[Construction and expression of the eukaryotic expression plasmids of human TSHR extracellular domain]
Zi-Qin Zhao1, Lan-Ying Li, Feng-Xian Lu
1Department of Clinical Laboratory, Tianjin Gong-an Hospital, Tianjin 300042, China. zhaoziqingzzq@163.com
Aim:
To construct the eukaryotic expression plasmids 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 into pcDNA3.1(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 HindIII 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 plasmids 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.
