Preparation Of Temperature-responsive Nanobody And Its Application In Sample Pretreatment Of Aflatoxin B₁

Aflatoxin（AF）is a double furan ring toxin produced by filamentous fungi such as Aspergillus flavus and Aspergillus parasiticus.There are more than 20 derivatives of AF,the most toxic of which is aflatoxin B₁（AFB₁）.AFB₁ can cause human liver cancer and esophageal cancer.AF mainly pollutes grain,oil and its products.The detection methods of AFB₁ mainly include thin-layer chromatography（TLC）,enzyme-linked immunosorbent assay（ELISA）,high-performance liquid chromatography（HPLC）,fluorescence resonance energy transfer（FRET）and biosensors.HPLC and ELISA are still the main detections in the current national standards.Due to the complex sample matrix,sample pretreatment is essential for separating AFB₁efficiently,reducing or avoiding matrix interference,and achieving accurate qualitative and quantitative detection.At present,the extraction methods of various mycotoxin samples include liquid-liquid extraction（LLE）,solid-phase extraction（SPE）,immunoaffinity chromatography（IAC）.These methods have the disadvantages of complex operation,environmental pollution,low specificity,and high cost.In this study,Elastin-like polypeptides（ELPs）were fused with anti-AFB₁ nanobody to establish a non-column affinity precipitation method for the extraction of AFB₁ in a homogeneous reaction system rapidly.The preparation of raw materials for this method only depends on the biosynthesis of microorganisms.A large number of cheap recombinant proteins can be obtained without the organic coupling of solid-phase carriers,which greatly reduces the extraction cost of mycotoxins.The conclusions are as follows:We synthesized the repeat sequence ELP20,ELP40,ELP60,ELP80 by recursive directional ligation（RDL）,and their lengths were 300,600,900 and 1200 bp respectively.The expression vectors p ET30a-G8-ELP20,p ET30a-G8-ELP40,p ET30a-G8-ELP60 and p ET30a-G8-ELP80 were transformed into E.coli BL21（DE3）cells.SDS-PAGE analysis showed that the four fusion proteins were expressed in the form of inclusion bodies.The inclusion bodies were dissolved in denaturing buffer and refolded by dilution,inverse transition cycling（ITC）purification,dialysis and column refolding.The SDS-PAGE analysis showed that the purity of the four proteins is similar.The ITC showed the highest yield for the refolding of inclusion body,which was 83%,90.7%,89.5%,and 88.3%for the four fusion proteins respectively.Indirect competitive ELISA showed that the IC₅₀ of G8-ELP80 obtained by dilution refolding was the lowest（4.35 ng/m L）.The phase transition temperature（Tt）of nanobodies by turbidity analysis was in line with expectations.As the length of ELP increases,the value of Tt decreases.The secondary structures of the four fusion proteins were mainlyβ-sheet andβ-turn which were detected by the circular dichroism spectroscopy.The research we have done suggests that the ELP tag fused with the anti-AFB₁ nanobody can realize the temperature-responsiveness of the nanobody.The effects of different renaturation methods on protein characteristics were compared,which provided a basis for subsequent AFB₁ detection and analysis.The inclusion body protein in the precipitate from 800 m L bacterial solution was collected after crushing and centrifugation and fully dissolved with a denaturation buffer.The fusion protein can refold through add the same volume of renaturation buffer every 2 h.The Minimate TM EVO system was used to concentrate the 250 m L protein solution after renaturation.After concentration,25 mg of recombinant protein G8-ELP80 with a storage concentration of 766μg/m L was obtained,and the protein yield was 31.25 mg/L.The standard curve was established by indirect competitive ELISA to determine the recovery rate of AFB₁.The factors affecting the capture efficiency such as ELP label length,antigen-antibody reaction ratio,methanol tolerance,salt ion type,salt ion concentration and p H were optimized.The experimental results showed that the optimal number of repeat units of ELP tag was80,the optimal reaction ratio was 3:1,the maximum methanol tolerance concentration of recombinant protein was 10%,the optimal salt ion for recombinant protein phase change coagulation was 0.6 M（NH₄）₂SO₄,and the optimal reaction p H of the system was 7.0.Under the condition of single factor optimization,the particle sizes of G8-ELP80 before and after phase transition were measured,which were 1μm and 4μm respectively.The particle size increased by 3 times after phase transition,which further proved that G8-ELP80 had good phase transition ability.We determined the protein sedimentation rate of G8-ELP80 at different concentrations under the above phase transition conditions in order to ensure the complete sedimentation of the recombinant protein in the capture system.Four types of samples such as corn,rice,soy sauce and veg oil with low（10μg/kg）,medium（50μg/kg）and high（100μg/kg）AFB₁ were selected and tested in order to investigate the effect of the sample matrix.The spiked samples were purified by the immunoaffinity column method and non-column affinity precipitation method respectively.The purified solution was pre-column derivatized with n-hexane and trifluoroacetic acid.The AFB₁ content in the purified solution was determined by HPLC.The experimental results showed that the purification effect of the commercial immunoaffinity column was stable.The recovery rate of different samples at low concentrations was more than80%,and the recovery rate of different samples at medium and high concentration was more than 90%.The purification effect of the non-column affinity precipitation method increased with the increase of standard concentration.The recovery rate could reach more than 80%at high standard concentration and more than 70%at medium standard concentration.At the spiked concentration of as low as 10μg/kg,the recovery rate of AFB₁ in the samples purified by the non-column affinity precipitation method was still about 60%,suggesting that the AFB₁ non-column affinity precipitation method established in this study has great potential and utilization space in the field of small molecule sample treatment and rapid detection.