腭裂相关基因TTF-2转基因小鼠载体的构建及体外表达  

Construction and in Vitro Expression of Cleft Palate Relevant Gene TTF-2 Transgenic Mice Vector

作　　者：黄磊[1] 石冰[2] 郑谦[2] 王龑[2] 宋庆高[2] 

机构地区：[1]广州市第一人民医院口腔科,广州广东510016 [2]四川大学华西口腔医学院唇腭裂研究室,四川成都610041

出　　处：《昆明医学院学报》2011年第7期12-16,共5页Journal of Kunming Medical College

基　　金：国家自然科学基金资助项目(30171020)

摘　　要：目的扩增及克隆C57BL/6j小鼠titf-2基因并利用小鼠骨髓间充质细胞进行表达.方法从C57BL/6j小鼠肝脏组织中提取基因组DNA,用PCR从中扩增出titf-2基因片段.将titf-2基因片段插入载体pMD18-T中,经全自动序列分析仪测序正确后,再克隆至表达载体pBROAD3-mcs中,构建重组表达载体pBROAD3-mcs-titf-2,并转染小鼠骨髓间充质细胞进行表达.用羊抗鼠TTF-2多抗IgG对表达产物进行Western Blot签定.结果 DNA测序结果证实,获得titf-2基因片段,大小与理论值相符;Western Blot分析表明,表达出TTF-2约为4.2KDa大小的蛋白.结论成功扩增、克隆titf-2基因,并在小鼠骨髓间充质细胞中得到表达,为下一步建立titf-2转基因小鼠模型奠定了基础.Objective To amplify and clone the C57BL/6J mouse titf-2 gene and express its protein in the mouse marrow mesenchymal cell.Methods The DNA encoding the C57BL/6J mouse titf-2 gene was amplified by PCR from the mouse liver tissue.The titf-2 gene was inserted into plasmid pMD18-T.After sequencing,The titf-2 gene was cloned into eukaryotic expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-titf-2 and it was used to transfect the mouse marrow mesenchymal cell.The expression product was identified by Western Blot with specific TTF-2 antibody.Results The titf-2 gene amplified was confirmed by sequencing.The pBROAD3-titf-2 expression vector was shown by western blot to be the protein of Mr 4.2kDa.Conclusion The C57BL/6J mouse titf-2 gene has been successfully cloned and efficiently expressed in the mouse marrow mesenchymal cell and may be used as a tool of transgenic mice.

关 键 词：TTF-2 克隆 WESTERN Bloting 

分 类 号：R782[医药卫生—口腔医学]