传染性法氏囊病病毒vp3基因在昆虫细胞中的表达及其ELISA抗原反应性的分析  

Expression of vp3 gene of infectious bursal disease virus in insect cells and analysis of antigenicity for VP3-ELISA

作　　者：刘华洁[1,2] 杜希宁[2,3] 梅永杰 王晓丽[2] 欧阳伟[2] 毕振威[2] 潘群兴[2] 姚火春[1] 王永山[2] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,江苏南京210014 [3]南京师范大学生命科学学院,江苏南京210046

出　　处：《中国兽医科学》2015年第3期227-234,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31272537;31302070);江苏省自然科学基金项目(BK2012787;BK20141381);江苏省农业科技自主创新基金项目[CX(13)5032]

摘　　要：为分析传染性法氏囊病病毒(IBDV)重组VP3蛋白的ELISA抗原反应性,将IBDVvp3基因插入杆状病毒表达系统供体质粒pFastBacHTA中,构建重组供体质粒pFastBacHTA-vp3并转化大肠杆菌DH10Bac感受态细菌,经抗性筛选获得重组杆状病毒表达质粒pBac-vp3。用pBac-vp3转染Sf9昆虫细胞,获得重组杆状病毒vBac-vp3。对重组杆状病毒感染的Sf9细胞用Western-blot检测,在33ku处存在一条特异的蛋白条带;用间接免疫荧光试验检测时可见特异性荧光。以纯化的重组VP3蛋白为包被抗原建立了检测IBDV抗体的间接ELISA(VP3-ELISA),并与重组VP2蛋白包被抗原建立的VP2-ELISA和全病毒抗原ELISA(IDEXX-ELISA)进行了比较。对42份不同鸡血清中IBDV抗体的检测结果显示,VP3-ELISA的区分度低于VP2-ELISA和IDEXX-ELISA;VP3-ELISA阳性检出率低于VP2-ELISA和IDEXX-ELISA;VP3-ELISA与IDEXX-ELISA的相关系数低于VP2-ELISA与IDEXX-ELISA的相关系数。结果表明,在昆虫细胞中表达的重组VP3蛋白具有良好的特异性和抗原反应性,用它建立的ELISA的抗原反应性弱于用重组VP2蛋白和IBDV全病毒抗原建立的ELISA的抗原反应性。In order to analyze the antigenicity of infectious bursal disease virus(IBDV)recombinant VP3 protein for establishment of ELISA,one recombinant vBac-vp3 baculovirus was successfully achieved.The IBDVvp3 gene was inserted into pFastBacHTA donor plasmid of the baculovirus expression system.The recombinant donor plasmid pBac-vp3 was transformed into E.coli DH10 Bac and cultured in selective media.The pBac-vp3 plasmids were transfected into Sf9 insect cells.Approximately 33 ku protein band was detected by Western-blot.The specific fluorescence was also detected out by the indirect immunofluorescence assay(IFA)in Sf9 cells which were infected with vBac-vp3.An indirect ELISA based on the expressed VP3 protein as the coating-antigen was set up for the detection of the IBDV antibodies compared to the VP2-ELISA coated with recombinant VP2 protein and IDEXX-ELISA coated with whole IBDV.In result,the positive detection rate and the ability to distinguish between positive and negative sera of VP3-ELISA are lower than the VP2-ELISA and IDEXX-ELISA.The correlation coefficient of VP3-ELISA with IDEXX-ELISA is lower than that of VP2-ELISA with IDEXX-ELISA in the IBDV antibodies detection of42 chicken sera.The above-mentioned result indicated that the recombinant VP3 protein expressed in insect cells possessed specific antigenicity and could be used for detecting IBDV antibodies in ELISA.The VP2-ELISA is weaker than both VP2-ELISA and IDEXX-ELISA.

关 键 词：传染性法氏囊病病毒 VP3蛋白 杆状病毒 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]