Cloning,transcription profile analysis and prokaryotic expression of ORF115 gene of Anguillid herpesvirus

【Objective】To clone the ORF115 gene of Anguillid herpesvirus(AngHV)for the bioinformatic features of the sequence, and analyze the transcription profiles and prokaryotic expression of the gene in the process of virus invasion into cells to lay a foundation for further studies on characteristics and function of ORF115 gene as well as development of immunological diagnostic techniques and subunit vaccines. 【Method】Specific primers were designed to amplify ORF115gene based on the sequence of AngHV reference strain(NC_013668) in GenBank. ORF115 gene was amplified from the isolated AngHV-FJ Fujian strain(AngHV-FJ) and cloned into pMD19-T vector. Sequence of ORF115 gene was then obtained after the verification by enzyme digestion and sequencing. Bioinformatic features of the sequence were analyzed through online softwares such as Softberry,ProtParam, TMHMM, SignalP 5.0, PSORTⅡ and BepiPred. The transcription profiles of ORF115 gene were analyzed by reverse transcription PCR(RT-PCR). Then, the gene was cloned into the expression vector pET-32a and transformed into Escherichia coli BL21(DE3)competent cell. The expression of ORF115gene was induced by IPTG and the product obtained analyzed by SDS-PAGE and Western blotting. 【Result】The full length of ORF115 gene from AngHV-FJ cloned was 318 bp,the similarity to the reference sequence published by GenBank was 100%;bioinformatic prediction demonstrated that the molecular weight of AngHV-FJ ORF115 gene was 11.8 kD, the theoretical isoelectric point(pI) was 6.04, the instability coefficient was 16.38, and the total average hydrophilicity index was 0.172. ORF115 protein was an acidic and weak hydrophobic protein and was relatively stable. Moreover, it had transmembrane structure without signal peptide. It was mainly distributed in cytoplasm with long fragment antigenic epitopes. Transcription profile analysis indicated the transcription of ORF115 gene could be detected at 6 h after virus infection, the peak value was reached at 24 h after infection, and there was no significant change and belonged to AngHV late stage gene. SDS-PAGE and Western blotting analysis showed that ORF115 gene was successfully expressed in E. coli with expected size of expressed proteins around 32 kD. 【Conclusion】AngHV ORF115 is an acidic and weak hydrophobic protein with long fragment antigenic epitopes. It belongs to AngHV late gene and may play a role in the late stage of viral infection. The prokaryotic expression protein of ORF115 obtained can be used for preparation of polyclonal antibody and the development of subunit vaccine of AngHV.