从猕猴肝脏组织扩增黄嘌呤脱氢酶／氧化酶基因片段

PCR amplification of the xanthine dehydrogenase /oxidase gene fragment from the liver tissue of rhesus monkey

RT-PCR扩增猕猴黄嘌呤脱氢酶/氧化酶(XDH/XO)基因片段,为进一步开展相关研究提供实验资料。方法提取猕猴新鲜肝脏组织总RNA,用RT-PCR二步法进行XDH/XO基因片段扩增,对获得的目的片段进行序列测定,与GenBank上发表的人类(Homo sapiens)、小鼠(Mus musculus)、家鼠(Rattus norvegicus)、野猪(Sus scrofa)等物种XDH/XO基因进行该序列同源性比对分析,DNAMAN软件预测该段核苷酸的氨基酸序列,InterProScan及SWISS-MODEL工具进行XDH/XO的编码蛋白结构域及功能预测及三维结构构建。结果 RT-PCR产物电泳检测得到了与设计大小相一致的目的条带,序列测定共测到683个核苷酸,DNAMAN软件预测该段核苷酸的氨基酸序列包括了1个编码53个氨基酸的开放阅读框(ORF),通过该软件包中Multiple alignment对目的基因片段的核苷酸序列与NCBI报道的人类、小鼠、家鼠、野猪XDH/XO基因mRNA互补的cDNA核苷酸序列同源性进行同源性比较分析,结果显示所扩增得到的目的片段与人类同源性最高,为95.6%,与小鼠、家鼠、野猪的同源性分别为85.2%、84.3%、86.1%,说明获得的基因片段是猕猴的XDH/XO基因片段,且该基因在物种间具有较高的相似性。生物信息学预测该段XDH/XO编码蛋白含有醛氧化/脱氢酶的钼喋呤结合点结构域及黄嘌呤脱氢酶结构域。结论在体外成功扩增出猕猴XDH/XO基因片段,为进一步开展高尿酸血症致病机理研究,抗高尿酸血症新药研发奠定工作基础。

Objective To amplify the xanthine dehydrogenase/oxidase （XDH/XO） gene fragment from the liver tissue of rhesus monkey. Metheds Total RNA was extracted from the liver tissue for amplification of XDH/XO gene fragment by two-step RT-PCR. The sequence of amplified nucleotide acids was analyzed by multiple alignment of DNAMAN software after gel eleetrophoresis and sequencing. The sequences were compared with that of the other species （Homo sapiens, Mus musculus, Rattus norvegicus, Sus scrofa） searched from GenBank. Sequencing results by DNAMAN software predicted the amino acid sequence. Conserved domains and protein three-dimensional structure of XDH/XO was analyzed by InterProScan and SWISS-MODEL. Results The expected 683 bp fragment was obtained by two-step RT-PCR. This nueleotide acid sequence was predicted that it encoded an open reading frame with 53 amino acids by DNAMAN software. The amplified fragment sequence compared with the XDH/XO gene showed nucleotide 95.6% homology with Homo sapiens, 85.2% with Mus musculus,84.3% with Rattus norvegicus, and 86. 1% with Sus scrofa reported in GenBank. It had more similarity within different species. The bioinformatie analysis showed that XDH/XO-encoding protein contained aldehyde oxidase/xanthine dehydrogenase molybdopterin binding domain, and xanthine dehydrogenase domain. Conclusions The XDH/XO gene fragment has been successfully amplified from the liver tissue of rhesus monkey. It lays the foundation for further studies on pathogenetic mechanism of hyperuricemia and development of new drug for the treatment of hyperuricemia.