【作者】 张建辉；

【导师】 赵兴绪； 吴润； 柳纪省；

【作者基本信息】 甘肃农业大学 ， 临床兽医， 2006， 硕士

【摘要】 绵羊朊蛋白等位基因多态性与绵羊痒病具有相关性,了解中国绵羊朊蛋白等位基因的情况有助于评估我国绵羊罹患绵羊痒病的风险性和建立无绵羊痒病风险的羊群。本试验拟建立单链构象多态性技术和非变性聚丙烯酰胺凝胶技术的联合使用的方法来检测绵羊朊蛋白等位基因的多态性。实验从10只健康绵羊全血中获取总DNA,用所设计的引物以聚合酶链式反应(PCR)扩增出朊蛋白等位基因的片断。用单链构象多态性技术(SSCP)对基因片段进行分析以确定各只羊的朊蛋白等位基因的变异性。选取同种变异的羊组中的一只进行第二次的非变性聚丙烯酰胺凝胶电泳,分出杂合子的姊妹链分别进行纯化回收,并验证所回收的产物是清晰的两条。纯合子经琼脂糖凝胶纯化回收。产物分别克隆到PMD18-T载体中,提取质粒经PCR和EcolⅠ、HindⅢ双酶切后确定为克隆成功。菌液经测序后由DNAstar软件对序列进行比对分析。此外还对5只羊的朊蛋白等位基因全长进行了分析研究研究表明,在被研究的10只羊中,8只为纯合子,另外2只为杂合子。在对此有限的样本群进行SSCP电泳时发现纯合子羊为一种基因型,2只杂合子羊拥有另外一种基因型。纯合子羊的基因型为136AA154RR171QQ,杂合子羊的基因型是136AA154RR171HR。对朊蛋白等位基因全长的分析中发现,5只羊均为朊蛋白等位基因纯合子,存在与痒病发生明显关联的基因型VRQ(173),也存在与抗痒病发生相关的基因型ARR(7401、201)和低相关性的ARQ(175、133)。在被发现的7个氨基酸多态位点中,有6个是在先前的研究中报道过的(23L→V、25K→E、136 A→V、154H→R、171Q→R、189 Q→C),一个是没有报道过的(24 C→W)。被研究的羊朊蛋白等位基因与痒病相关基因占73%,预示在本试验的采样区,其绵羊可能存在有绵羊痒病潜在的高风险性。更多还原

【Abstract】 The polymorphism of PrP allele is associated with the scrapie and the study on the condition of the PrP alleles in Chinese sheep by the new method is necessary. The current research is to investigate the PrP alleles in sheep by using SSCP and PAGE methods and the results could be used in establishing a sheep herd without the scrapie risk.Total DNA was extracted from peripheral blood samples of 10 sheep. The PrP allele were amplified by polymerase chain reaction (PCR) with a pair primers. The product of PCR was analyzed by single-strand conformation polymorphism (SSCP) and PAGE .The difference of the product was determined. The silver-staining procedure using polyarylamide gel was performed after eletrophoresie.The product of heterozygote and homozygote were purified cloned into PMD18-T Vector, respectly. The clone were selected and amplified by polymerase chain reaction . After digestion with EcolⅠand HindⅢ, the entire clone PrP allele fragment was constructed. The nucleotides were analyzed and amino acid sequences of their homology by DNAstar. Similar methods were used to analyse the PrP allele from 5 sheep.The results indicated that SSCP and PAGE could be used to seek polymorphism PrP allele and PrP is encoded by a single-copy autosomal gene. In this study, it was found that 2 sheep genotype are heterozygote with the phenotypy of 136AA 154RR171HR and other 8 sheep are homozygote with the phenotypy of 136AA 154RR171QQ. It was found that all of 5 sheep are homozygote and 7 polymorphisms in PRNP gene,including 6 previously reported(23L→V、25K→E、136 A→V、154H→R、171Q→R、189 Q→C)and 1 was not reported(24 C→W). VRQ(173)and ARQ(175、133) are associated with susceptibility to scrapie; ARR(7401、201) are associated with resistance to scrapie in several genotype.It could be concluded from the above results that the samples containing the PrP allelic ARQ and VRQ at codons 136,154 and 171 are associated with susceptibility to scrapie 73% in the total. The risk of the occurrence of scrapie in the experimental areas of Chinease sheep was obtained from information of the result.更多还原