Anti-human Cd271 Monoclonal Antibodies

Objective:Produce Mabs against human CD271 molecules by DNA immunization in order to get a specific marker for isolating and identifying the MSCs.Methods:The full-length gene of human CD271 was amplified by reverse transcriptase polymerase chain reaction(RT-PCR).The gene was then cloned into the two expression vectors pcDNA3.1 and pET22b respectively.(1) The recombinant plasmid pcDNA3.1/CD271 was injected into BALB/C mice quadriceps muscle of thigh and we pretreat muscles as follows:①inject cardiotoxin seven days before DNA immunization.②inject high osmotic pressure sucrose solution.To boost the immunized mice,the recombinant plasmid was injected into spleen three days before fusion.Then monoclonal antibody was produced by conventional hybridoma technique.Serum for anti-serum titer evaluation was drawn every seven days after DNA immunization.The isotype of antibody was detected by sandwich ELISA.To test the affinity of antibody to natural membrane protein and denatured protein,we utilized FACS,fluorescence microscope and Western blot respectively.(2) The gene was obtained by PCR and cloned into prokaryotic expression vector pET22b. Result:(1) The 1281bp fμll-length gene was amplified,and the recombinant plasmid pcDNA3.1/CD271 was constructed.(2) The highest anti-serum titer was 1:160 after the third immunization,and the titer rose to 1:320 after boosting.(3) We obtained two monoclonal antibodies against human CD271 molecules,named 4B7E and 4B7D.(4) The isotypes of these two antibodies were both IgM.Light chains wereκtype. (5) The epitope of these antibodies,4B7E and CD271(BD) were different.(6) FACS,fluorescence microscope and Western blot experiments indicated the antibody could bind specificitily to nature membrane protein and denatured protein.(7) The prokaryotic expression vector pET22b/CD271 was successfully constructed.Conclusion:The anti-CD271 monoclonal antibodies had expected binding activity with MSCs that derived from bone marrow or umbilical cord,and the CD271 molecules may be a novel specific marker for isolating and identifying these mesenchymal stem cells.