Construction And Immune Response Of GN Genes Deletion Mutant Of Pseudorabies Virus

Pseudorabies virus (PRV) can cause an acute infections disease, which is characterized by extreme itching and encephalomyelitis in domestic animals and wildlife. Now vaccination is the main measures to control the spread of PRV. In the study, the gN gene deletion mutants rPRV-gE-/TK-/gN-and rPRV-Ba-gN-without reporter gene were constructed from the PRV mutant rPRV-gE-/TK-and the vaccine stain Bartha K61(PRV-Ba) using a Cre-loxp system, and the growth characteristic and immune response of the recombinant PRVs were investigated. This study provides a candidate strains for the development of recombinant attenuated PRV vaccine and its vectored vaccine.1Construction of gN gene deletion mutants and their growth characteristicThe gN genes were amplified by PCR from PRV-JSZ strain as recombinant arms, enhanced green fluorescent protein (EGFP) was chosen as a reporter gene, and then the transfer vector pSKgNRL-GFP with two loxP sites was constructed by insertion of the EGFP gene into gN gene. The transfer plasmid pSKgNRL-GFP was co-transfected with the genomic DNA of rPRV-gE-/TK-into PK15cells. After agar overlay, plaques with fluorescence caused by the mutant rPRV-gE-/TK-/gN-/GFP+were collected, then the gene deletion mutant was purified by limitd dilution method. The EGFP gene was knocked out by using Cre recombinase, and the gN gene deletion mutant rPRV-gE-/TK-/gN-was also purified by limitd dilution method. The gN gene deletion mutant rPRV-Ba-gN-was obtained by the same method. The deletion of195bp segment in gN gene of rPRV-gE-/TK-/gN-or rPRV-Ba-gN-was confirmed by PCR and sequence analysis.Compared with parental strain, the growth speed of rPRV-gE-/TK-/gN-was slightly slow in the early and the titer of recombinant virus was not affected in PK15cells, but the growth speed of rPRV-gE-/TK-/gN-was significantly slower and the titer of recombinant virus was reduced10-fold lower in BHK21cells. The results showed that a certain impact in vitro proliferation characteristics of virus was caused by the further deletion of gN gene,but the influence on growth of recombinant virus was diverse in different cell lines.Compared with the parental strain PRV-Ba, the growth speed of rPRV-Ba-gN-was significantly slower and the titer of recombinant virus was100-fold lower in both PK15cells and BHK21cells. The results demonstrated that a certain impact in vitro proliferation characteristics of virus was caused by the deletion of gN gene in PRV-Ba strain.2Immune response of deletion mutantsCompared with the wild type strain PRV-JSZ, The LD50results showed that the deletion of gE, TK and gN genes could cause a significant attenuation in virulence. Since the LD50s of both strain rPRV-gE-/TK-/gN-and rPRV-gE-/TK-were more than1×106TCID50, the virulence of the mutant and the partantal strain need to be further evaluated. Compared with the partental strain PRV-Ba, the virulence of rPRV-Ba-gN-was attenuated, indicating that the gN gene deletion may further reduce the virulence of the PRV-Ba virus.Compared with the survival rates of immunized mice after challenge with the virulent PRV-JSZ, the protection rate of rPRV-gE-/TK-/gN-group was higher than rPRV-gE-/TK-group when once immunization was adopted. The two groups had the similar protection rate when twice immunization was adopted. The protection rates of rPRV-gE-/TK-/gN-group and rPRV-gE-/TK-group were both higher than that of PRV-Ba group. The protection rate of rPRV-Ba-gN-group was slight higher than that of PRV-Ba group. As the immune protection and immune dose were positively correlated, and the protection rates of groups with tiwce immunization were higher than the group with once immunization, and groups immunized with106TCID50had similar protection rates in both once and twice immunization.The results of serum antibody showed that rPRV-gE-/TK-/gN-group induced significantly higher antibody levels (p<0.05) when compared with rPRV-gE-/TK-group. The rPRV-Ba-gN-group induced slight higher antibody levels (p>0.05) when compared with PRV-Ba group. The antibody levels was higher when the mice were immunized with higher dosage. The antibody levels in the twice immune groups were higher than that in the once immune groups. The higher antibody levels of sera were correlated with higher protection against challenge. In conclusion, the two gN mutants could provide effective protection for mice against virulent PRV challenge, and the recombinant rPRV-gE-/TK-/gN-has a higher protection rate.