[Preparation of anti-human indoleamine 2,3-dioxygenase polyclonal antibody].
OBJECTIVE
Indoleamine 2,3-dioxygenase (IDO), a cytosolic hemoprotein, catalyzes the rate-limiting step in tryptophan catabolism along the kynurenine pathway in mammals, arrests the growth of pathogens, and suppresses T-cell responses, therefore, leads to IDO-dependent tumor immune tolerance. This study was to express and purify His-hIDO fusion protein and to generate rabbit anti-human IDO polyclonal antibody, which was used to analyze IDO expression in tumor cells.


METHODS
Human IDO cDNA was cloned into pET30a(+). The recombinant vector pET30a(+)-hIDO was transformed into BL21 after sequencing. The expression of His-hIDO protein was induced by IPTG. The anti-human IDO polyclonal antibody was obtained by immunizing rabbits with purified His-hIDO protein. The quality of the antibody was identified by Western blot. IDO expression in human fibroblast cancer cell line A431 and liver cancer cell line HepG2 induced by interferon-gamma (IFN-gamma) was also analyzed using the antibody.


RESULTS
His-hIDO fusion protein was specifically combined with His-probe polyclonal antibody. The rabbit anti-human IDO polyclonal antibody was of high titer with high specificity. It could recognize IDO expression induced by IFN-gamma in A431 and HepG2 cells.


CONCLUSIONS
The rabbit anti-human IDO polyclonal antibody could recognize IDO expression in tumor cells in vitro effectively, therefore, provides a tool to study the role of IDO in tumor immune tolerance.
Xie
Bai-Lu
BL
Department of Microbiology and Biopharmaceutics, School of Pharmaceutical Sciences,Sun Yat-sen University, Guangzhou, Guangdong, PR China.
Liu
Peng
P
Ou
Xue-Ling
XL
Du
Jun
J
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D015153:Blotting, Western; D045744:Cell Line, Tumor / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins; D014169:Transformation, Bacterial
D000906:Antibodies; D018076:DNA, Complementary; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D011994:Recombinant Proteins; D007371:Interferon-gamma