Biochemistry: Production of recombinant Rh blood group antigens for detection of alloimmunisation

Abstract

Due to the importance of the antigen/ antibody reaction, all hospitals and blood banks worldwide rely on routine blood group phenotyping of samples. Using recombinant antigens removes the disadvantage of the current antibody screening when using the human red blood cells, which can complicate the detection of antibody in di erent cases: in the case of multi-transfused individuals and in the case of weak antigens. Although constructing recombinant antigens for some blood group antigens has been achieved successfully, such as; Kell, Du y and Lutheran blood group antigens, no studies have been published describing successful production of RH recombinant antigens in a prokaryotic expression system. Rh proteins are conserved across many different species. The aim was to construct a hybrid Rh protein comprised of a Nitrosomonas europaea (N. europaea) Rh50 backbone containing di erent external loops of the human RhD protein. Human RhD external loop 6 was the most suitable candidate to start with due to its location between highly conserved domains of the protein. The hybrid gene for the NeRH50"human RHD was sub-cloned into an expression vector (pET) and transformed in BL21 (DE3) competent E. coli. We constructed three other hybrid genes with RhD human external loops on a NeRh50 backbone: loops 4, 6; loops 3, 4, 6 and loops 2, 3, 4, 6. 14 Hybrid recombinant constructs were successfully expressed in the E. coli membrane as shown with western blot, ow cytometry, and transmission electron microscopy by the use of di erent commercial/ customized monoclonal and polyclonal antibodies against D epitopes in human RhD proteins and NeRh50 like protein. Due to a lack of time, testing human sera against the recombinant antigens was not possible. However, the approach utilised in this study may lead to a new diagnostic assay whereby the expression of human RhD external loops in E. coli may be capable of detecting speci c anti-Rh antibodies in patient serum.