers were designed according to the B2L protein gene of sheep aphthoea virus published in GenBank. The B2L gene of suspected sheep mouth sore virus was amplified by PCR, and observed by negative staining transmission electron microscope after differential centrifugation. Based on this, three pairs of primers were designed according to the B _ 2LN _ (F1L) VIR gene of sheep mouth sore virus in GeneBank. Sheep aphtha virus was amplified by DNA and ligated to pMD19-T vector and transformed into DH5a receptive cells. The positive plasmids and recombinant plasmids were screened by blue and white spot and sequenced. The nucleotide homology was analyzed by DNAstar software. The results of sequencing were compared and analyzed by using the method of BLAST on NCBI and the nucleotide sequence of Orfv gene B2LF1L1 / VIR published in GenBank. Finally, the Meg Align software package of DNA Star program was used to analyze the nucleotide sequence of VIR gene. Phylogenetic tree was constructed by Clustal-V Method. The results showed that obvious cytopathic changes appeared in the first and second passages of calf testicular cells and MDBK cells inoculated from three different regions, but the cytopathic changes were not obvious from the third generation. But the cell culture could still amplify the obvious bands by PCR, and the typical sheep aphtha virus particles were observed by transmission electron microscope. These results indicate that Orfv-C and Orfv-Q.The nucleotide homology of B2LF1L and VIR genes between the three isolates were 94.8-97.4% and 95.1-100%, respectively. The nucleotide homology of the three isolates was 94.8% -97.4% and 95.1-100%, respectively. The nucleotide homology of the three isolates was 94.8% -97.4% and 95.1-100% respectively with the Orfv virus strains B2LLF1L and VIR genes from different sources at home and abroad. The homology of glycoside was 98.0% and 99.1.The phylogenetic tree analysis of B2LF1L and VIR genes showed that Orfv-W strain was closely related to goat isolate and Orfv-Q strain was closely related to sheep isolate, and Orfv-C strain was closely related to sheep strain. It was also closely related to goat isolates, and the three gene branches were close to those of the isolates in northwestern China.
【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】：2015
【分类号】：S852.654

【参考文献】