Ⅱ型鲤疱疹病毒ORF6多克隆抗体的制备与鉴定

【目的】制备Ⅱ型鲤疱疹病毒（CyHV-2）ORF6多克隆抗体，为深入探究ORF6在CyHV-2急性感染或潜伏感染过程中的作用机制提供技术支持。【方法】利用MEGA 6.0、Adobe Illustrator CS6及BepiPred-2.0等在线软件分析CyHV-2的ORF6基因序列信息，采用PCR从CyHV-2基因组中扩增ORF6基因片段，将其连接至原核表达载体pET-28a后转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导和尿素洗涤纯化获得融合蛋白。以纯化的融合蛋白ORF6免疫健康Babl/c小鼠制备ORF6多克隆抗体，并通过Western blotting和间接免疫荧光技术鉴定ORF6多克隆抗体的特异性。【结果】CyHV-2的ORF6基因全长3003 bp，与CyHV-3和CyHV-1的ORF6氨基酸序列相似性均高于30.00%，其中与CyHV-3的相似性高达39.36%；其抗原表位最可能存在于第1~300位氨基酸序列中。将PCR扩增获得的ORF6基因片段连接至原核表达载体pET-28a可成功构建重组质粒pET-28a-ORF6，转化BL21（DE3）感受态细胞后经IPTG诱导4 h即获得融合蛋白ORF6；原核表达的融合蛋白ORF6主要以包涵体形式进行表达，其分子量为17.13 kD，经6 mol/L尿素洗涤纯化后的浓度为0.1 mg/mL。以纯化融合蛋白ORF6免疫Babl/c小鼠制备获得的ORF6多克隆抗体能特异性识别融合蛋白ORF6及CyHV-2感染的异育银鲫尾鳍细胞（GiCF）；利用制备的ORF6多克隆抗体对CyHV-2感染GiCF细胞进行间接免疫荧光分析，结果在CyHV-2感染GiCF细胞周围能观察到特异性绿色荧光，而在未感染CyHV-2的GiCF细胞周围未观察到特异性绿色荧光，进一步说明ORF6多克隆抗体可特异性识别CyHV-2。【结论】制备获得的ORF6多克隆抗体能与CyHV-2感染GiCF细胞发生特异性免疫反应，即可用于鉴定CyHV-2感染，为探究ORF6蛋白功能是否与CyHV-2潜伏感染相关打下了基础。

Preparation and identification of polyclonal antibody against cyprinid herpesvirus-2 ORF6

【Objective】 The polyclonal antibody of ORF6 of cyprinid herpesvirus-2(CyHV-2)was prepared to provide a technical means for further investigation of the function of ORF6 in CyHV-2 acute infection or a latent infection process.【Method】 The ORF6 gene sequenceof CyHV-2 was analyzed by online softwares such as MEGA 6.0,Adobe Illustrator CS6 and BepiPred-2.0. The ORF6 gene was amplified from the CyHV-2 genome by PCR and cloned into the prokaryotic expression vector PET-28a. The recombinant expression plasmid was transformed into Escherichia coli BL21(DE3),after that,expression was induced by IPTG. The recombinant protein was purified by urea method,Babl/chealthy mice were immunized with purified recombinant ORF6 protein,serum was collected to prepare polyclonal antibody against ORF6 protein. Finally,the specificity of antibody was identified by Western blotting and indirect immunofluorescence technique(IFA).【Result】 The full-length ORF6 gene of CyHV-2 was 3003 bp,and the amino acid sequence similarity with CyHV-3 and ORF6 of CyHV-1 was higher than 30.00%,and the similarity with CyHV-3 was 39.36%. Its antigenic epitope was most likely to exist in amino acid sequences 1-300. ORF6 gene fragment obtained by PCR was ligated to the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-ORF6,which was transformed into BL21(DE3)competent cells and induced by IPTG for 4 h to obtain the fusion protein ORF6,it was mainly expressed in the form of inclusion bodies,and its molecular weight was 17.13 kD. The concentration of ORF6 purified by 6 mol/L urea washing was 0.1 mg/mL. The ORF6 polyclonal antibody prepared by immunizing Babl/c mice with purified fusion protein ORF6 could specifically recognize the fusion protein ORF6 and the Carassius auratus gibelio caudal fin cell(GiCF) infected by CyHV-2. The prepared ORF6 polyclonal antibody was used for indirect immunofluorescence analysis of GiCF infected with CyHV-2. The results showed that specific green fluorescence could be observed around GiCF infected with CyHV-2,while no specific green fluorescence was observed around GiCF cells not infected with CyHV-2,which further indicated that ORF6 polyclonal antibody could specifically recognize CyHV-2.【Conclusion】 The prepared ORF6 polyclonal antibody can specifically react with CyHV-2 infected GiCF,which can be used to identify CyHV-2 infection,and lay a foundation for later exploration of whether ORF6 protein function is related to CyHV-2 latent infection.