Construction and identification of recombinant adenovirus Ad5F35 containing prodynorphin gene

Objective To construct and identify recombinant adenovirus expressing prodynorphin gene (PDP) in vector Ad5 F35.Methods Restriction fragment of PDP from pUC57-PDP was inserted in the vector of pDC316.The insertion and the direction of the insert were confirmed by polymerase chain reaction (PCR) and restriction enzyme digestion.The constructed pDC316-PDP was co-transfected with adenovirus backbone pBHG-fiberS/35 into 293 cells to establish the recombinant adenovims Ad5F35-PDP, which was confirmed by PCR.Results The gene sequence coding PDP was identical with that of GenBank by DNA sequencing, and the construction of pDC316-PDP was completed.The significant virus plaques was observed in the 293 cells co-transfected with pDC316-PDP and pBHG-fiber5/35, indicating the successful homologue recombination and virus packaging of the interest fragment in 293 cells.The successful construction of the recombinant adenovirus Ad5F35-PDP with PCR was confirmed, and the titer of Ad5F35-PDP was approximately 1 × 1012 v.p./ml.Conclusion The high-titer,purified recombinant adenovirus of Ad5F35-PDP which contains the full-length coding region of PDP has been established, which could be used in the gene therapy for morphine addiction, and to test the bio-safety and biological function of Ad5F35-PDP in the future. Key words: Dynorphin;  Recombinant adenovirus;  Gene therapy