Construction of Recombinant Adenovirus Plasmid Encoding Human nm23-H1 Gene by AdEasy-1 System

Objective To construct the recombinant adenovirus vector encoding human nm23-H1 gene by AdEasy-1 adenovirus vector system.Methods A pair of DNA primers with KpnⅠand XhoⅠ restriction enzyme site were designed.Nm23-H1 cDNA was cloned from pMD18-T-nm23 plasmid by PCR technology and cloned into pShuttle-CMV in correct direction and the recombinant plasmid was called pShuttle-CMV-nm23-H1.It was identified by double digestion with KpnⅠand XhoⅠrestriction enzymes and DNA sequencing.The replication-defective adenovirus type 5 backbone plasmid pAdEasy-1 was transformed into competent bacteria BJ5183 by electroporation,pShuttle-CMV-nm23-H1 was linearized by PmeⅠand dephosphorylated and transformed into competent bacteria BJ5183 which contained pAdEasy-1 backbone plasmid.Then the product was selected by kanamycin and identified by digestion with PacⅠ.The recombinant adenovirus vector plasmid was amplified broadly in XL10-Gold competent bacteria and identified by PCR.Results A fragment size about 460bp was got by digestion with KpnⅠand XhoⅠ.Its sequence was identical to that of gene nm23-H1 by DNA sequencing.The recombinant advenoviral plasmid could produce a fragment whose size was 3.0kb or 4.5kb when digested with PacⅠ.The 460bp fragment was appeared after PCR identification.Conclusion The recombinant adenovirus plasmid encoding human nm23-H1 gene by AdEasy-1 adenovirus vector system was constructed successfully.