副猪嗜血杆菌间接ELISA抗体检测试剂盒的研制与应用  

Development and Application of Indirect ELISA Kits for Antibody Detection of Haemophilus parasuis

作　　者：张鹏云 陈敏[1] 刘明星 周红[1] 蔺辉星[1] 范红结[1] ZHANG PengYun;CHEN Min;LIU MingXing;ZHOU Hong;LIN HuiXing;FAN HongJie(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《中国农业科学》2023年第8期1606-1614,共9页Scientia Agricultura Sinica

基　　金：江苏省农业科技自主创新资金项目(CX(19)2020);南京农业大学大学生创新训练项目(202117XX07,202117XX22);江苏高校优势学科建设工程专项资金项目(PAPD)。

摘　　要：【背景】副猪嗜血杆菌(Haemophilus parasuis,HPS)是猪的上呼吸道病原菌,引起格拉瑟氏病,主要引起断奶前后和保育阶段的猪发病,通常见于5—8周龄的青年猪,发病率一般为10%—15%。该菌有15种血清型,目前主要养猪国家流行的血清型为4型、5型、12型和13型,是严重危害养猪业发展的主要细菌性病原之一。目前国内还没有检测该菌抗体的商品化试剂盒。【目的】研制一种针对副猪嗜血杆菌的快速、敏感和特异性强的抗体检测试剂盒,为格拉瑟氏病的有效防控提供技术支持。【方法】表达并纯化OppA、DppA、HbpA 3种不同的副猪嗜血杆菌ABC转运体周质底物结合蛋白,使用副猪嗜血杆菌阴、阳性参考血清筛选以上3种蛋白中免疫反应性和反应特异性好的蛋白。以筛选的蛋白为包被抗原,建立检测HPS抗体的间接ELISA方法,优化间接ELISA反应条件,并组装试剂盒。在此基础上,确定该试剂盒的敏感性、特异性;通过对不同时间、不同猪场采集的2000份临床猪血清样本进行检测,评价该试剂盒的实用性;在以上临床血清样本中随机选取200份,分别以研制的试剂盒、间接血凝试验以及进口商品化试剂盒进行检测,比较检测结果,验证该试剂盒的符合率;最后,使用研制的试剂盒检测免疫和攻毒猪采集的血清,评价该菌免疫后的抗体消长规律。【结果】成功表达并纯化了OppA、DppA、HbpA 3种蛋白,发现OppA免疫反应性和反应特异性最好。经过反应条件优化,确定OppA包被浓度为1μg·mL-1,封闭液为0.5%的BSA-PBS溶液,样品稀释液为1%的BSA-PBST溶液,样品孵育时间为30min,样品稀释度为1﹕50,酶标二抗孵育时间为30min,底物作用时间为15 min,临界值为0.18;试剂盒的敏感性和特异性为96.67%,能与国内常见血清型HPS阳性血清反应而不与猪其他常见病原阳性血清发生交叉反应;2000份临床血清样本阳性检出率为34.65%;随机抽取【Background】Haemophilus parasuis(HPS)is the pathogen of upper respiratory tract of pigs,causing Glaser’s disease,mainly in pigs before and after weaning and in the nursery stage,which is usually seen in young pigs aged 5-8 weeks,and the incidence rate is generally 10%-15%.There are 15 serotypes of the bacteria and the serotypes currently prevalent in major pig-raising countries are 4,5,12 and 13,and it is one of the main bacterial pathogens affecting the development of pig industry.At present,there is no commercial kit for detecting antibody of the bacteria in China.【Objective】The development of a rapid,sensitive and specific antibody detection kit could provide the technical support for the effective prevention and control of the disease.【Method】Three different periplasmic substrate binding proteins of OppA,DppA and HbpA were expressed and purified.The positive and negative serum was used to screen one of the above three proteins with sound immunoreactivity and specificity.Using the screened protein as the coating antigen,an indirect ELISA method for detecting HPS antibody was established,the reaction conditions of indirect ELISA were optimized,and the kit was assembled.On this basis,the sensitivity and specificity of the kit were evaluated;the practicability of the kit was evaluated by testing 2000 clinical pig serum samples collected at different times and from different pig farms;based on the above clinical serum samples,200 samples were selected randomly and tested with this kit,indirect hemagglutination test,and imported commercial kit,respectively,and the test results were compared to verify the compliance rate of the kit;finally,the kit developed in this study was used to detect immune and challenge pigs.The collected serum was used to evaluate the growth and decline of antibodies against the bacteria after immunization.【Result】Three proteins of OppA,DppA and HbpA were successfully expressed and purified,and it was found that OppA had the best immunoreactivity and specificity.After optim

关 键 词：副猪嗜血杆菌 ABC转运体周质底物结合蛋白 间接ELISA 免疫评价 

分 类 号：S858.28[农业科学—临床兽医学]