Construction of recombinant pcDNA3.1/hTSHR_(1043～1354 bp) and its expression in CHO cells

Objective To construct the recombinant pcDNA3.1/hTSHR1043～1354 bp and its expression in CHO cells.Methods Total thyroid RNA was prepared from normal human thyroid tissue.Then RNA was reverse transcripted and cDNA was subjected to PCR amplification.PCR product was cloned into pcDNA3.1 and then the recombinant was transformed into TOP10 E.coli.Constructed pcDNA3.1/hTSHR1043～1354 bp was identified by PCR amplifying,restrictied enzyme digestion analysis and DNA sequencing.CHO cells were transfected by pcDNA3.1/hTSHR1043bp-1354bp and Western blot was applied to detect target protein expression in CHO.Results A 312 bp fragment encoding hTSHR ectodomain amino end was obtained by PCR amplification.PCR amplifying,HindⅢ restriction enzyme digestion and DNA sequencing confirmed that pcDNA3.1/hTSHR1043～1354 bp had been constructed successfully,with the correct sequence and direction of hTSHR1043～1354 bp.CHO cells were transfected by pcDNA3.1/hTSHR1043～1354 bp and a 15 900 band was detected by Western blot,which is consistent with expectation.Conclusion The recombinant pcDNA3.1/hTSHR1043～1354 bp is constructed.The target protein TSHR1043～1354 bp,with 15 900 molecular weight,is expressed in CHO cells transfected by pcDNA3.1/hTSHR1043～1354 bp.