Cloning,Expression And Related Functional Analysis Of Pseudorabies Virus UL12 Gene

Pseudorabies virus(PRV)is an important pathogen of pigs and is widely spread around the world.For a long time,PRV has been widely concerned and studied by veterinarians,virologists and neurobiologists.The current research on PRV has provided a comprehensive reference for the pathogenesis of herpes virus.In 2018,Shanghai,China reported the first case of human infection with PRV,which attracted the attention of relevant researchers.Herpes Simplex Virus(HSV)The UL12 gene encodes an alkaline nucleic acid enzyme(Alkaline nuclease,AN),which is encoded by a co-conserved sequence in the herpesviridae genome.Studies have reported that HSV UL12-induced mtDNA stress response triggers innate antiviral response.In this study,the PRV UL12 gene was used as a research object,and its related functions were initially explored.The main content is:1.Construction of pEGFP-N1-UL12 recombinant plasmid and bioinformatics analysis of PRV UL12 proteinThe specific primers were designed with reference to the PRV UL12 gene sequence in GenBank.The PRV-XJ strain was used as a template to PCR-amplify the UL12 target gene and successfully construct the pEGFP-N1-UL12 recombinant plasmid.The sequence of PRV UL12 amino acid was analyzed by bioinformatics method.prediction.2.PRV UL12 eukaryotic expression and its cellular localizationpEGFP-N1-UL12 was transfected into BHK21 cells,Western blot was used to verify the expression of UL12 protein,and the target protein was detected in the culture supernatant and cells.UL12-GFP fusion protein was detected by cell localization,and the results showed that UL12 gene The product is distributed in both the nucleus and the cytosol.3.Research on the related functions of PRV UL12 geneAfter transfection of PRV UL12,the expression of UL12-GFP green fluorescent protein was observed by flow cytometry.The expression of caspase-3 gene wasdetermined by ?-actin as an internal reference.The expression level of gB after transfection of UL12-expressing BHK21 cells with PRV was drawn,and a one-step growth curve was drawn to monitor the proliferation dynamics of PRV.The results showed that the fluorescence morphology of the experimental group was significantly rounded compared with the empty group;the apoptosis rate of the experimental group was significantly increased;the caspase-3 was not significantly differently expressed;the virus content of the experimental group was slightly lower than that of the control group.In summary,this study was the first to use the PRV-XJ strain as a template to amplify the UL12 gene,construct a recombinant plasmid pEGFP-N1-UL12,and make a preliminary study on the partial function of the PRV UL12 gene,and also explore the future of HSV UL12 gene.The direction provides partial reference basis.