利用噬菌体展示技术筛选传染性脾肾坏死病毒吸附蛋白及中和抗体识别表位

Infectious spleen and kidney necrosis virus（ISKNV）is confirmed as the pathogen of Siniperca chuatsi fulminant infectious disease and the vaccine immunization is regarded as a preferred measure to control the disease. In this project we intend to construct the phage display library of ISKNV and show completely the peptides that virus gene encodes on the surface of the phage with their original configurations, then screen them respectively with the fish antibody against ISKNV, so as to find ISKNV epitope peptides recognized by neutralizing antibody. The sequences of positive clones will be subcloned into recombinants and expressed. If possible, the healthy Siniperca chuatsi will be immunized with purified recombinant proteins(r-proteins) and then artificially infected with ISKNV to estimate the protectiveness of r-proteins. Moreover，the immunological methods for the inspection of ISKNV will be established supposing the time is enough.

传染性脾肾坏死病毒（ISKNV）被确定为鳜暴发性传染病的病原。疫苗免疫是防治该病的首选措施。本研究通过构建ISKNV的噬菌体展示文库，将病毒基因组编码的蛋白多肽以完整的空间结构充分展示于噬菌体表面，然后用鱼抗病毒抗体进行多轮筛选，确定出ISKNV的中和抗体识别表位肽谱，克隆出相关基因序列，力争重组表达后获得纯化蛋白，免疫鳜鱼，通过人工感染实验确定其免疫保护性，并建立针对ISKNV的免疫学检测方法。

免疫预防是防治鳜鱼传染性脾肾坏死病毒（ISKNV）病的有效措施。本研究从暴发传染性病的鳜鱼体内成功分离到病原，经电镜观察，PCR等鉴定为传染性脾肾坏死病毒（ISKNV），并用蔗糖密度梯度离心初步分离到ISKNV病毒悬液。PCR扩增了覆盖ISKNV全基因组的大片段，成功构建了ISKNV的噬菌体展示文库，库容达6.2×108。采集鳜鱼血清，纯化获得其免疫球蛋白IgM，制备了兔抗鳜IgM多抗，并用辣根过氧化物酶标记，建立了针对ISKNV的免疫学检测方法，测定鳜鱼抗ISKNV多抗血清的效价在1:5120以上，纯化后用以筛选ISKNV噬菌体展示文库中的阳性抗原表位。