抗猪胸膜肺炎放线杆菌ApxIV毒素单克隆抗体的制备及初步应用  

DEVELOPMENT AND PRIMARY APPLICATION OF MONOCLONAL ANTIBODIES AGAINST APXⅣ OF ACTINOBACILLUS PLEUROPNEUMONIAE

作　　者：张志妮[1] 张伟娟[1] 林燕清[1] 顾万军[2] 黄良宗[2] 朱军[1] 姚丰华[1] 朱国强[1] 

机构地区：[1]扬州大学兽医学院,扬州225009 [2]佛山科学技术学院生命科学学院,佛山528231

出　　处：《中国动物传染病学报》2009年第4期36-42,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家十一五科技支撑项目(2006BAD06A12)

摘　　要：据猪胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae,App）apxⅣ毒素基因5′端的保守区域设计一对特异性引物,应用PCR方法扩增出致病性App1~12血清型菌株的保守5′端序列片段,构建的重组质粒pETapxⅣN经IPTG诱导表达出分子量大小为35.3kDa的可溶性重组蛋白。以亲和层析试剂盒纯化的重组蛋白免疫BALB/c小鼠制备抗ApxⅣ毒素单克隆抗体（McAb）。以间接ELISA法筛选到两株分泌稳定、抗体亚类均为IgGl的杂交瘤细胞5B7和5C11,其培养上清和小鼠腹水抗体效价分别为1∶64、1∶128和1∶64 000、1∶128 000.两株单抗与临床猪瘟病毒、猪圆环病毒、猪呼吸道繁殖障碍综合征病毒、猪黄、白痢产肠毒素大肠杆菌和猪肺疫多杀性巴氏杆菌感染阳性血清均不发生交叉反应,显示出良好的特异性.竞争性结合试验表明两株单抗识别不同的抗原结合表位.以（NH4）2SO4盐析法纯化的5C11小鼠腹水单抗包被酶标板,生物素标记纯化的5B7单抗建立了检测ApxⅣ毒素的双抗体夹心ELISA法,其包被单抗最佳工作浓度为4μg/ml,生物素标记单抗最佳工作浓度为0.8μg/mL,对重组表达ApxⅣ毒素（rApxⅣ）的最低检出量为60pg/mL。从10份临床病猪血清样本中检出6份ApxⅣ毒素阳性,与细菌分离鉴定和PCR结果相符合,结果表明此法可用于App感染的临床诊断。The N-terminal conserved sequences of apxⅣ gene from Actinobacillus pleuropneumoniae（App） serotypes 1~12 were amplified by PCR and cloned into the prokaryotic plasmid pET-22b（＋） to construct recombinant plasmid pET apxⅣN.A 35.3 kDa soluble recombinant protein（rApxⅣN） were expressed and purified by Ni-TED（tris-carboxymethyl ethylene diamine） immobilized metal ion affinity chromatography（IMAC）.BALB/c mice were immunized with purified rApxⅣN and two hybridoma cell lines secreting monoclonal antibodies（MAbs） of 5B7 and 5C11 were identified.The ELISA titers of 5B7 and 5C11 were 1∶64 and 1∶128 in culture supernatants,and 1∶64 000 and 1∶128 000 in ascites.Both MAbs showed no cross-reactions with porcine sera of classical swine fever virus,porcine circorvirus,porcine reproductive and respiratory syndrome virus,enterotoxigenic Escherichia coli and Pasteurella multocida.The MAbs 5B7 and 5C11 were IgG1 but recognized different epitopes as demonstrated in competitive binding ELISA.A double MAb-mediated sandwich ELISA was developed by using 5C11 as a coating antibody（4 μg/mL） and 5B7 as a biotin-labeled antibody（0.8 μg/mL）.The sandwich ELISA detected as low as 60 pg/mL of rApxⅣ.Furthermore,10 clinical porcine sera samples were tested and 6 were valued as positive samples,which were consistent with results of bacterial isolation and PCR.The preliminary application of the double MAbs-mediated sandwich ELISA suggests that it could be a valuable method for detection of App.

关 键 词：猪胸膜肺炎放线杆菌 ApxⅣ毒素 表达 单克隆抗体 

分 类 号：S858.282.619[农业科学—临床兽医学] Q785[农业科学—兽医学]