Constructing recombinant adenovirus carrying rat interleukin-10 gene using AdEasy system / 中国组织工程研究

ABSTRACT

AIM:The homologous recombination takes place in E.coli BJ5183 between shuttle vector and adenoviral backbone vector.Recombinants are selected for kanamycin resistance,and the linearized recombinant plasmid is transfected into 293 cells.In this study,AdEasy system was used to generate recombinant adenovirus vector carrying rat interleukin-10(IL-10) gene.METHODS:The experiment was conducted in Department of Microbiology,Qingdao University Medical College from August 2006 to May 2007.①The materials included five clean-grade SD rat,a shuttle vector pAdTrack-CMV,an adenoviral backbone plasmid pAdEasy-1,E.coli.BJ5183 and DH10B,and human embryo kidney 293 cells,which were given as a present by professor Luo.All animal treatment was accorded with the ethical standards.②Total RNA was extracted from spleen cells of rat stimulating by lipopolysaccharide.IL-10 cDNA was amplified by using RT-PCR.The gene of interest was firstly cloned into a shuttle vector pAdTrack-CMV.The resultant plasmid was linearized by digesting with restriction endonuclease Pme Ⅰ,and subsequently transformed into E.coli.BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1.Finally,the linearized recombinant plasmid was transfected into adenovirus packaging cell lines 293 cells.Recombinant adenovirus vAd-IL-10 was obtained.The expression of green fluorescent protein(GFP) was observed under fluorescent microscope.293 cells were cultured in 96-well culture plate with 1 ?104 cells per well.Condensed virus stock solution was added into the plate at different ratios.Recombinant adenoviruses titer was determined.Three days later,total RNA was extracted from 293 cells by TRIzol one-step method,and contaminant DNA was digested by DNaseⅠ.Electrophoresis detected the expression of IL-10 mRNA.RESULTS:①GFP expression was observed 16 hours after packing of the linearized recombinant adenoviruses in 293 cells.The amplification product of replicationdeficient Ad-IL-10 virus was transfected into adenovirus packaging cell lines 293 cells.When the fourth and fifth generations were seeded in virus for 24-48 hours,fluorescence was found in almost cells,and 5.5?108 pfu/mL titer of Ad-IL-10 was obtained.Titer of vAd-GFP was 9.0?108 pfu/mL.②Total RNA was extracted from transfected 293 cells.Electrophoresis showed that 580bp amplification product was expressed obviously and recombinant adenovirus was duplicated in 293 cells.CONCLUSION:The recombinant adenoviral vector carrying IL-10 gene is successfully constructed using AdEasy system.Moreover,vAd-IL-10 recombinant adenovirus with high titer is prepared and transcribed in 293 cells.