【作者】 王勇；

【导师】 彭大新； 陈义平； 陈素娟；

【作者基本信息】 扬州大学 ， 预防兽医学， 2013， 硕士

【摘要】 布鲁氏菌病(Brucellosis)是由布鲁氏菌(Brucella)侵入机体引起的人畜共患传染病,感染动物主要表现为流产和不孕不育等症状。近年来,布鲁氏菌病在我国部分地区仍然对当地的畜牧业和人类健康造成较大的危害,及时做出快速准确的诊断是防治和根除该病的关键。目前,我国在布鲁氏菌病防控工作中主要采用虎红平板凝集试验、试管凝集试验、补体结合试验等传统血清学方法进行检测,因此有必要建立一种高通量的、快速准确的检测布鲁氏菌抗体的ELISA方法。本研究通过原核表达系统表达了布鲁氏菌的BP26、 OMP16、CP39、SP41和eryC这5个特异性蛋白,并对其作为检测用抗原的可行性进行了研究,最终选定重组周质蛋白BP26作为包被抗原,初步建立了检测牛布鲁氏菌病血清抗体的间接ELISA方法。一、布鲁氏菌BP26、OMP16、CP39、SP41和eryC基因的原核表达根据羊种布鲁氏菌M5(B.melitensis M5)的BP26、OMP16、CP39、SP41和eryC基因序列设计引物,用PCR方法扩增目的片段,大小分别为868bp、556bp、732bp、1329bp和985bp,然后把目的片段分别连接到克隆载体pMD18-T,测序确认后再连接到表达载体pET-28a(+)。将酶切鉴定正确的重组质粒转入大肠杆菌BL21,经IPTG诱导表达,得到重组蛋白,大小分别约为32kDa、21kDa、30kDa、51kDa和38kDa,且均为包涵体表达。优化的表达条件为：37℃下,220rpm摇振培养,IPTG终浓度1mM,诱导5h。用His亲和层析法对BP26、OMP16、CP39、SP41和eryC这5种重组蛋白进行纯化,收获的纯化产物浓度分别为1.0mg/mL、2.4mg/mL、3.5mg/mL、1.3mg/mL、1.7mg/mL。用Western blotting试验鉴定5种重组蛋白的免疫反应性,结果表明5种重组蛋白均能与牛布鲁氏菌病阳性血清发生特异性反应,只是反应的强度有所不同,其中以重组蛋白BP26反应信号最强。二、检测牛血清中布鲁氏菌抗体的间接ELISA方法的建立将亲和层析纯化的重组蛋白BP26、OMP16、CP39、SP41和eryC分别以0.5μg/mL、4μg/mL、3μ/mL、4μg/mL、2μg/mL包被酶标板,检测48份牛血清样品,并与Pourquier(?)ELISA试剂盒的平行检测结果进行比较,敏感性分别为90.9%、90.1%、97.%、90.1%和90.1%,特异性分别为80%、80%、73.3%、73.3%和53.3%。5种重组蛋白间接ELISA分别检测由虎红平板凝集试验、试管凝集试验、Pourquier(?) ELISA试剂盒检测均为阳性牛血清样品20份、阴性牛血清样品8份,重组蛋白BP26检测的阳性OD450平均值与阴性OD450平均值的比值P/N明显大于其它4个重组蛋白的P/N值,确定重组蛋白BP26作为检测用抗原。运用重组蛋白BP26作为抗原,并对各反应条件进行优化,初步建立了用于检测牛布鲁氏菌病血清抗体的间接ELISA方法。抗原最佳包被浓度为500ng/mL,待检牛血清样品最佳稀释度为1：100,血清样品的OD45o≥0.2×ODP+0.8×ODN判为阳性,反之判为阴性(ODp表示阳性对照血清OD450的平均值,ODN阴性对照血清OD450的平均值)。用建立的间接ELISA方法分别检测牛结核阳性血清、牛大肠杆菌阳性血清、牛口蹄疫阳性血清和牛病毒性腹泻阳性血清,结果均为阴性,说明该方法具有高度的特异性。取8份OD值不同的血清样品,分别进行8次批内重复性试验和2次批间重复性试验,重复性良好。用该方法对174份临床牛血清样品进行检测,并与IDEXX公司的牛布鲁氏菌病Pourquier(?) ELISA试剂盒平行检测结果进行比较,Pourquier(?) ELISA试剂盒阳性检出率为70.11%,本实验建立的间接ELISA的阳性检出率为68.97%。该方法敏感性、特异性、符合率分别为94.2%(113/120)、83.3%(45/54)和90.8%(158/174)。上述研究表明建立的基于重组周质蛋白BP26的间接ELISA方法具有较好特异性和敏感性,而且重复性好,可用于牛布鲁氏菌病血清抗体的检测。更多还原

【Abstract】 Brucellosis is a zoonotic disease caused by members of the genus Brucella, and the main syptoms of the infected animals are abortion and dysgenesia. Recently, Brucellosis is prevalent in some fields of China, which brings a great threat to the development of livestock industry and human health. To prevent and eradicate the disease effectively, it’s very important to diagnose it rapidly and accurately. Rose bengal plate agglutination test (RBT), serum tube agglutination test (SAT) and complement fixation test (CFT) are still the main methods for diagnosing brucellosis in China. Thus, it is necessary to develop ELISA methods to detect serum antibody against Brucella, which may complete a high-throughput and precise detection. In our study,5specific proteins of Brucella were expressed by prokaryotic system, and their feasibilities for diagnosis were estimated. Finally, the recombinant protein BP26was selected, and an indirect ELISA for diagnosing brucellosis was established.1. The expression of recombinant proteins BP26, OMP16, CP39, SP41and eryCAccording to the sequence of BP26,OMP16, CP39, SP41and eryC gene of B. melitensis M5, five pairs of primers were designed. The target fragments with expected sizes of868bp,556bp,732bp,1329bp and985bp were amplified by PCR, and were cloned into pMD18-T vector respectively. Then, they were subcloned into the downstream of T7prometer of pET-28a(+) vector. The recombinant plasmids were transformed into E.coli BL21, and the recombinant proteins were induced by IPTG. Various expression conditions were optimized systematically, and then the expression products were analyzed by SDS-PAGE. The results showed that the molecular weights of the expression products were about32kDa,21kDa,30kDa,51kDa and38kDa, respectively. The best expression conditions of recombinant proteins were as following:The recombinat E.coli BL21was induced with1mM IPTG at37℃for5 hours.The expression products were purified from bacterial inclusion bodies by His·Bind affinity chromatography, and the concentrations of purified BP26,OMP16, CP39, SP41and eryC were1.0mg/mL,2.4mg/mL,3.5mg/mL,1.3mg/mL and1.7mg/mL, respectively. The result of western-blotting showed that all the recombinant proteins could react with the cattle antiserum against Brucella.2. The establishment of an indirect ELISA for the detection of antibodies against BrucellaFive purified recombinant proteins BP26, OMP16, CP39, SP41and eryC were used to coat microtiter plates with concentration of0.5μg/mL,4μg/mL,3μg/mL,4μg/mL and2μg/mL, respectively.Forty-eight cattle serum samples were tested. The sensitivities of the methods were90.9%,90.1%,97%,90.1%and90.1%, respectively, when compared with. Pourquier(?) ELISA Kit of IDEXX, and their specificity were80%,80%,73.3%,73.3%and53.3%, respectively. Then twenty positive sera and eight negative serum, which were detected by RBT、SAT and Pourquier(?) ELISA Kit were further detected by ELISA coated with the five proteins. By analysis of the data, the P/N value of recombinant protein BP26was the highest one among the five proteins. Therefore, BP26was selected as a coating antigen.The optimal coating concentration of antigen was500ng/mL and the dilution of serum was1:100, which were determined by checkerboard titration. The cutoff value was determined as0.2×ODP+0.8×ODN. When cattle sera against Mycobacterium tuberculosis, Escherichia coli, Foot-and-mouth disease virus and bovine viral diarrhea virus were tested, the results of the iELISA were all negative. The results of reproducibility showed the coefficient of variation (CV) was less than10%. Finally, a total of174clinical cattle sera were detected. The positive rates detected by the established iELISA and Pourquier(?) ELISA Kit were68.97%and70.11%, respectively. Compared to Pourquier(?) ELISA Kit, the sensitivity, specificity and coincidence rate of the iELISA were94.2%(113/120),83.3%(45/54) and90.8%(158/174), respectively.All these results indicated the iELISA based on recombinant periplasm protein BP26was sensitive and specific, and reproductive. The iELISA could be used to detect the antibody of cattle sera against Brucella.更多还原