Human CD96 gene cloning, expression and identification.
- Author:
Jian-ming ZENG
1
;
Fei LIU
;
Ping-hai TAN
;
Li-na WANG
;
Mo LI
;
Zhong-hua CHEN
;
Song LI
;
Yi-fei LONG
;
You-qiang LI
;
Cha CHEN
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies; immunology; metabolism; Antigens, CD; biosynthesis; genetics; immunology; Base Sequence; Cloning, Molecular; Escherichia coli; genetics; metabolism; Humans; Immune Sera; biosynthesis; Immunization; Leukemia, Myeloid, Acute; immunology; Molecular Sequence Data; Neoplastic Stem Cells; immunology; Rabbits; Recombinant Proteins; biosynthesis; genetics; immunology
- From: Journal of Southern Medical University 2011;31(7):1232-1235
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct and express human CD96 gene outer membrane domain (hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.
METHODShCD96om 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.
RESULTShCD96om 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.
CONCLUSIONThe CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells, and its rabbit polyclonal antibody has been obtained.
