Study On The Effect Of P53 On The Replication And Pathogenicity Of Pseudorabies Virus In Mice

Pseudorabies virus(PRV)is one of the most important pathogen that disturbs our local swine industry in China.From the 1980s,PRV began to outbreak at each part of the nation one after another,and according to the results of sequencing,the popular PRV strains outbreak in China are different from which in Europe and the United States,which is in another branch of evolution tree.Once the pig is infected by PRV,it is hard to wipe it outside of the body,causing the virus lifelong emission for the pig,and the death of the piglets.It just give a huge hit to the development of agricultural economy of China.P53 plays an important role as "the guardian" of the animal genome.P53 gene,also known as TP53 gene,has a molecular weight of about 43.7Ku and is regulated by phosphorylation.P53 locates in the 12th chromosome of the pig and regulates the transmission of various biological signals in the cell.The P53 gene has the function of anti-virus,and it can interact with p53 after it invades the body.1.The establishment of PRV gB gene real-time PCR detection methodFirst we design a pair of primers that specific amplificate PRV virus(Bartha strains)gB gene in RT-qPCR,and then we get the purpose gene amplification,we purifying PCR product and put it into the pMD19T plasmid in T-A clone,finally we successfully build the recombinant plasmid pMD-gB.we dilute the recombinant plasmid by 10 times dilution,using SYBR Green fluorescence qPCR established gB gene amplification standard curve,the curve equation is:y = 3.8212 + 41.587 x,R2 = 0.9997,linear relationship of standard curve is good,can be used as the:method for the following experiment.2.p53 affects PRV's replication and pathogenicity in miceBefore the challenge,we prepare a bunch of PRV viral materials and successfully checkup the p53 gene knockout c57BL/6 mice by western blotting.And then challenge wild type(WT)and p53 type knockout(p53 KO)mice at about 1.0 ×104 TCID50 PRV,and then we harvest two groups of mouse's brain.The following experiment is what we have done:Viral loads and viral TCID50,survival rate experiment,tissue's pathological section for H.E staining and observation,determination of inflammatory cytokines' mRNA and antibody levels.We found that the p53 gene knockout can reduce PRV replicate in mice,and weakened its pathogenicity.3.Differential transcriptomic profile of PRV-infected mouse brainAfter challenge PRV into WT mice and p53 KO mice by intracerebral injection,we harvest its brain tissue of each group to do the different expresed genes(DEGs)analysis,and validate the microarray's 16 DEGs by using quantitative PCR,determine the transcriptome data's accuracy.The results found that the p53-KO mice after PRV challenge,the interferon and chemokine gene or other antiviral related genes were significantly down regulation than WT mice.through the experiment of WT mice challenge vs control,it shows that the p53 KO mice is not susceptible to PRV,further verify the result of the second part.In conclution,this study demonstrated that the knockout of p53 gene inhibited the replication and pathogenicity of PRV in mice.