The Expression Of The Orf Virus ORFV112 Gene

Orf,also known as Contagious ecthyma(CE),is a contactous,epithelial and infectious zoonosis caused by the Orf virus(ORFV).ORFV mainly infects sheep and goats,causing serious economic losses to the sheep industry and threatening human health.The ORFV genome has about 131 genes,consisting of a central coding region and two identical inverted terminal repeat regions.ORFV112 gene is located in the inverted terminal repeat region and encodes the Chemokine Binding protein(CBP).As an early gene,ORFV112 mainly plays a role in the early stage of virus infection and can effectively bind to the surface receptors of chemokines,thereby interfering with the immune defense of host,inhibiting the activity of immune factors,and playing a role in virus immune evasion.1.The whole genome sequencing of the ORFV-JL strain was performed using the Illumina Hiseq PE150 sequencing platform,and we studied the physicochemical properties of the ORFV-JL strain.The results showed that the ORFV-JL strain had a full-length nucleotide sequence of 139 279 bp with a GC content of 64.87%and a total of 131 ORFs.All ORFs accounted for 83.69%of the total genome length;ORFV-JL strain was sensitive to temperature,pH,and chloroform,and different in cell infectivity for different strains.2.The ORFV112 fragment was amplified by PCR using ORFV genome as a template.The ORFV112 fragment was double-digested and the digested ORFV112 gene fragment was ligated with pET28a(+)to construct the recombinant plasmid pET28a-ORFV112.Double enzyme digestion and sequencing were performed to identify correctly transformed E.coli BL21(DE3),IPTG induced the expression of the strain,and detected by SDS-PAGE and Western Blot.The expressed ORFV-CBP fusion protein was purified by a Ni-NTA column,and the purified expression product was detected by SDS-PAGE.The results showed that the pET28a-ORFV112 was successfully constructed.After being induced by IPTG,the His-tagged fusion protein was successfully expressed in the strain containing pET28a-ORFV112.The results of SDS-PAGE and Western Blot showed that the molecular weight of the fusion protein was approximately 36 ku,which is consistent with the expected size.SDS-PAGE electrophoresis results showed that the fusion protein was present in inclusion bodies after ultrasonic disruption.3.The purified ORFV-CBP fusion protein was used as an antigen to immunize rabbits with multiple subcutaneous injections to prepare rabbit anti-ORFV-CBP polyclonal antibodies.The titer of multi-antibody was detected by indirect ELISA.The results showed that the obtained polyclonal antibody titer was above 1:25600.4.ORFV112 gene was amplified by PCR and ligated with pEGFP-N1 to construct pEGFP-N1-ORFV112,transfected into 293T cells,and was identified by fluorescence microscope,flow cytometry,qRT-PCR detection and Western blotting.The results showed that the pEGFP-N1-ORFV112 was successfully constructed,and the ORFV-CBP fusion protein was expressed,which will be helpful for the study of function in the ORFV-CBP.5.RNA-seq sequencing of 293T cells overexpressing the ORFV-CBP fusion protein was performed using the Illumina Hiseq sequencing platform,and the effect of ORFV-CBP on the cells was examined at the transcript level.After sequencing,192 significant differential transcripts were obtained for expressing of the ORFV-CBP,and GO functional classification was performed.A total of 11 differential transcripts involved in the immune process were obtained.qRT-PCR further validates differential transcripts.The results showed that there were 9 differential transcripts.Our findings laid the foundation for the subsequent study on the effect of ORFV-CBP on the expression of genes.