摘要
目的 克隆人膀胱逼尿肌中肾上腺素能 β2 受体基因全长 ,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 β2 AR的全长cDNA序列 ,上游引物 5′端加有BamHⅠ及ClaⅠ酶切位点 ,下游引物加有HindⅢ酶切位点 ,将该片段插入pUC19载体中 ,用ClaI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。对重组子进行琼脂糖凝胶电泳和测序鉴定。 结果 所克隆的目的基因约为 1.2kb ,并成功连接到逆转录病毒载体pLNCX上 ,测序证明 ,插入的目的片段碱基序列与Genebank报道的完全一致 ,且插入载体方向正确。 结论 成功克隆了人逼尿肌中肾上腺素能 β2 受体基因的全长cDNA序列 ,构建了反义真核表达载体 ,为研究 β2 AR亚型的功能及进一步研究膀胱功能障碍的药物治疗提供了实验依据。
Objective To clone human beta-2 adrenoceptor gene from human bladder smooth muscle and to construct its antisense eukaryotic expression vector. Methods The β_2-AR full length cDNA was cloned from human detrusor cells through RT-PCR and subcloned into clone vector (pUC18).The objective gene was then cut from ClaⅠ/HindⅢ sites of pUC18 with restriction endonulcease and subcloned into pLNCX vector in trans-direction.Finally the constructed β_2-AR gene antisense expresstion vector was identified through restriction endonuclease analysis and sequencing. Results The sequence of cloned β_2-AR full length cDNA was certified by comparison with the database of the Genebank.The constructed antisense eukaryotic expression vector was proved to be same with designed by restriction endonuclease analysis and sequencing. Conclusions β_2-AR full length cDNA was cloned and its antisense eukaryotic expression vector was successfully constructed.This technique establishs the foundation for the further research on drug treatment of bladder dysfunction.
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
2004年第6期368-370,共3页
Chinese Journal of Urology
基金
国家自然科学基金项目资助 (3 0 170 3 5 2 )
