Prokaryotic Expression Of Sheep Aphthous Virus ORF019?ORF020 Gene And Preparation Of Polyclonal Antibody Against ORF019 Protein

Sheep's sore,also known as contagious ecthyma(CE),is a zoonotic disease caused byinfection with the member of the parapoxvirus,Orf virus(ORFV),Human,sheep andgoats can be exposed to infections by acute infectious skin diseases that have developeda series of mechanisms to evade the monitoring of host and animal host immunesystems The study found that the proteins encoded by the ORFV ORF019 and ORFV ORF020 genes are orthologs to the E2 L and E3 L proteins of VACV(vaccinia virus).It was confirmed that the E3 L protein inhibits the INF system by isolating dsRNA,and E2 L is present inmature virions of infected cells.The transport of virions plays a crucial role However,there are few studies on the functions of E2 L and E3 L proteins encoded by ORFVORF019 and ORF020 genes,respectively.Therefore,the expression of proteinsencoded by ORFV ORF019 and ORF020 genes can be used to create conditions for immunodiagnosis and immune prevention of sheep's sores.1.ORFV ORF019 and ORF020 cloning and bioinformatics analysisRefer to the genomic sequence of ORFV OV-SA00 strain registered in GenBank(accession number: AY386264),and design a pair of specific primers based on ORF019 and ORF020 gene sequences using Oligo6.0 bioinformatics software to extract ORFV/QH02/ The genomic DNA of 2010 strain was used as a template,and ORF019 and ORF020 were amplified by PCR.Then,the prokaryotic expression vector pET-32a(+)was constructed and transformed into DH5? competent cells.The recombinant strains positive by PCR and restriction enzyme digestion were sequenced.The results showed that the recombinant plasmids pET-32a-ORF019 and pET-32a-ORF020 were successfully constructed.The ORF019 gene and ORF020 gene are located in the central conserved region of the ORFV genome,and the 19 th and 20 th open reading frames encode approximately 84 ku,20 ku size proteins.By homology analysis,it was found that the two genes were highly conserved in the domestic ORFV strain and had low homology in the parapoxvirus genus.2.Prokaryotic expression and purification of ORFV E2 L protein and E3LproteinThe constructed prokaryotic expression recombinant plasmids pET32a-ORF019 and pET32a-ORF020 were transformed into E.coli BL21(DE3)pLys competent cells.The expression time,IPTG final concentration and temperature were optimized to maximize expression.The soluble analysis fusion protein pET32a-ORFV 019 exists in the form of inclusion bodies,and the fusion protein pET32a-ORF020 is present in the supernatant.The target protein was purified by nickel affinity chromatography,and finally the Western-blot was used to identify the target protein which was induced to express with good reactogenicity.3.Preparation and identification of polyclonal antibodies against ORFV E2 L proteinThe purified E2 L fusion protein was used to prepare the immunogen,and the rabbit polyclonal anti-ORFV E2 L protein polyclonal antibody was prepared by subcutaneous injection of New Zealand rabbits.The indirect ELISA method was used to detect the E2 L polyclonal antibody titer.The results showed that the titer of the rabbit antipolyclonal antibody was up to 1:128,000,and the E2 L protein was confirmed to havegood immunogenicity by Western-blot,indicating that the recombinant protein of ORFV E2 L has good antigenicity.