Study On The Function Of ORF059 Gene Of ORF Virus

ORF is a zoonotic disease,distributed widely.It causes serious economic loss to the animal husbandry and also threatens human health.ORFV belongs to the genus Parapoxvirus of the Poxviridae family.ORF059 gene is located in the highly conserved region of the viral genome,which is the main heparin binding active protein.It can bind to cell surface heparan sulfate receptors,which is related to the adsorption and invasion of ORFV.It is also a component of the envelope protein of the ORFV,which can make the host produce neutralizing antibodies.In the first experiment,the recombinant plasmid pEGFP-ORF059-N1 was constructed with pEGFP-N1 as vector.Use pEGFP-ORF059-N1 to transfect 293T cells.Fluorescence microscopy was used to observe the fluorescent protein.The rabbit anti-ORFV-059 polyclonal antibody prepared by our laboratory was used as the primary antibody for Western blot verification.Then,the experimental group eGFP-ORF059 and the control group eGFP were subjected to transcriptome sequencing to analyze differential genes and their enrichment pathways.The results showed that green fluorescence was observed in 293T cells transfected with the recombinant plasmid pEGFP-ORF059-N 1 under fluorescence microscope,and fluorescence was observed in 293T cells transfected with the empty plasmid pEGFP-N1,but no fluorescence was observed in 293T cells.Western blot only detected the fusion protein in the 293T cells transfected with the recombinant plasmid pEGFP-ORF059-N1,with a size of 64 kDa.Analysis of transcriptome sequencing revealed 499 differentially expressed genes,mainly enriched in immune response protein binding?receptor binding?protein dimerization activity?PI3K-Akt signaling pathway?B cell receptor signaling pathway?chemokine signaling pathwayand and other pathways.In the second experiment,lentivirus was used as vector to construct recombinant plasmid to infect MDBK cells.The results were verified by fluorescence microscopy and Western blot.Transcriptome sequencing was performed to analyze differential genes and enrichment pathways.The results showed that MDBK cells were not fluorescent and that MDBK cells infected with GFP-tagged bovine kidney cells and ORF059 with GFP could detect fluorescence.Western blot detected only MDBK cells with GFP ORF059 exogenous gene fusion protein,the size of 64kDa.Analysis of transcriptome sequencing revealed 72 differentially expressed genes,mainly enriched in positive regulation of DNA damage response?signal transduction by p53?regulation of transcription from RNA polymerase ? promoter?regulation of protein phosphorylation?cell adhesion.PI3K-Akt signaling pathway.B cell receptor signaling pathway and other pathways.In the third experiment,the goat fibroblast cells were infected with the lentivirus of second experiment and were detected by fluorescence microscopy and Western blot.Then the transcriptional group was sequenced.The results showed that goat fibroblast cells were not fluorescent,infected with goat fibroblast cells with GFP tags,and goat fibroblast cells with GFP ORF059 gene could detect fluorescence.Westem blot only detected the fusion protein of goat fibroblast cells with ORF059 exogenous gene with GFP,and the size of the fusion protein was 64kDa.Analysis of transcriptome sequencing revealed 407 differentially expressed genes,mainly enriched in regulation of transcription?cell adhesion?negative regulation of apoptotic process?regulation of transcription from RNA polymerase ? promoter?PI3K-Akt signaling pathway and other pathways.In this experiment,the function of ORF059 gene was studied from the protein level and the molecular level,which laid the foundation for the development of a subunit vaccine for genetic engineering of orf,and also provided a basis for a more in-depth study of the pathogenic mechanism of orf.