鲤疱疹病毒3型ORF136基因编码蛋白多克隆抗体的制备与鉴定

摘要

为了对鲤疱疹病毒3型(Cyprinid herpesvirus 3, CyHV-3)ORF136基因编码蛋白进行功能研究和血清学诊断,本实验通过对ORF136基因推导的第31~157位氨基酸序列进行PCR扩增,并与原核载体pET-32a(+)连接,转化至大肠杆菌Rosetta(DE3)感受态后进行IPTG诱导表达,将纯化后的重组蛋白免疫新西兰白兔(Oryctolagus cuniculus)以制备ORF136多克隆抗体, 运用Western blot和间接免疫荧光技术对抗体进行鉴定.结果表明, 重组融合表达蛋白大小与预期一致, 约为35 kD, 且主要分布在包涵体中.Western blot分析显示, 免疫兔后获得的纯化ORF136多克隆抗体能特异性识别纯化的CyHV-3和感染CyHV-3的KS细胞; 间接免疫荧光分析进一步表明ORF136多抗能识别感染CyHV-3的KS细胞.ORF136多克隆抗体的制备为ORF136蛋白功能研究和CyHV-3血清学诊断方法的建立提供了重要基础.%Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiologic agent of koi herpesvirus disease(KHVD)causing morbidity and mortality in common carp and koi Cyprinus car-pio L.populations around the world.Recently,knowledge about diagnosis and detection based on nucleic acid has been reported, but validation of serological techniques for virus infection is limited due to lack of effective anti-bodies. Additionally, fundamental research on the function of structural proteins from CyHV-3 is necessary to understand the relationship between host and virus. ORF136 is one of the predicted envelope proteins incorporated into mature virions. To study the function of proteins encoded by the ORF136 gene of CyHV-3 and to establish serological methods for detection of CyHV-3, the predicted antigenic determinant based on amino acid sequences of proteins encoded by ORF136 was amplified using PCR. Then, the recombinant prokaryotic expression vector was constructed by connecting the cloned fragment to prokaryotic expression vector pET-32a (+), used for trans-formation of E.coli Rosetta (DE3) and protein expressions induced by IPTG. New Zealand white rabbits were immunized with the recombinant proteins of pET-32a-ORF136 four times. Furthermore, the antiserums were col-lected and affinity purification was used to obtain polyclonal antibody after 66 days. Characterization of the poly-clonal antibody against the ORF136 protein was further analyzed using western blot and indirect immunofluores-cence assays. The expected product size of 381 bp was obtained and the recombinant prokaryotic expression vec-tor pET-32a-ORF136 was confirmed to be constructed as expected using enzyme digestion. As SDS-PAGE analy-sis indicated that the molecular weight of recombinant protein pET-32a-ORF136 was consistent with the expected size (about 35 kD) and was distributed in the inclusions. Western blot analysis further showed that the purified polyclonal antibody could specifically recognize purified CyHV-3 virions and KS cells infected with CyHV-3. Moreover, immunofluorescence staining showed that specifically green fluorescence was present in the cytoplasm of KS cells infected with CyHV-3, but not in the negative control, further suggesting that CyHV-3 was recognized by the polyclonal antibody. The purified recombinant proteins and effective antibodies are necessary to develop serological methods to detect antigens, except nucleic acid detection method. The purified recombinant proteins could be used as antigens to capture antibodies against CyHV-3 from common carp or koi exposed to KHVD. Similarly, development of a double antibody sandwich ELISA for detection of CyHV-3 also requires an effective polyclonal antibody to capture antigens released from the host. Antibodies produced by recombinant proteins from rabbit or mouse provides the foundation for functional research into structural and nonstructural proteins. In this study, the polyclonal antibody against the ORF136 protein was prepared and analyzed using immunoblotting and immunofluorescence techniques, which provides an essential and valid tool for further research into CyHV-3.