Construction and identity of recombinant adenovirus co-expressing NK4 and PUMA gene

Abstract

Objective To construct replication deficient recombinant adenovirus co-expressing PUMA and NK4 genes using AdEasy-1 system,observe the expression of PUMA and NK4 genes in pancreatic cancer cell after andenovirus infection.Methods PUMA and HGF/NK4 gene were cloned to adenovirus shutter plasmid for the construction of recombinant plasmid pAdTrack-PUMA/NK4.The recombinant plasmid digested by PmeI was transformed into bacteria B J5183 containing backbone vector pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral vector pAd-PUMA-NK4.The recombinant adenoviral vector was then transfected into HEK293 package cells to produce virus particles.The expression of NK4 and puma gene in cells were detected in human pancreatic cancer cells by qRT-PCR after adenovirus infection.Results The recombinant adenoviral vector was identified by PCR and restriction endonuclease digestion.High level expression of NK4 and PUMA mRNA was maintained for 6 days in SW1990 cells after adenovius infection.Conclusion The infection of Recombinant adenovirus Ad-PUMA-NK4 can mediate high level expression of NK4 and PUMA,it provides an important tool for further gene therapy of pancreatic cancer with them. Key words: Adenovirus; PUMA gene; NK4 gene; Pancreatic cancer