摘要
目的旨在构建超极化激活环核苷酸门控阳离子通道基因亚型4(HCN4)重组腺病毒载体并鉴定其离子通道功能。方法全长人HCN4基因酶切后亚克隆到腺病毒穿梭载体pAdTrack-CMV,形成含目的基因和绿色荧光蛋白基因的穿梭载体。穿梭载体和病毒骨架载体pAdEasy-1经电穿孔法在BJ5183大肠杆菌中同源重组,重组后的腺病毒载体经PacⅠ酶切线性化后,利用脂质体介导转染到HEK293细胞进行病毒的包装和扩增。用多聚酶链反应(PCR)方法对病毒上清中的HCN4进行检测,并将重组腺病毒感染COS-7细胞,用免疫荧光染色检测HCN4蛋白质的表达,利用全细胞膜片钳方法测定转HCN4基因细胞的电生理功能。结果通过酶切、测序、PCR等证实HCN4通道基因重组腺病毒载体构建正确,免疫荧光检测证实转染AdHCN4的COS-7细胞中HCN4通道基因的表达,膜片钳实验也在转基因细胞中检测到超极化激活的非选择性内向阳离子电流(If)。结论HCN4通道基因重组腺病毒载体的构建成功为进一步研究该离子通道的电生理功能及在心律失常疾病基因治疗方面的潜在应用价值奠定了基础。
Objective To construct recombinant adenovirus that express the human hyperpolarization activated cyclic nucleotide gated cation channel 4 （HCN4） and study the electrophysiological function of HCN4 in COS-7 cells. Methods Human HCN4 gene was ligated into a shuttle vector pAdTrack-CMV. Homologous recombination was performed in BJ5183 bacteria by cotransforming linearized shuttle plasmid and adenovirus backboned vector pAdEasy-1. The positive recombinant adenovirus plasmid was digested with Pac Ⅰ and transfected into HEK 293 cells for the packaging and amplification of adenovirus particles. After infection of AdHCN4 in COS-7 cells, HCN4 protein expression was detected by immunofluorescence cytochemistry and the functional characteristics of HCN4 channel in cells was determined by patch clamp experiments. Results The results showed that the recombinant adenovirus was successfully constructed. HCN4 protein was detected in infected cells by immunofluorescence cytochemistry. Patch clamp recorded a Cs^＋ -sensitive inward current from - 40mV to - 140mV in response to hyperpolarization activation in infected cells. Conclusions These findings demonstrate that HCN4 can be successfully delivered by an adnovirus vector and functionally expressed in infected cells. This may present a platform for further investigation in electrophysiological function and potential value of HCN4 for gene therapy.
出处
《中国心脏起搏与心电生理杂志》
2006年第1期58-62,共5页
Chinese Journal of Cardiac Pacing and Electrophysiology
基金
国家高技术研究发展计划(863计划)基金(2001AA2160313)
北京市科技计划重大项目基金(H020220010490)
教育部211工程和北京大学985基金
关键词
分子生物学
超极化激活环核苷酸门控阳离子通道
起搏电流
腺病毒
Molecular biology
Hyperpolarization activated cyclic nucleotide gated cation channel
Pacemaker current
Adenovirus
