论文部分内容阅读
目的以白念珠菌Csa2蛋白为靶标,建立双抗体夹心ELISA检测体系,评价该方法的检测灵敏度和特异性。方法构建pPIC9K-Csa2重组表达载体,电转化毕赤酵母GS115,甲醇诱导表达、纯化重组蛋白rCsa2。rCsa2蛋白分别免疫新西兰大白兔和豚鼠制备免疫血清。以纯化兔多抗和豚鼠抗血清配对,利用棋盘滴定法,确定最佳实验条件,建立双抗体夹心ELISA检测系统。检测rCsa2和白念珠菌不同培养时间的上清,确定检测方法的灵敏度;检测其他3种念珠菌、5种曲霉、新型隐球菌和马尔尼菲青霉的培养上清,评价检测方法的特异性。结果成功构建表达载体并获得真核表达重组蛋白rCsa2,经SDS-PAGE鉴定相对分子质量(Mr)为13 300,符合预期值;Western blot法证实该蛋白可与特异性抗体结合。免疫动物获得高效价免疫血清,并以此成功建立双抗体夹心ELISA,可检测rCsa2蛋白的灵敏度约为240 pg/mL,并最早可检测到培养18 h的白念珠菌培养上清,与其他10种临床常见真菌的培养上清无交叉反应。结论建立了有较高的检测灵敏度和特异性的Csa2的双抗体夹心ELISA,为区别诊断白念珠菌感染提供新的方法。
OBJECTIVE To establish a dual antibody sandwich ELISA system for the detection of C. albicans Csa2 protein and to evaluate the detection sensitivity and specificity of this method. Methods The recombinant plasmid pPIC9K-Csa2 was constructed and transformed into Pichia pastoris GS115 by electroporation. The recombinant protein rCsa2 was purified by induction with methanol. rCsa2 protein were immunized New Zealand white rabbits and guinea pigs to prepare immune sera. The purified rabbit polyclonal antibody and guinea pig antiserum were matched by chessboard titration method to determine the best experimental conditions and establish double antibody sandwich ELISA detection system. The supernatant of rCsa2 and Candida albicans at different culture time was detected to determine the sensitivity of the detection method. The culture supernatants of 3 other Candida, 5 kinds of Aspergillus, Cryptococcus neoformans and Penicillium marneffei were detected to evaluate the specificity of the detection method . Results The recombinant plasmid rCsa2 was successfully constructed and its molecular weight (Mr) was 13 300 by SDS-PAGE. The result was consistent with the expected value. Western blot confirmed that the protein could bind with specific antibody. Immunization of animals to obtain high titer immune serum, and thus the successful establishment of double antibody sandwich ELISA rCsa2 protein detection sensitivity of about 240 pg / mL, and the earliest detection of 18 hours of culture of Candida albicans culture supernatant, and other 10 The common clinical fungus culture supernatant without cross-reaction. Conclusion Double antibody sandwich ELISA with high sensitivity and specificity of Csa2 was established, which could provide a new method for the differential diagnosis of Candida albicans infection.