Human Erythrocyte Glycophorin B Of Monoclonal Antibodies

Blood group has been defined as 29 blood systems and more than 600 antigens on the surface of red blood cells, which plays significant role in blood transfusion, medical examination and hereditary diease diagosise. 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, the later has two subtypes: GPB-S(48%) and GPB-s(52%). The difference between them is that the 48th amino acid of GPB is Met in the S antigen, while Thr in the s antigen. Anti-S and Anti-s are all warm IgG antibodys which are often produced after blood transfusion and could cause serious haemolyticus transfusion response.We have cloned gene GPBs from K562 cell line by RT-PCR. Subcloned it to pTZ57R/T then to eukaryotic expression vector pcDNA3.1-His-Myc-A. The CHO cell was transfected with the recombinant plasmid, then screened with G418. The stable transgenic cell line is identified with RT-PCR and Western blot. Meanwhile, we amplified the extracelluar region of GPBs and cloned it into the prokaryotic expression vector pGEX 4T-2. The fusion protein GST-GPBs was expressed in the E.coli BL21 by inducing with IPTG.8 week-old female SPF BALB/c mice were immunized with RBC, transgenic GPBs-CHO cells and purified GST-GPBs extracellular region fusion protein. Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells, and positive hybridoma clones were screened by RBC hemagglutination test, ELISA, Western blot, we abtained 1 hybridoma strain which anti extracellular region of GPBs specially. The titer of supernatant and ascitic fluid were tested by direct and indirect agglutination, the titer of supernatant was 1×26, and the titer of ascitic fluid was1×213. The subclasses and isotypes were identified by mouse monoclonal antibody isotyping kit, the immunoglobulin subclasses of the mAb is IgG. The mAb anti-GPB-s provides an useful tool for the study of MNSs blood group and the serological diagnosis.