Human CD96 gene cloning, expression and identification / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct and express human CD96 gene outer membrane domain (hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.</p><p><b>METHODS</b>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 polyclonal antibody was prepared by immunization of rabbits with the fusion protein. The specificity of anti-hCD96 antibody was determined by Western blotting.</p><p><b>RESULTS</b>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 kD hCD96om fusion protein.</p><p><b>CONCLUSION</b>The CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells, and its rabbit polyclonal antibody has been obtained.</p>