重组人RAMP-1基因腺病毒载体的构建及鉴定

目的构建携带人受体活性修饰蛋白-1（RAMP1）基因的腺病毒表达载体，为进一步深人研究RAMPl的功能奠定基础。方法通过基因工程技术将RAMP1基因克隆到穿梭质粒pShutde—GFP—CMV中；I-CeuI＋I—SceI双酶切穿梭质粒pShuttle—GFP—CMV—RAMPl，将回收的GFP-CMV—RAMP1片段，亚克隆至腺病毒骨架载体pAdxsi得到重组腺病毒质粒；重组腺病毒质粒酶切线性化后应用脂质体法转染293细胞进行包装扩增，得到重组腺病毒Ad—RAMP1并进行PCR鉴定及滴度测定。结果构建的重组穿梭质粒pShuttle—GFP—cMVl-RAMP1双酶切后，得到O．8kb（RAMP1）和5．1kb（pShuttle—GFP—CMV）两个片段；重组腺病毒质粒pAdxsi—GFP—CMV—RAMP1用XhoI酶切得到7个片段，而作为对照的空腺病毒质粒只得到6个片段；重组腺病毒Ad—RAMP1 PCR鉴定可见阳性扩增条带；病毒滴度检测达4．5×10^11PFU／ml。结论成功构建了携带人RAMP1基因的重组腺病毒载体，为进一步研究RAMPl的功能研究奠定了基础。

Construction and identification of human RAMP1 gene recombinant adenovirus

Objective To construct and identify human RAMP1 recombinant adenovirus. Methods Human RAMP1 gene segment was subcloned into pShuttle-GFP-CMV. PShuttle-CMV-RAMP1 was digested by I-Ceu I ＋I-Sce I double enzyme and subcloned into adenovirus backbone vector to form recombinant adenovirus plasmid. Recombinant adenovirus plasmid was transfected into 293 cell lines by liposome. Human RAMP1 recombinant adenovirus was detected by polymerase chain reaction, the titer of virus was analyzed. Results Cloned sequence 0.8kb （RAMP1） and 5.1kb （pShuttle-GFP-CMV）was obtained by double enzyme after RAMP1 cDNA segment was cloned into pShuttle-GFP-CMV. DNA sequencing results indicated that the clone location was correct. Recombinant adenovirus plasmid was cut into seven fragments while empty vector gained only six fragments after digested by XhoI RAMPlcDNA （0.8kb） was amplified by PCR with virus titer of 4.5×10^11PFU/ml. Conclusions The recombinant adenovirus containing human RAMP1 gene was established successfully. The wiU be helpful for further study of the function of human RAMP1.