Structure-activity Relationship Of Framework Region Of Anti-aflatoxin B₁ Single Chain Fv Antibody Fragment

Mycotoxins are highly toxic secondary metabolites produced by toxigenic fungus that pollute crops in suitable temperature and humidity,which are also harmful to human beings and animals.Aflatoxins are commonly fungal toxins mainly produced by Aspergillus flavus,Aspergillus parasiticus and Aspergillus nomius,and contaminate a vast array of foods and agricultural commodities,like beans,nuts,peanuts and corns,especially in hot and humid area.At present,aflatoxin and its derivatives have at least 20 kinds,including AFB1,AFB2,AFG1,AFG2,AFM1,AFM2 and so on.Among those metabolites,AFB1 has the highest toxicity,and has been classified in Group I carcinogen by International Agency for Research on Cancer of World Health Organization.In order to avoid the pollution risks of AFB1,now almost all the countries and regions have set the maximum limits standard for AFB1.It is significant to detect the AFB1 in human foods and animal feeds for risk prevention.Up to now,a host of methods have been developed to detect AFB1 in foods and feeds,mainly including thin layer chromatography,liquid chromatography and immunoassays techniques.Among these methods,immunoassays techniques have been widely used to detect AFB1 because they are rapid,selective and sensitive,which require high sensitivity and specificity antibodies.With the rapid development of molecular biology technology,recombinant antibodies,especially single chain Fv antibody fragment(ScFv)obtained the broad attention of researchers because of the advantages of easy modification,short preparation process and high expressing yield.However,ScFv still has lower affinity and stability than its natural antibodies and thus its application is limited.Studying the reaction between antigen and antibody can elucidating the affinity effect mechanism of ScFv,which are helpful for reforming ScFvs in the genetic level and thus provide theoretical guidance to improve its affinity in the future.In the present study,the three-dimensional structure model of ScFv-2C10 was obtained by homology modeling based on the amino acid sequence of ScFv-2C10.Then,molecular docking between ScFv-2C10 with AFB1 had been completed and the CDR and FR of ScFv-2C10 were classified to predict the binding sites between ScFv-2C10 and AFB1.According to their interaction,the site-directed mutagenesis experiments have been performed.Subsequently,wild type and mutant were expressed in Escherichia coli(E.coli)BL21(DE3),sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and enzyme-linked immunosorbent assay(ELISA)were employed to characterize ScFvs.The main results were as follows:1 Homology modeling and molecular docking of anti-AFB1 ScFv-2C10Taking VH and VL amino acid sequence of ScFv-2C10 obtained previously by our research group as the foundation,three-dimensional structure of ScFv-2C10 was obtained by homology modeling.In order to explore the amino acid which played a key role in the process of the antigen-antibody binding reaction,molecular docking between ScFv-2C10 and AFB1 was done.The result of molecular docking showed that the Arg-67?Asn-87 and Lys-237 of ScFv-2C10 had formed hydrogen bond with AFB1.Subsequently,the CDR and FR of ScFv-2C10 has been divided,heavy chain and light chain contain 3 CDR and 4 FR respectively.Based on these,the amino acids that formed hydrogen bonds in docking results and the polar amino acids of the CDR3 were chosen to be mutated for further study.2 Constructing the mutant of anti-AFB1 ScFv-2C10Taking pET-30a-pelB-ScFv-VH-linker-VL-2C10 as raw material,recombinant plasmid which contained mutant sites was obtained by PCR amplification through primers which are fully matched,reverse complementary and containing mutant.The PCR product was treated with Dpn I endonuclease to digest the parental DNA template and then was transformed into E.coli trans5a competent cells.The DNA sequences of the mutants were confirmed by DNA sequencing.The sequencing results showed that 14 mutants had been obtained.3 Characterization of anti-AFB1 ScFvs produced in E.coli BL21(DE3)The ScFv-2C10 and mutant have been transferred into E.coli BL21(DE3)competent cells to induce the expression of the objective protein.After protein purification,the quantitative examination was taken by coomassie brilliant blue method,and the characterization of anti-AFB1 ScFvs were measured by SDS-PAGE and ELISA.The results showed that ScFvs had already been successfully expressed in E.coli BL21(DE3)and existed in the form of an active soluble protein.The affinity constant and sensibility of R67L was approximately 3 and 1 time of that of the parent strain,respectively.