[Preparation,Identification,and Application of Monoclonal Antibody against Orf Virus 118 Protein].

Abstract

This study was designed to prepare a monoclonal antibody against Orf virus 118 protein and explore the biological properties of ORFV118 using this antibody. We constructed a recombinant plasmid pET33b-ORFV118 that contained a full-length ORFV118 gene. The plasmid was transformed into E.coli BL21,and the expression of ORFV118 was induced by isopropyl-β-d-thiogalactoside (IPTG).Prokaryotic ORFV118 was purified via Ni-NTA affinity chromatography and was subsequently used as an antigen to immunize mice. An anti-ORFV118 antibody was prepared using hybridoma technology. The titer and specificity of this antibody were tested by an indirect ELISA and Western blot/immunohistochemistry, respectively. We successfully obtained three antibody-secreting hybridomas,1A2,3B5,and 5D10.The titers of the three hybridomas were 1:10000,1:6400,and 1:8000.The monoclonal antibody (mAb),1A2 and the highest titer was selected for further research. The mAb 1A2,an IgG1 type antibody was bonded to its immunizing antigen, both the eukaryotic and natural ORFV118 with high specificity. The immunohistochemical analysis showed that the focal specific staining was restricted to the epidermal layer and subcutaneous tissue, which conformed to the characteristics of an ORFV infection. The mAb 1A2 recognized ORFV118with high specificity. Further study of mAb 1A2 will facilitate our understanding of ORFV118 and provide potentially novel methods for the diagnosis, prevention, and treatment of Orf.