Construction and idenfication of replication deficient recombinant adenovirus of NK4 protein

Objective To construct replication deficient recombinant adenovirus vector expressing human NK4 protein by homologous recombination in bacteria using AdEasy system,and produce adenoviral particles.Methods The Coding regions(CDS) of NK4 gene was obtained from pcDNA3/hNK4 plasmid by restriction endonuclease digestion,and was inserted into the downstream of CMV promoter of a shutter plasmid pAdTrack-CMV.The recombinant transfer plasmid pAdTrack-CMV-NK4 was linearized by Pme I and co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for homologous recombination to obtain the recombinant adenoviral plasmid.The recombinant adenoviral plasmid was linearized and was then transfected into HEK293 package cells to produce virus particles.The expression of NK4 was examined by RT-PCR after infection of HepG2.Results The recombinant adenoviral plasmid was successfully established and confirmed by restriction endonuclease digestion.Green fluorescent protern(GFP) expression was observed on the 2th day after transfection.Expression of NK4 gene mRNA after infection was observed in HepG2 cells.Conclusion The achievement of recombinant adenoviral plasmid and recombinant adenovirus of NK4 provides evidences for further investigation of its function and application.