pcDNA3.1/hTSHRa重组体的构建  

Construction of recombinant plasmid pcDNA3.1/hTSHRa

作　　者：朱云娟[1] 姚智[1] 赵紫琴[2] 李兰英[3] 陆凤先[2] 叶静[2] 

机构地区：[1]天津医科大学免疫学教研室,天津300070 [2]天津医科大学病理生理学教研室,天津300070 [3]天津医科大学内分泌学研究所,天津300070

出　　处：《天津医科大学学报》2006年第4期489-491,共3页Journal of Tianjin Medical University

基　　金：国家自然科学基金资助项目(30370682);天津市科技发展计划项目(05YFGDSF02700)

摘　　要：目的:构建重组体pcDNA3.1/hTSHRa。方法:提取人正常甲状腺组织总RNA,逆转录后进行PCR,将纯化的扩增产物hTSHRa与表达载体pcDNA3.1TOPO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进行鉴定。结果:PCR扩增得到hTSHR膜外区N端753bp的基因片段hTSHRa。该片段与表达载体pcDNA3.1TOPO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hTSHRa片段插入序列和方向正确。结论:hTSHRa片段插入真核表达载体pcDNA3.1TOPO,插入序列和插入方向正确,可用于后续基因表达。Objective： To construct the recombinant eukaryotic expression vector pcDNA3.1/hTSHRa. Methods： Total thyroid RNA was prepared from normal human thyroid tissue. Then RNA was reversely transcripted and cDNA was subjected to PCR amplification. PCR product was cloned into pcDNA3.1 and then the recombinant vector was transformed into TOP10 E.coli. Constructed pcDNA3.1/hTSHRa was identified by PCR amplifying, restricting enzyme digestion analysis and DNA sequencing. Results： A 753 bp fragment encoding hTSHR ectodomain amino end was obtained by PCR amplification. PCR amplifying, Hind Ⅲ restriction enzyme digestion, and DNA sequencing confirmed that pcDNA3.1/hTSHRa with the correct sequence and direction had been constructed successfully. Conclusion： hTSHRa with correct sequence and direction is cloned into the eukaryotic expression vector pcDNA3.1/hTSHRa, and it can be applied to protein expression in the future.

关 键 词：促甲状腺激素受体 基因表达 PCDNA3.1 Graves’病 

分 类 号：Q78[生物学—分子生物学]