【作者】 吴润；

【导师】 陈怀涛； 谢庆阁；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2004， 博士

【摘要】 朊粒病(prion disease)是一类遗传性、传染性或散发性的致死性神经变性疾病,其中牛海绵样脑病(BSE)、绵羊痒病(scrapie)和人克-雅氏病(CJD)是最为关注的朊粒病,其始终涉及一个宿主常染色体PrP基因编码的细胞型PrP(PrPC)经转译后修饰而被转变成致病型PrP(PrPSc)的过程。虽然对朊粒病的传染性、遗传性有所认识,但至今对正常朊蛋白的生理功能、朊粒病传染的种间屏障、朊粒增殖、朊粒构象转变等机制还不完全清楚,这严重制约着对朊粒病发病机理和朊粒致病机制的进一步认识。本研究的主要目的是为偶蹄动物朊蛋白基因变异性和多态性的遗传规律、氨基酸变异与其蛋白构象变化的关系、种间屏障的遗传学基础等研究提供实验材料和理论依据并建立实验技术平台,进行了下述研究和结果讨论。分别从9头牛(牦牛,荷斯坦牛和秦川黄牛各3头)、6只羊(藏绵羊和山羊各3只)、4峰双峰驼和1只马麝的全血中提取基因组总DNA,用所设计特异引物以聚合酶链式反应扩增出朊蛋白基因,并克隆到pMD 18-T载体。序列分析表明所克隆的PrP基因片段大小分别为:牛基因795 bp,绵羊、山羊和马麝基因771 bp,双峰驼基因768 bp、792 bp和795 bp,均为包含在单一外显子内的完整开放阅读框,与国外报道的同种、同属或同科动物PrP序列基本相同。这些PrP基因含有6个或5个短而富含G-C的重复元件,可编码八肽Pro-His-Gly-Gly-Gly-Trp-Gly-Gln或九肽Pro-Gln/His-Gly-Gly-Gly-Gly- Trp-Gly-Gln,牛基因含6个重复元件(4个八肽重复和2个九肽重复),绵羊、山羊和马麝基因含5个重复元件(3个八肽重复和2个九肽重复),双峰驼的2个基因克隆含6个重复元件(4个八肽重复和2个九肽重复)而另2个基因克隆含5个重复元件(4个八肽重复和1个九肽重复);其氨基酸序列含有24个氨基酸的N-端信号肽和23个氨基酸的C-端信号肽(除3个双峰驼基因含22个氨基酸的C-端信号肽外)。牦牛、双峰驼和马麝PrP基因序列在国内外未见报道。就以上动物PrP基因的核苷酸和其推导氨基酸序列同源性和变异比较分析可见,牛PrP基因间核苷酸和氨基酸序列的同源性分别在99.0 %～100.0 %和99.2 %～100.0 %之间,共发现18个碱基替换,其中5个替换引起氨基酸突变。发现牦牛朊粒基因在126(A→G)、234(G→A)和678(T→C)位的特征性核苷酸替换,但均为同义码替换;绵羊和山羊PrP基因间核苷酸和其推导氨基酸序列的同源性分别在99.0～100.0 %和98.1～100.0 %之间,共发现17个碱基替换,其中11个为异义突变。6个绵羊和山羊PrP基因均为密码子136、154和171的PrPARQ等位基因;双峰驼PrP基因间核苷酸和其推导氨基酸序列同源性在91.0 %～100.0 %和94.2 %～100.0 %之间,共存在133个碱基替换,其中26个为异义突变;马麝PrPC与白尾鹿(white-tailed deer)和麋鹿(red deer)的PrP基因相比,其核苷酸和推导氨基酸序列同源性分别为97.4%、97.9%和98.1%、97.7%,共发生15个碱基替换,其中5个为异义突变。分别利用DNAstar和Clustalx程序及treev32软件进行PrP基因序列的同源性分析、多重排比和构建进化树,并对其变异与结构、功能和种间屏障的关系以及系统发生关系作了比较和描述。22种偶蹄动物的PrP基因大小有所差异,为768～795 bp,可编码255～264个氨基酸的朊蛋白。核苷酸和氨基酸序列的同源性,偶蹄动物间≥88.6%和≥93.3%,反刍动物间≥95.4%和≥96.5%。虽然骆驼属内的核苷更多还原

【Abstract】 Prion diseases consists of a group of genetic, infectious, or sporadic fatal neurodegenerative diseases, of which Bovine spongiform encephalopathy (BSE), sheep scrapie, and human Creutzfeldt-Jakob disease (CJD) are the most notable ones, all of which invariably involve a post-translational modification process of the cellular PrP (PrPC) encoded by the host euchromosome PrP gene during the period it converted into the pathogenic PrP (PrPSc). Although there are some insights in the infectious and inheritable character of the prion diseases, it has been unclear what is really the physiological function of normal cellular PrP, species barriers for transmission of the diseases and the mechanism of prion propagation and its conformational change yet, and these questions highly restricted to further interpret the pathogenesis of prion diseases and the pathogenic mechanism of prion. The main aims of the current study was to provide the experimental materials and the theoretical evidences to further study the genetical rule of variability and polymorphism, the relationship between amino acid variation and its protein conformational change of artiodactyl PrP gene, as well as the genetic basis of the species barrier, and to set up the experimental technology flats. The following investigations were carried and the results are discussed.The total DNAs were extracted from peripheral blood of nine cattle (three yaks, three Holstein/Friesian and three Qinchuan cattle), three Tibetan sheep and three goats, four bactrian camels and one musk deer respectively. The PrP genes were amplified by polymerase chain reaction using two pairs of special primers, then cloned into pMD 18-T Vector. The sequencing analysis showed that the sizes of genes were 795 bp in the cattle, 771 bp in the sheep, goat and musk deer, 768 bp, 792 bp and 795 bp in the bactrian camels respectively. The coding sequences of the genes had the complete ORFs contained within a single exon and were basically same as those of the published PrP genes of the same animal species, genus or family. The cloned PrP genes in this experiment contained five or six copies of a short G-C-rich element, which encoded the octapeptide Pro-His-Gly-Gly-Gly-Trp-Gly-Gln or nonapeptide Pro-Gln/His-Gly-Gly-Gly-Gly-Trp-Gly-Gln. There were six repeating elements in the cattle genes (four octorepeats and two nonarepeats), five repeating elements in the sheep, goat and musk deer (three octorepeats and two nonarepeats), meanwhile, of the four camels studied, two of them had six repeating elements (four octorepeats and two nonarepeats) and the other two had five repeating elements (four octorepeats and one nonarepeat). All the amino acid sequence of these genes included a N-terminal signal peptide with 24 amino acids and a C-terminal signal peptide with 23 amino acids (except three camel genes which had a C-terminal signal peptide with 22 amino acids). The PrP gene sequences of yak, bactrian camel and musk deer had not been reported previously.Comparison analysis of these PrP genes revealed the identities and variations of the nucleotide sequence and its putative amino acid sequence. It was indicated that the homology within cattle ranged from 99.0 to更多还原