Construction Of A ORFV009 Gene-deleted Mutant Based On CRISPR/Cas9 Gene Editing Technology And Evaluation Of Immune Effect

Contagious Ecthyma,also known as Orf,is an acute,high-contact infectious disease caused by Orf virus(ORFV)in sheep,goats,and small wild ruminants.Animals' lips,breasts,reproductive organs,etc infected with the disease are most vulnerable,and skin ulcers,blisters,crusts usually appear in the affected area.Orf is also a zoonotic disease that not only causes huge economic losses to the sheep breeding industry,but also threatens the lives and health of related personnel.There is no effective treatment for Orf.Vaccination is the most effective means of prevention and control.However,due to the unique immune evasion mechanism of Orf virus,there are still many deficiencies in the commercially available inactivated vaccines and attenuated vaccines in terms of immune protection effects.As a new type of vaccine,the gene-deleted vaccine has a very broad prospect in the development of a vaccine for Orf.Homologous recombination gene knockout technology is a commonly used method for constructing gene-deleted strains,but traditional homologous recombination methods have a low success rate and take a long time.In this study,we used CRISPR / Cas9 gene editing technology to cause DNA double-strand breaks(DSBs)in the target region of the gene deletion in advance,which greatly increased the probability of homologous recombination in the target region.We constructed a homologous recombination shuttle plasmid pUC57-vv7.5-EGFPORFV009;sgRNA expression vector plasmids gRNA1,gRNA2,gRNA3,gRNA4;Cas9 expression vector plasmid these three vectors by PCR amplification,oligonucleotide sequence synthesis,vector ligation,and other methods.We co-transfected the sgRNA expression vector plasmid and Cas9 expression vector plasmid into OFTu cells through co-transfection experiments,and then inoculated the cells with Orf virus strain SY17 virus,causing double-strand breaks(DSBs)in the ORFV 009 gene region.Next,the homologous recombination shuttle plasmid was transfected,and the probability of homologous recombination was increased by means of homologous recombination repair(HDR).We observed the green fluorescence under a fluorescence microscope to obtain the ORFV 009 gene deletion strain.Subsequently,the limiting deletion method was used to purify and screen for the gene-deleted virus,and perform PCR identification and gene sequencing identification of the screened gene-deleted virus.A strain of ORFV 009 gene deletion was successfully obtained.Moreover,it was proved that the success rate of constructing the ORFV 009 gene deletion strain based on CRISPR / Cas9 gene editing technology was as high as 91.6%,which improved the efficiency by more than 20 times compared with the traditional homologous recombination method.In this study,the ORFV 009 gene deletion strain was used as a vaccine candidate strain to determine the cytopathic(CPE)ability,one-step growth curve and other biological characteristics of the gene deletion virus.The results showed that the virus of the ORFV 009 gene deletion strain could normally infect cells and had no significant difference in replication ability from the original virus.The ORFV 009 gene-deficient strain was then inoculated with the original virus and PBS control to a 3-month-old lamb,which proved that the pathogenicity of the ORFV 009 gene-deficient strain was significantly reduced compared with the original virus.The ORFV 009 gene deletion strain and PBS control were used to immunize 3-month-old lambs,and booster immunization was performed 3 weeks after the inoculation.Lamb serum was collected weekly to measure its specific antibodies and cytokine levels using indirect ELISA and cytokine detection kits.The results showed that the ORFV 009 gene-deficient strain can induce cellular and humoral immunity,but the level of humoral immunity is not high.It is dominated by cellular immunity,especially Th1 type immune responses.After 6 weeks of immunization,Orf virus strain SY17 strain was used to test the protection of the lambs in the experimental group and the PBS control group.The PBS control group all had the disease and the experimental group had no disease.Experimental immune protection rate is 100%The above research results provide an important method basis and material basis for the development of a gene-deficient vaccine for Orf,and have important significance for the prevention and control of Orf.