本文選題：牛病毒性腹瀉病毒 + 全基因測(cè)序　； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：牛病毒性腹瀉病毒(Bovine viral diarrhea virus,BVDV)是牛病毒性腹瀉-黏膜病(Bovine viral diarrhea-mucosal disease,BVD/MD)的病原體,可引起牛、鹿、羊等動(dòng)物發(fā)病,表現(xiàn)出廣泛的臨床體征,包括輕微的上呼吸道征象,循環(huán)白細(xì)胞的短暫降低和短期低燒,嚴(yán)重呼吸道疾病,胃腸病癥,出血綜合征和肺炎,繁殖障礙等,給世界各國(guó)的畜牧業(yè)造成了嚴(yán)重的危害和經(jīng)濟(jì)損失。主要研究?jī)?nèi)容分為以下2部分:1.鹿源BVDV Y2分離株的全基因測(cè)序及遺傳變異分析以實(shí)驗(yàn)室分離的1株鹿源BVDV 1b型Y2分離株為研究對(duì)象,進(jìn)行全基因測(cè)序。參照Gen Bank中的BVDV 1b型的全基因組序列的保守區(qū)域,設(shè)計(jì)了8段分段引物。通過(guò)Y2病毒增殖,提取病毒RNA,RT-PCR擴(kuò)增Y2的8個(gè)片段,并將各片段分別克隆到p LB simple vector后轉(zhuǎn)化至Top10感受態(tài)細(xì)胞后進(jìn)行測(cè)序;參考Genbank發(fā)表的BVDV 1b型的基因序列對(duì)Y2的全基因組進(jìn)行拼接和分析;對(duì)Y2的5’UTR、和全基因、Npro基因、E2基因及編碼的氨基酸進(jìn)行分析和比對(duì),構(gòu)建遺傳進(jìn)化樹(shù)。本實(shí)驗(yàn)完成了對(duì)鹿源BVDV Y2株全基因測(cè)序,基因組全長(zhǎng)12306bp。其中5’UTR381bp,3’UTR 228bp,CDS區(qū)11697bp,能編碼3898個(gè)氨基酸,Genbank登錄號(hào)KY964311。Y2囊膜結(jié)構(gòu)蛋白有潛在的N-糖基化位點(diǎn)有11個(gè)。根據(jù)Y2 5'UTR基因序列構(gòu)建進(jìn)化樹(shù),Y2株與BVDV-1b型的Osloss、INSP4ncp、GX4株等處于同一個(gè)分枝上,為BVDV 1b亞型。通過(guò)構(gòu)建進(jìn)化樹(shù)發(fā)現(xiàn):Y2的5’UTR與VEDEVAC、Osloss的親源距離近,與CP7最遠(yuǎn)。Y2的全基因及氨基酸與IBSP4ncp、Osloss、GX4和VEDEVAC親緣距離近,與3156株最遠(yuǎn)。Y2的Npro基因及氨基酸與Osloss、IBSP4ncp、VEDEVAC、GX4親緣距離近,Npro基因與JL-1親緣距離最遠(yuǎn),Npro氨基酸AU526、AU526、HP-KY-RK13。Y2的E2基因及氨基酸與Osloss、BSP4ncp、GX4、VEDEVAC近,E2基因與JL-1親緣距離最遠(yuǎn),E2氨基酸與3156株的差異性最大。結(jié)果表明Y2株與BSP4ncp、Osloss、VEDEVAC和GX4親緣關(guān)系較近。Y2的全基因測(cè)序?qū)VDV的反向遺傳技術(shù)的研究、BVDV的持續(xù)性感染研究和疾病防控等研究方面有較大的應(yīng)用前景。2.BVDV分型熒光定量PCR方法的建立實(shí)驗(yàn)通過(guò)構(gòu)建BVDV-1、BVDV-2、BVDV-3標(biāo)準(zhǔn)的陽(yáng)性質(zhì)粒和標(biāo)準(zhǔn)陽(yáng)性RNA,以標(biāo)準(zhǔn)陽(yáng)性RNA為模板建立一步法實(shí)時(shí)熒光RT-PCR。建立的3個(gè)方法在分別在分別檢測(cè)豬瘟病毒、小反芻獸疫病毒、BVDV-1、BVDV-2、BVDV-3、綿羊邊界病毒、牛輪狀病毒、偽狂犬病毒8種病毒時(shí)均無(wú)交叉反應(yīng),具有良好的特異性。本實(shí)驗(yàn)建立分型方法靈敏性好,檢測(cè)BVDV-1和BVDV-2的檢測(cè)限最低拷貝數(shù)為102copies RNA,檢測(cè)BVDV-3熒光方法的最低檢測(cè)拷貝數(shù)為103 copies RNA;建立BVDV分型的一步實(shí)時(shí)RT-PCR在107~105 copies RNA 3個(gè)梯度范圍內(nèi)是組內(nèi)和組間重復(fù)性較好(CV0.02)。并從80個(gè)已知樣品中檢測(cè)出了45個(gè)BVDV-1陽(yáng)性感染,5株BVDV-3陽(yáng)性感染,但未檢測(cè)出BVDV-2感染的樣品。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65
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本文編號(hào)：1885428