Identification,Expression,and Characterization Analysis Of The Immunogenic Protein ORF36 Of Anguillid Herpesvirus

Anguillid herpesvirus(Ang HV)is an important viral pathogen of eel,which causes “mucus sloughing and hemorrhagic septicemia disease” on cultured eels.The study of immunogenic protein of Ang HV is of great significance for the development of immunological diagnostic technology and subunit vaccine of Ang HV.In order to obtain the immunogenic proteins of Ang HV,the virus particles were purified and the immunogenic protein ORF36 was identified by mass spectrometry.The sequence of ORF36 was cloned from the genome of Ang HV-FJ,and further analyzed by bioinformatics software;After that,ORF36 was expressed by prokaryotic system,and purified to prepare a rabbit polyclonal antibody;The titer of the polyclonal antibody was tested,followed by neutralizing effect test of the antibody against the virus.The specific test using the antibody was conducted in vivo and in vitro,respectively,as well as the conduction of sensitivity test in vivo.The location of ORF36 on the virus particles was also determined.The main findings are listed as follows:1.Identification of main immunogenic proteins ORF36 of Ang HV:The virion were purified by sucrose density gradient centrifugation.New Zealand white rabbits were immunized with purified virion to prepare a rabbit polyclonal antibody;The purified structural protein of virion was analyzed by Western blot using the antibody.Through comparison with the protein bands separated by SDS-PAGE,the band with high immunogenicity was determined and recovered by cutting.It was then enzymolyzed and identified by LC-MS/MS mass spectrometry.The protein was identified to be ORF36 of Ang HV by being blasted with the protein sequences in NCBI database.The result indicated that ORF36 is not only a structural protein but also the main immunogenic protein of Ang HV.2.Sequence feature analysis and prokaryotic expression of ORF36:The primers for ORF36 gene amplification were designed based on the genomic sequence of Ang HV reference strain.After gel recovery,the product was linked to p MD19-T vector and transformed into Escherichia coli DH5α.The plasmid T-ORF36 was obtained after plasmid DNA extraction and sequencing validation.Then the amplified sequence and its encoded amino acid sequence were analyzed by bioinformatics online tools.The result indicated that the homology between the amplified sequence and the reference strain Ang HV-1 ORF36 was 99.74%.The encoded ORF36 protein has stable property,but is short of transmembrane domain and signal peptide.13 B cell epitopes were predicted and thus the protein has good immunogenicity.The primers were designed,and PCR amplification was conducted using T-ORF36 as the template.The sequence was cloned into the expression vector p ET-30 a and transformed into E.coli BL21(DE3).Through IPTG induction,ORF36 was largely expressed in the E.coli BL21(DE3).SDS-PAGE and Western blot analysis showed that the expressed ORF36 protein mainly existed in the form of inclusion bodies,and consistent with the expected size of 40 k Da.The purified fusion expression protein was recovered in the method of gel cutting.3.Preparation and application of anti-ORF36 polyclonal antibody and localization of ORF36 on virus particle: The anti-ORF36 polyclonal antibody was prepared by immunizing New Zealand white rabbits with the purified ORF36.The titer of the prepared polyclonal antibody was measured using ELISA method.The results showed that the titer was 1:8000.The results of antibody neutralization test showed that the polyclonal antibody could significantly reduce the virus titer of Ang HV,indicating that it had neutralization ability on Ang HV.The specific tests on antibody were conducted in vitro and in vivo using EO cells infected with Ang HV-FJ,as well as gills,fins,and skin mucus tissues of eels,respectively.The results showed that polyclonal antibodies could specifically recognize EO cells and the tissues of eels infected with Ang HV-FJ both in vitro and vivo.The results of antibody sensitivity test showed that the minimum virus quantity detected in vivo by this antibody was 1000 PFU.Then localization of ORF36 on the virus particles was determined by the separated envelope protein and nucleocapsid protein.It was revealed that ORF36 was a structural protein of Ang HV and located on the nucleocapsid of the virus.The main immunogenic protein ORF36 of Ang HV virion was identified,and the polyclonal antibody against ORF36 with virus neutralization effect was prepared.The antibody could recognize Ang HV infection both in vitro and vivo,and with high sensitivity.ORF36 was identified as the nucleocapsid structural protein of Ang HV.These results would lay a research foundation for elucidating the role of ORF36 in Ang HV infection and development of the viral subunit vaccine.