The Expression Of Divalent Nanobody And Polyvalent Nanobody Against Ochratoxin A

Ochratoxin is a class of mycotoxin mainly produced by several Penicillium and Aspergillus fungal species under certain conditions,including seven different analogues.Ochratoxin A has the strongest perniciousness among the seven kinds of different ochratoxin analogues.Ochratoxin A mainly contaminates cereal,corn,peanut,cottonseed,nuts,coffee bean and fruits.The toxic effect of ochratoxin A contains nephrotoxic,hepatotoxicity and immunosuppressive effects.The mainly detection methods of detecting ochratoxin A include HPLC,HPLC-MS,ELISA,colloidal gold immunochromatography and some other methods.Nanobody is derived from the variable domain of the heavy-chain antibody in camelids,and it’s the smallest antigenbinding unit so far.Nanobody has good heat stability and water solubility,and can be produced in different kinds of expression system such as Escherichia coli express system.Nanobody has been widely used in many fields such as medical diagnostics and food safety analysis.Present work performed nanobody polyvalent expression by building fusion protein using glutathione S-transferases and cholera toxin B subunit,and established a ELISA method using CTB-VHH to detect ochratoxin A in cereal.The results are shown as follows:1.The expression and activity analysis of divalent nanobody and polyvalent nanobody.The construction of GST-VHH prokaryotic expression strain and CTB-VHH prokaryotic expression strain was successful.GST-VHH and CTB-VHH were successfully prokaryotic expressed.After the purification of GST-VHH and CTB-VHH,the result of SDS-PAGE analysis show that monovalent GST-VHH and polyvalent CTB-VHH were collected.The result of competitive ELISA shows that the IC50 of GST-VHH and CTB-VHH are 2.11 ng/m L and 0.65 ng/m L,respectively.Fifty percent of the antigen-binding capacity of GST-VHH and CTB-VHH lose after heated for 5 minutes at the temperature of 50.21 ℃ and 65.64 ℃,respectively.2.The application of polyvalent nanobody CTB-VHH.The indirect-competitive ELISA based on CTB-VHH for the detection of OTA in cereal and feed was developed with an IC50 value of 0.65 ng/m L,linear range of 0.27 ~ 1.58 ng/m L,LOD of 0.095 ng/m L and no cross-reactivity with other mycotoxins.The OTA recovery of intra-assay ranged from 80.0 % to 108.4 % with the coefficient variations of 2.51 ~ 7.48 %.The results of inter-assay show that OTA recovery ranged from 84.7 % to 99.6 % with the coefficient variations of 2.61 ~ 4.79 %.A good correlation was obtained from the OTA content detected by the indirect-competitive ELISA based on CTB-VHH and commercial ELISA kit.