本文關鍵詞： 傳染性海綿狀腦病 朊蛋白 綿羊 山羊 系譜樹 多態(tài)性分析　出處：《中國農業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：傳染性海綿狀腦病(Transmissible Spongiform Encephalopathie, TSEs)是一組可引起神經退行性變性的致命疾病,包括鹿和麇鹿的慢性消耗性疾病(Chronic Wasting Disease, CWD),人的克雅氏病(Creutzfeldt-Jakob disease, CJD),水貂中的傳染性水貂腦病(TME),牛的瘋牛病和綿羊及山羊的羊癢病等。異常形式的PrPC的蓄積是引起TSE疾病的主要原因。這種異常形式的PrP被稱為PrPSc,其具有部分蛋白酶抗性和傳染性。因此,正常朊蛋白一級結構的改變對于構象異常朊蛋白的產生會有影響。為了闡明巴基斯坦綿羊和山羊不同品種朊蛋白一級結構的多態(tài)性,本研究對12個品種的綿羊和11個品種的山羊朊蛋白結構進行測序分析,從而為綿羊和山羊的抗病育種提供理論基礎,也為癢病的發(fā)生提供詳盡的基礎數(shù)據。首先,本研究從巴基斯坦所有省份選取的涉及十二個品種129只綿羊,進行朊病毒蛋白的基因序列分析,并比較其與他哺乳動物物種的基因多態(tài)性。首先,從羊頸靜脈無菌采取正常血液樣本。采用標準流程從血液樣本中提取基因組DNA,然后,對基因組DNA進行了771 bp prion基因片段的PCR擴增。對PCR產物進行測序。通過軟件Codon Code aligner來進行序列分析,并檢測識別新的單核苷酸多態(tài)性。通過軟件Codon Code aligner和MEGAv.6來對PrP基因序列進行編輯和匹配,進而構建系譜樹,并探查巴基斯坦山羊種群與其他哺乳動物群的系統(tǒng)演化。使用“R’統(tǒng)計軟件包用于繪制多維標度圖,測算遺傳距離并進行主成分分析。單核苷酸多態(tài)性分析結果表明在379,380,404,407,414,428,437,455,461,511,512,513,566,572,691,711和718位點發(fā)生突變。這些突變表現(xiàn)為轉移突變和顛換突變。序列分析表明,綿羊PrP基因出現(xiàn)7個氨基酸多態(tài)性位點。這些氨基酸多態(tài)性將這些品種綿羊的基因型劃分為13個等位基因和15個基因型。羊癢病最耐受基因突變(Q171R)出現(xiàn)的頻度為0.107。羊癢病最敏感基因突變(A136 V)在所檢測羊樣品里沒有出現(xiàn)。在接下來的研究中,從巴基斯坦所有省份的11種山羊品種中選取了94只山羊,對其進行朊蛋白基因的檢測。通過對PrP基因中771 bp編碼區(qū)域的PCR擴增,觀察了氨基酸亞基的六個多肽位點(一個新的)：126(A-G)、304(G-T)、379 (A-G)、414(C-T)、428 (A-G)和718(C-T)。位點C.428與其它10個品種相比較,具有高度的突變性。比特拉羊(Beetal)與其他10個品種相比,在此位點具有更多的變異性�；谶@些PrP變體,NJ系譜樹顯示鹿與所有山羊種群,連同家綿羊和摩弗倫羊共處一個分支并共享其最近共同祖先(most recent common ancestors, MRCA)。蛋白分析表明這些多態(tài)性可以導致不同的一級、二級和三級蛋白結構。多維標度圖清晰地表明山羊種群與其他哺乳動物相比較,有較遠的生物學相近性并且明顯已經分離為特殊的種群。通過主分量分析,綿羊和山羊中朊蛋白基因的這些新發(fā)現(xiàn),將為對羊癢病的抗性和易感性、飼養(yǎng)管理、跨物種傳播、發(fā)病機制的進一步研究提供幫助。綜上所述,本研究對巴基斯坦所有省份的綿羊和山羊進行了調研,涉及12個品種的綿羊和11個品種的山羊,采集了大量的樣本,基本反映了巴基斯坦地區(qū)山羊及綿羊的基因分布情況,為相關部門防控動物疫病提供重要的依據,尤其是對防控朊病毒在這些動物中的傳播具有重要的參考價值和指導意義。這些在山羊和綿羊中朊蛋白基因多態(tài)性的發(fā)現(xiàn),為巴基斯坦加強家山羊和家綿羊對癢病的防控提供分子理論依據。
[Abstract]:Transmissible spongiform encephalopathy (Transmissible Spongiform, Encephalopathie, TSEs) is a group of diseases can cause fatal neurodegenerative diseases, including chronic consumption of deer and deer (Chronic Wasting Disease, the CWD), human Creutzfeldt Jakob disease (Creutzfeldt-Jakob disease, CJD), mink in transmissible mink encephalopathy (TME), bovine the BSE and scrapie in sheep and goats. The accumulation of abnormal forms of PrPC is the main cause of TSE disease. The abnormal form of PrP called PrPSc, which is part of the protease resistant and infectious. Therefore, the primary structure of normal prion protein to produce abnormal prion protein conformational change. There will be affected. In order to elucidate the polymorphism of Pakistan sheep and goat prion protein of different varieties of the primary structure of goat prion protein in this study, 12 sheep and 11 varieties of structure sequence analysis, In order to provide a theoretical basis for the breeding of sheep and goat, also provide the basic data for the detailed occurrence of scrapie. Firstly, this study selected from Pakistan province to all twelve varieties of 129 sheep prion protein gene sequence analysis, and compare it with his mammalian species polymorphism firstly. And take the normal blood samples from sheep jugular vein. The standard aseptic process of extracting genomic DNA from blood samples and genomic DNA of the 771 BP prion gene fragment was amplified by PCR. The PCR products were sequenced. Sequence analysis was performed by software Codon Code aligner, and the detection and identification of new single nucleotide polymorphism through. The software Codon Code aligner and MEGAv.6 of PrP gene sequences were edited and matched, and construct phylogenetic tree, and exploration of Pakistan goat population and other mammalian animal group The use of "R" system evolution. The statistical package used to draw a multidimensional scaling map, principal component analysis and calculation of genetic distance. Single nucleotide polymorphism analysis showed that occurred in 379380404407414428437455461511512513566572691711 and 718 mutations. These mutations showed metastasis mutation and transversion mutation. Sequence analysis showed that 7 amino acids of sheep PrP gene polymorphism site. These amino acid polymorphism divides the genotypes of these sheep breeds was 13 alleles and 15 genotypes. The scrapie resistance gene (Q171R) mutation frequency of 0.107. for scrapie sensitive gene mutation (A136 V) in the detection of samples in sheep did not appear. The following research, from 11 goat breeds in Pakistan province were selected in all 94 goats, detection of prion protein gene by PrP based on it. Because of amplification of 771 BP encoding region of PCR, a six amino acid polypeptide sites to observe subunit (a new):126 (A-G), 304 (G-T), 379 (A-G), 414 (C-T), 428 (A-G) and 718 (C-T). The sites of C.428 compared with other 10 varieties phase has mutagenicity. High bit pull the sheep (Beetal) compared with the other 10 varieties, with more variability at this locus. The PrP variant based on NJ family tree of deer and goat populations show all, together with domestic sheep and mouflon coexistence of a branch and share the most recent common ancestor (most recent common ancestors, MRCA). Protein analysis showed that these polymorphisms can lead to different level, two level and three level of protein structure. Multidimensional scaling map clearly and other mammals Mingshan sheep population compared with biological far similar and has clearly separated into special populations. Through principal component analysis, sheep And these new findings of prion protein gene in goats, for feeding and management of scrapie resistance and susceptibility, and cross species transmission, to provide help for further research in pathogenesis. In summary, the study of sheep and goats in Pakistan province all conducted an investigation, involving 12 species and 11 varieties of sheep the goat, collecting a large number of samples, basically reflects the distribution of goats and sheep in Pakistan gene, provide an important basis for the relevant departments to prevention and control of animal diseases, especially has important reference value and guiding significance for the prevention and control of the spread of prions in these animal. These in goats and sheep in the prion protein gene polymorphism the discovery of the goat and sheep home, provide molecular basis on the prevention and control of scrapie for Pakistan to strengthen.

【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S826;S827

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