鸡新城疫病毒HN基因片段的原核表达及其单抗制备  

Prokaryotic Expression of Chicken NDV-HN Gene Fragment and Preparation of Its Monoclonal Antibody

作　　者：王贤[1] 陈芳芳[1] 余为一[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230027

出　　处：《安徽农业科学》2011年第20期12223-12224,共2页Journal of Anhui Agricultural Sciences

基　　金：国家自然基金项目(30671537)资助

摘　　要：[目的]研究NDV-HN蛋白抗原表位在动物免疫应答中的作用特点。[方法]根据HN基因和载体pGEX-4T-1的多克隆位点自行设计1对引物,以该实验室保存的重组质粒为模板,进行PCR扩增,再将其所得片段定向插入pGEX-4T-1,构建重组质粒,并对该重组质粒进行鉴定;再将构建的重组质粒在大肠杆菌中表达;最后,鉴定了相应单克隆抗体。[结果]PCR结果表明,得到了与预期结果一致的长度为850 bp的片段;重组质粒鉴定结果表明,成功构建了包含目的基因片段的重组表达质粒pGEX-4T-HN23;重组质粒经原核表达获得大小为57 kD的融合蛋白,免疫Balb/c小鼠试验表明,用免疫鼠脾细胞与骨髓瘤细胞融合,经ELISA筛选和3次亚克隆获得2株稳定传代(可传20代以上)和分泌抗NDV-HN抗体的杂交瘤细胞株。[结论]获得的鸡新城疫病毒HN单克隆抗体,为进一步研究HN在致病过程的作用及特异性诊断提供了重要试验材料。[Objective] The aim was to study characteristics of the NDV-HN protein epitope in the animal immune response.[Method] A pair of specific primers was designed according to the HN gene sequence and multiple clone sites of vector PGEX-4T-1;and then PCR amplification was conducted by using a recombinant plasmid as template.The amplified fragment was inserted into pGEX-4T-1 to construct a new recombinant plasmid.After the recombinant plasmid was identified,it was expressed in prokaryotic cells.The monoclonal antibody of NDV-HN was also prepared and identified.[Result] PCR result showed that a DNA fragment of 850 bp in length was amplified as expected;identification of recombinant plasmid revealed that the target gene was successfully constructed into the plasmid pcGEX-4T-HN23,by which a fusion protein with 57kD was expressed.The spleen cells of the mouse immunized with the purified fusion protein were fused with SP2/0 cells.After ELISA screening and subcloning for three times,two strains which could generate stably more than 20 times and produce antibody to HN protein were obtained.[Conclusion] The resulting monoclonal antibody against NDV-HN protein provided important materials for investigating the role of HN in pathopoiesis process and its specific diagnosis.

关 键 词：新城疫病毒 HN 原核表达 单抗 

分 类 号：S852.657[农业科学—基础兽医学]