一种基于Ⅱ型鲤疱疹病毒衣壳蛋白72的免疫学检测方法

Ⅱ型鲤疱疹病毒(cyprinid herpesvirus 2,Cy HV-2)是引起养殖异育银鲫(Carassius auratus gibelio)造血器官坏死症的致病病原。在临床筛查中基于病毒核酸的PCR和real time PCR技术已经建立,但是稳定性更强的免疫学诊断技术国内外尚无报道。本研究目的是利用Cy HV-2编码的ORF72基因(Gen Bank登录号:AFJ20502.1)所编码的衣壳蛋白作为捕获抗原,通过识别感染病毒的鱼体中的相应抗体,从而对样本进行临床免疫学检测。首先采用PCR方法从纯化的Cy HV-2基因组中扩增ORF72基因,并把该基因克隆至原核表达载体PGEX-4T-3,并转化到大肠杆菌中诱导表达,诱导表达的产物通过SDS-PAGE进行鉴定,对表达的重组蛋白进行纯化。用已纯化的72重组蛋白对小鼠进行免疫,制得72重组蛋白的抗体。Western blot检测表明所制备的多克隆抗体既能识别原核表达的重组蛋白,也可以识别Cy HV-2病毒粒子上的衣壳蛋白72。在上述基础上建立了基于Western blot技术的Cy HV-2抗体检测技术:用纯化的72重组蛋白作为检测抗原,鲫鱼血清用作一抗,兔抗鲫Ig M多克隆抗体作为二抗,酶标羊抗兔作为三抗鉴定鲫鱼是否存在Cy HV-2特异性抗体。在对急性感染期的临床样本检测中,本方法能在所有样本中检测出ORF72特异性抗体存在,表明72重组蛋白作为相应抗体捕获原可以用于确诊鲫鱼是否感染Cy HV-2。本研究建立的实验室免疫学检测方法为商品化免疫学检测技术的开发奠定了基础,对Cy HV-2的检验检疫具有一定的临床应用价值。

An immunological method to detect CyHV-2 based on capsid protein 72

Cyprinid herpesvirus II (CyHV-2) is the main pathogen causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid-based diagnostic methods have been applied, no stable and sensitive im-munological diagnostic methods have been reported. In this study, to detect CyHV-2 in clinical samples by immu-nological methods, recombinant protein 72, encoded by the CyHV-2 ORF72 gene, was used as a capture antigen to identify specific antibodiesagainst CyHV-2 in the serum of infected fish. First, ORF72 was amplified from the purified CyHV-2 genome, and cloned into expression vector PGEX-4t-3 to produce recombinant GST-ORF72 inEscherichia coli. The recombinant protein was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The puri-fied recombinant protein 72 was used to immunize mice and polyclonal antibodies were obtained. Western blotting showed that the polyclonal antibody could not only identify the recombinant protein, but could also recognize protein 72 in virus particles. A Western blotting protocol was established for the specific detection of antibodies against protein 72 protein of CyHV-2 and then applied to detect CyHV-2 infection in clinical fish sera samples. In conclusion, the Western blotting technique established in this study could be used for specific serodiagnosis of CyHV-2 infection.