贝氏柯克斯体重组抗原P1表达纯化及胶体金免疫层析方法的建立

Preparation of the Coxiella burnetii recombinant antigen and establishment of the colloidal gold immunochromatographic method

目的制备贝氏柯克斯体的重组外膜蛋白P1,并建立一种快速检测贝氏柯克斯体抗体的胶体金免疫层析方法。方法利用贝氏柯克斯体主要的表面蛋白P1的良好免疫原性和免疫反应性,对已构建的表达P1抗原的重组工程菌,异丙基-β-D-硫代吡喃半乳糖苷诱导表达P1重组蛋白,通过蛋白纯化、复性、浓缩等过程获得目的蛋白。纯化的P1重组抗原包被硝酸纤维素膜,胶体金标记SPA,建立检测贝氏柯克斯体抗体的免疫层析方法,评价该方法的特异性、灵敏性、稳定性及应用性。结果纯化后获得分子质量(Mr)为52000的目的蛋白,纯度为94%。建立了快速检测贝氏柯克斯体特异性抗体的方法,对阳性参照样本检测,目测灵敏度可达1∶105,采用金标仪半定量检测灵敏度可达116ng/ml;对94份鼠血清和90份健康人血清检测,无阳性检出;对布鲁氏菌、普氏立克次体、军团菌、土拉弗朗西斯菌等传播途径和临床特征相似的病原抗体进行检测,未发生交叉反应。结论利用重组抗原建立的贝氏柯克斯体抗体胶体金免疫层析方法具有较好的敏感性和特异性,可用于传染病快速筛查。

Objective To establish a colloidal gold immunochromatography test （ICT） for rapid detection of Coxiella burnetii antibody by prepared recombinant outer membrane protein P1 protein. Methods Isopropyl-beta-D-thiogalactoside （IPTG） was used to induce the constructed recombinant engineering to express recombinant C. burnetii P1 protein, which was then purified, renaturated and concentrated to get the target protein nitrocellulose membrane coated by purified recombinant P1 antigen and SPA labeled by colloidal gold was used for the establishment of the ICT method, whose specificity, sensitivity, stability and applicability were then evaluated. Results After purification, a recombinant P1 protein product of 52 000 with a purity of 94% was obtained, which was then used to establish the ICT method for rapid detection of C. burnetii antibody, the sensitivity of which was up to 1∶105 in the detection of the positive reference samples with a sensitivity of 116 ng/ml in semi-quantitative detection. Ninety-four mouse sera and 90 healthy human sera were tested for specificity with no positive results and no cross-reaction was found in the antibodies of Brucella, Rickettsia prowazekii, Legionella and Francisella tularensis. Conclusion The ICT method established with recombinant C. burnetii P1 antigen is of good sensitivity and specificity, which is applicable to the rapid screening of communicable diseases.