Construction And Characteristics Of Gene-deleted Strain JL14-?gI/gE/TK Of Pseudorabies Virus

Pseudorabies?PR?caused by pseudorabies virus?PRV?is an acutely infectious disease in swines,bovines,caprids and other animals,of which swine is its major host.PR has spread to most parts of the country,causing huge economic losses in the pig industry.There is no specific medicine for PR,so that preventive vaccination serves as the main way to control the rise and epidemic of PR.In this study,an epidemic virus strain PRV JL14 was isolated.The recombinant virus PRV JL14-?gI/gE/TK was constructed by deleting its gI,gE and TK genes with homologous recombination technique.Then,the characteristics of the recombinant virus were preliminarily studied contributing to the development of porcine pseudorabies vaccine and virus vector vaccine.The results are as follows:1.PRV JL14 isolation and identificationIn 2014,PRV was isolated from the brain of aborted piglets in Jilin City.The virus particles were observed by transmission electron microscope,which showed typical herpesvirus structure.The gB and gC gene fragments of the virus were amplified by PCR.After sequencing and phylogenetic tree analysis,the strain was identified as the domestic epidemic variant strain after2011 and named as PRV JL142.Construction of recombinant virus PRV JL14-?gI/gE/TKIn this study,the homologous regions LgI/gE,RgI/gE and LTK,RTK flanking the gI/gE and TK gene were amplified by PCR using PRV JL14 as template after these PCR productswere inserted into pUC19 vector to obtain the transfer vectors pUC-?gI/gE and pUC-?TK.Then,I amplified the EGFP expression cassette by PCR and inserted the products into the space between the left and right arms through In-Fusion enzyme in order to obtain transfer vectors pUC-?gI/gE-EGFP and pUC-?TK-EGFP.PRV-JL 14 and Transfer vector pUC-?gI/gE-EGFP transfected the BHK21 cells via Lipofectamin?2000.Following cell lesions,the recombinant virus PRV JL14-?gI/gE-EGFP was obtained by plaque purification under fluorescence microscope.PRV JL14-?gI/gE-EGFP and pUC-?gI/gE were cotransfected into BHK-21 cells to obtain recombinant virus PRV JL14-?gI/gE by reverse screening.Based on PRV JL14-?gI/gE,pUC-?TK-EGFP and pUC-?TK,we obtainedPRV JL14-?gI/gE/TK in the same way.The knockoutis was confirmed by PCR and sequenced.The results showed that 2681 bp and 912 bp were respectively deleted at the gI/gE and TK.3.Characterization of recombinant virus PRV JL14-?gI/gE/TKIn this study,the one-step growth curves of recombinant virus PRV JL14-?gI/gE/TK and PRV JL14 on BHK-21 cells were plotted,the proliferation titers of two strains can both reach10^7.5TCID₅₀/mL in 30h and show no significant difference in the growth.Inoculating different doses of PRV JL14-?gI/gE/TK into piglets,the piglets produced corresponding antibodies without symptoms,indicating that the strain is safe to the target animal.The piglets immunized with PRV JL14-?gI/gE/TK showed no symptoms after PRV JL14 attack,and the specific gE antibody was not negative.The results showed that PRV JL14-?gI/gE/TK strain obtained in this study has a good safety and immunogenicity to the pig,which is characterized by PRV vaccine candidate strains,and it is of great significance for the epidemic prevention and control of PRV variant strain.