Construction And Immunogenicity Of Recombinant Adenovirus Containing Double Genes Of ORFV Shanghai Strain

ObjectiveOrf is a contagious zoonosis caused by orf virus affecting sheep and goats.It is widespread in the world,which seriously harms the development of sheep industry and human health.This study intends to determine the characteristics of the orfv epidemic strain of Chongming area of Shanghai by isolation and identification of orf virus in this region and sequence analysis of isolated impartant genes.Then,the recombinant adenovirus of orf was constructed with the double gene,and the immunological evaluation was carried out to establish a theoretical foundation for the development of the nucleic acid vaccine of orf.MethodsChlamydial tissue was collected from lambs infected with suspected orf virus in Chongming area of Shanghai.The virus was isolated and identified by PCR,MDBK cell isolation,electron microscopy,and indirect immunofluorescence techniques.Two strains of Shanghai isolate protective antigen genes F1L and B2L genes were cloned and sequenced,and phylogenetic tree was constructed.The target genes of F1L and B2L of ORFV-F416 strain were amplified by PCR to construct a recombinant adenovirus shuttle plasmid.The shuttle plasmid and the backbone plasmid were co-transfected into HEK-293 cells,and the orf recombinant adenovirus was packaged.The recombinant adenovirus was used to infect MDBK cells,the titer of virus was measured by Karber method.The expression of recombinant adenovirus was detected by PCR,IFA and Western-blotting.Meanwhile,the mice were immunized,and the immune effect was evaluated by immune index test and toxin protection test.Results1.Two strains of ORFV were successfully isolated and named as ORFV-F416 and ORFV-F429 respectively.Compared with reference strains,nucleotide sequence homology of F1L genes from the two isolates was 95.2%⁹9.4%and amino acid sequence homology was94.4%⁹9.4%.There was no nucleotide deletion in the 44⁶6 position of the amino acid.Phylogenetic tree analysis showed that two strains belonged to the same branch,and had the closest relationship with Fujian strain FJ-YX?KC568410?.Compared with reference strains,nucleotide sequence homology of B2L genes from two isolates was 96.7%⁹9.6%and amino acid sequence homology was 94.7%⁹8.9%.Phylogenetic tree analysis showed B2L genes had close genetic relationship with Guangxi strain GX-YB?JQ904793?and distant genetic relationship with vaccine strain.F1L and B2L genes of two isolated share 99.9%and 99.0%nucleotide sequence,respectively.2.The pAV-CMV-F1L-T2A-B2L-P2A-EGFP recombinant adenovirus was successfully obtained and its TCID₅₀ was 10^-5.375/mL.PCR,IFA,and Western blot confirmed that the target gene was expressed at both gene and protein levels.After immunizing mice with orf recombinant adenovirus,IgG antibody level of the recombinant adenovirus group in the first week was significantly higher compared with the control group and empty vector group and the difference was significant?P<0.01?in the second to the fourth week.IgG antibody level of the recombinant adenovirus group reached the peak at in the fourth week and began to decline after the fifth week.After stimulation without stimulation,non-specificity and specificity,the lymphocyte proliferation ability of the orf recombinant adenovirus group was significantly increased?P<0.01?,and the protection rate of the challenge test was 80%.It was shown that the recombinant adenovirus of orf can stimulate humoral and cellular immune responses in mice and has good immunogenicity.Conclutions1.Two orf virus,ORFV-F416 and ORFV-F429,were successfully isolated in Chongming,Shanghai.There were some variations in F1L and B2L genes of the two isolates,and they were closely related to the strains of epidemic strains in southern China.2.The successful construction of the recombinant adenovirus of orf can promote humoral and cellular immune responses in mice and establish a foundation for the development of orf vaccine.