【摘要】 目的构建重组体pcDNA3.1/hTSHR1043～1354bp及在中国仓鼠卵巢细胞(CHO)表达hTSHR膜外区氨基酸314～418蛋白分子。方法提取人正常甲状腺组织总RNA,反转录后进行聚合酶链反应(PCR),将纯化的扩增产物hTSHR1043～1354bp与表达载体pcDNA3.1连接后转化TOP10E.coli感受态菌。经PCR、HindⅢ酶切和核苷酸序列测定方法鉴定插入序列和方向正确的重组质粒转染CHO细胞,并用Westernblot方法鉴定表达的蛋白。结果PCR扩增得到hTSHR膜外区C端312bp的基因片段hTSHR1043～1354bp。该片段与表达载体pcDNA3.1的连接后转化TOP10E.coli感受态菌。重组质粒经PCR、HindⅢ酶切和核苷酸序列测定方法鉴定证实hTSHR1043～1354bp片段插入序列和方向正确。重组质粒转染的CHO细胞裂解液经Westernblot可以检测出15900的蛋白条带,与预期的蛋白相对分子质量相符。结论成功构建了重组体pcDNA3.1/hTSHR1043～1354bp,其转染CHO细胞后表达159000的目的蛋白(hTSHR氨基酸314～418蛋白片段)。更多还原

【Abstract】 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.更多还原