An improved system for constructing marker-free recombinant goatpox viruses to express foreign proteins

Goatpox virus (GTPV), belonging to the genus Capripoxvirus in the family Poxviridae, causes a contagious disease affecting goats and sheep. Homologous recombination as a conventional method is commonly used to construct recombinant GTPVs but generally with genetic markers, such as enhanced green fluorescent protein (eGFP) and guanine phosphoribosyl transferase (gpt). We have previously constructed a recombinant GTPV, which can efficiently express the EG95 antigen of Echinococcus granulosus, but contains eGFP and gpt markers in viral genome. In this study, our previous GTPV-generating system was modified by reconstruction of a Loxp-containing transfer plasmid for deleting markers using the Cre/Loxp system. Meanwhile, the previous method was significantly improved by introduction of an immortalized goat testis cell line as a substitute for primary cells. Based on the latest system, a marker-free recombinant GTPV was reconstructed for expressing the EG95 antigen, and showed neither a significant difference in replication kinetics from its parental virus nor mutations in the foreign sequence during serial 10 passages in vitro.