Abstract: The aim of this study was to investigate the potential of canine adenovirus type 2 (CAV-2) as an oncolytic virus and to further explore the value of sperm adhesion factor (SPAM1) synergizing with CAV-2 to remodel the tumor microenvironment in tumor immunotherapy. Based on the P15-CAV-2 reverse genetic manipulation platform, the RED/ET homologous recombination system was used to construct an intermediate vector carrying the exogenous gene of SPAM1, and then the intermediate vector was used to deletion the E3 region of the CAV-2 backbone vector and replace the expression cassette of the exogenous gene of SPAM1. Then the synthetic primers was used to knockdown the kmccdB reverse screening expression cassette to construct the P15A-CAV-2-mCMV-SPAM1-SV40 polyA infectious clone plasmid and rescue the recombinant lysogenic viruses in the MDCK-E1A cell line, then the recombinant viruses' in vitro tumor lysis effect was validated. The results of sequencing and digestion showed that, a recombinant lyssavirus strain stably expressing the exogenous gene SPAM1 had been successfully constructed and rescued, and the IFA assay proved that the recombinant strain could stably express the exogenous gene at a high level, and the exogenous protein had the characteristics of diffusely distributed in the intercellular region. The recombinant lysovirus with the deletion of the E3 region expressing the exogenous gene SPAM1 was found to have a strong killing effect on the canine cancer cell line A72 by light microscopy and CCK8 assay. In conclusion, the current study successfully constructed a recombinant CAV-2 strain with a good tumor lysis effect, which lays the foundation for subsequent application in pet tumor therapy.

Key words: oncolytic virus, sperm adhesion factor, virus reverse genetic operation, canine adenovirus-2