Preparation Of Ochratoxin A Nanobody And Study On Ochratoxin A Detection Technology Based On Magnetic Separation Immuno-Nanobody

Ochratoxin A（OTA）is the most toxic mycotoxin in ochratoxin.OTA contamination is often found in unprocessed agricultural products.OTA has immunotoxicity,neurotoxicity,nephrotoxicity and hepatotoxicity,posing A serious threat to human health.Currently,OTA is classified as potential carcinogenic agents（Group 2B）according to the International Agency for Research on Cancer（IARC）.Many countries in the world have strict limit standards for OTA in agricultural products.Therefore,establishing a quick and simple detection method for early identification of OTA can avoid food safety problems caused by OTA.Nanobody（Nb）has attracted extensive attention in the research of medicine,therapy,biological imaging and environmental detection due to its inherent small molecular weight,stable physical and chemical properties,high affinity and easy recombination expression.In this study,anti-OTA nanoantibodies were prepared,and a rapid and simple immunoassay method was constructed with anti-OTA nanobodies as the core,which realized the rapid detection of OTA in agricultural products.The main contents of this study are as follows:1.Construction,screening and identification of anti-OTA phage display nanobody library:An initial library of 2.2×10⁶cfu nanobody was constructed by immunizing camels with OTA-BSA antigen,and a 3.45×10¹³ cfu/m L anti-OTA nanobody phage display library was obtained after rescue by helper phage M13KO7.The solid dating and panning techniques were used to develop a reasonable panning scheme.Twenty positive phage clones were randomly selected for sequencing,and three different nanobody sequences were obtained by Bioedit software.2.Prokaryotic expression and binding activity identification of anti-OTA nanobody:The Avi-Nb gene was successfully amplified and cloned into the p TRX prokaryotic expression vector.Colony PCR was used to identify the PTRX-VHH positive clones.The PTRX-VHH plasmid was transformed into the susceptible cells of E.coli expression strain Trans B（DE3）,which was expressed by IPTG and purified by Ni column.Finally,three kinds of bioactive nanobody,Nb-2C,Nb-2G and Nb-2B,were obtained.The binding activity to OTA antigen was determined by indirect binding ELISA,in which Nb-2G had the best binding activity to OTA antigen.The competitive inhibition standard curve of Nb-2G was established based on indirect competitive ELSIA,and the IC50 was 22.5ng/m L.Finally,homology modeling and molecular docking techniques were used to analyze the interaction mechanism between the nanobody and OTA.3.Establishment of rapid OTA detection method by MBS-ELISA based on Avi-Nb:OTA-MBs immunomagnetic beads were successfully prepared.The matrix effect was evaluated by optimizing the concentration of OTA-MBs and Avi-Nb,the concentration of organic solvent in the reaction system,incubation time,washing times,p H value and other conditions.OTA MBS-ELISA of Nb-2G/streptavidin-ALP nanoparticles and magnetic beads based on Avi labeling was established,which realized the goal of one-step detection,improved the detection sensitivity and significantly simplified the detection procedure.LOD was 0.07ng/m L（IC90）,IC50 was 1.17 ng/m L,and linear range was 248.8 pg/m L to 5.28 ng/m L（IC20 to IC80）.At the same time,the method is easy to prepare and a large number of prokaryotic expression and gene manipulation,low cost,and easy to mass production.In conclusion,this study established a method for OTA detection based on phage display technology,biotin-Streptavidin system and immunomagnetic bead technology,which has high response to OTA and has been successfully used for the detection of actual samples.