Construction Of Recombinant Adenovirus Of Overexpressed Human Receptor Activity-modifying Protein 1 Gene And Expression Of Infected Rabbit Marrow Mesenchymal Stem Cells

Objective To construct recombinant eukaryotic expression adenovirus vector pAdxsi-EGFP-hRAMPl and investigate the expression of hRAMP1 in rabbit MSCs infected with adenovirus vector encoding hRAMP1 gene(Ad-EGFP-hRAMP1). Methods:hRAMP1 cDNA segment was liberated from the cloning vector of pOTB7-hRAMPl via EcoRI+XhoI, and subcloned into pShuttle-EGFP-CMV, which was digested by I-CeuI+I-SceI double enzyme and subcloned into adenoviral plasmid to form recombinant adenovirus plasmid. The recombinant adenovirus plasmid was transfected into 293 cell lines by liposome. After observing the coming out of adenovirus, then harvested the adenovirus, and then freezed, intreased and rereceived the adenovirus, we centrifugaled, dialyzed and puried the adenovirus for its titre determnation. Rabbit bone marrow derived mesenchymal stem cells(MSCs) were isolated using Ficoll desity gradient method and according to the adherent property of MSCs.Then we infected rabbit MSC with adenovirus vector encoding enhanced green fluorescent protein(EGFP) and hRAMP1.The efficiency of infection and expression of human Receptor activity-modifying protein 1 gene (hRAMPl)were detected by converted fluorescence microscope. The mRNA and protein expression of hRAMPl was detected in BMSCs,which were infected with Ad-EGFP-hRAMP1 at the most appropriate MOI, were detected by RT-PCR and Western-Blot respectively. Results:Cloned sequence about 0.8kbp was obtained by EcoRI+XhoI digestion after hRAMPl cDNA segment was cloned into pShuttle-EGFP-CMV. Recombinant adenovirus plasmid was cut into seven fragments while empty vector gained only six fragments after digested by Xho I. The recombinant adenovirus was pathogenic to 293 cells after recombinant adenovirus plasmid was packaged in it. hRAMP1 cDNA (164bp) was amplified by PCR with virus titer of 2.5×1011PFU/ml after transfected 293 cells with supernatant. The efficiency of recombinant adenovirus infecting rabbit MSCs was about 80% when multiplicity of infection was 800. Rabbit MSCs expressed green fluorescence after transfection. The effective expression of hRAMPl in infected cells has been proved by RT-PCR and WesternBlot. Conlusion:The recombinant adenovirus containing hRAMP1 gene with enhanced green fluorescent protein was successfully constructed. MSCs are ideal cells for cell-mediated hRAMP1 gene therapy or cell therapy.