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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 titf-2 gene of C57BL / 6j mouse and express it in mouse bone marrow mesenchymal cells.Methods Genomic DNA was extracted from the liver tissue of C57BL / 6j mice and the titf-2 gene was amplified by PCR The fragment of titf-2 gene was inserted into vector pMD18-T and sequenced by the automatic sequence analyzer. The titf-2 gene fragment was cloned into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-mcs-titf-2 Mouse bone marrow mesenchymal cells were expressed by Western Blot with goat anti-mouse TTF-2 polyclonal antibody.Results DNA sequencing results confirmed that the titf-2 gene fragment size consistent with the theoretical value; Western Blot Analysis showed that the expression of TTF-2 protein was about 4.2KDa.Conclusion The titf-2 gene was successfully amplified and cloned, and expressed in mouse bone marrow mesenchymal cells, for the next step to establish titf-2 transgenic mice The model laid the foundation.