甲状腺转录因子-1融合表达载体的构建及体外活性鉴定

Contruction and Expression of Eukaryotic Expression Vector pcDNA3.1/myc-His(-)C-TTF1 Carrying the Gene Encoding Thyriod Transcription Factor 1

目的:构建人甲状腺转录因子-1(TTF1)编码基因的真核表达载体pcDNA3.1/myc-His(-)C-TTF1,并观察其蛋白质体外转录翻译情况。方法:据已知的TTF1基因序列,应用巢式PCR技术,从人肺腺癌细胞株SPC-A1中扩增出TTF1基因,通过TA连接将其克隆入pGEM-T-easy载体,经测序鉴定后,双酶切插入真核表达质粒pcDNA3.1/myc-His(-)C中;重组质粒经XhoⅠ和BamHⅠ双酶切鉴定后,进行蛋白质体外翻译观察TTF1体外表达情况,用电泳迁移率变动分析(EMSA)验证获得的体外翻译蛋白TTF1是否具有与下游靶基因UGRP1启动子结合的能力。结果:成功构建了含TTF1编码基因的真核表达载体pcDNA3.1/myc-His(-)C-TTF1,并能在体外表达TTF1蛋白。结论:能方便地获得TTF1体外翻译蛋白,为进一步研究TTF1蛋白与相应的DNA反应元件及其他转录因子的相互作用奠定了基础。

Objective： To construct the eukaryotic expression vector pcDNA3.1/myc-His（-）C-TTF1 and to dectet expression of human thyriod transcription factor 1（TTF1） in vitro. Methods： According to humqn TTF1 gene sequence in GenBank（BC006221）, eDNA of human TTF1 gene was obtained by RT-PCR from SPC-A1 cell line of human lung adencarcinoma. The PCR products were subcloned into pGEM-T-easy vector. After comfirmed by DNA sequencing, the pGEM-T-easy- TTF1 was digested by Xho I/BarnH I and then the TFF1 gene was cloned into the expression vector pcDNA3.1/ myc-His（-）C. A recombinant pcDNA3.1/myc-His（-）C-TTF1 was cornfirmed by Xho [/BamH I disgesttion. From the recombinant pcDNA3.1/myc-His （-）C-TTF1, The human TTF1 protein was successfully expressioned by using transcription and translation kit. The electrophoretic mobility shift assay（EMSA） indicated that the obtained TFFI protein in vitro could be bind with UGRP1 gene promoter sequence. Results： The eukaryotie expression vector pcDNA3.1/myc-His（-）C-TTF1 was successfully constructed and human TTF1 protein was successfully expressed. Conclusion： Expression of TTF1 in vitro can be used in the further research of interaction of TTF1 and its DNA response elements and others transcription factors.