人吲哚胺2,3-双加氧酶多克隆抗体的制备

背景与目的:吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)是一种哺乳动物细胞质中的血红素蛋白,是催化色氨酸沿犬尿氨酸途径代谢的限速酶.它的表达不仅能抑制病原微生物的生长,还能抑制T细胞的应答,从而介导肿瘤免疫耐受.本研究的目的是表达和纯化His-hIDO融合蛋白,制备兔抗人IDO多克隆抗体,并用此抗体检测IDO在肿瘤细胞中的表达.方法:将编码人IDO全长cDNA克隆入pET30a( ),测序鉴定表达载体pET30a( )-hIDO,转化大肠杆菌BL21,IPTG诱导表达目的蛋白His-hIDO;用纯化的目的蛋白免疫新西兰大白兔,获得兔抗人IDO多克隆抗体;用Western blot检测该多克隆抗体与重组蛋白His-hIDO的反应性,并用该抗体分析表皮癌细胞A431和肝癌细胞HepG2经IFN-γ诱导前后的IDO表达情况.结果:His-hIDO融合蛋白可与抗His多克隆抗体特异性结合;兔抗人IDO多克隆抗体的效价高,特异性好,能检测肿瘤细胞经IFN-γ诱导后的IDO的表达.结论:兔抗人IDO多克隆抗体能够有效地识别体外表达的IDO蛋白,为研究IDO在肿瘤免疫耐受中的作用提供有力工具.

Preparation of anti-human indoleamine 2,3-dioxygenase polyclonal antibody

BACKGROUND & 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. CONCLUSION: 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.