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【目的】从鼠源合成噬菌体抗体库中,快速分离获得微囊藻毒素LR单链抗体。【方法】采用磁珠筛选和负筛选方法,设计两轮微囊藻毒素LR单链抗体的筛选方案。用噬菌体产出投入比和多克隆噬菌体免疫分析,对每轮筛选后微囊藻毒素LR特异性噬菌体的富集效果进行鉴定。再将第2轮筛选获得的次级库提取单链抗体基因,转入可溶性表达载体;诱导表达后,采用时间分辨荧光免疫法,对单个噬菌体克隆鉴定并测序。【结果】两轮筛选的噬菌体产出投入比分别为4.8×10-8和2.88×10-6。第2轮筛选后的次级库,经多克隆噬菌体免疫分析得到的荧光信噪比,比原始抗体库提高了22.8倍。最终鉴定得到5株不同的阳性噬菌体克隆,其中最佳的克隆对微囊藻毒素LR的检测灵敏度(IC10)为13 ng.mL-1,抑制中浓度(IC50)为435 ng.mL-1,线性范围在31—5 952 ng.mL-1。【结论】本研究筛选到的单链抗体,有潜力应用于微囊藻毒素LR的免疫学检测或免疫亲和柱的制备,同时也探索了一种高效的微囊藻毒素LR单链抗体的库筛选、鉴定方法。
【Objective】 The microcystin LR scFv was isolated rapidly from murine synthetic phage antibody library. 【Method】 Two rounds of microcystin LR single-chain antibody screening programs were designed using magnetic bead screening and negative screening methods. Phage output ratio and polyclonal phage immunoassay were used to identify the enrichment effect of microcystin-LR-specific phage after each round of screening. Secondly, the single-chain antibody extracted from the secondary library obtained in the second round of screening was transferred into the soluble expression vector. After the expression was induced, the single phage clones were identified and sequenced by time-resolved fluorescence immunoassay. 【Result】 The results showed that the ratio of input to output of two rounds of phage was 4.8 × 10-8 and 2.88 × 10-6, respectively. The second round of screening of the secondary library, polyclonal phage immunoassay fluorescence signal-to-noise ratio, compared with the original antibody library increased by 22.8 times. Five different positive phage clones were finally identified. The best clone was 13 ng.mL-1 for microcystin LR, 435 ng.mL-1 for IC50, The linear range was 31-5 952 ng.mL-1. 【Conclusion】 The single-chain antibodies screened in this study have potential application in the immunological detection of microcystin LR or the preparation of immunoaffinity column. At the same time, an efficient microcystin LR scFv Library screening, identification methods.