Construction Of Genetic Engineering Vaccine Against Pseudorabies Virus And Transcriptome And Proteomic Analysis Of Prv Infected PK-15 Cells

Pseudorabies virus(PRV)is a type of herpes virus that can infect livestocks,pets,fur animals and wild animals,causing fever,itching(except pigs)and encephalomyelitis.PRV can infect pigs of all ages,mainly causing reproductive disorders of breeding pigs,nervous system and respiratory symptoms of piglets.Since 2011,the emergence of PRV mutant strains has reduced the protection level of vaccines such as Bartha-K61.Pseudorabies epidemic occurred in large-scale pig farms immunized with PRV vaccine in many regions of China,which seriously threatened the healthy and sustainable development of the pig breeding industry in China.Therefore,in this study,the etiology,diagnostic technology,new genetic engineering vaccine,transcriptomic and proteomics of PRV mutant strains were studied to shed a light on the effective prevention and control of the disease.The main research findings were summarized as follows:1.In this study,a wild PRV strain was isolated and identified from a pig farm immunized with the Bartha-K61 vaccine in Shandong.The PRV SD-2017 strain was successfully isolated and identified,and the biological characteristics was analyzed.Based on genetic evolution analysis of g E,TK and g B genes,this strain was identified as the current popular variant of PRV in China.Transmission electron microscopy showed that the diameter of the virus particles was about 140?180 nm,and the virions had thick envelope with radiate spikes on the surface of envelope.The core of the virions was compact,showing the typical structure and morphology of herpes virus.The titer of the strain on PK-15 cells was 10^9.0TCID₅₀/m L,and the LD₅₀was3.2×10^2.0TCID₅₀/m L on rabbits.2.In order to differentiate and diagnose PRV vaccine strains from wild strains,the enzymatic recombinant isothermal amplification(ERA)fluorescence detection method for g B and g E genes of PRV was successfully established.In this assay,the sensitivity of g B and g E genes was 10copies/?L and 10³copies/?L,respectively.Compared with fluorescence quantitative PCR,the coincidence rates of g B and g E gene detection of this assay were 100%and 92.31%,respectively.The reaction conditions of this method were maintained at 38?for 25 min,and the specific differential detection of g B and g E genes of PRV could be realized.The reaction system is pre-freeze-dried and can be assembled into a test kit for field application,which can be effectively used for the rapid differential diagnosis of PRV wild virus and vaccine virus.3.With the PRV variant strain SD-2017 as the parent strain,the g E/g I/TK virulence genes were deleted using homologous recombination technology,and PRV SD-2017?g E/g I strain and SD-2017?g E/g I//TK-EGFP strain were constructed respectively.One-step growth curve and pathogenicity of the constructed virus strain were studied,and the virus yield on PK-15 cells could reach 10^8.0TCID₅₀/m L.The LD₅₀ of the SD-2017?g E/g I/TK to rabbits was greater than 1.0×10^6.0 TCID₅₀/m L,which provided an ideal candidate vaccine strains for the development of gene-deleted inactivated vaccine and attenuated live vaccine against PRV mutant strains.4.Using DCpep as an intramolecular adjuvant and Por B as a novel vaccine adjuvant,recombinant baculovirus Ac-g B-DCpep and Ac-Por B containing gp67 protein secretion signal peptide were constructed respectively.The fusion expression of PRV g B-DCpep and the single expression of Por B protein were performed using the eukaryotic expression system of insect cells.Based on the PRV virulence gene deletion strain constructed in the previous study,a three-gene defective live vaccine and a double-gene defective inactivated vaccine of PRV SD2017 strain were prepared respectively.These two vaccine strains,together with g B-DCpep genetic engineering subunit vaccine and Bartha K61 live vaccine,were used to evaluate the immune effect against PRV mutant strains in rabbits.Live attenuated SD2017?g E/g I/TK vaccine,PRV-g B+Por B subunit vaccine and SD2017?g E/g I+Por B inactivated vaccines all showed good immune protection in rabbits.DCpep can be used as a potential molecular adjuvant,and Por B protein can be used as a new vaccine adjuvant,which can induce and stimulate humoral immunity and cellular immunity significantly,and then can be used in the preparation of new genetic engineering vaccines.5.Based on transcriptomic sequencing technology,the transcriptomic differences of PK-15 cells infected with PRV SD-2017 strain,SD-2017?g E/g I/TK strain,and Bartha K61 strain were comprehensively analyzed,and the result showed that genes with up-regulated transcription were mainly concentrated,including cell cycle,m TOR signaling pathway,autophagy-animal,PI3K-Akt signaling pathway,cell aging,cancer pathway,endocytosis,HTLV-I infection,ap1 signaling pathway,MAPK and other signaling pathways.The differentially down-regulated genes are mostly concentrated in signaling pathways such as ribosomes,oxidative phosphorylation of RNA transport,m RNA monitoring pathways,oocyte meiosis,protein processing in the endoplasmic reticulum,propionate metabolism,gene replication,Parkinson's disease,Alzheimer's disease,health Signal pathways such as heat.The biological significance of related genes needs further research to reveal.6.Based on TMT quantitative proteomics technology,the proteomic differences of PK-15 cells infected with PRV SD-2017 strain,SD-2017?g E/g I/TK strain,and Bartha K61 strain were comprehensively analyzed.Signals related to biological processes,molecular functions and cell localization in PRV-infected cells with different virulence were analyzed.Differential proteins folllowing PRV infection are associated with major signaling pathways including antigen processing and display,primary bile acid synthesis,ether lipid metabolism,mineral absorption,peroxisomes,HTLV-I infection,oxytocin signaling pathway,herpes simplex infection,Kaposi's sarcoma-associated herpes virus infection,cell aging,chemokine signaling pathway,notch signaling pathway,etc.The mechanism of PRV virus-host cell interaction is worthy of further study,especially how the key genes involved in proteome differences regulate PRV infection,which will help to better understand the specific infection mechanism of PRV.In addition,this study analyzed the correlation between the transcriptome and proteome GO function enrichment of PRV-infected PK15 cells and found that the two are closely related in the cell part,cell,organelle part of the macromolecular complex,catalytic activity,binding,cellular process,metabolic process,etc.The significant differences in these aspects are related,which provides favorable conditions for the follow-up in-depth study of the pathogenic mechanism of different PRV strains.