Abstract: 【Objective】 The polyclonal antibody of ORF6 of cyprinid herpesvirus-2(CyHV-2)was prepared to provide a technical means for further investigation of the function of ORF6 in CyHV-2 acute infection or a latent infection process.【Method】 The ORF6 gene sequenceof CyHV-2 was analyzed by online softwares such as MEGA 6.0,Adobe Illustrator CS6 and BepiPred-2.0. The ORF6 gene was amplified from the CyHV-2 genome by PCR and cloned into the prokaryotic expression vector PET-28a. The recombinant expression plasmid was transformed into Escherichia coli BL21(DE3),after that,expression was induced by IPTG. The recombinant protein was purified by urea method,Babl/chealthy mice were immunized with purified recombinant ORF6 protein,serum was collected to prepare polyclonal antibody against ORF6 protein. Finally,the specificity of antibody was identified by Western blotting and indirect immunofluorescence technique(IFA).【Result】 The full-length ORF6 gene of CyHV-2 was 3003 bp,and the amino acid sequence similarity with CyHV-3 and ORF6 of CyHV-1 was higher than 30.00%,and the similarity with CyHV-3 was 39.36%. Its antigenic epitope was most likely to exist in amino acid sequences 1-300. ORF6 gene fragment obtained by PCR was ligated to the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-ORF6,which was transformed into BL21(DE3)competent cells and induced by IPTG for 4 h to obtain the fusion protein ORF6,it was mainly expressed in the form of inclusion bodies,and its molecular weight was 17.13 kD. The concentration of ORF6 purified by 6 mol/L urea washing was 0.1 mg/mL. The ORF6 polyclonal antibody prepared by immunizing Babl/c mice with purified fusion protein ORF6 could specifically recognize the fusion protein ORF6 and the Carassius auratus gibelio caudal fin cell(GiCF) infected by CyHV-2. The prepared ORF6 polyclonal antibody was used for indirect immunofluorescence analysis of GiCF infected with CyHV-2. The results showed that specific green fluorescence could be observed around GiCF infected with CyHV-2,while no specific green fluorescence was observed around GiCF cells not infected with CyHV-2,which further indicated that ORF6 polyclonal antibody could specifically recognize CyHV-2.【Conclusion】 The prepared ORF6 polyclonal antibody can specifically react with CyHV-2 infected GiCF,which can be used to identify CyHV-2 infection,and lay a foundation for later exploration of whether ORF6 protein function is related to CyHV-2 latent infection.