Human CD96 gene cloning,expression and identification

Objective To construct and express human CD96 gene outer membrane domain(hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.Methods hCD96om was amplified by RT-PCR from the peripheral blood of patients with acute myeloid leukemia and inserted into prokaryotic expression vector pET32a(+) to construct the recombinant plasmid pET32-CD96.The expression of hCD96om was induced by IPTG in BL21(DE3) cells,and the expression product was identified by Western blotting.The anti-hCD96 polyclona1 antibody was prepared by immunization of rabbits with the fusion protein.The specificity of anti-hCD96 antibody was determined by Western blotting.Results hCD96om protein was expressed in E.coli BL21(DE3) cells in the form of inclusion body,with a relative molecular mass around 37 kD.Western blotting showed a specific reaction of the prepared antiserum with the 70 kD protein extracted from human leukemia cell line HL-60 cells and with the 37 000 hCD96om fusion protein.Conclusion The CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells,and its rabbit polyclonal antibody has been obtained.