Construction, Expression And Biological Activity Identification Of A Novel Genetic Engineering Antibody ScFab

Antibody is a special kind of immunoglobulin produced by vertebrates against foreign objects(antigen),and research on it has three stages(polyclonal antibody, monoclonal antibody and genetic engineering antibody).Genetic engineering antibody can be antibody or antibody fragment obtained by expressing recombined genes or gene fragments of antibody in various expression systems and includes chimeric antibody,reshaped antibody,bispecific antibody and small molecular antibody fragments,and so on.Small molecular antibodies are antibody fragments of low relative molecular weight but with antigen-binding activity,including antigen-binding fragment(Fab), variable fragment(Fv),single chain variable fragment(scFV),and so on.Now Fab and scFv are key antibodies to the study of small molecular antibody fragments.They have advantages of brief preparation method,low immunogenicity and high penetrating capacity,which make them widely used in targered tumor treatment,viral disease treatment and tumor detection by radioimmunoimaging.Based on the advantages and disadvantages of Fab and scFv,coding genes(Lc and Fd)of a McAb against Sudan I was to be cloned and constructed into a scFab antibody with a synthesized Linker and an expression vector pET24a-scFab will be constructed.At last,a novel small molecular antibody was expected to be abtained and has the advantages of scFv and Fab.This paper can be divided into four parts:A.Lc and Fd genes were cloned and constructed into clone vector.After identification and DNA sequence,their sequences and coding proteins are analyzed, which demonstrated that both genes cloned and their coding proteins had characters that conform to IgG of mouse.B.Based on the analisis of gene fragments and vector pET24a,expression vector pET24a-scFab was designed and constructed by recombining suitable Lc and Fd fragments with a 102bp synthesized DNA Linker.Then pET24a-scFab was transformed into E.coli BL21(DE3).C.After induction by IPTG,SDS-PAGE and western blot demonstrated scFab antibody expressed in the form of inclusionbody in BL21(DE3).Large quantity scFab inclusionbody was harvest after expression optimization.D.The inclusive body was solved by urea and renaturation was done by diluting with renaturation solution and dialysis.After renaturation,binding ability and specificity of scFab antibody to Sudan I was shown by indirect ELISA and competitive ELISA.Conclusion:A novel scFab antibody was constructed and it may lay a foundation for the further study of novel genetic engineering antibody.