鳗鲡疱疹病毒ORF87基因克隆及其多克隆抗体的制备

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

【目的】克隆鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)ORF87基因,分析其序列特征并制备多克隆抗体,为揭示ORF87基因在AngHV侵染过程中的作用机制提供技术支持。【方法】根据GenBank已公布AngHV参考株(NC_013668)的ORF87基因序列设计引物,从AngHV-FJ株基因组中扩增出ORF87基因,构建重组质粒pMD19-ORF87经酶切鉴定和测序验证后,采用Softberry、ProtParam、CDD、TMHMM、SignalP 5.0、PSORT及BepiPred等在线软件进行生物信息学分析;将ORF87基因克隆至p ET-32a表达载体上,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;以纯化的融合蛋白免疫新西兰大白兔,制备ORF87多克隆抗体。【结果】克隆获得的AngHV-FJ株ORF87基因为2127 bp,与GenBank已发布的参考序列高度同源(相似性为99.72%),仅存在4个碱基突变;其编码蛋白分子量为80.1 kD,理论等电点(pI)为7.62,不稳定系数为42.38,总平均亲水性为-0.271。ORF87蛋白具有蛋白激酶C超家族(PKC-like Superfamily)的保守结构域,但不具信号肽和跨膜结构域;其亚细胞定位可能位于细胞核和细胞质膜,存在19个潜在B细胞抗原表位,即具有良好的免疫原性。AngHV-FJ株ORF87基因可在大肠杆菌中高效表达,表达获得的融合蛋白约100.0 kD,主要以包涵体形式存在;以融合蛋白免疫新西兰大白兔制备获得的ORF87多克隆抗体效价达1∶16000,能特异性识别AngHV的ORF87蛋白。【结论】AngHV-FJ株源ORF87基因经原核表达载体诱导表达获得的融合蛋白ORF87具有Serine/threonine蛋白激酶活性,以其免疫新西兰大白兔制备获得的ORF87多克隆抗体具有高效价,能特异性识别AngHV感染,可作为亚细胞定位及表达时相分析等蛋白特性研究的工具,用于解析ORF87基因在AngHV侵染过程中的作用。

【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-32 a 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.