鳗鲡疱疹病毒ORF115基因克隆、转录时相分析及原核表达

Cloning,transcription profile analysis and prokaryotic expres⁃sion of ORF115 gene of Anguillid herpesvirus

【目的】克隆鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)ORF115基因,获得ORF115基因序列生物信息学特征,分析其在病毒入侵细胞过程中的转录时相并进行原核表达,为解析ORF115基因的特性和功能及开发免疫学诊断技术和亚单位疫苗打下基础。【方法】根据GenBank已公布AngHV参考株(NC_013668)的基因序列,设计特异性引物扩增ORF115基因,从分离的AngHV-FJ福建株(AngHV-FJ)基因组中扩增出ORF115基因,克隆至pMD19-T载体,经酶切鉴定和测序验证,获得ORF115基因序列。采用Softberry、ProtParam、TMHMM、SignalP 5.0、PSORTⅡ及BepiPred等在线软件进行生物信息学分析;采用反转录PCR(RT-PCR)对ORF115基因进行转录时相分析;将ORF115基因克隆至表达载体p ET-32a,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达后进行SDS-PAGE检测及Western blotting鉴定。【结果】克隆获得的AngHV-FJ ORF115基因全长318 bp,与GenBank已发布的参考序列相似性为100%;生物信息学预测表明,AngHV-FJ ORF115基因编码蛋白的分子量为11.8 kD,理论等电点(p I)为6.04,不稳定系数为16.38,总平均亲水性指数为0.172,属酸性弱疏水蛋白,较稳定;OR115蛋白存在跨膜结构,无信号肽,主要分布于细胞质中,有长片段抗原表位;转录时相分析结果表明,ORF115基因的转录本在病毒感染细胞后6 h即可检出,于感染24 h达峰值,随后无明显变化,属于病毒晚期基因。SDS-PAGE及Western blotting分析结果显示,ORF115基因可在大肠杆菌中表达,表达蛋白大小与预期一致,约32 kD。【结论】AngHV ORF115基因属于病毒晚期基因,其编码蛋白属酸性弱疏水蛋白,有长片段抗原表位,可能在病毒感染晚期发挥作用。获得的ORF115原核表达蛋白可用于制备多克隆抗体及AngHV亚单位疫苗开发。

【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 ORF115 gene 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 ob-tained 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,PSORTⅡand BepiPred.The transcrip-tion 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 ORF115 gene 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 hydro-philicity index was 0.172.ORF115 protein was an acidic and weak hydrophobic protein and was relatively stable.More-over,it had transmembrane structure without signal peptide.It was mainly distributed in cytoplasm with long fragment an-tigenic epitopes.Transcription profile analysis indicated the transcription of ORF115 gene could be detected at 6 h after vi-rus 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 hy-drophobic 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.