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目的建立一种快速诊断莱姆病螺旋体的胶体金免疫层析试纸条检测方法。方法以莱姆病伯氏疏螺旋体标准株B31DNA为模板,PCR扩增OspA基因,构建重组质粒pET-30a-OspA,以IPTG诱导原核表达并用亲和层析方法对重组蛋白OspA进行纯化;用柠檬酸钠还原法以OspA作为检测线,优化试纸条制备条件,制备莱姆病诊断试纸条,检测其特异性和敏感性;用该方法对20份绵羊血清进行检测,并用WB验证结果的准确性。结果构建了外膜基因OspA的原核表达体系,获得高纯度的OspA蛋白;用40nm金颗粒制备的莱姆病胶体金试纸条,最适胶体金标记pH值为6.2,SPA蛋白的最佳标记量是6μg/ml,该蛋白不与布鲁氏菌、大肠杆菌等阳性血清反应,能重复多次与莱姆病阳性血清特异性反应。用该方法检测20份动物血清样品2份阳性(10%),与WB结果一致。结论以纯化的OspA蛋白为包被抗原制备的免疫层析试纸条特异性强,敏感性高,与WB验证结果符合率高,且具有准确、快速、方便的优点,可用于临床样品的检测。
OBJECTIVE To establish a colloidal gold immunochromatographic test strip for rapid diagnosis of Lyme disease. Methods The OspA gene was amplified by PCR using B31 DNA of Borrelia burgdorferi as a template. The recombinant plasmid pET-30a-OspA was constructed and induced by IPTG. The recombinant protein OspA was purified by affinity chromatography. Sodium acid reduction method OspA as a test line to optimize the conditions for the preparation of test strips, preparation of diagnostic test strips Lyme disease, to test its specificity and sensitivity; using this method on 20 sheep serum was detected and confirmed by WB accuracy. Results The prokaryotic expression system of the outer membrane gene OspA was constructed and the high purity OspA protein was obtained. The Lyme disease colloidal gold test strip prepared with 40 nm gold particles showed the best colloidal gold labeling value of 6.2 and the best labeling of SPA protein The amount of 6μg / ml, the protein does not react with the positive serum of Brucella, Escherichia coli and repeated multiple Lymphomyelitis-positive serum-specific reactions. Two of 20 animal serum samples tested positive (10%) using this method, consistent with the WB results. Conclusion The immunochromatographic strip with purified OspA protein as coating antigen has strong specificity, high sensitivity, high coincidence rate with WB verification results, and has the advantages of accuracy, speed and convenience, and can be used for the detection of clinical samples .