摘要
目的本研究旨在克隆人KCNE5(hKCNE5)基因,构建其重组腺病毒表达载体,以便研究hKCNE5基因过度表达对心房颤动的防治作用。方法首先利用PCR技术克隆hKCNE5基因,并将hKCNE5基因亚克隆到腺病毒穿梭质粒pShuttle-CMV;然后将该重组质粒线性化后电转化含有复制缺陷型腺病毒pAdEasy-1(Ad)的BJ5183-AD-1细胞,经过同源重组,获得复制缺陷型重组腺病毒hKCNE5-Ad,并经酶切鉴定、序列分析证实无误;接着将其转染超级感受态细胞XL10-Gold扩增、纯化、线性化,再感染AD-293细胞进行包装便可获得大量复制的重组腺病毒hKCNE5-Ad。结果hKCNE5基因被成功克隆后正确插入腺病毒载体Ad,其序列与GenBank公布的一致;hKCNE5-Ad可感染AD-293细胞并能在AD-293细胞内进行高效复制;收集含有hKCNE5-Ad的AD-293细胞悬液,-80℃保存备用。结论本研究成功地克隆了hKCNE5基因,构建了hKCNE5基因重组腺病毒表达载体,为进一步研究hKCNE5基因过度表达对心房颤动的防治作用奠定了基础。
Objective To clone human KCNE5 （hKCNE5） gene and construct a recombinant adenoviral vector bearing hKCNE5 gene so as to investigate the prophylactic and therapeutic effects of overexpressing hKCNE5 gene on a subset of atrial fibrillation. Methods An approximate 0.6 kb of hKCNE5 gene fragment including the whole coding sequence was amplified from human genomic DNA by polymerase chain reaction （PCR） and subcloned into pShuttle-CMV vector. Once constructed, the hKCNES-pShuttle-CMV vector was linerized with PmeI and transformed into BJ5183-AD-1 competent cells transfected with adenoviral backbone plasmid pAdEasy-1 （Ad）. Transfonnants were selected for kanamycin resistance and subsequently identified by restriction endonuclease digestion and PCR-sequencing. After a recombinant hKCNE5-Ad was confirmed, it was promptly produced in bulk using the recombination-deficient ultracompetent XL10-Gold strain. Purified recombinant hKCNF.5-Ad was cut with PacI to expose its inverted terminal repeats, and was then utilized to transfect AD-293 cells where deleted viral assembly genes were complemented in vitro. Results hKCNE5 gene was cloned and correctly inserted into Ad and the sequence of hKCNE5 inserted was consistent with what publicized in GenBank. hKCNES-Ad could be appropriately packaged in AD-293 cells because the evident cytopathic effect （CPE） resulted from it was observed. The supematant of hKCNES-Ad derived from AD-293 cells was collected for the coming application. Conclusion hKCNE5 gene is triumphantly cloned and the recombinant adenovirus carrying hKCNE5 gene is successfully constructed, which paves the way for the research into the preventive and remedial effects of hKCNE5 gene on a subgroup of atrial fibrillation.
出处
《同济大学学报（医学版）》
CAS
2006年第5期1-5,共5页
Journal of Tongji University(Medical Science)
基金
国家杰出青年科学基金(30425016)
国家自然科学基金(3057076830330290)
海外青年学者合作研究基金(30528011)
教育部科学技术研究重点项目(104081)
上海市优秀学科带头人计划项目(05XD14014)
