人甲状腺刺激激素受体cDNA的克隆及真核表达载体的构建

目的克隆人甲状腺刺激激素受体(hTSHR)基因cDNA并构建其真核表达载体。方法以人甲状腺组织cDNA为模板,聚合酶链反应(PCR)扩增hTSHR基因编码区的全部序列,克隆入pGEM-T载体中,经限制性内切酶、DNA序列分析鉴定目的基因后,定向亚克隆到真核细胞表达载体pcDNA3.1中,并进行双酶切鉴定。结果PCR扩增的特异性片段长度为2 337 bp,以此构建的pGEM-T-hTSHR克隆载体,经限制性内切酶酶切及DNA序列分析证实载体中带有hTSHR基因编码区的目的片段,重组质粒pcDNA3.1-hTSHR经KpnⅠ和XbaⅠ双酶切后显示5 1000 bp和2 300 bp左右的2个条带,DNA序列分析显示与GenBank中的hTSHR基因cDNA序列一致,证明hTSHR基因已成功克隆入真核细胞表达载体pcDNA3.1中。结论成功构建了野生型pcDNA3.1-hTSHR重组真核表达载体。

Cloning of human thyroid stimulating hormone receptor gene and construction of its eukaryotic expression vector

Objective To clone human thyroid stimulating hormone receptor（hTSHR） gene and construct its eukaryotic expression vector. Methods The whole coding sequence of hTSHR gene was amplified by polymerase chain reaction （PCR） applied with human thyroid cDNA. The fragment was inserted into cloning vector pGEM-T and the recombinant pGEM-T-hTSHR was identified by double digestion with restriction enzymes and sequencing. The hTSHR cDNA fragment was subcloned into pcDNA3.1 plasmid. The recombinant pcDNA3. 1-hTSHR was identified by double digestion with restriction enzymes and sequencing. Results The length of the specific fragment by PCR was 2337bp,and the pGEM-T-hTSHR plasmid was identified by the same way. The sequence was identical to that of hTSHR cDNA in GenBank. The recombinant pcDNA3.1-hTSHR plasmid was separated into two bands, approximately 5 100 kb and 2300bp, by using respective restriction enzymes Kpn I and Xba I ,and the sequence was identical to that of hTSHR cDNA in GenBank, suggesting that hTSHR gene fragment had been cloned into pcDNA3.1 vector correctly. Conehsion The wild-type recombinant eukaryon expression vector pcDNA3.1-hTSHR was successfully constructed.