Abstract：Anguillid herpesvirus (AngHV) is an important viral pathogen of eel (Anguilla), it causes“mucus sloughing and hemorrhagic septicemia disease”on cultured eels, which brought huge economic losses to the farmers. The study of immunogenic protein of AngHV is of great significance for the development of immunological diagnostic technology and subunit vaccine of AngHV. In order to obtain the immunogenic proteins of AngHV, in this study, mass spectrometry analysis on the viron proteins was conducted. The obtained protein ORF36 was further analysed by bioinformatics software. Then the sequence of ORF36 was cloned into expression vector pET-30a, transformed into Escherichia coli BL21, and induced by isopropyl β-D-thiogalactoside (IPTG) for prokaryotic expression. After that, the anti-ORF36 polyclonal antibody was prepared by immunizing rabbits (Oryctolagus cuniculus) using the expressed recombinant protein. The titer of the polyclonal antibody was tested, followed by the specific detection on the prepared antibody by the eel ovary cell line (EO) cells and host tissues infected by AngHV. The sensitivity and neutralization effect of the antibody on AngHV was evaluated, then the virion localization analysis of ORF36 was conducted. According to the results of mass spectrometry, the main immunogenic protein of the virion was identified to be ORF36. Bioinformatics analysis showed that ORF36 protein had no transmembrane domain or signal peptide. Thirteen B cell epitopes were predicted, which indicated that ORF36 had good immunogenicity. ORF36 was cloned into prokaryotic expression vector pET-30a, and induced for protein expression. SDS-PAGE results showed that high level of expressed ORF36 was achieved in E. coli BL21 (DE3), with the size of 40 kD and mainly existed in the form of inclusion body. ELISA results showed that the titer of the anti-ORF36 polyclonal antibody was 1: 8 000. Western blot results showed that the prepared antibody could specifically recognize infected EO cells, as well as the gill, fin and skin mucus tissues of eels infected by AngHV. Sensitivity evaluation results showed that the antibody could detect a minimum level of 1 000 PFU virions. The neutralization effect test results showed that the antibody significantly reduced the viral titer, revealing the neutralizing effect of the antibody on AngHV. Western blot on the envelope, nucleocapsid, and purified virions demonstrated that ORF36 was the structural protein of virion and localized on the nucleocapsid. In conclusion, the main immunogenic protein ORF36 of AngHV virion was identified, and the polyclonal antibody against ORF36 with virus neutralization effect was prepared, and ORF36 was identified as the nucleocapsid structural protein of AngHV. These results would lay a research foundation for elucidating the role of ORF36 in AngHV infection and development of the viral subunit vaccine.
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