Isolation, Cloning, Expression and Purification of Recombinant RhD Antigen from Cord Blood

Background: rh (rhesus) is a highly complex blood group system in man deeply rooted in transfusion medicine. isolationof rhd from cord blod, cloning and expression of recombinant rhd antigen in bacterial expression system was the aim ofthis study.methods: total rnas were extracted from cord blood (o+). the quality of rna was determined by electrophoresis. in orderto obtain coding sequence of rhd antigen cdna was synthesized and rh d gene was amplified by rt-pcr. the isolatedrhd gene was cloned to puc18 vector and transformed to dh5á. the confirmed construct was sub cloned intoexpression vector, pbadgiii/a, and expressed in top10 e.coli. the expressed protein was characterized by sds-pageand western blot analysis. antigenicity of the expressed protein was assessed by elisa using commercially available humananti-rhd polyclonal antibody with peroxidase conjugated goat anti-human igg, igm, iga as secondary antibody.results: rhd gene was successfully cloned and expressed. the expected size of recombinant rhd protein was detected insds-page, and confirmed by dot and western blot analysis. rhd antibody reacted with recombinant rhd antigen as wellas with rhd polypeptide extracted from rbcs membrane.conclusion: the recombinant rhd may be helpful to further investigate the molecular basis of rhd protein and could beapplicable for production anti- d antibody in an animal model