副猪嗜血杆菌不同血清型稳定表达蛋白的筛选及间接ELISA检测方法的建立  

Screening of stably-expressed proteins in different serotypes of Haemophilus parasuis and establishment of an indirect ELISA

作　　者：朱晓明[1] 邹垚[1] 陈攀[1] 王丽敏[1] 李玉峰[1] 

机构地区：[1]南京农业大学农业部细菌学重点开放实验室,江苏南京210095

出　　处：《中国兽医科学》2015年第1期68-74,共7页Chinese Veterinary Science

基　　金：公益性行业(农业)科研专项(201203039)

摘　　要：为了建立一种能够检测不同血清型副猪嗜血杆菌抗体的间接ELISA,选用了在4、5、12型副猪嗜血杆菌中稳定表达的3种蛋白经原核表达后作为候选抗原和全菌裂解物同时包被酶标板,对临床猪血清进行副猪嗜血杆菌抗体检测,发现以锰依赖的超氧化物歧化酶(MSD)作为包被抗原得到的结果最接近用副猪嗜血杆菌全菌裂解物进行ELISA检测的结果,故确定以MSD蛋白为抗原建立间接ELISA。通过方阵滴定对ELISA反应条件进行筛选优化,确定了最佳反应条件:包被抗原的质量浓度为1.5μg/mL,4℃过夜;50g/L脱脂奶粉溶液37℃封闭2h;血清稀释度1∶160,37℃作用90min;酶标二抗的稀释度为1∶15 000,37℃作用45min;底物显色时间为37℃6min。抗体临界值D450nm≥0.245 1判为阳性,D450nm<0.208 3判为阴性,介于两者之间为可疑。特异性和重复性试验证明,该方法与猪繁殖与呼吸综合征病毒、猪瘟病毒、伪狂犬病病毒、猪圆环病毒2型、口蹄疫病毒、红斑丹毒丝菌、马链球菌、猪链球菌、大肠杆菌、肠炎沙门菌血清抗体无交叉反应,批内、批间重复性较好。用建立的间接ELISA对114份临床血清进行检测,与4、5、12型全菌裂解物的ELISA检测结果比较,阳性符合率分别为80.77%、80.00%和90.00%。结果表明,建立的间接ELISA可被用于4、5、12型副猪嗜血杆菌的抗体检测和流行病学调查。In order to establish an indirect ELISA for detecting antibodies against different serotypes of Haemophilus parasuis,three stably expressed proteins in serotypes 4,5and 12 expressed in Escherichia coli and lysis products of bacteria were used as candidate antigens to coat ELISA plates.Compared with the results of ELISA using lysis products of bacteria as antigens,the recombinant manganese-dependent superoxide dismutase(MSD)protein was confirmed to be the optimal coating antigen to establish ELISA.The optimized ELISA conditions were as follows:antigen protein was coated at 4℃ overnight with a concentration of 1.5μg/mL,the plates were blocked with 50g/L skim milk at 37℃for 2h;the sera samples were incubated at 37℃for 1.5hwith 1∶160dilution and secondary antibody was incubated at 37℃for 45 min with 1∶15 000 dilution.The cutoff values of positive and negative results were D450nm≥0.245 1and D450nm<0.208 3,respectively.The specificity test results showed that this ELISA method did not react with positive sera of other 10pathogens(porcine reproductive and respiratory syndrome virus,classical fever virus,pseudorabies virus,porcine circovirus type 2,foot-and-mouth disease virus,Erysipelothrix rhusiopathiae,Streptococcus equi,Streptococcus suis,E.coli,Salmonella enteritidis)and the repeatability of intra-and inter-assay were excellent.A total of 114 sera samples obtained from swine farms were detected by the indirect ELISA using MSD protein and lysis products of serotypes 4,5and 12 H.parasuis as antigen,the positive coincidence rates were 80.77%,80.00% and 90.00%,respectively.So the assay can be used as a tool for diagnosis and epidemiological surveys on serotypes 4,5and 12 H.parasuis.

关 键 词：副猪嗜血杆菌 MSD重组蛋白 间接ELISA 

分 类 号：S852.612[农业科学—基础兽医学]