Study On Improving The Immunity Effect Of Porcine Pseudorabies Virus By GM-CSF

Pseudorabies（Pesudorabies,PR）is a highly pathogenic,high mortality and severe infectious disease caused by Pseudorabies Virus（PRV）.It can infect a variety of domestic and wild animals.Pigs are the only survivors after PRV infection.Most of the animals,infected pigs have persistent infections.my country’s prevention and control of PR mainly uses PRV gene-deletion vaccines,which have achieved certain immunization effects.However,since 2011,many immunized pig farms have seen PR epidemics.Later studies have confirmed that new mutations in PRV have occurred.The immune effect of traditional vaccines has declined,so in order to effectively prevent and control PR,it is necessary to improve the immune effect of PR.GM-CSF is the most widely used cytokine used to enhance the immune effect of vaccines.This study tried to useGM-CSF to enhance the immune effect of PR.The PRV gene manipulation platform was successfully constructed using CRISPR/Cas9 technology,and the PRV TK gene was knocked out and replaced.For GM-CSF,the recombinant strain PRV TK^-/eGFP⁺/GM-CSF⁺was constructed and the immune effect was evaluated.The following results were obtained:1.Construction of recombinant PRV TK^-/eGFP⁺Construct the sgRNA plasmid targeting the parental PRV SX-10-2015 TK gene;then amplify the homology arms of the TK gene by overlap PCR,construct the recombinant plasmid pUC19 TK-eGFP,and transfect the recombinant plasmid and sgRNA plasmid into BHK-21 cells according to a certain ratio.After BHK-21 cells were inoculated with PRV,cell clones expressing eGFP were picked and purified to obtain recombinant PRV TK^-/eGFP⁺.Through TCID₅₀,the virus titer was determined to be 1?10^6.43TCID₅₀/0.1 m L;the virus growth curve was drawn and it was found that the growth pattern of PRV TK^-/eGFP⁺and the parent strain were similar,but the virus titer was reduced;the mouse pathogenicity test showed that the lethality rate of PRV TK^-/eGFP⁺in mice was 20%.Histopathology of tissue were slight hemorrhage in the liver and slight thickening of the lung interstitium.2.Construction of recombinant PRV TK^-/eGFP⁺/GM-CSF⁺Construct the recombinant plasmid pUC19 TK-CMV-GM-CSF-eGFP,transfect the recombinant plasmid and sgRNA plasmid into BHK-21 cells according to a certain ratio,inoculate PRV,and pick and purify the cell clones expressing eGFP to obtain the recombinant PRV TK^-/eGFP⁺/GM-CSF⁺.Through TCID₅₀,the virus titer was determined to be 1?10^5.76TCID50/0.1 m L;the virus growth curve was drawn and it was found that the growth pattern of PRV TK^-/eGFP⁺/GM-CSF⁺and the parent strain were similar,but the virus titer was significantly reduced;the mouse pathogenicity test showed that,PRV TK^-/eGFP⁺/GM-CSF⁺is not pathogenic to mice,and the tissues have no obvious lesions.3.Evaluation of immune effect of recombinant PRV TK-/eGFP+/GM-CSF+The mice were immunized with PRV TK^-/eGFP⁺/GM-CSF⁺and PRV TK^-/eGFP⁺,and blood was collected from the mice every 7 days after immunization to monitor the changes in gE antibody levels.It was found that PRV TK^-/eGFP⁺/GM-CSF⁺produced higher levels of gE antibody than PRV TK^-/eGFP⁺.After immunization,the mice were challenged and infected by 1?10⁵TCID₅₀/0.1 m L and 1?10³TCID₅₀/0.1 m L of PRV,and it was found that PRV TK^-/eGFP⁺can provide 60%protection against 1?10⁵TCID₅₀/0.1 m L of PRV infection in mice,and 1?10³TCID₅₀/0.1 m L of PRV infection provides 100%protection,and PRV TK^-/eGFP⁺/GM-CSF⁺can provide 100%protection against mouse 1?10⁵TCID₅₀/0.1 m L and 1?10³TCID₅₀/0.1 m L PRV infection.The mice on the 0th,7th,and 21st day of the challenge were dissected,pathological sections were made,and each was measured.The viral load in the tissue and the expression of related cytokines in the spleen.Pathological sections show that after PRV TK^-/eGFP⁺immunization,1?10⁵TCID₅₀/0.1 m L infection can still produce mild pathogenicity in mice.1?10³TCID₅₀/0.1 m L of PRV infection is not pathogenic,PRV TK^-/eGFP⁺/GM-CSF⁺is not pathogenic to1?10⁵TCID₅₀/0.1 m L and 1?10³TCID₅₀/0.1 m L of PRV infection;Viral load results it was shown that the viral load of the brain tissue of the immunized mice was significantly reduced.On the 7th day of the challenge,the viral load in each tissue reached the maximum;the expression level of cytokine showed that GM-CSF can significantly enhance the expression of IFN-γand IL-4 and does not significantly enhance the expression of IL-6 and TNF-α.In summary,the study has successfully established a PRV gene knockout platform using CRISPR./Cas9 technology,and constructed a recombinant PRV TK^-/eGFP⁺/GM-CSF⁺expressing GM-CSF on this basis.The immunization results showed that GM-CSF has enhanced the immunity effect and has the poten tial to be developed as a vaccine.