Effect Of Deletion Of ORFV122 Gene On Virus Replication And Virulence

Contagious ecthyma(CE),also known as "Orf",is an acute and highly contact disease caused by Orf virus(ORFV)which mainly infects sheep,goats and human.The disease is characterized by papule,vacuole,pustule,ulcer and scab on the lips,mucosa,the upper jaw,nasal cavity,and so on.ORFV mainly infects goats and sheep,especially in lambs.As a member of the Poxviridae family,the ORFV genome is large and consists of inverted terminal repeats(ITRs)(ORFs001-008 and ORFs112-134)and a central core region(ORFs009-111)at both ends.The terminal ITRs may be related to virus virulence,infection,and regulation of host immune response and inflammatory response.In view of this,this study intends to study the function of the unknown functional gene 122 in the terminal region by constructing a gene deletion mutant virus.However,the function of some ORFV-encoding proteins in the terminal region is still unclear.In order to study the function of the protein encoded by the ORF122 gene of the ORFV terminal region,this study first used the ORFV-SY17 strain as experimental material,amplified the ORFV122 gene by PCR technology,and connected the gene to the prokaryotic expression vector PET-28 a to construct PET-28a-122 recombinant expression plasmid;Then,the recombinant plasmid was transformed into E.coli BL21(DE3)competent state,induced expression by IPTG induction,and the ORFV122 protein was separated and purified by nickel ion affinity chromatography column.The purified ORFV122 protein was immunized into BALB / c mice to prepare corresponding polyclonal antibodies.Western blot detection results showed that the polyclonal antibody could react specifically with ORFV whole virus protein;the antibody titer was detected by ELISA it is about 1: 80000;the successful preparation of this polyclonal antibody provides important experimental materials for the subsequent research on the function of ORFV122 gene.Using homologous recombination technology to construct the ORFV-SY17 strain ORF122 gene deletion virus strain(ORFV-SY17 ? 122),using PCRtechnology to respectively amplify the ORFV122 gene left and right homology arm nucleotide sequences,and the pSPV-EGFP plasmid after connecting,the shuttle plasmid pSPV-L-EGFP-R was successfully constructed.The shuttle plasmid was transfected into OFTu cells inoculated with ORFV-SY17,using the green fluorescent protein EGFP as a selection marker,and the ORFV-SY17 ? 122 deletion virus strain was screened using a fluorescence microscope.After multiple rounds of screening and PCR identification,the ORFV-SY17 ? 122 deletion virus was successfully obtained.To clarify the effect of 122 gene deletion on virus virulence,wild-type(WT)strains and ORFV-SY17 ? 122 deletion strain were inoculated into rabbits and lambs.The results showed that the lesion area of ORFV-SY17?122 gene-deficient strains inoculated in rabbits and lambs was significantly smaller than that of wild strains,and the disease duration was significantly shorter than that of wild strains.This result indicates that 122 gene is a virulence gene of ORFV.In addition,the growth curves of ORFV-SY17 and ORFV-SY17 ?122 deletion strain showed that deletion of ORF122 gene would significantly reduce the virus replication,which indicated that the 122 gene was an essential gene for ORFV replication.A thorough understanding of the function of the ORFV122 gene will not only help to fully understand the pathogenic mechanism of ORFV,but also provide a theoretical basis for the development of ORFV vaccine.