Construction Of Pseudorabies Virus GXLB-2015,GXGG-2016 GI/gE Gene Deletin With Egfpexpressing Strain And Its Biological Characteristics

Pseudorabies virus(PR)is an acute infectious disease caused by pseudorabies virus(PRV),Causing number of animals infected including carnivores,ruminants,rodents and other animals.Since 2011,the outbreak of pseudorabies caused by variant strains has makedhuge economic loss to the pig breeding industry in China..The gI/gE gene is the main virulence gene of pseudorabies virus,which can reduce the virulence of the virus by deleting its function.The investigation found that pseudorabies virus infection was common in some large-scale pig farms in Guangxi.The recombinant strain GXLB-2015(Recombinant of mutants and vaccine strains)and the natural Tk gene deletion strain of GXGG-2016,were isolated from the samples collected in Guangxi.In this experiment,the plasmid pMD18T-LR was constructed using the pseudorabies virus GXLB-2015 strain and GXGG-2016 strain as templates,and EGFP expression sequence(fluorescent labeling)was inserted by restriction endonuclease digestion and T4 DNA ligase ligation.The recombinant plasmid pMD18T-L-EGFP-R corresponding to GXLB-2015 strain and GXGG-2016 strain was constructed;GXLB-2015 and GXGG-2016 gI/gE gene double deletedand EGFP expressing strain rGXLB-2015-? gI/gE-EGFP and rGXGG-2016-? gI/gE-EGFP were successfully constructed by homologous recombination method.In order to understand the growth characteristics and pathogenic to mice of recombinant strain(GXLB-2015 and GXGG-2016 gI/gE double gene deletion and EGFP expressing strain),Plaque assays and the multi-step growth curve was performed on;The mice were challenged with differen titer of four virus and determine the viral content in the brain and lungs.Result show:gI/gE double gene deletion and EGFP-expressing recombinant strains shows smaller in plaque size and lower growth characteristics.gI/gE double gene deletion shows differen influence to the time to began to proliferate rapidly of rGXGG-2016-gI/gE-EGFP and r GXLB-2015-? gI/gE-EGFP;rGXGG-2016-? gl/gE-EGFP shows no pathogenic to mice as GXGG-2016 does,under the experiment doses;r GXLB-2015-? gI/gE-EGFP display a lower median lethal dose than GXLB-2015 but the onset time is longer,the condition is lighter and the course of disease is longer,This work preliminarily understood the effect of gI/gE double gene deletion and EGFP-expressing on the growth characteristics of GXLB-2015 and GXGG-2016.Providing a reference for the construction of gI/gE double gene deletionin in the next.Laying the foundation for the expresses other virues protein as a potential carrier.