Screening And Identification Of AFB1-binding Proteins

Aflatoxin B1(AFB1)is the one of the most toxic fungal secondary metabolite ever known.AFB1 can cause acute and chronic poisoning,and it is harmful to human and livestock due to its three pathogenic roles:carcinogenic,teratogenic,and mutagenic.There have been a lot of reports on the mechanism of AFB1 biosynthesis and pathogenesis,but the mechanism of how AFB1 was transferred into animal cell remained unknown.A method based on immobilized affinity chromatography technique to screening AFB1-binding proteins was designed in the study.Firstly AFB1 coupling protein(AFB1-BSA)was prepared,which was used to incubate with total proteins from mice liver.The AFB1-binding proteins were harvested through non-specificity washing and specific elution,analyzed by SDS-PAGE,and identified by mass spectrometry.Finally,8 possible AFB1-binding proteins were identified.After further bioinformatic analysis,estradiol betadehydrogenase 5(Akrlc6)and 40S ribosomal protein A(Rpsa)were figured out for further study.The Rpsa protein is a membrane binding protein for laminin,Ruan virus,bacteria,and so on.Then,rpsa and akr1c6 were amplified,and inserted into the expression vector pET28a respectively.The recombinants were then transformed into E.coli BL21(DE3).After induced by IPTG,Rpsa and Akr1c6 were expressed and purified by Ni-column protocol.The interaction between AFB1 and proteins(Rpsa or Akrlc6)were identified by ELISA.More specific,AFB1-BSA was coated on 96-wells,Incubated with the purified proteins,detected with His-monoclonal antibody,and the result showed there was a perty high Interaction between AFB1 and those proteins.For Rpsa is a protein that is high expression on the cell membrane,we conducted cellular co-localization experiments to study the intuitive interaction between Rpsa and AFB1.It was found in cellular co-localization experiments that there was a co-located phenomenon between Rpsa and AFB1 in HELA cells.The purified proteins were used to immunize Balb/c mice.Polyclonal antibodies against Rpsa and Akrlc6 were acquired respectively which laid a foundation for the next protein function research on the cellular level.This study has successfully screened and verified two AFBi-binding proteins.At present,further expriments about the biological role of those two proteins on the absorption and distribution of AFB1 in cells is on the way,which would lay a solid foundation for the research of AFB1 pathogenesis in animal cells.