人膀胱逼尿肌中肾上腺素能β_3受体基因的克隆及其载体的构建  

β_3-AR gene cloning from human bladder detrusor smooth muscle and construction of its antisense eukaryotic expression vector

作　　者：李昕[1] 王平[1] 李刚[1] 刘屹立[1] 

机构地区：[1]中国医科大学附属第四医院泌尿外科,辽宁沈阳110032

出　　处：《第四军医大学学报》2006年第13期1199-1201,共3页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30170325)

摘　　要：目的:克隆人膀胱逼尿肌中肾上腺素能β3受体基因全长,并构建其反义真核表达载体.方法:采用RT-PCR法从人膀胱逼尿肌中扩增出β3-AR的全长cDNA序列,上游引物5′端加有BamH I及C laI酶切位点,下游引物加有HindIII酶切位点,将该片段插入pUC18载体中,摇菌扩增后,用C laI和HindIII切下目的基因,然后将目的片段反向插入逆转录病毒表达载体pLNCX上.最后对产生的重组子进行琼脂糖凝胶电泳和测序鉴定.结果:经琼脂糖凝胶鉴定,所克隆的目的基因大约为1.2 kb,并成功地连接到逆转录病毒载体pLNCX上,经测序证明,插入的目的片段其碱基序列与Genbank上报道的完全一致,且插入载体的方向完全正确.结论:成功克隆了人逼尿肌中肾上腺素能β3受体基因的全长cDNA序列和构建了其反义真核表达载体.AIM： To clone human beta-3 adrenoceptor （β3- AR） gene from human bladder detrusor smooth muscle and construct its antisense eukaryotie expression vector. METHODS： The β3-AR full-length eDNA was cloned and amplified from human detrusor muscle cells through RT-PCR （BamHI and ClaⅠ sites were added into the 5＇ end of upstream primer, and HindⅢ into downstream） and subeloned into clone vector （pUC18 ）. The target gene was then cut from ClaⅠ/HindⅢ sites of pUC18 with restriction endonuelease and inserted inversely into pLNCX vector. Finally, the constructed β3-AR gene antisense expression vector was identified though restriction endonuelease analysis and sequencing. RESULTS： Target gene of about 1. 2 kb was successfully cloned into retroviral vector pLNCX. The sequence of cloned β3-AR full-length eDNA was identical to the reported one in GenBank. The constructed antisense eukaryotie expression vector was proved the same as designed by restriction endonuelease analysis and sequencing. CONCLUSION： β3-AR full- length eDNA was cloned from human bladder smooth muscle and their antisense eukaryotie expression vectors were constructed successfully.

关 键 词：受体 肾上腺素能Β3 载体 

分 类 号：R730.51[医药卫生—肿瘤] R587.106[医药卫生—临床医学]