Construction Of A GI/gE Gene-deleted And Dual Expressing GC Gene Pseudorabies Virus And Evaluation Of Its Immune Efficacy

Pseudorabies virus?PRV?is also called SuidHerpesvirus 1,and it belongs to Herpesviridae,Alphaherpesvirinae.It can cause porcine pseudorabies.Vaccine immunization is the most effective way to prevent and control this disease.But since2011,many pig herds in C hina have been suffering from the epidemic of pseudorabies even after the use of PRV vaccine which showed that current PRV vaccines have been unable to provide adequate protection against the infection of epidemic strains,it is necessary to develop new vaccines.In this study,rPRV-AH-gI^-/gE^-/EGFP⁺was used as the parent strain with the gI and gE gene-deleted.PRV gC gene was inserted into the deletion region to construct the recombinant virus rPRV-AH-gI^-/gE^-/gC⁺by homologous recombination.On this basis,the growth characteristics,pathogenicity and immunogenicity of rPRV-AH-gI^-/gE^-/gC⁺were evaluated.The detailed contents are as follows:1.Construction of the recombinant virusrPRV-AH-gI^-/gE^-/gC⁺The EGFP coding sequence in pEGFP-N1 plasmid was replaced by gC gene of PRV AH-China-2013.The P_CMV-gC-SV40 polyAexpression cassette was obtained by PCR amplification.And then,the recombinant plasmid pMD-LA-gC-RA was successfully constructed by inserting P_CMV-gC-SV40 polyAexpression cassette into pMD-LA-RA.The plasmid p MD-LA-gC-RA was transfected into BHK-21 cells and then inoculated with rPRV-AH-gI^-/gE^-/EGFP⁺to make the plasmid and viral genome homologously recombine in the cell.Recombinant virus rPRV-AH-gI^-/gE^-/gC⁺was obtained by single cell isolation and plaque purification.By PC R detection and Fluorescence microscopy,it was proved that the gC gene has been successfully recombined into the virus and the EGFP ge ne has been removed.2.Study of the biological characteristics of the recombinant virus rPRV-AH-gI^-/gE^-/gC⁺ThegrowthcurveofPRV-AH,rPRV-AH-gI^-/gE^-/EGFP⁺and rPRV-AH-gI^-/gE^-/gC⁺were determined.The results showed that the three kinds of viruses have similar growth kinetics and the highest titer of them was10^-7.63TCID₅₀/mL,10^-7.00TCID₅₀/mL and 10^-7.38 TCID₅₀/mL,respectively.The difference of gC gene in the transcription level of mRN A between rPRV-AH-gI^-/gE^-and rPRV-AH-gI^-/gE^-/gC⁺was tested byreal-time fluorescence quantitative RT-PCR.The results showed that the amount of gC gene in the recombinant virus rPRV-AH-gI^-/gE^-/gC⁺is 1.85 times of it in rPRV-AH-gI^-/gE^-at 6h after inoculated BHK-21 cell,about 35.93 times at 12h and 40.64 times at 24h.3.Evaluation of the immune efficacy of the recombinant virus rPRV-AH-gI^-/gE^-/gC⁺The immune efficacy ofinactivated rPRV-AH-gI^-/gE^-/gC⁺vaccine was evaluated in K unming mice.The mice were immunized with the dosage of 10⁵ TCID₅₀ and 10⁶TCID₅₀two times with the interval of 28d respectively.After immunization,the mice were infected with lethal dosage of the classical pseudorabies virus strain or PRV-AH strain.The protective rate was 100%in mice treated with 10⁶ TCID₅₀ dosage of the inactivated vaccine against two strains.The mice immunized with 10⁵ TCID₅₀ dosage of the inactivated vaccine had a protective rate of 87.5%when attacked by classical pseudorabies virus.In conclusion,a gE/gI gene-deleted and dual expressing gC gene pseudorabies virusrPRV-AH-gI^-/gE^-/gC⁺wasconstructedsuccessfully.Comparedwith rPRV-AH-gI^-/gE^-,the gC gene of rPRV-AH-gI^-/gE^-/gC⁺was increasedsignificantly in the mRN A transcription level.The recombinant virus rPRV-AH-gI^-/gE^-/gC⁺had good immunogenicity and provided good protective efficacy against the classical pseudorabiesvirusstrainandPRV-AHstraininmice.Therefore,rPRV-AH-gI^-/gE^-/gC⁺can be a new vaccine candidate to control the current epidemic of pseudorabies in C hina.