Construction And Immunogenicity Evaluation Of GI/gE Genes Deletion Mutant Of Pseudorabies Virus

Pseudorabies(PR)is an acute infectious disease caused by pseudorabies virus(PRV)in domestic animals and wild animals.A pig is the main host,and it does the greatest harm to pigs.The pig PRV is one of the major viruses that caused great economic loss in pig farms.At present,there is no effective drug of PR in China.In our lab,we isolated PRV-GD2013 and constructed the gI/gE double-deletion strain without marker gene(EGFP)by homologous recombination.Then,we study the immunogenicity of recombinant PRV preliminarily,which is to provide material to study pseudorabies vaccine.The results are as follows:(1)This study was collecting the suspected pseudorabies-infected pigs' brain tissue in a pig farm in Guangdong province in March,2013.The virus was isolated from the brain tissue and then identified by transmission electron microscopy(TEM)and the gB and gE sequences were amplified by PCR.The results of TEM showed that the virus particles have a typical herpes virus structure and the gB and gE genes of pseudorabies virus could be amplified by PCR.The sequence analysis and biological characteristics comfirmed that the isolated strain was a PRV mutant compared with the popular strains in China in recent years,named as PRV-GD2013.(2)The construction of double-deletion vector of g I/gE of PRV.The g I and gE genes were PCR amplified from GD2013 as the upper and lower arm of transfer vector respectively.At the same time,CMV-EGFP-SV40 ployA singal sequence were amplified from pEGFP-C3.Then,gI-EGFP-gE and gI-gE were amplified by Over-lap PCR,and they were combined with pBluescript KS(+)by homologous recombination.So pBLE-gI-EGFP-gE transfer vector and pBLE-gI-gE transfer vector were constructed successfully.BHK-21 cells were co-transfected with PRV-GD2013 virus,pBLE-gI-EGFP-gE transfer vector and Lipofectamin 2000 transfection reagent.The viruses were harvested when 80% cells were infected.PRV-GD2013-?gI/gE-EGFP recombinant viruses were obtained by plaque purification.Finally,BHK-21 cells were co-transfected with PRV-GD2013-?gI/gE-EGFP,pBLE-gI-gE transfer vector and Lipofectamin 2000 transfection reagent.Then PRV-GD2013-?gI/gE recombinant viruses were obtained by plaque purification.The result of PCR indicated that there is a 2169 bp deletion within the gI and gE genes of PRV-GD2013-?gI/gE.(3)Growth Characteristic and immune response of deletion mutant.The highest titers of PRV-GD2013-?gI/gE and PRV-GD2013 in BHK-21 cells were both 108.00TCID50,indicating that the PRV-GD2013-?gI/gE mutant is of a similar growth speed with its parental strain.The piglets doesn't shown any clinical symptoms after being inoculated by PRV-GD2013-?g I/gE of Different doses(n=5),and the PRV specific antibody levels was significantly higher than that in the swines immunned by the Bartha-K61 vaccine.In addition,the toxicity attack test also shows that the swine doesn't show any clinical symptoms and pathological changes after the vaccination with PRV-GD2013-?gI/gE.But the swines vaccinated by Bartha-K61 showed clinical symptoms after being challenged,so they were not completely protected.In conclusion,PRV-GD2013-?gI/gE had good immunogenicity to pigs,and it was a good candidate for a PRV vaccine.