Cloning,Expression,Subcellular Localization Of 115 Gene Of ORFV-FJND Strain And Establishtion Of ELISA Detection Method

Orf virus(ORFV)belongs to the genus Parapoxvirus of the poxviridae family.The virus is an enveloped virus with a large double-stranded DNA genome.ORFV is highly infectious and mainly infects ruminants such as goats,sheep,camels,and cattle.Orf is a zoonotic infectious disease caused by ORFV.The affected sheep show pimples,vesicles,pustules,and scabs on the lips,muzzles,nostrils,and skin and mucous membranes around the breast,the disease is easily confused with diseases such as sheep pox clinically.Orf is prevalent globally,including in China's Xinjiang,Inner Mongolia,Gansu,Hubei,and Fujian.It has been reported all year round,and the disease is most frequent in spring and autumn,causing serious economic losses to the sheep industry.At present,the prevention of Orf mainly depends on vaccine immunization,but the current vaccine effect is not satisfactory,and there is no clinically effective antiviral drug in treatment of Orf,after the onset of Orf,the secondary bacterial infections are treated by drugs and feeding management is improved to promote body recovery.The Orf is still one of the major disease affects the sheep industry in China.Therefore,the establishment of accurate and rapid detection methods is of great significance to prevention and control of the disease.In order to investigate the immunological characteristics of 115 gene of ORFV-FJND strain and its encoded protein,in this study,a pair of specific primers were designed based on the 115 gene sequence of ORFV in Gen Bank to amplify the ORFV 115 gene of ORFV-FJND strain by PCR technology.The gene was cloned into the pMC100-M cloning vector and sequenced.The bioinformatics software DNAMAN was used to analyze the sequence homology of ORFV 115 gene and the encoded amino acid,and the genetic evolution tree was constructed using software MEGA7.0.The results show that the ORFV 115 gene of ORFV-FJND strain is 450 bp in length and encodes 149 amino acids,which has 84.4%?98.9%homology with the115 genes of 15 ORFV strains in Genbank,and shares 98.9%homology with the 115gene of ORFV-YX(No.KP010353.1)strain isolated in Fujian in 2012.Phylogenetic tree analysis shows that ORFV-FJND strain is closest to ORFV-YX strain.The Prot Scale,SOPMA,Signal P and TMHMM programs in Ex PASy software were used to predict and analyze the ORFV 115 protein's hydrophobicity,secondary structure,signal peptide and transmembrane domain online.The analysis results showed that the protein encoded by the ORFV 115 gene is a hydrophilic protein,and its secondary structure includes?-helix(Hh),?-sheet(Ee),?-turn(Tt),and random coil(Cc).It is dominated by random coils.The protein has no signal peptide and no transmembrane domain.On this basis,the ORFV 115 gene was subcloned into the pGEX-6P-1 vector and transformed into E.coli Rosettaganmi B(DE3)for inducible expression.It was determined that the temperature of 37?,the induction expression time of 4 h,and the final concentration of IPTG 1 mmol�L^-1 were the optimal induction expression conditions for ORFV 115 recombinant protein.SDS-PAGE results showed that the expressed protein was about 42 kDa in size,which was consistent with the expected size.The solubility analysis results of the recombinant protein showed that the protein was in the form of inclusion bodies.The recombinant protein was purified using an inclusion body protein purification kit,and its concentration was determined to be0.823 mg�mL^-1.SDS-PAGE results showed a single-purpose band.The purified target protein was identified correctly by Western Blot,indicating that 115 protein was successfully expressed and had good reactogenicity.The eukaryotic expression plasmid pEGFP-N1-115 was successfully constructed and transfected into 293T cells.The results of subcellular localization showed that green fluorescence accumulated in the nucleus,suggesting that the protein mainly performs its biological function in the nucleus.The results laid a foundation for further research on the mechanism of interaction between ORFV and the host.The purified ORFV 115 recombinant protein was used as the coating antigen,and the reaction conditions were optimized to establish an indirect ELISA method for detecting ORFV antibodies.It was determined that the optimal coating concentration of the ORFV 115 protein was 2.0?g/hole,the optimal serum dilution was 1:100,the optimal enzyme-labeled secondary antibody dilution was 1:30000,and the blocking time was 2 h,the optimal incubation time for serum and secondary antibody was all45 min,and the optimal substrate coloration time was 10 min.This ELISA method has good specificity,sensitivity and repeatability.327 clinical samples from Fuzhou,Sanming and Ningde city were detected using this method,the positive rate of ORFV antibody was 87.46%.The results provided a new practical method for ORFV antibody detection,laid a foundation for further research on the function of ORFV115 gene,and have important significance for the diagnosis and prevention of sheep's aphthous ulcer.