用于自然感染低龄绵羊抗体检测的禽分枝杆菌副结核亚种抗原筛选与评价

Screening and evaluation of antigens for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in naturally infected young sheep

目的分析禽分枝杆菌副结核亚种(Mycobacterium avium subspecies paratuberculosis,MAP)10个血清反应性抗原(MAP1138c、MAP2121c、MAP0150c、MAP0862、MAP0209c、MAP2120c、MAP0038、MAP3420c、MAP 2154c和MAP-2751)在自然感染低月龄绵羊体内的抗体反应,评估其诊断价值。方法采集有副结核(paratuberculosis,PTB)病史羊群中无明显PTB症状的6月龄绵羊血清和肛拭子样品,采用PTB抗体检测ELISA试剂盒检测血清,基于MAP F57元件的荧光定量PCR检测肛拭子,根据检测结果对羊只进行分组。采用PCR检测阳性肛拭子中的MAP 10个抗原基因。将抗原基因克隆至pET-28a载体,在大肠埃希菌BL21(DE3)中用IPTG诱导表达,Ni-Sepharose纯化重组抗原。将纯化的重组抗原包被ELISA板,用于检测采集的血清,以分析所选抗原在自然感染绵羊体内的抗体反应。分析不同抗原血清抗体的检出率,以评估其诊断价值。结果所采样的72只绵羊被分为肛拭子阳性-抗体阳性(34只)、肛拭子阳性-抗体阴性(23只)、肛拭子阴性-抗体阴性(15只)3组。10个抗原基因均能在阳性肛拭子中检测到,且每个基因序列高度一致。经ELISA检测,MAP1138c、MAP2121c、MAP0150c、MAP0862在感染绵羊体内产生了抗体反应。在57只感染羊中,分别有30、34、24和31只检测到针对MAP1138c、MAP2121c、MAP0150c和MAP0862的抗体。在肛拭子阳性-抗体阳性组中,抗MAP1138c的抗体检出率(76.47%)最高;在肛拭子阳性-抗体阴性组中,抗MAP2121和MAP0862的抗体检出率(52.17%和47.83%)高于其他2种蛋白。在72份血清样品的检测中,包被MAP2121c和MAP0862的ELISA检测结果的符合率超过91%。结论MAP1138c、MAP2121c和MAP0862可能是诱导自然感染低龄绵羊产生MAP抗体反应的显性生物标志物,MAP2121c和MAP0862可弥补商品化ELISA试剂盒在PTB早期诊断上敏感性不足的缺陷。

Objective To analyze the antibody responses of 10 serum reactive antigens(MAP1138c,MAP2121c,MAP0150c,MAP0862,MAP0209c,MAP2120c,MAP0038,MAP3420c,MAP 2154c and MAP2751)of Mycobacterium avium subspecies paratuberculosis(MAP)in naturally infected young sheep and evaluate their diagnostic value.Methods Serum samples and anal swab samples were collected from 6-month-old sheep without obvious PTB symptoms in the flocks with paratuberculosis(PTB)history.The serum samples were tested by PTB antibody detection ELISA kit,and anal swab samples were detected by the fluorescent quantitative PCR based on MAP F57 element.The sheep were grouped according to the test results.PCR was used to detect 10 MAP antigen genes in positive anal swabs.The antigen genes were cloned into pET-28a and induced to be expressed in E.coli BL21(DE3)strain by IPTG.The recombinant antigens were purified by Ni-Sepharose,and then coated on the ELISA plate for testing the collected serum samples to analyze the antibody reaction of the selected antigens in naturally infected sheep.The detection rates of serum antibodies against different antigens were analyzed to evaluate the diagnostic value of the antigens.Results The 72 sheep sampled were divided into three groups:anal swab positive-antibody positive(n=34),anal swab positive-antibody negative(n=23),and anal swab negative-antibody negative(n=15).All 10 antigen genes were detected from positive anal swabs,and sequences of each gene were highly consistent.Through ELISA detection,MAP1138c,MAP2121c,MAP0150c and MAP0862 produced antibody reactions in infe-cted sheep.Antibodies against MAP1138c,MAP2121c,MAP0150c and MAP0862 were detected in 30,34,24 and 31 of the 57infected sheep,respectively.In the anal swab positive-antibody positive group,the detection rate of anti-MAP1138c antibody was the highest(76.47%).In the anal swab positive-antibody negative group,the detection rates of antibodies against MAP2121 and MAP0862(52.17%and 47.83%)were higher than those of the other two proteins.In the detection of 72serum samples,the overlap of ELISA coated with MAP2121c and MAP0862 exceeded 91%.Conclusion MAP1138c,MAP-2121c and MAP0862 may be dominant biomarkers to induce MAP antibody response in naturally infected young sheep.MAP2121c and MAP0862 can make up for the deficiency of sensitivity of commercial ELISA kits in early diagnosis of PTB.