hTSHR胞膜外区片段真核表达载体的构建和表达

Construction and expression of the eukaryotic expression plamids of human TSHR extracellular domain

目的：构建人类促甲状腺激素受体（hTSHR）胞膜外区氨基端的两个片段hTSHRf（aa29-100）、hTSHRe（aa101-278）的真核表达载体pcDNA3.1-hTSHRf和pcDNA3.1-hT-SHRe,并在CHO细胞中进行表达。方法：RT-PCR法从人甲状腺正常组织的cDNA中扩增hTSHRf和hTSHRe,定向插入真核表达载体pcDNA3.1（D）/V5-His-TOPO中,经酶切、PCR和测序鉴定后通过脂质体介导转染至CHO细胞中进行表达,RT-PCR扩增转染细胞的cDNA、Western blot分别鉴定hTSHR在CHO细胞中mRNA和蛋白水平的表达。结果：RT-PCR分别扩增两个片段的阳性克隆株,所得片段大小与预期一致,经Western blot鉴定,相对分子质量（Mr）分别为11900、23600左右,与预期的大小相符。结论：真核表达载体pcDNA3.1-hTSHRf和pcDNA3.1-hTSHRe构建成功,RT-PCR和Western blot检测证实重组质粒能在CHO细胞中高效表达,为进一步研究pcDNA3.1-hTSHRf和pcDNA3.1-hTSHRe在体内的基因表达,建立Graves＇病的动物模型奠定了基础。

AIM： To construct the eukaryotic expression plamids of hTSHR extracellular domain and study their expression in CHO cells.METHODS： The human TSHR extracellular domain cDNAs,which were 188-403 bp and 407-904 bp,were amplified from human normal thyroid by RT-PCR.Two fragments were inserted intopcDNA3.1（D）/V5-His-TOPO.Then the recombinant plasmids pcDNA3.1-hTSHRf and pcDNA3.1-hTSHRe were transfected into CHO cells by Lipofectin after they were identified by restricting enzyme Hind Ⅲ digestion analysis,PCR amplifying and DNA sequencing.RT-PCR and Western blot analysis were used to analyse hTSHR expression on mRNA and at protein levels.RESULTS： Two bands of 220 bp and 540 bp were amplified from CHO cells transfected by the recombinant plamids pcDNA3.1-hTSHRf and pcDNA3.1-hTSHRe,respectively.Western blot analysis revealed that CHO cells transfected by pcDNA3.1-hTSHRf and pcDNA3.1-hTSHRe had strong bands with molecular weight of about 11 900 and 23 600,respectively.CONCLUSION： The recombinant plasmids have been successfully constructed.The transcription on CHO cells transfected by the recombinant plasmids has been proved by RT-PCR and eukaryotic expression has been confirmed by Western blot analysis.Our research will contribute to further study on gene expression in vivo and the establishment of animal models of Graves＇ disease.