鲤春病毒血症病毒核蛋白、磷蛋白与基质蛋白的表达、抗体制备及免疫原性比较

Recombinant expression and polyclonal antibody preparation of the nucleoprotein,phosphoprotein and matrix protein of spring viremia of carp virus

鲤春病毒血症病毒(SVCV)能够引起鲤科鱼类大量死亡,被世界动物卫生组织(OIE)列为必须申报的重要疫病,也是我国唯一被列为一类疫病的鱼类传染病。为建立SVCV的快速免疫学诊断方法,研究其主要结构蛋白间的免疫原性差异,实验首先采用原核表达系统克隆并诱导表达,纯化SVCV的核蛋白(N)、磷蛋白(P)和基质蛋白(M),并进一步免疫新西兰白兔制备抗血清,抗血清经Protein A柱进一步纯化获得3种蛋白的多克隆抗体。利用间接酶联免疫吸附测定(ELISA)和免疫印迹实验(Western blot)对抗体效价和特异性进行分析验证。结果发现,SVCV的N、P和M重组蛋白均在原核表达系统获得大量表达,且表达的蛋白经纯化后免疫实验动物产生了相应的多克隆抗体。Western blot结果显示,3种蛋白抗体均与SVCV重组蛋白及天然蛋白发生特异性的免疫反应。ELISA结果显示,针对P蛋白制备的抗体效价最高,可达409 600;针对N和M蛋白制备的抗体效价也均大于204 800。同时,特异性检测实验结果显示,制备的3种蛋白抗体均仅与SVCV发生特异性免疫反应,而与SVCV宿主其他易感病毒均不发生交叉反应。实验结果将对SVCV的快速诊断及疫苗开发提供新的手段和思路。

Spring viremia of carp virus（SVCV） is an OIE listed highly pathogenic agent of several economically important Cyprinidae fish species. Currently, there is no effective vaccine or drug available for this virus, and prevention of SVC mostly relies on prompt diagnosis. Previously, detection methods based on the G gene or the G glycoprotein have been developed. However, the highly genetic diversity of the G gene seriously limits the reliability of those methods. To develop a rapid immunological detection method for SVCV, the N, P and M genes of SVCV were cloned into a p RSET-A prokaryotic expression vector. Recombinant proteins were then induced with 1 mmol/L IPTG at 37 °C. Next, recombinant proteins were purified and used to immunize New Zealand rabbits. Titers and specificities of antibodies were verified by indirect ELISA and Western blot. Our results indicated the obtained antibodies were highly specific to SVCV and the titers could reach at least as high as 204 800.Besides, indirect ELISA assays based on prepared antibodies showed there was no cross reaction with other aquatic viruses. Our results will be useful for the rapid immunological detection of SVCV and provide a new insight into the vaccine development of SVCV.