Construction Of TK/gI/gE Gene-deleted Recombinant Pseudorabies Virus With Double Copy Of GC Gene And Evaluation Of Its Immune Efficacy

Pseudorabies is an acute infectious disease caused by animals infected with Pseudorabies virus（PRV）.There is no effective drugs for pseudorabies currently,and vaccine still is an effective means of preventing and controlling the disease.Since2011,pseudorabies has occurred in some vaccinated farms,and swept across the whole country quickly.A new epidemic strain was isolated from a swine farm infected with pseudorabies,and it was identified as a pseudorabies mutant strain.This indicated that the vaccine represented by Bartha-K61 can no longer provide effective protection against the epidemic strains.Therefore,it is urgent to develop vaccines for the epidemic of pseudorabies.In this study,with homologous recombination technology,the TK genes of the rPRV-AH-gI^-/gE^-genome were deleted to obtain rPRV-AH-TK^-/gI^-/gE^-and the PRV gC gene expression cassette was inserted at the gI/gE gene deletion position of rPRV-AH-TK^-/gI^-/gE^-to obtain rPRV-AH-TK^-/gI^-/gE^-/gC⁺.On this basis,biological characteristics and immunogenicity of rPRV-AH-gI^-/gE^-/gC⁺were evaluated.The main research is described as follows:1.Construction of rPRV-AH-TK^-/gI^-/gE^-and rPRV-AH-TK^-/gI^-/gE^-/gC⁺.TK genes deletion homologous arms LA1 and RA1 were amplified with PRV-AH strain as a template by PCR and then inserted into the pMD-18 T vector to construct the plasmid pMD-LA1-RA1.EGFP expression cassette was amplified with pEGFP-N1 as a template by PCR to construct a transfer plasmid pMD-LA1-EGFP-RA1 expressing green fluorescent protein.The plasmid pMD-LA1-EGFP-RA1 was transfected into BHK-21 cells by the lipofection,and then inoculated with the rPRV-AH-gI^-/gE^-.With green fluorescent protein as a screening marker,fluorescent recombinant virus was screened to obtain rPRV-AH-TK^-/EGFP⁺/gI^-/gE^-.To delete the EGFP gene,the same method was used to make homologous recombination of rPRV-AH-TK^-/EGFP⁺/gI^-/gE^-and plasmid pMD-LA1-RA1 to obtain rPRV-AH-TK^-/gI^-/gE^-without EGFP gene with screening.The gE-deficient transfer plasmids pMD-LA-RA and pMD-LA-EGFP-RA were constructed with methods described above.Homologous recombination was occurred into rPRV-AH-TK^-/gI^-/gE^-and the plasmid pMD-LA-EGFP-RA to obtain rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺with screening.Based on the plasmid pgC-His-N1constructed in the early stage of the laboratory（replace the EGFP gene of the pEGFP-N1 plasmid with the gC gene coding region of PRV-AH strain）,the His tag was introduced into the 5’end of the gC coding region sequence（CDS）to construct plasmid pgC-His-N1.The CMV-gC-SV40 poly A expression cassette was amplified by PCR and inserted into the plasmid pMD-LA-RA to construct the plasmid pMD-LA-gC-RA.Homologous recombination was occurred into rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺and the plasmid pMD-LA-gC-RA to obtain rPRV-AH-TK^-/gI^-/gE^-/gC⁺.2.Study of the biological characteristics of recombinant virusesrPRV-AH-TK^-/gI^-/gE^-/gC⁺and rPRV-AH-TK^-/gI^-/gE^-were passaged continuously and the 1st,10th,and 15th generations were subjected to PCR identification.The results showed recombinant viruses are genetically stable.The growth curve of rPRV-AH-TK^-/gI^-/gE^-/gC⁺,rPRV-AH-TK^-/gI^-/gE^-and PRV-AH were determined.The results showed that the growth kinetics of the two recombinant viruses were basically similar with parental strains and there was no significant difference among them.The protein expression of the inserted PRV gC expression cassette was identified with Western blot and His-tag was used as the primary antibody.Western blot results showed that the inserted gC genes was normally expressed.3.Evaluation of the immune efficacy and protection of recombinant virusrPRV-AH-TK^-/gI^-/gE^-/gC⁺and rPRV-AH-TK^-/gI^-/gE^-were vaccinated with4-week-old Kunming mice at a dose of 10⁵TCID₅₀,10⁶TCID₅₀and 10⁷TCID₅₀respectively.The safe dose test results showed that no mice died and no clinical symptoms of PRV were observed,which indicated that the two recombinant viruses were safe for mice.4-weeks-old Kunming mice were immunized with rPRV-AH-TK^-/gI^-/gE^-/gC⁺and rPRV-AH-TK^-/gI^-/gE^-at doses of 10⁵TCID_50,10⁶TCID₅₀respectively.The group of mice vaccinated with commercial vaccine was set as a control group.Booster immunization was performed 21d later.At 4 weeks post-booster immunization,all the mice were challenged intramuscularly with 10⁶TCID₅₀PRV-AH.The results showed that the neutralizing antibodies of rPRV-AH-TK^-/gI^-/gE^-/gC⁺immunized groups were higher than that of rPRV-AH-TK^-/gI^-/gE^-immunized groups at the same immunization dose（p<0.05）.After challenge,the protection rates of 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺and 10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺were 75.0%,which was the highest among all the vaccinated groups.In summary,rPRV-AH-TK^-/gI^-/gE^-/gC⁺elicits stronger neutralizing antibodies response,and it can provide better protection for immunized mice.Therefore,rPRV-AH-TK^-/gI^-/gE^-/gC⁺could be a new vaccine candidate strain to lay the foundation for the control of current domestic pseudorabies.