Cloning,prokaryotic expression and polyclonal antibody preparation of Anguillid herpesvirus ORF87

【Objective】Anguillid herpesvirus(AngHV) ORF87 was cloned,analyzed on sequence features,and prepared for polyclonal antibody,so as to provide technical support for revealing the role of ORF87 gene on the AngHV infection.【Method】The ORF87 sequence was amplified from the genome of AngHV-FJ strain by PCR with primers based on the AngHV reference strain(NC_013668) in GenBank to construct recombinant plasmid pMD19-ORF87. The gene was verified by restriction enzyme digestion and sequencing. The bioinformatics information was analyzed by online softwares such as Softberry,ProtParam,CDD,TMHMM,SignalP 5.0,PSORT and BepiPred. Then,ORF87 was cloned into the expression vector pET-32a and transformed into Escherichia coli BL21(DE3) competent cells The transformed E. coli was induced by IPTG,and the protein expression of ORF87 gene was validated by SDS-PAGE and Western blotting analysis. The expressed fusion protein was purified,and used to immunize New Zealand rabbits to prepare antiORF87 polyclonal antibody.【Result】The AngHV-FJ ORF87 was 2127 bp with only four base mutations,which shared high similarity(99.72%) with the reference sequence in GenBank. The molecular weight of its encoded protein was 80.1 KD,the theoretical isoelectric point(PI) was 7.62,the instability coefficient was 42.38,and the total average hydrophilicity was -0.271. ORF87 contained a conserved domain of PKC-like Superfamily,but the protein had no signal peptide and transmembrane domain. The subcellular localization of the protein was predicted to localize on the nucleus and cytoplasmic membrane with good immunogenicity due to 19 potential B-cell antigen epitopes. It was indicated that the AngHV-FJ ORF87 could be highly expressed in E. coli. The fusion protein was expressed at the size of 100 kD and mainly in the form of inclusion body. The prepared rabbit anti-ORF87 polyclonal antibody by fusion protein could specifically recognize AngHV ORF87 with serum titer of 1:16000.【Conclusion】The fusion protein ORF87 of AngHV-FJ induced by prokaryotic expression vector has Serine/threonine protein kinase activity. The anti-ORF87 polyclonal antibody obtained by immunizing the rabbits has high titer and specific recognition with AngHV infection. The antibody can be used as a tool to study protein characteristics such as subcellular localization and expression profile,which is helpful to reveal the role of ORF87 gene on the AngHV infection.