双峰驼朊蛋白基因的克隆和序列分析

Cloning and sequence analysis of prion protein gene from Camelus bactrianus

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.

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.