抗黄曲霉毒素M1纳米抗体的筛选与初步鉴定OA北大核心CSTPCD

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 x 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.