Purification Of Recombinant Virus Deleting Goat Pox Virus KLP2 Gene And Construction Of ANK Gene Deletion Virus

Goat pox is a worldwide epidemic,and vaccination immunization is a more effective way to prevent this disease.However,due to the use of attenuated vaccines,some side effects are often caused during use,such as abortion of pregnant sheep and toxic recovery.In order to further reduce the toxicity of the vaccine,the recombinant sheep pox virus vaccine strain lacking the virulence factor KLP2 obtained by the cell recombination method was purified to obtain a safer sheep pox vaccine.At the same time,in order to study the role of another virulence factor ANK of goat pox,an expression vector lacking the gene virus was constructed,which provided a basis for further research on safer and more effective sheep pox vaccine.Firstly,2-3 months old healthy ram testicles and healthy developmental fetal sheep were taken aseptically,and testicular tissue and fetal sheep skin tissues were separated,and cultured with shredded tissue and trypsin-digested dispersed cells to obtain well-grown testes.Primary cells of fetal sheep skin and their culture characteristics were studied.When the cells were adherently grown to 80%-90%,the goat pox virus vaccine strain was inoculated,and the cytopathic effect was observed.Next,the KLP2 deleted transfer vector plasmid was extracted and transfected into goat poxvirus-infected sheep testicular primary cells with Lipofectamine ~TMM 2000,and the recombinant virus with green fluorescence was observed under a microscope.Picking up primary cells with fluorescent cells to inoculate recombinant virus by four methods.The fluorescence density and PCR method were used to determine whether the recombinant virus was purified.Once again,the purified recombinant virus was inoculated into the primary testis cells,and the growth rate and degree of cytopathic effect were observed to evaluate the effect,and the vaccine strain and the prevalent strain were used as controls;the skin was scraped and combined with subcutaneous injection.The recombinant virus was used in mice,and the vaccine strain and the epidemic strain were used as controls.The clinical changes of the mice were observed,and the effect of the recombinant virus was evaluated at the animal level.In addition,the homologous arm of both sides of the ANK gene was ligated to the GFP side of the reporter gene by fusion PCR,and the expression vector of ANK gene deletion was obtained by ligation with the pET42b vector,and transfected into the sheep infected with the sheep pox virus vaccine strain.Primary testicular cells were microscopically examined for recombinant virus with green fluorescence.Results The primary cells of lamb testis and the primary cells of fetal sheep skin were successfully prepared.The culture characteristics of testicular cells were superior,and they were used as cells for subsequent experiments in this study.The purification method of the KLP2gene-deleted recombinant virus is the most effective in the fourth type,and the purified virus can be obtained after 3 to 4 generations.Compared with vaccine strains and epidemic strains,the virulence effect of recombinant virus has a macroscopically weakening effect at the cellular level,and there is no obvious clinical effect in the mouse test.In this experiment,a transfer vector containing two gene deletions of ANK gene goat pox virus THX strain GTPV-138 and GTPV-141.2 was successfully constructed,and a recombinant virus lacking the target gene was obtained.In this study,a faster method for constructing a sheeppox gene deletion recombinant virus and a fluorescently labeled virus was developed to lay the foundation for the study of the biological characteristics of late sheep pox virus.