Construction And Immunological Efficacy Of A Pseudorabies Virus GI/gE/TK Gene Deletion Strains With Double Fluorescent Tag

Pseudorabies Virus(PRV)is the second type of animal disease in OIE,which is popular in China.At present,it is mainly to use Bartha K61 vaccine to prevent immunity.However,more and more reports indicate that the vaccine immunization piggery appears to be PRV or pig serum to the positive.Gene deletion vaccine of PRV native strains is the study for further prevention of PRV infection.First,we reconstructed the Puc57 plasmid and added the TK gene homologous arm and red fluorescence expression cassette.We successfully constructed Puc57-TK-RED transfer vector containing red fluorescence expression cassette and TK homologous arm.Subsequently,the PRV XJ gE/gI double deletion virus genome DNA and transfer vector plasmid DNA were co-transfected into the cells,and some TK genes were deleted by homologous recombination.The missing strains were repeatedly screened and purified by the red fluorescence label.After purification of20 generations,the specificity of gE,gB,purification of virus TK gene PCR deletion virus detection,and to observe the lesions using a fluorescence microscope.Finally the identification results show that the experiment successfully constructed the deletion of PRV gE,a plant can be red and green fluorescence gB,TK gene PRV XJ gI/gE/TK three gene deletion strains.Subsequently,the stability of the PRV XJ gI/gE/TK three deletion mutant was identified.The results showed that the deletion process was stable without exogenous virus contamination,and the virulence declined.Then the virus titer of PRV XJ gI/gE/TK three deletion mutant was detected,and the growth curve of the deleted virus was drawn.The growth characteristic of the virus was similar to that of the parental PRV XJ strain,PRV XJ gE/gI double gene deletion strain.The experimental results showed that the PRV XJ gI/gE/TK three gene deletion strain had good safety,good immunogenicity,and could stimulate the mice to produce high effective neutralization antibody.In conclusion,PRV XJ gI/gE/TK three gene deletion strain can be used as an excellent vaccine candidate strain,which is significant for further development of new PRV gene deletion vaccine.