基因Ⅰ型乙脑病毒EDⅢ结构域蛋白的原核表达及其抗体制备与鉴定  

Identification of EDⅢ protein from the genotypeⅠJapanese encephalitis virus

作　　者：于瑞明 田占成[1] 独军政[1] 高闪电[1] 刘光远[1] 罗建勋[1] 殷宏[1,2] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2017年第12期1475-1480,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31201899);中国农业科学院农业科技创新工程项目(2014ZL010)

摘　　要：为了分析和验证作为乙脑病毒(Japanese encephalitis virus,JEV)亚单位疫苗研制的主要候选抗原EDⅢ结构域的免疫原性及其所产生的抗体对病毒的抑制效果,本研究利用RT-PCR方法扩增基因Ⅰ型JEV GS株EDⅢ结构域基因,构建原核重组表达载体p ET-30a-EDⅢ,转化大肠杆菌BL21感受态细胞,挑取阳性克隆进行诱导表达和纯化;将纯化的EDⅢ重组蛋白免疫家兔以制备多克隆抗体,利用Western-blot和间接免疫荧光试验验证制备的家兔抗EDⅢ多克隆抗体与全病毒抗原的特异性反应,并通过噬斑减少中和试验(PRNT50)测定家兔抗EDⅢ多克隆抗体的中和抗体效价。结果表明,重组EDⅢ蛋白以包涵体的形式表达,并获得了较高纯度的EDⅢ重组蛋白,制备的家兔抗EDⅢ多克隆抗体与全病毒抗原具有良好的特异性反应,中和抗体效价为133±46.2。以上结果表明,基因Ⅰ型JEV GS株EDⅢ结构域具有良好的免疫原性,免疫动物所产生的抗体具有较高的中和抗体效价,EDⅢ结构域可作为JEV亚单位疫苗研制的候选抗原。The study was conducted to identify the immunogenicity of EDⅢ protein and the inhibitory effects on the invasion of JEV produced by the polyclonal antibodies against the EDⅢ protein. The EDⅢ gene from the genotype I JEV was amplified by RT-PCR and constructed into the expression vector PET-30a. The recombinant plasmids verified by sequencing were transformed into L coli BL21 and the positive clone was induced for expression and purified. New Zealand white rabbits were immunized with the purified recombinant EDⅢ protein（rEDⅢ）.The specific reactivity between the rabbit polyclonal antibodies against rEDⅢ and JEV was determined by Western-blot and the indirect immunofluorescence test,and the neutralizing antibody titer of the rabbit polyclonal antibodies against rEDⅢ was measured by the plaque-reduction neutralization test（PRNT50）. The results showed that the rEDⅢ was expressed in a inclusion body form. Rabbit polyclonal antibodies against the rEDⅢ specifically recognize the native antigen of JEV and its neutralizing antibody titer was 133±46.2. These results indicated that EDⅢ protein of genotype I JEV GS strain had strong immunity and played the critical roles in the invasion of JEV.From the above,the EDⅢ prote in couod be a better candidate antigen for JEV subunit vaccines.

关 键 词：乙脑病毒 EDⅢ结构域 原核表达 多克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]