黄曲霉毒素M1(aflatoxin M1,AFM1)对人类和动物具有致癌性、诱变性、致畸性等,广泛存在于哺乳动物乳汁及其乳制品中。因此,制备特异性强、灵敏度高的抗体来检测AFM1至关重要。该研究以AFM1-BSA为抗原免疫双峰驼,采血分离淋巴细胞,提取总RNA反转录为cDNA,通过巢式PCR获得重链可变区(variable domain of heavy chain,VHH),与pComb3XSS载体连接电击转化至Escherichia coli TG1,构建AFM1特异性噬菌体展示文库。以AFM1-OVA为靶向抗原,使用4轮酸洗脱和竞争洗脱相结合的策略对文库进行淘选,并对抗AFM1纳米抗体进行初步鉴定,最后预测了精准的三维模型结构。结果显示,抗AFM1纳米抗体文库库容量为2.27×109 CFU,多样性丰富,插入率为100%。经淘选共获得6条独特序列的纳米抗体。其中VHH-M4和VHH-M6具有较高的灵敏度,初步测定半抑制浓度分别为0.415 ng/mL和1.877 ng/mL,线性范围分别为0.141~1.652 ng/mL和0.800~3.811 ng/mL。一级和二级结构结果显示VHH-M4和VHH-M6均为亲水性蛋白,框架区(framework region,FR)和互补决定区(complementarity determining region,CDR)区符合典型纳米抗体的结构特征。该研究为AFM1免疫学检测提供核心元件,并为探究AFM1与VHH的分子结合机制奠定基础。
Aflatoxin M1 (AFM1) is carcinogenic, mutagenic, and teratogenic to humans and animals, and is found in mammalian milk and dairy products.As a result, it is critical to develop specific and sensitive antibodies for detecting AFM1.In this study, AFM1-BSA was used to immunize Bactrian camel, blood was collected to isolate lymphocytes, total RNA was extracted, and reverse transcribed into cDNA.The heavy-chain variable region (VHH) was obtained by nested PCR.Then, it was ligated with the pComb3XSS vector and transformed into Escherichia coli TG1, the anti-AFM1 phage-display library was constructed.The library was eluted using AFM1-OVA in combination with four rounds of acid elution and competitive elution.The anti-AFM1 nanobodies were preliminarily identified and highly accurate three-dimensional models were predicted finally.The results showed that the library capacity was 2.27×109 CFU, with a 100% insertion rate and a wide range of diversity.Six unique sequences of nanobodies were obtained after screening.Among VHH-M4 and VHH-M6 had higher sensitive, with IC50 of 0.415 ng/mL and 1.877 ng/mL, and linear ranges of 0.141-1.652 ng/mL and 0.800-3.811 ng/mL, respectively.The primary and secondary structure prediction revealed that both VHH-M4 and VHH-M6 were hydrophilic proteins.The FR and CDR regions were consistent with the structural characteristics of typical nanobodies.This research provides core components for the immunoloassay of AFM1, and lays the foundation for exploring the molecular recognition mechanism of AFM1 and VHH.
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