鹿源BVDV Y2分离株全基因序列分析及BVDV分型荧光定量PCR检测方法的建立

发布时间：2018-05-14 00:08

  本文选题：牛病毒性腹泻病毒 + 全基因测序　； 参考：《吉林农业大学》2017年硕士论文

【摘要】：牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)是牛病毒性腹泻-黏膜病(Bovine viral diarrhea-mucosal disease,BVD/MD)的病原体,可引起牛、鹿、羊等动物发病,表现出广泛的临床体征,包括轻微的上呼吸道征象,循环白细胞的短暂降低和短期低烧,严重呼吸道疾病,胃肠病症,出血综合征和肺炎,繁殖障碍等,给世界各国的畜牧业造成了严重的危害和经济损失。主要研究内容分为以下2部分:1.鹿源BVDV Y2分离株的全基因测序及遗传变异分析以实验室分离的1株鹿源BVDV 1b型Y2分离株为研究对象,进行全基因测序。参照Gen Bank中的BVDV 1b型的全基因组序列的保守区域,设计了8段分段引物。通过Y2病毒增殖,提取病毒RNA,RT-PCR扩增Y2的8个片段,并将各片段分别克隆到p LB simple vector后转化至Top10感受态细胞后进行测序;参考Genbank发表的BVDV 1b型的基因序列对Y2的全基因组进行拼接和分析;对Y2的5’UTR、和全基因、Npro基因、E2基因及编码的氨基酸进行分析和比对,构建遗传进化树。本实验完成了对鹿源BVDV Y2株全基因测序,基因组全长12306bp。其中5’UTR381bp,3’UTR 228bp,CDS区11697bp,能编码3898个氨基酸,Genbank登录号KY964311。Y2囊膜结构蛋白有潜在的N-糖基化位点有11个。根据Y2 5'UTR基因序列构建进化树,Y2株与BVDV-1b型的Osloss、INSP4ncp、GX4株等处于同一个分枝上,为BVDV 1b亚型。通过构建进化树发现:Y2的5’UTR与VEDEVAC、Osloss的亲源距离近,与CP7最远。Y2的全基因及氨基酸与IBSP4ncp、Osloss、GX4和VEDEVAC亲缘距离近,与3156株最远。Y2的Npro基因及氨基酸与Osloss、IBSP4ncp、VEDEVAC、GX4亲缘距离近,Npro基因与JL-1亲缘距离最远,Npro氨基酸AU526、AU526、HP-KY-RK13。Y2的E2基因及氨基酸与Osloss、BSP4ncp、GX4、VEDEVAC近,E2基因与JL-1亲缘距离最远,E2氨基酸与3156株的差异性最大。结果表明Y2株与BSP4ncp、Osloss、VEDEVAC和GX4亲缘关系较近。Y2的全基因测序对BVDV的反向遗传技术的研究、BVDV的持续性感染研究和疾病防控等研究方面有较大的应用前景。2.BVDV分型荧光定量PCR方法的建立实验通过构建BVDV-1、BVDV-2、BVDV-3标准的阳性质粒和标准阳性RNA,以标准阳性RNA为模板建立一步法实时荧光RT-PCR。建立的3个方法在分别在分别检测猪瘟病毒、小反刍兽疫病毒、BVDV-1、BVDV-2、BVDV-3、绵羊边界病毒、牛轮状病毒、伪狂犬病毒8种病毒时均无交叉反应,具有良好的特异性。本实验建立分型方法灵敏性好,检测BVDV-1和BVDV-2的检测限最低拷贝数为102copies RNA,检测BVDV-3荧光方法的最低检测拷贝数为103 copies RNA;建立BVDV分型的一步实时RT-PCR在107~105 copies RNA 3个梯度范围内是组内和组间重复性较好(CV0.02)。并从80个已知样品中检测出了45个BVDV-1阳性感染,5株BVDV-3阳性感染,但未检测出BVDV-2感染的样品。
[Abstract]:Bovine viral diarrhea virus (BVV VV) is the pathogen of bovine viral diarrhea-mucosal disease (BVD / MDV), which can cause disease in cattle, deer, sheep and other animals, showing a wide range of clinical signs, including mild upper respiratory signs. The transient decline of circulating white blood cells and short-term low fever, severe respiratory diseases, gastrointestinal diseases, hemorrhage syndrome and pneumonia, reproductive disorders, etc. have caused serious harm and economic losses to animal husbandry around the world. The main research content is divided into the following two parts: 1. The whole Gene sequencing and genetic variation Analysis of Deer BVDV Y2 strain A BVDV 1b type Y2 isolate isolated from a laboratory was used as the research object, and the whole gene sequencing was carried out. According to the conserved region of the whole genome sequence of BVDV 1b in Gen Bank, 8 segments of primer were designed. Eight fragments of Y2 were amplified by RT-PCR from Y2 virus. Each fragment was cloned into p LB simple vector and transformed into Top10 receptive cells for sequencing. Referring to the gene sequence of BVDV 1b published by Genbank, the whole genome of Y2 was spliced and analyzed, and the E2 gene of Y2 gene was analyzed and compared with that of Npro gene, and the phylogenetic tree was constructed. The whole gene of BVDV Y2 strain was sequenced and the genome was 12306bp. Among them, 5 UTR381bpH3UTR228bpPU CDS region can encode 3898 amino acids KY964311.Y2 envelope structural protein with 11 potential N-glycosylation sites. According to the sequence of Y2 5'UTR gene, the Y2 strain was constructed on the same branch as the BVDV-1b type of Oslossia INSP4ncpGX4 strain, and was a subtype of BVDV 1b. By constructing the phylogenetic tree, we found that the 5'UTR of 1: Y2 was closely related to VEDEVAC Osloss, and the whole gene and amino acid of CP7 farthest. Y2 was closely related to IBSP4ncpOsloss-GX4 and VEDEVAC. The distance between the Npro gene and the amino acid of the farthest. Y2 gene and the amino acid of the Npro gene and the Amino acid of the Oslossn IBSP4ncpAVEDEVACV GX4 is the most distant between the Npro gene and the JL-1 gene. The E2 gene of the Npro amino acid AU526HP-KY-RK13.Y2 and the amino acid of the Npro gene and the amino acid of the OslossBSP4ncpC4ncpGX4VAC and JL-1 have the greatest difference between the amino acid Amino acid of 3156 strains and the Amino acid of the Npro amino acid AU526HP-KY-RK13.Y2. The results showed that Y2 strain had a close relationship with BSP4ncpOsloss.VEDEVAC and GX4. The reverse genetic technique of BVDV was studied by whole gene sequencing of Y2 strain. 2. The fluorescent quantitative PCR of BVDV typing can be used in the study of persistent infection and disease prevention and control of BVDV. Methods by constructing the positive plasmid and standard positive RNAs of BVDV-1, BVDV-2 and BVDV-3, a one-step real-time fluorescence RT-PCR was established using standard positive RNA as template. The three methods had no cross reaction in the detection of swine fever virus, BVDV-1 and BVDV-2 BVDV-3, sheep boundary virus, bovine rotavirus and pseudorabies virus respectively, and had good specificity. The sensitivity of the method was good. The lowest copy number of BVDV-1 and BVDV-2 was 102copies RNAs, the lowest copy number of detecting BVDV-3 fluorescence method was 103 copies RNAs, and the one step real time RT-PCR for BVDV typing was good reproducibility within and between groups within the range of 107 ~ 105 copies RNA gradient. 45 BVDV-1 positive strains were detected from 80 known samples, but no BVDV-2 positive samples were detected.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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本文编号：1885428