Biopanning,Preparation And Application Of A Novel Affibody Targeting Aristolochic Acid Ⅰ

Aristolochic acids（AAs）are a group of nitrophenanthrene organic acids naturally occurring in Aristolochic plants.Aristolochia plants have a wide range of pharmacological effects and high medicinal value.However,it has been found that the exposure of aristolochia acid can cause renal injury and gene mutagenicity.Aristolochic acids was classified as a class I carcinogen by the World Health Organization in 2012.Aristolochic Acids I（AAⅠ）and its metabolite Aristolochic lactams are the most toxic components in Aristolochic acids.Therefore,how to effectively remove AAⅠ from herbal products is of great importance.In this study,the affibody targeting AAⅠ was successfully selected by phage-display technology and its application potential in selective adsorption to AAⅠ was further studied.The main contents and results are as follows:（1）Two complete antigens of AAⅠ were synthesized by using BSA and OVA as carrier proteins,respectively.Alternately,BSA-AAⅠ and OVA-AAⅠ were used as target proteins.An affibody named Z_AAⅠ-A5with specific binding to AAⅠ was obtained after 7 rounds of biopanning.Z_AAⅠ-A5 was expressed in E.coli and successfully purified.The interaction between Z_AAⅠ-A5 and AAⅠ was studied by fluorescence quenching experiment.At room temperature,the K_D of Z_AAⅠ-A5 and AAⅠ is 14.80μM,and the intermolecular interaction were mainly through hydrogen bond and van der Waals force.（2）Two self-assembling peptides（SAPs）tagged Z_AAⅠ-A5recombinant protein,Z_AAⅠ-A5-ELK16,Z_AAⅠ-A5-L6KD were designed and successfully produced in E.coli.The ZAAⅠ-A5-L6KD showed better assemble,higher productivity and purity than Z_AAⅠ-A5-ELK16.The dissociation constant（K_D）of Z_AAⅠ-A5-L6KD with AAⅠ was 12.43μM at room temperature.The intermolecular interaction was still through hydrogen bond and van der Waals force.These results indicated that the protein aggregation has no effects on the interaction between Z_AAⅠ-A5 and AAⅠ.Z_AAⅠ-A5-L6KD was characterized by SEM and DLS.The affibody aggregate was granular,and the particle size was in the range of 300 nm-900 nm.Protease hydrolysis experiments showed that ZAAⅠ-A5-L6KD aggregates retained biological activity,which was different from ordinary inclusion bodies.Meanwhile,Z_AAⅠ-A5-L6KD showed higher stability than Z_AAⅠ-A5.（3）The isothermal static adsorption behavior of Z_AAⅠ-A5-L6KD on AAⅠ was studied based on Langmuir model.The simulation results showed that the adsorption capacity of ZAAⅠ-A5-L6KD on AAⅠ was consistent with the model.The maximum adsorption capacity of ZAAⅠ-A₅-L6KD was 9.89 mg/g,which was significantly higher than that of negative control affibody aggregates Z_J2-L6KD.It is proved that ZAAⅠ-A5-L6KD has the adsorption capacity of AAⅠ.At the same time,the results of selective adsorption experiments showed that Z_AAⅠ-A5-L6KD had poor adsorption capacity for benzoic acid and nitrobenzene,which indicated that ZAAⅠ-A5-L6KD had selective adsorption to AAⅠ.In conclusion,this project successfully developed the affibody molecule specifically binding to AAⅠ,and a novel active aggregate material was designed and produced in E.coli.,The selective adsorption capacity of the material for AAⅠ was also studied.These results provide important theoretical support and research experience for the development of novel technology of"detoxification and efficiency preservation"of Chinese herbal products.