Constrction And Biological Characteristic Identification Of Goatpox Virus Gene Deleted Mutant

Goat Pox caused is an acute feverish and highly contagious disease by goatpox virus (GTPV), has a very high morbidity and mortality. In China, the live attenuated GTPV vaccine strain AV41 is widely used to control goat pox. Some reports showed that it may cause generalized skin nodules and abortions in local breeds of goats and imported pure breeds and crossing breeds of goat in southern China, Therefore, it is necessary to develop a more safe and effective engineering vaccine of GTPV.In this study, the attenuated GTPV virus vaccine strain AV41 was regarded as templates. Two pairs of primers were designed, and four genome fragments of AV41 ORF3～ORF5 and ORF19～ORF20, ORF148～ORF149 and ORF150～ORF151 serving as flanking sequences of homologous recombinant arm were amplified by PCR, respectively. Then, two shuttle vector plasmids, pLF-Eg and pRF-Eg, were constructed by ligation of ORF3～ORF5 and ORF19～ORF20, ORF148～ORF149 and ORF150～ORF151, respectively, and insertion of the expression cassettes of EGFP gene and gpt gene. Vero cells pre-infected with GTPV AV41 were subsequently transfected with two resulting plasmids, respectively. Finally, through homologous recombination, selection culture, purification and identification, recombinant GTPV with 15 ORFs deleted and another recombinant GTPV of whose 3 ORFs were deleted were obtained. Results of further biological characteristics identification demonstrated that two recombinant viruses can keep genetic stability at least 15 passages, and they share similar growth character and pathogenic character with their parental virus, AV41, in cell culture. This study lay a firm basic for further development on more safe and efficient live attenuated GTPV vaccine strain, and provided the researches on live vector vaccines basing on recombinant GTPV with valuable information.