蓝舌病病毒VP2蛋白展示口蹄疫病毒中和表位的研究  

Display expression of food-and-mouth disease virus neutralizing epitope on bluetongue virus VP2 protein

作　　者：罗小暖[1,2] 王芳[2] 崔玉东[1] 于力[2] 

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/牛羊传染病研究创新团队,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第9期731-734,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31201941)

摘　　要：为在蓝舌病病毒(BTV)VP2蛋白中鉴定展示表达外源B细胞表位的位点,本研究通过同源建模预测VP2蛋白的结构,采用SOE-PCR方法,将O型口蹄疫病毒编码保守的中和表位(8E8)序列分别插入BTV-8 VP2基因编码的两个不同loop区序列中,并分别构建了两种嵌合VP2基因。将嵌合表位编码序列的VP2基因克隆于杆状病毒转移载体pFastBac HTb中,转化感受态细胞DH10Bac,筛选阳性重组杆粒rBacmid-VP2-8E8,转染昆虫细胞Sf9,获得重组杆状病毒rBac-VP2-8E8。经western blot和间接免疫荧光验证,嵌合8E8表位的VP2蛋白在Sf9细胞中正确表达,并且8E8表位能够正确展示在VP2蛋白表面;VP2蛋白三聚体分析表明,嵌合8E8表位的VP2蛋白仍能形成三聚体。以上结果表明,BTV VP2蛋白至少存在两个插入位点,能够容纳12个氨基酸短肽的插入。该结果为BTV病毒样颗粒展示8E8表位的研究提供实验依据。To identification of the sites on the bluetongue virus （BTV） VP2 for surface expression of foreign peptides, the 3D structure of BTV-8 VP2 and the sites for accommodating foreign peptides were predicated based on homology modeling. Then, two chimeric genes of BTV-g VP2 were amplified by SOE-PCR, which contained an inserted sequence encoding 12 amino acid residues of the O serotype food-and-mouth disease virus neutralizing epitope （8E8） within two different loop encoding sequences of BTV-8 VP2 gene, and the chimeric genes were cloned into pFastBac HTb vectors, respectively. The recombinant plasmids were transformed into competent cells of E.col~ DH10Bac to construct recombinant bacmids. Two recombinant baculovirus were generated by transfection of the recombinant bacmids into Sf9 cells, respectively. The epitope-chimeric VP2 were correctly expressed in insect cells and the antigenic activity of the 8E8 epitope was detected by western blot and the indirect immunofluorescence assay. Moreover, the two epitope-chimeric VP2 still had the ability to form the multimetric structure. These data demonstrated that the two loops in VP2 tolerated the insertion of 12-amino acid peptide and the chimeric VP2 was potential to be used as vector for development of polyvalent vaccine.

关 键 词：蓝舌病病毒 VP2蛋白 O型口蹄疫病毒8E8表位 杆状病毒表达 

分 类 号：S852.65[农业科学—基础兽医学]