Construction,Screening And Characteristics Of Anti-AFB₁ Single Chain Fv Fragments Phage Library

Aflatoxin B1(AFB1)which has strong toxicity and carcinogenicity,is the secondary metabolite mainly produced by the fungi of A.parasiticus and A.flavus.Owing to its stable structure,high-temperature heating is generally not able to destroy it.Therefore,this toxin threatens the lives of humans and animals seriously.Even now,there are no effective methods to protect food from contaminating by AFB1.So,most countries in the world have set the maximum amount of AFB1 that could exist in food and feed,and have been actively exploring the rapid and efficient methodologies for testing the concentration of AFBi.Among these methods,using immunological method to detect AFB1 has been a hot topic because of its low cost,high sensitivity,simple operations and other advantages.Immunological detection depends on the high-affinity and high-specificity antibody.The processes of preparing conventional antibodies are time-consuming,expensive and potentially hazardous,all of which encourage people to vigorously develop genetic engineering antibodies.As the third generation of genetic engineering antibodies,single chain Fv fragment(ScFv)has attracted widely public attention due to its low immunogenicity,small molecular weight,easy transformation and expression.Phage display antibody library technology is famous for its convenient operation,enormous capacity and high throughput screening.The antibodies with specific binding ability can be screened directly from the single chain Fv fragment antibody library,which provides strong technical support for people to acquire high-quality antibodies.In this study,taking AFB1 as the research object,ScFv genes were amplified from spleen cells of AFB1-BSA immunized mice,and then were inserted into pCANTAB5e vector.Subsequently,the anti-ABF1 ScFv were screened by phage library and characterized.The main results are followed.1.Construction of anti-AFB1 ScFv gene libraryBalb/c mice were immunized with complete antigen AFB1-BSA for five times.After that,antisera was separated and their titers and sensitivities were detected via ELISA.The data shows that the antisera titer could be up to 25600 and the highest sensitivity is 35.5 ng/mL.RNA of mice spleen cell RNA was extracted and then cDNA was produced by reverse transcription.All the mouse antibody heavy chain variable regions and light chain variable regions was amplified with degenerate primers.The gene splicing by overlap extension PCR(SOE-PCR)was employed to connect the two variable regions to be the complete ScFv gene.Two restriction sites(SfiI and Notl)were engineered into the ScFv gene to ligate to the pCANTAB5e vector that was transformed into E.coli TGI.Based on the number of transformants on resistant plate,the storage capacity of ScFv gene library was calculated of 5×106 cfu/mL.2.Construction,screening and identification of anti-AFB1 ScFv phage libraryThe E.coli TG1 that contained ScFv gene library was infected with helper phage M13K07 to get the primary ScFv phage library and the capacity of this library is approximately 2×1013 pfu/mL.After five rounds of elution,enrichment and screening,two positive single-chain phage antibodies,ScFv-A and ScFv-B were obtained by phage ELISA with antigen AFB1-OVA.Then,the plasmids were isolated and sequences were determined by DNA Sequencing.3.Characterization of anti-AFB1 ScFvThe amino acid sequences of anti-AFB1 ScFv deduced accroding to nucleic acid sequences were input to SWISS-MODEL service system for getting high homology.The high similarity polypeptide sequences were set as the template of ScFv-A or ScFv-B protein to simulate three-dimensional structure of them using Discovery Studio(DS)software.Then these molecular structures were docked with antigen AFB1.The results indicate that the Trp33 and Serl06 in the heavy chain variable region of ScFv-A and Tyr33,Ser52 and Tyr102 in the heavy chain variable region of ScFv-B play the key roles in combination with AFB1,respectively.Recombinant expression vector of pET-30a-ScFv-A and pET-30a-ScFv-B were constructed and transformed into E.coli BL21(DE3).The purified recombinant proteins were detected via SDS-PAGE and ELISA.The results show that the two single-chain antibodies could be expressed in E.coli BL21 in the form of the active and soluble protein.The molecular weight of two recombinant proteins are approximately 30 kDa.Indirect competitive ELISA analysis illustrated that the sensitivity of ScFv-A and ScFv- B were approximately 72 ?g/mL and 60 ?g/mL,respectively.