II型鲤疱疹病毒ORF4的多克隆抗体制备及其组织分布

Prokaryotic expression, polyclonal antibody preparation and tissue-tropism analysis of cyprinid herpesvirus II non-structural protein ORF4

【目的】制备II型鲤疱疹病毒(Cy HV-2)ORF4多克隆抗体,利用免疫学方法研究ORF4编码蛋白在病毒感染过程中的组织表达特征。【方法】利用在线软件SMART和BLASTx分析Cy HV-2 ORF4序列,采用PCR技术从Cy HV-2基因组中扩增得到ORF4基因,克隆至表达载体PGEX-4T-3中,将获得的重组表达质粒转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达,表达产物经过SDS-PAGE和Western blot鉴定,再利用亲和层析法纯化出重组蛋白。用纯化的重组ORF4蛋白免疫新西兰兔,收集兔血,分离兔血清获得抗ORF4蛋白的多克隆抗体,再利用Western blot检测抗体与ORF4蛋白之间的特异性。提取攻毒后的异育银鲫肌肉、脑、鳃、脾脏、肝胰脏、心脏、肾脏等组织DNA,用荧光定量PCR技术监测其病毒在各组织的复制水平。使用RIPA裂解液提取上述各组织总蛋白,再利用Western blot技术检测ORF4在感染Cy HV-2的异育银鲫各组织中的表达情况。【结果】原核表达的重组ORF4蛋白分子量为65 k D,与预期大小一致;获得的ORF4抗血清能特异性识别重组ORF4蛋白。病毒感染的异育银鲫体内ORF4主要表达在肾脏、脾脏和鳃上;荧光定量PCR证明Cy HV-2主要在肾脏、脾脏和鳃上富集。【结论】Cy HV-2的非结构蛋白ORF4可能参与病毒的复制,是病毒复制感染周期的特征性指示蛋白之一,这为深入研究ORF4在Cy HV-2感染过程中的作用机制提供参考。

[Objective] Acquire the polyclonal antibody against non-structural protein ORF4 of cyprinid herpesvirus 2, and reveal the tissue distribution pattern of ORF4 protein during the virus infection. [Methods] The ORF4 structure was analyzed by SMART and BLASTx. The ORF of ORF4 was amplified from viral genomic DNA and cloned into plasmid PGEX-4T-3. The recombinant plasmid was transformed into Escherichia coli BL21（DE3） for recombinant protein production after induction by IPTG. The expressed recombinant protein was then purified by affinity chromatography and the purified protein was used to immunize New Zealand rabbits to obtain the anti-ORF4 polyclonal antibody. The specificity of the anti-ORF4 polyclonal antibody was confirmed by Western blot. Furthermore, real time PCR analysis was employed to monitor the genomic replication level in various tissues of virus-infected Carassius auratus gibelio including muscle, brain, gill, spleen, liver pancreas, heart, kidney. And the tissue distribution pattern of ORF4 in infected Carassius auratus gibelio was subsequently analyzed by Westem blot. [Results] The expressed recombinant ORF4 protein in bacteria was detected at 65 kD as expected. Immunoassays suggested that the home-made antibody can specially recognize either the recombinant protein or endogenous ORF4. It proved that the ORF4 was mainly distributed in the kidney, spleen and gill in vivo, which was in consistence with the high level of virus replication in these tissues revealed by real time PCR assay. [Conclusion] Non-structural viral protein ORF4 might involve in efficient viral replication, which could serve as a marker protein for active virus infection. This study paved the way for studying the mechanism of ORF4 in the process of cyprinid herpesvirus 2 infection.