白念珠菌感染特异性Csa2蛋白双抗体夹心ELISA体系的建立与评价

发布时间：2019-06-13 22:03

【摘要】：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。
[Abstract]:Objective to establish a double antibody sandwich ELISA detection system with Csa2 protein of candida albicans as target and to evaluate the sensitivity and specificity of the method. Methods the recombinant expression vector pPIC9K-Csa2 was constructed and induced by electrotransformation of Pichia pastoris GS115, methanol. The purified recombinant protein rCsa2.rCsa2 protein was immunized to New Zealand white rabbits and guinea pigs to prepare immune serum. By pairing purified rabbit polyclonal antibody and guinea pig antiserum, the optimum experimental conditions were determined by chessboard titration, and a double antibody sandwich ELISA detection system was established. RCsa2 and candida albicans were detected for different culture time to determine the sensitivity of the detection method, and the culture smears of the other three species of candida, five kinds of Aspergillus, Cryptococcus neoformans and Penicillium marneffei were detected, and the specificity of the detection method was evaluated. Results the expression vector was successfully constructed and the eukaryotic expression recombinant protein rCsa2, was identified by SDS-PAGE as the relative molecular weight (Mr) of 13.300. The predicted; Western blot method confirmed that the protein could bind to the specific antibody. The high valence immune serum was obtained from immunized animals, and the sensitivity of double antibody sandwich ELISA, for detecting rCsa2 protein was about 240 pg/mL,. The culture medium of candida albicans cultured for 18 hours could be detected at the earliest, and there was no cross reaction with the culture medium of other 10 kinds of common clinical fungi. Conclusion A double antibody sandwich ELISA, with high sensitivity and specificity for Csa2 detection is established to provide a new method for the differential diagnosis of candida albicans infection.
【作者单位】：南方医科大学珠江医院检验医学部;突发传染病病原微生物医学重点实验室;海南省人民医院检验科;香港大学微生物系;
【基金】：广东省高等学校珠江学者岗位计划(2009 GDUPS) 广东省高校感染性疾病诊断试剂工程技术研究中心建设项目(GCZX-A1106) 国家自然科学基金青年项目(81101299,81101320)
【分类号】：R392-33

【共引文献】