鱼类弹状病毒重组多表位肽的构建及免疫效应研究

Rhabdovirus is a highly infectious viral pathogen of fish and other aquatic animals. Based on the complete genome sequence and monoclonal antibodies of Siniperca chuatsi rhabdovirus (SCRV), as well as the antigenic analysis of viral proteins, we will construct recombinant multi-epitope peptides of fish rhabdovirus, and investigate their effects on the antiviral immune responses in mandarin fish. The B cell epitopes of SCRV G protein will be identified by using monoclonal antibodies with pepscan, and the T cell epitopes will be analyzed by epitope prediction programs. The multi-epitope peptides coding gene fragments will be obtained with recombinant DNA technology, and then subcloned into the prokaryotic expression vector, or into the eukaryotic expression vector to construct multi-epitope DNA vaccines against SCRV. The protective effects of different multi-epitope peptides will be analyzed and compared in mandarin fish vaccinated with the purified recombinant proteins or multi-epitope DNA vaccines, and the peptides offering good protection would be screened out. In order to understand the antiviral immune responses elicited by multi-epitope peptides, we will further analyze the virus-specific antibody titers, the proliferation of lymphocytes, and the expression patterns of immune related genes in vaccinated fish.Taken together, the work will provide prospective candidate antigens for the development of epitope-based vaccines against fish rhabdovirus.

弹状病毒是危害鱼类及多种水生动物的烈性、传染性病毒病原。申请者在已获得鳜鱼弹状病毒全基因组序列及多株单克隆抗体、以及对病毒免疫相关蛋白的抗原性进行初步分析的基础上，拟进行鱼类弹状病毒重组多表位肽的构建及免疫效应研究。利用单抗、生物信息学、肽扫描技术等对鳜鱼弹状病毒糖蛋白（G）与核蛋白（N）蛋白的T、B细胞抗原表位进行预测和筛选；然后将候选抗原表位基因经不同组合、串联，表位之间以柔性短肽进行间隔，获得多表位肽编码基因片段，分别构建原核与真核表达系统；进一步以纯化的多表位融合蛋白或重组真核表达质粒免疫鱼体，经攻毒实验测试不同多表位肽的免疫抗病效果，并从鱼体特异性抗体的效价、淋巴细胞增殖情况、以及免疫相关基因表达变化等方面，了解其诱导宿主的免疫应答作用，揭示免疫效应机制。通过上述研究，以期获得免疫原性强、可诱导高效免疫保护效果的多表位肽，从而为鱼类弹状病毒新型高效疫苗的研发提供候选抗原。

弹状病毒是一类引起鱼类及多种水生动物致死性和流行性病害的重要病原。本项目针对鳜鱼弹状病毒，开展了其重组多表位肽的构建及免疫效应研究。设计了16个截短的鳜鱼弹状病毒糖蛋白（G）基因片段，经PCR扩增，克隆至原核表达载体pET-32a。经IPTG诱导，在大肠杆菌BL21中表达出16个重组截短G蛋白，并经Ni-NTA亲和层析柱对融合蛋白进行了纯化。利用间接ELISA和Western blot方法对已制备的鳜鱼弹状病毒单克隆抗体1B2和2C1所识别的表位进行精确定位，结果显示：单抗1B2 能够识别G蛋白的 182SEAPRESCK190 ，单抗2C1识别 G蛋白的366SSEDVTGEIV375。同时，利用生物信息学和Western blot方法鉴定了鳜鱼弹状病毒核蛋白（N）的2个抗原表位，分别为128GQADVQDK135和342TSAELVKA349。将筛选的G蛋白抗原表位与N蛋白抗原表位进行串联，并在表位间加入Ala-Ala-Tyr（AAY）氨基酸间隔序列，经人工合成，获得了2条重组多表位肽，分别为GN-1（SEAPRESCKAAYSSEDVTGEIVAAYGQADVQDK）和GN-2 （SEAPRESCKAAYSSEDVTGEIVAAYTSAELVKA）。将合成的重组多表位肽经腹腔注射鲫鱼，并从鱼体特异性抗体水平、淋巴细胞增殖情况、以及免疫相关基因表达变化等方面，测试分析了其诱导宿主的免疫应答反应。结果显示：（1）在免疫后第14d和21d， GN-1和GN-2免疫组的鳜鱼弹状病毒特异性ELISA抗体水平明显高于PBS对照组。（2）脾脏淋巴细胞在特异性抗原（GN-1 和GN-2）的刺激下，刺激指数均显著高于 PBS 对照组。（3）在GN-1 和GN-2免疫组鱼脾脏组织中，免疫相关基因IRF-7、Mx、IgM不同程度上调表达。该研究结果表明，重组多表位肽GN-1和GN-2均能有效诱导鱼体产生细胞和体液免疫应答，为基于表位的鱼类弹状病毒新型高效疫苗研发奠定基础。