中国旱獭（M.himalayana）GAPDH分子的部分cDNA克隆及序列分析

Cloning and Sequence Analysis of Partial cDNA of Chinese Marmot GAPDH

目的对新近建立的乙肝研究动物模型喜马拉雅旱獭3-磷酸甘油醛脱氢酶（GAPDH）的部分cDNA序列进行了克隆和序列分析．为该模型在HBV感染研究中的应用奠定基础。方法根据Genbank的人GAPDHeDNA的序列设计特异性引物．以提取的旱獭脾组织总RNA为模板,RT-PCR扩增旱獭GAPDH cDNA序列。纯化的PCR产物连接至T载体（pMD18-T），然后构建重组质粒pMD18-T-mhGAPDH。对重组质粒进行PCR初筛、酶切鉴定，将阳性克隆测序。对序列进行同源性以及种系进化分析。结果扩增的旱獭GAPDH序列为535bp，编码序列为533bp（nt3-335），编码177个氨基酸。获得的序列和其它类哺乳动物GAPDH的同源性均在88％以上，其中与土拨鼠GAPDH的同源性最高（99．62％），氨基酸序列同源性高达100％。构建种系进化树，分析结果提示喜马拉雅旱獭GAPDH与土拨鼠GAPDH的亲缘关系最接近。结论成功的克隆了喜马拉雅旱獭GAPDH的部分序列。序列分析结果提示喜马拉雅旱獭GAPDH与土拨鼠GAPDH的亲缘关系最近，同源性最高。

Objective To characterize the partial cDNA sequence of GAPDH in M himalay- ana, a newly established animal model for HBV study. Methods The primers were designed based on the eDNA sequence of human （Homo sapiens GAPDH. Total RNA was isolated from spleen tissues of M. himalayana, and served as the template for RT-PCR amplification. PCR product was cloned into TA cloning vector with pMD18-T. Positive clones were selected and identified by PCR and restricted enzyme digestion. The recombinant plasmid pMD18-T-mhGAPDH was sequenced commercially. The homologies of the eDNA sequences of GAPDH among M. himalayana, wood- chuck, and other mammalian species were compared, and the phylogenetic tree was conducted. Results Sequence analysis indicated the partial sequence （535 bp） of GAPDH in hi. himalay- ana was obtained, including the coding sequence of 533 bp （referred to nt3-535 ） . Homology and phylogenetie analysis indicated that GAPDH in M. himalayana had high homology （up to 88 % ） and close relationship to the counterpart of other mammalian species, with the highest homology （99. 62 % ） and the closest relationship to the counterpart of woodchuck. Cortdusion The par-tial sequence of GAPDH in M. himalayana was successfully obtained. The sequence analysis indi-cated GAPDH in M. himalayana had the highest homology and the closest relationship to the coun-terpart of woodchuck.