Expression Of Ochratoxin A Pentavalent Nanobody And Preparation Of Immunoaffinity Column

Ochratoxin is a secondary metabolite produced by certain species of Aspergillus and Penicillium including seven kinds of similar structure compounds.Ochratoxin A?OTA?has the strongest toxicity.It has renal toxicity,liver toxicity,teratogenicity,carcinogenesis,and mutagenicity.Nanobody?Nb?is also known as single domain of heavy chain antibody.It consists of a heavy chain variable region that naturally depletes the light chain.It is the smallest fragment that naturally exists and binds to antigens.It has the advantages of small molecular mass,easy modification,easy expression,and good stability.With the development of science and technology and the deepening of research,the combination of genetic engineering technology and the modification of nano-antibodies carrying specific structures have more obvious advantages.Multivalent nanobodies,fusion nanobodies,and multispecific nanobodies have characteristics than unmodified nanobodies.It provides the basis for building more sensitive detection methods.The immunoaffinity column technique is a method for separating and purifying by the high specificity and affinity existing between the biological antigen and the antibody.Compared with other extraction methods,immunoaffinity column technology has the advantages of fewer toxic reagents,convenience and simplicity,and effective elimination of matrix interference.This study was based on the prokaryotic expression vector pET-CTB-VHH28,which was transformed into E.coli BL21?DE3?for prokaryotic expression.A high-purity pentavalent nanobody is obtained,and an ochratoxin A immunoaffinity column is prepared by using the purified CTB-VHH28.The main results are as follows:1.Expression and analysis of pentavalent nanobody against ochratoxin AThe prokaryotic expression vector pET-CTB-VHH28 was transformed into E.coli BL21?DE3?for prokaryotic expression and successfully expressed pentavalent Nanobody.By optimizing the concentration of IPTG,the expression temperature and the expression time,the optimal expression conditions was:the concentration of IPTG was 0.05 mmol/L,the induction temperature was 16?and the induction time was 10 h.After purification by nickel column,high-purity pentavalent nanobody was successfully obtained and the production yield was 20 mg/L.By the activity assay,results showed that the fusion proteins CTB-VHH28 could specifically bind to OTA and the IC₅₀ of indirect competitive inhibition ELISA was 0.38 ng/mL under the condition of 2.5%methanol.Thermal stabilization results showed that the pentamer structure begun to be destroyed when CTB-VHH28 was heated at 70°C.When the heating temperature was higher than 85°C,the antibody is basically inactivated.The critical temperature for inactivation of CTB-VHH28 was between 55⁷5°C.For the methanol tolerance analysis,when the concentration of methanol in the reaction system was not higher than 10%,the binding capacity of the antigen and CTB-VHH28 did not change significantly.It helps to improve the repeatability and stability of the detection method.2.Preparation of immunoaffinity column based on Nanobody CTB-VHH28The ochratoxin A nanobody immunoaffinity column was successfully prepared by randomly coupling CTB-VHH28 with cyanogen bromide activated agarose microspheres in coupling buffer.The best coupling time for ligand CTB-VHH28 was 3 h.The optimal conditions for the loading buffer are 10%-methanol PBS buffer,pH=7.2,and an ionic strength of0.1M.Affinity column was washed with 90%methanol solution and the washing volume was 4 ml.Its performance was evaluated,the results show that affinity column is used continuously for 4 times and the recovery rate can still reach 75%.Recovery rate remained at 80%when stored at 37°C for 20 days.The grain samples were spiked and indirect competition ELISA was used to determine the extracts purified using the affinity column.The results showed that the recoveries of the OTA standards range from 85.4%to 106.7%,and the inter-assay coefficient of variation ranges from 2%to 5%.