Construction Of Cbp/gif Double Gene-deleted Contagious Ecthyma Virus And Its Biological Characteristics

Contagious ecthyma CE(Contagious ecthyma CE),also known as Orf(Orf),is a contagious and epithelial infectious disease of goats caused by Contagious ecthyma virus(CEV).The disease has a great impact on the feeding of goats,causing the sick goats to become weak and even die.The disease is prevalent in all goats-raising countries in the world,seriously affecting the economic benefits of the goats-raising industry.There is still no specific cure for CE,and vaccine immunization is an ideal means to prevent and control the disease,but there is no commercial CEV vaccine in China.Immunity against CEV is mainly cellular immunity,so traditional inactivated vaccines have limited or even ineffective effects.However,the attenuation mechanism of passaged live attenuated vaccines is still unclear,and there is a risk of returning to strong virulence,which makes it difficult to guarantee the safety of the vaccine.Therefore,the use of gene knockout technology to delete the main virulence genes of CEV and construct an attenuated candidate vaccine strain with missing virulence genes is an important research direction for CEV's vaccines.Chemokine binding protein(CBP)and granulocyte-macrophage colony stimulating factor inhibitory protein(GIF)are two important virulence proteins of CEV.CBP is a protein expressed in the early stage of infection,while GIF is in the middle and late stages of viral infection.Expressed,these two virulence proteins play an important role in evading the surveillance and clearance of the host immune system by CEV.Studies have shown that deletion of the cbp gene can significantly weaken CEV.In addition,there is a significant difference between the gif gene of the CEV vaccine strain and the virulent strain.Therefore,this study intends to use homologous recombination technology to knock out the cbp gene and gif gene of CEV,and construct a CEV cbp/gif double gene deletion strain rGS14?CBP?GIF,in order to obtain a safe and effective vaccine candidate strain for the ovine aphtha virus gene deletion live vaccine,to provide technical support for the prevention and control of the CE epidemic in our country.In this study,the DNA of the CEV GS14 strain was used as a template to amplify the upstream and downstream homology arms of the cbp and gif genes,and the GFP expression box containing the lox P site in the same direction was amplified from the EGFP-N1 plasmid,and the fusion PCR method was used to After the above three sequences were connected,they were transfected into CEV-infected goat testis primary cells,and the fluorescent single-gene deletion strains rGS14 GFP?CBP and rGS14GFP?GIF were screened and purified.Cre recombinase was used to knock out the GFP expression frame,and the non-fluorescent recombinant viruses rGS14?CBP and rGS14?GIF were screened and purified.The obtained recombinant virus rGS14?CBP was used to knock out the gif gene according to the same strategy.After screening,purification and identification,the obtained CEV double gene deletion recombinant virus was named rGS14?CBP?GIF.The biological characteristics of the double-gene deletion strain rGS14?CBP?GIF were studied,including the determination of the TCID₅₀ of the recombinant virus,the observation of its in vitro CPE,the determination of a one-step growth curve,the establishment of a real-time fluorescent quantitative PCR method to draw a virus content curve,and the determination of the recombinant virus's effect on goats.Safety and immune challenge protection test by establishing disease model.The results showed that the TCID₅₀ of the recombinant virus rGS14?CBP?GIF strain was 10^-6.3/0.1m L,and its in vitro CPE,growth curve and virus content were not significantly different from those of the parent strain GS14;Inoculate CEV antibody-negative 1-month-old goats with different concentrations of the diseased lamb crust grinding disease liquid and observe for 21 days to determine the lowest concentration of 10^-2 to establish a model of infectious pustular dermatitis in goats;Single-gene deletion strains rGS14?CBP strain,rGS14?GIF strain,and double-gene deletion strain rGS14?CBP?GIF with 10⁶TCID₅₀ were used to scratch and inoculate CEV antibody-negative 1-month-old goats'lips.Each strain was inoculated with5 goats.Observed for 21 days,the mental state and feeding of all goats were normal,and there were no local or systemic adverse reactions,which proved that the 3 strains have good safety;20 1-month-old goats were divided into 4 Groups,each with 5 heads,among which groups 1-3 are immunized groups,respectively immunized with 2 single gene deletion strains and 1 double gene deletion strain with 10⁵TCID₅₀,and the fourth group is the unimmunized control group.On the next 21 days,the smallest concentration of goats'infectious pustular dermatitis clinical material was used to challenge the virus.As a result,all the unimmunized control groups were affected,and the protection rate of the rGS14?CBP strain immunized group was 40%(2/5)The protection rate of rGS14?GIF strain immune group was 50%(2/4),and the protection rate of rGS14?CBP?GIF strain immune group was 100%(4/4),which proved that rGS14?CBP?GIF strain has good immunogenicity.The results of this study show that the CEV cpb/gif double gene deletion strain rGS14?CBP?GIF is an ideal vaccine strain candidate for the CEV gene deletion live vaccine.