鸡传染性支气管炎病毒HH06株囊膜蛋白多克隆抗体制备及生物学功能检测  

Preparation of Polyclonal Antibody against Recombinant E Protein of IBV HH06 Strain and Its Biological Function

作　　者：白妍[1] 赵蕾[1] 杜威威[1] 潘龙[1] 黄小丹[1] 杨贵君[1] 李广兴[1] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030

出　　处：《中国畜牧兽医》2016年第8期1967-1974,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31172295;31272569)

摘　　要：采用RT-PCR方法扩增鸡传染性支气管炎病毒（infectious bronchitis virus,IBV）HH06株E基因全长,经抗原性和亲水性分析,将E基因部分序列（193~327bp）亚克隆于pET-32a（＋）和PVAX1载体中。将阳性重组质粒pET-32a-E1转化E.coli Rosetta（DE3）感受态细胞,诱导表达获得大小约为23ku的重组截短E1蛋白,Western blotting检测重组蛋白与IBV全病毒多克隆抗体能特异性反应。以纯化后的E1蛋白免疫新西兰白兔制备多克隆抗体,ELISA检测抗体效价达到220;Western blotting分析表明,E1多克隆抗体与重组表达蛋白具有良好的反应性;间接免疫荧光试验显示,多克隆抗体可检测到PVAX-E1转染Hela细胞表达的E1蛋白和感染鸡胚肾细胞（CEK）中的HH06株IBV,抗E蛋白多克隆抗体的制备为E蛋白功能的深入研究奠定了基础。In this study,IBV HH06 complete Egene was firstly cloned and sequenced.According to the hydrophilicity and antigenic index analysis,its partial gene（193to 327bp）was subcloned into prokaryotic expression vector pET-32a（＋）and eukaryotic expression vector PVAX1.The recombinant plasmid pET-32a-E1 was transformed into E.coli Rosetta（DE3）and induced with IPTG.The recombinant IBV truncated E1 protein with molecular weight of 23 ku was observed as expected.It could be recognized by positive IBV antisera in Western blotting with high reactivity.Then the purified recombinant protein was used as antigen for immunization of rabbit to prepare polyclonal antibody.Indirect ELISA showed that the titer of polyclonal antibody was 220,and it had high reactivity and specialty with recombinant protein.Furthermore,IFA test demonstrated that this polyclonal antibody could react with Hela cells transfected with PVAX-E1 plasmid and IBV-infected CEK cells.The IBV E polyclonal antibody obtained in this study laid a foundation for further functional research of E protein in IBV pathogenesis.

关 键 词：传染性支气管病毒 囊膜蛋白 多克隆抗体 免疫印迹试验 间接免疫荧光试验 

分 类 号：S852.65[农业科学—基础兽医学]