猪伴侣蛋白10基因克隆和鉴定

Cloning and identifying of porcine chaperonin 10

目的克隆猪伴侣蛋白 10 (chaperonin 10 ,cpn10 )基因并比较其在不同种属间的序列同源性。方法利用抑制消减杂交技术 (SSH)、DNA序列测定技术、表达序列标记 (EST)拼接技术、生物信息学分析、RT -PCR等进行研究。结果首次成功获得全长的猪cpn10cDNA序列。与人、牛、大鼠、小鼠的cpn10相比 ,在核苷酸水平上的同源性分别为 94 %、94 %、88%、88% ,在氨基酸水平上的同源性分别为 10 0 %、10 0 %、99%、96 %。功能区分析发现 ,猪cpn10蛋白由 10 2氨基酸组成 ,分子量为 10 930 .97,等电点为 8.90 ,无N端信号肽序列 ,C端无核定位信号 (NLS)。RT -PCR结果显示 ,在未灭活补体人血清 (C -HS)作用下 ,猪内皮细胞cpn10基因表达上调。结论cpn10是进化上高度保守的基因 ,可能与猪到人异种移植排斥反应相关。

Objective To clone porcine chaperonin 10 gene and analyze the sequence homology among the different species. Methods Suppression subtractive hybridization (SSH), DNA sequencing, expressed sequence tag (EST) sequencing alignment method, bioinformaties analysis, RT-PCR were used for the cDNA cloning of porcine cpn10. Results The full length cDNA of porcine cpn10 was successfully cloning and sequenced. There were 94%, 94%, 88%, 88% homology between porcine and human, bovine, rat, mouse at the nueleotide level, and there were 100%, 100%, 99%, 96% homology at the amino acid level. Porcine cpn10 is predicted to consist of 102 amino acids. The molecular weight is 10930.97. The isoelectric point is 8.9. The protein does not contain N-terminal signal sequence and C-terminal nuclear localization signal (NLS). RT-PCR analysis showed an up-regulation of cpn10 on porcine endothelial cells treated with complement-containing human serum (C-HS) . Conclusion The data demonstrate porcine cpn10 was highly conserved and may have relationship with pig-to-human xenotransplantation.