双峰驼朊蛋白基因的克隆和序列分析

分别从4峰双峰驼全血中提取基因组总DNA,用所设计的引物扩增了细胞型朊蛋白(PrP)基因,并克隆到pMD 18-T载体。序列分析表明,所克隆的4个双峰驼PrP基因片段大小分别为768、768、792和795 bp,包含了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,均与国外报道的同属单峰驼PrP序列基本相同。4个双峰驼PrP基因含5个或6个短而富含G-C的元件,可编码5个或6个八肽(九肽)重复序列Pro-His-Gly-Gly-Gly-Trp-Gly-Gln或Pro-Gln/His-Gly-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22个氨基酸(除LT200302为23个氨基酸外)的C-端信号肽。4个双峰驼PrP基因之间相比较,其核苷酸和推导氨基酸序列的同源性为91.0%~100.0%和94.2%~100.0%。共发生133个碱基替换,其中107个为同义码替换,26个为异义突变。

Cloning and sequence analysis of prion protein gene from Camelus bactrianus

Genomic DNA was extracted from peripheral whole-blood of four Camelus bactrianus respectively.The PrP gene was amplified by polymerase chain reaction using a pair of primers,and then cloned into pMD 18-T Vector.Sequencing showed that the four Camelus bactrianus genes were 768 bp,768 bp,792 bp and 795 bp in length respectively.All the entire PrP coding sequences had the complete ORFs contained within a single exon and were very similar to the published gene sequences of Camelus dromedarius by and large.The sequences of PrP gene 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 the nonapeptide Pro-Gln/His-Gly-Gly-Gly-Gly-Trp-Gly-Gln.All the amino acid sequences of these genes had a N-terminal signal peptide of 24(amino) acids,and a C-terminal signal peptide of 22 amino acids(with the exception of the LT200302 clone which contained a C-terminal signal peptide of 23 amino acids).Comparison among these genes revealed that the sequence(identities) of nucleotide and their putative amino acid ranged from 91.0% to 100.0 % and from 94.2% to 100.0 %,respectively.Of the total 133 base substitutions,107 substitutions were synonymous mutation,and twenty-six produced amino acid mutation.