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-CeuⅠ+I-SceⅠdouble 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.RAMP1cDNA(0.8kb) was amplified by PCR with virus titer of 4.5×1011PFU/ml.Conclusions The recombinant adenovirus containing human RAMP1 gene was established successfully.The will be helpful for further study of the function of human RAMP1.