pcDNA3.1/hTSHR真核表达载体的构建及其在COS-7细胞中的表达

Construction of pcDNA3.1(+)/ hTSHR and its expression in COS-7 cells

目的 :构建pcDNA3.1(+)人促甲状腺激素受体 (hTSHR)基因真核表达载体 ,并在COS 7细胞中进行表达。方法 :以限制性内切酶EcoRI和XbaI双酶切pBluescriptSK(- ) /hTSHR ,获得hTSHRcDNA的全长片段 ,在以低熔点琼脂糖回收纯化后 ,定向插入真核表达载体pcDNA3.1(+)的多克隆酶切位点中 ,构建重组体pcDNA3.1/hTSHR ,进行酶切、PCR及测序鉴定。通过脂质体介导 ,转染COS 7细胞进行瞬时表达 ,并用RT PCR和免疫细胞化学染色法检测hTSHR的表达。结果 :双酶切pBluescriptSK(- ) /hTSHR得到约 2 70 0bp含TSHR全长cD NA的片段 ,同预期片段的大小相符。所构建的pcDNA3.1/hT SHR经双酶切及PCR鉴定同预期大小相符 ;测序结果与Gen Bank中收录的hTSHR全长序列一致 ,表明真核表达质粒构建正确。以脂质体转染COS 7细胞后 ,用RT PCR和免疫细胞化学染色法检测表明 ,细胞可表达hTSHR。结论 :成功地构建真核重组表达质粒pcDNA3.1/hTSHR ,为进一步研究TSHR的功能 ,以及利用hTSHR真核表达载体建立Graves实验动物模型奠定了基础。

AIM: To construct the recombinant eukaryotic expression vector pcDNA3.1(+)hTSHR and express it in COS-7 cells. METHODS: The full length cDNA sequence of hTSHR was obtained via the plasmid vector pBluescript SK(-)/hTSHR cut with EcoR I and Xba I, and then subcloned into eukaryotic expression vector pcDNA3.1(+). Constructed pcDNA3.1/hTSHR was identified by restricting enzyme digestion analysis, PCR amplifying and DNA sequencing. The recombinant expression plasmid was transfected into COS-7 cells by Lipofectin method. Human TSHR protein expression on COS-7 cells was detected by RT-PCR and immunocytochemical staining. RESULTS: Obtained full-length sequence of hTSHR gene was identical with that included in GenBank. Restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that pcDNA3.1/hTSHR had been constructed successfully. The recombinant plasmid could express hTSHR protein with activity on COS-7 cells. CONCLUTION: The pcDNA3.1/hTSHR has been successfully constructed, which will contribute to further studies on the TSHR function and to the establishment of a good animal model for Graves’ disease.