中国旱獭IL-10分子的克隆和序列分析  

Cloning and Sequence Analysis of Chinese Marmot Interleukin-10

作　　者：李安意[1] 王宝菊[1] 田拥军[1] 江敏[2] 鲍俊杰[1] 郝友华[1] 喻植群[1] 陆蒙吉[2] 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室 [2]华中科技大学同济医学院病原生物学系,武汉市430030

出　　处：《医学分子生物学杂志》2006年第5期344-346,共3页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.30271170)~~

摘　　要：目的克隆旱獭白细胞介素-10(IL-10)全长cDNA序列进行序列分析,为IL-10分子的表达和在土拨鼠肝炎病毒(woodchuckhepatitisvirus,WHV)感染中的应用奠定基础。方法根据Genbank的土拨鼠IL-10cDNA序列,在5′非编码区和3′非编码区设计特异性引物,提取旱獭脾组织总RNA作为模板,RT-PCR扩增旱獭IL-10cDNA。PCR产物纯化后连接至T载体(pMD18-T),构建重组质粒pMD18-T-cmIL-10。对重组质粒进行酶切鉴定,选择阳性克隆测序。对所获得的序列应用分析软件进行分析。结果RT-PCR扩增产物为685bp。重组质粒pMD18-T-cmIL-10经EcoRⅠ与PstⅠ双酶切提示含目的基因片段。序列分析提示旱獭IL-10的编码序列为537bp,与土拨鼠IL-10的同源性为100%。结论成功克隆旱獭IL-10分子,并构建重组质粒pMD18-T-cmIL-10。Objective Cloning and sequence analysis of Chinese Marmot Interleukin-10 cDNA were conducted for the further study of IL-10 in Chinese Marmot model of WHV infection. Methods According to the woodchuck interleukin-10 sequence reported in Genbank, primers of marmot interleukin-10 cDNA were designed and synthesized. Marmot Interleukin-10 cDNA was amplified from RNA of marmot spleen by reverse transcriptase polymerrase chain reaction （RT-PCR）. The production of amplification was ligated to pMD18-T vector and then transformed into the competent bacteria DH5ot to construct the recombinant plasmid pMD18-T-cmIL-10. The recombinant plasmid was digested with EcoR I and Pst I , and positive clones were sequenced. Results The marmot Interleukin-10 cDNA fragment containing 685 bp was amplified by RT-PCR, including 537 bp complete coding sequence. Restriction endonuclease mapping using EcoR I and Pst I showed that the targetgene was inserted into the recombinant plasmid. The complete coding seqence ot marmot lnterleukin-10 was consistent with that of woodchuck and highly homologous to its counterpart of other mammalian species through DNA sequencing. Conclusion The full length of marmot Interleukin-10 cDNA was successfully cloned and the recombinant plasmid pMD18-T-cmlL-10 was constructed.

关 键 词：中国旱獭 白细胞介素-10 序列分析 

分 类 号：R34-33[医药卫生—基础医学]