马麝朊蛋白(PrP)基因的克隆和序列分析

从马麝的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(PrPC)基因,并克隆到pMD18-T载体。序列分析表明所克隆的马麝PrP基因片段大约为771bp,包含了朊蛋白基因的完整编码区序列,即包含在单一外显子内的完整开放阅读框,与国外报道的同科动物PrP基因序列基本相同。马麝PrP基因含5个短而富含G-C的元件,可编码5个八肽(九肽)重复Pro-His-Gly-Gly-Gly-Trp-Gly-Gln或Pro-Gln/His-Gly-Ala/Gly-Gly–Gly-Trp-Gly-Gln,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。与白尾鹿(Odocoileusvirginianus)和麋鹿(Cervuselaphus)的PrP基因相比,其核苷酸序列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10个为同义码替换,5个为异义突变,即G57A、S100N、N173S、T177N和M208I。

Cloning and sequence analysis of prion protein (PrP) genes from musk deer

Total genomic DNA were extracted from whole-blood of a musk deer. The PrP genes were amplified by polymerase chain reaction using a pair of primers, and then these genes were cloned into pMD 18-T vectors. The sequence showed that Prp gene of musk deer was 771 bp long, including complete coding region of Prp gene, which was a complete ORF contained within a single exon, and was basically same as the published gene sequences of animals in same family. The sequence of PrP gene 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- Ala/Gly-Gly-Gly-Trp-Gly-Gln. The amino acid sequence of the gene had a N-terminal signal peptide containing 24 amino acids and a C-terminal signal peptide containing 23 amino acids. The comparison of the gene with white-tailed deer and red deer PrP gene revealed that the homogeneity of their nucleotide and putative amino acid sequences were 97.4 % and 97.9 %, as well as 98.1 % and 97.7 %, respectively. Within the total 15 base pair substitutions, ten base pair substitutions were synonymous mutation, and five produced amino acid mutation, such as G57A, S100N, N173S, T177N and M208I.