大鼠FXYD5基因的克隆及重组腺病毒载体的构建.docVIP

大鼠FXYD5基因的克隆及重组腺病毒载体的构建 　　作者：李小旺 作者单位：温州医学院第一附属医院 心血管内科 　　【摘要】 目的:克隆大鼠离子通道相关蛋白(FXYD5)基因全长cDNA，构建携带FXYD5基因的重组腺病毒载体，为进一步研究其在自发性高血压大鼠(SHR)中的功能作准备。方法:采用cDNA末端快速扩增的方法获得全长cDNA，并插入到载体pGEM-T Easy中测序分析。将FXYD5基因cDNA克隆至穿梭载体pAdTrack-CMV中，随后在BJ5183 细菌中与骨架载体AdEasy-1同源重组。筛选阳性克隆，酶切、测序鉴定正确后，重组载体磷酸钙-DNA共沉淀法转染AD293细胞，获得携带FXYD5基因的重组腺病毒。结果:大鼠FXYD5基因全长cDNA被成功地克隆;FXYD5重组腺病毒载体被成功地构建。结论:运用细菌内同源重组的方法可以在大肠杆菌中快速高效地构建腺病毒载体;FXYD5重组腺病毒载体的成功构建为探讨FXYD5的生物学功能奠定了基础。 　　【关键词】 FXYD5 腺病毒 基因 　　Cloning of rat FXYD5 gene and construction of recombinant adenoviral vector LI Xiao-wang，FANG Fei，SHI Xiang-xiang，HUANG Xiao-yan，ZHANG Huai-qin，YANG De-ye. Department of Cardiology，the First Affiliated Hospital of Wenzhou Medical college，Institute for Cardiovascular Biology and Gene of Wenzhou Medical college，Wenzhou，325000 　　Abstract: Objective: To clone rat FXYD5 gene full-length cDNA and construct its recombinant adenoviral vector for its function study in SHR. Methods: FXYD5 gene full-length cDNA was amplified using rapid amplification of cDNA end(RACE) method and inserted into pGEM-T Easy vector and confirmed by sequence analysis. The cDNA was cloned into the shuttle vector pAdTrack-CMV. Then the resultant vector was recombinated with pAdEasy-l backbone vector in BJ5183 cells. The positive clones were selected. Finally， the recombinant vector was transfected into AD293 cells by calcium phosphate method of DNA transfection. Results:The full length cDNA of FXYD5 was cloned，and its recombinant adenoviral vector was constructed successfully. Conclusion:The results indicate that the recombinant adenoviral vector can be constructed quickly and efficiently in E.coli with homologous recombination protocol. A recombinant adenovirus Ad-FXYD5 are successfully constructed. The adenovirus will be used to investigate the biologic role of FXYD5 in hypertension. 　　Key words: FXYD5; adenovirus; gene 　　FXYD5属于FXYD家族单跨膜I型膜蛋白。既往研究发现，FXYD5在某些肿瘤细胞中有大量表达，能下调E-钙粘素和促进肿瘤转移[1]。近来Lubarski 等[2]发现，FXYD5多表达在肾脏皮质、十二指