The Expression and Characterization of a Bifunctional Protein in E. Coli for Autologous Erythrocyte Agglutination Test

Abstract

H antigen, the precursor of A and B antigens, belongs to Hh blood system in which it is the only antigen. H antigen distributes on all the human RBC surface except for Bombay phenotype and the copy number of H antigen on the surface of an adult RBC is approximately 1.7 × 10⁶. These characteristics made H antigen the potential target molecule for the immunoassay and immunotherapy. A monoclonal antibody 2E8 against H antigen on the surface of erythrocyte had been prepared in previous work. Based on this antibody, the variable region genes of heavy and light chains (V_H and V_L) from 2E8 had been cloned by 5' RACE. The two variable region genes were spliced by overlap extension and assembled ScFv (V_H-linker-V_L) gene encoding the anti-H antigen named ScFv_2E8. According to the prediction of the three-dimension structure of ScFv_2E8 and C_H1 fragment from 2E8 and HIV-1 gp41 antigen peptide, we further constructed the ScFv_2E8-C_H1-gp41 fusion molecule. The recombinant ScFv_2E8-C_H1-gp41 gene was cloned into pET-his vector and expressed in BL21(DE3)plysS cells. The fusion protein was purified from the inclusion bodies. In a series of subsequent analyses, this fusion protein showed identical antigen binding site and activity with the parent antibody. Meanwhile, in mimic test, as the main ingredient of reagent for autologous erythrocyte agglutination test, the bifunctional protein could agglutinate the RBCs in the presence of HIV-1 gp41 antibodies using sera from HIV-infected individuals.