Construction of the recombinant adenovirus carrying porcine interferon gamma (poIFNgamma) and identification of its antiviral activity

The total RNA was extracted from peripheral blood mononuclear cells(PBMC) which was isolated from Meishan porcine and induced with concanavaline A(ConA),then the porcine interferon gamma gene(PoIFNγ,501bp) was amplified by RT-PCR.The result of sequencing demonstrated that the amplified PoIFNγ had 100% nucleotide homology with the other porcine IFNγ sequence published on GenBank.The objective gene(PoIFNγ) was inserted into adenoviral shuttle vector,pShuttleCMV,to construct recombinant plasmid pSh-PoIFNγ.And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37℃ for 24h on kanamycin resistance plate and selected for smaller colonies.Then,the extracted recombinant plasmid was named pAd-Sh-PoIFNγ,which was confirmed by PacⅠ digestion,and transformed into XL10-Glod(r) for copious preparation.pAd-Sh-PoIFNγ linearized with PacⅠwas co-transfected with liposome into 293 package cellline.After 7d-10d,the typical cytopathic effect indicated that recombinant adenoviral genome(deleted with E1 and E3 genes) carrying PoIFNγ was successfully packaged into intact virion.The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR.The antiviral activity of PoIFNγ was tested by CPE50 method.The results showed that the PoIFNγ expressed by adenovirus had high antiviral activity,which was 1.3×106 U/mL against VSV in MDBK cells.The results demonstrated that the recombinant adenovirus carrying PoIFNγ could be stably passaged.