Construction Of Anti-GPA/GPB ScFv And Fusion Express With Streptavidin

The blood group has been defined as 29 blood systems and more than 600 antigens on the surface of red blood cells. Glycoprotein is one class of main antigens which contains A,B,C,D and E. Glycoprotein A(GPA) and Glycoprotein B(GPB) belong to main molecular signs and constitutes MNSs blood group which are 60% homolog. The cloned mouse hybridoma cell line which can excrete anti-GPA/GPB is the basic foundation of monoclonal antibody.Based on the mAb of anti-GPA/GPB, we amplify its VH and VL by RT-PCR .Construct single chain Fv(scFv) with a linker between VH and VL . Cloned scFv to pTZ57R/T then to expression vector pBAD/gIIIA . Activity of fusion protein scFv-myc can be detected by SDS-PAGE and Western Blotting. Indirect agglutination assay also can show its activity . We clone the streptavidin(SA) from pSTMP plasmid and subclone to pBAD/gIIIA-scFv by recombination technology . The final fusion protein scFv-SA-w(whole) and scFv-SA- t(truncated) both have activity. The former effects in direct agglutination and the later effects in indirect agglutination .They can combine with biotin . The fusion protein scFv-SA-t can be used in autologous erythocyte agglutination(AGEN) to detect specific antibody in a man's whose blood who is to be inspected .It can combine the advantages of AGEN and biotin-avidin system.