羊朊蛋白基因的克隆和序列分析

分别从6只羊（3只藏绵羊和3只山羊）全血中提取基因组总DNA，用所设计引物以聚合酶链式反应扩增出细胞型朊蛋白（PrP^c）基因，并克隆到pMD 18-T载体。序列分析表明所克隆的羊PrP基因片段大小为771bp，包含了羊朊蛋白基因的完整编码区序列，为包含在单一外显子内的完整开放阅读框，其与国外报道的已知序列基本相同。所克隆的羊PrP基因含5个短而富含G-C的元件，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。这些PrP基因序列相比较，其核苷酸序列和推导氨基酸序列同源性分别在99．0％～100．0％和98．1％～100．0％之间。共发现17个碱基替换，其中6个为同义码替换、11个为异义突变。异义突变中，除SY200301～SY200303的S240P和MY200301的M112T外，其余均位于PrP氨基酸125～228的C-端球形结构区，分别为MY200301的G129S突变、MY200302的T191R突变、MY200303的G127S突变及SY200302和SY200303的H143R和R211G。6个羊PrP基因均为密码子136、154和171的PrPARQ等位基因。

Cloning and Sequence Analysis of Prion Protein (PrPC) Genes from Sheep and Goat

The total DNAs were extracted from peripheral whole-blood of three Tibetan sheep and three goats respectively. The PrP~c genes were amplified by PCR using two pairs of primers, and then were cloned into pMD 18-T Vector. The sequencing showed that the genes were 771 bp in length. All the entire PrP coding sequences had the complete ORFs contained within a single exon and were basically conserved with the published gene sequences. The sequence of the sheep and goat PrP genes cloned in this experiment contained five copies of a short, G-C-rich element, which encoded the octapeptide Pro-His-Gly-Gly- Gly-Trp-Gly-Gln or the nonapeptide Pro-Gln/His-Gly-Gly-Gly-Gly-Trp-Gly-Gln. The comparison of these genes each other revealed that the nucleotide and putative amino acid sequence identities ranged from 99.0% to 100.0 % and from 98.1% to 100.0 %, respectively. Of the total 17 base substitutions in the genes, six substitutions were synonymous mutation, and eleven produced amino acid mutation. Except for S240P of SY200301,SY200302 and SY200303 as well as M112T of MY200301, all other amino acid mutations including MY200301's G129S, MY200302's T191R, MY200303's G127S, SY200302 and SY200303's H143R and R211G, respectively were lied in residues 125231 of spherical structural domain on the C-termination. All the six PrP genes were PrPARQ alleles at codons 136,154 and 171.