免疫蛋白质组学方法对内脏利什曼病人血清识别主要抗原的鉴定

Use of an immunoproteomic approach to identify the dominant antigens reacting with serum patients with visceral leishmaniasis

目的鉴定我国内脏利什曼病人血清识别的主要抗原蛋白。方法培养杜氏利什曼原虫前鞭毛体并体外转化成无鞭毛体,提取总蛋白,经2-DE电泳,以内脏利什曼病人血清为一抗进行2-D Western blot,对强免疫识别点相应抗原蛋白进行MALDI-TOF/TOF串联质谱鉴定。结果等量的杜氏利什曼原虫前鞭毛体与无鞭毛体蛋白经2-DE电泳均可呈现约700个蛋白点,内脏利什曼病人血清识别的主要无鞭毛体抗原蛋白为β-微管蛋白、磷酸多糖β-1,3半乳糖基转移酶、PA7为磷酸多糖β-1,3阿拉伯糖基转移酶及延伸因子1等,识别的主要前鞭毛体抗原蛋白为热休克蛋白70及延伸因子1等。结论人内脏利什曼病患者血清能识别多种杜氏利什曼原虫蛋白,有利于筛选抗内脏利什曼病疫苗抗原和新的血清学诊断抗原。

Objective To identify the dominant antigens reacting with serum from patients with visceral leishmaniasis.Methods The promastigotes of Leishmania donovani were cultured and transformed into axenic amastigotes in vitro. The total proteins of promastigotes and axenie amastigotes were separated using two dimensional eleetrophoresis （2-DE）, proteins were identified using 2-D Western blotting, and proteins that reacted strongly to serum were analyzed with MAL- DI-TOF/TOF-MS. Results 2：DE yielded appropriately 700 protein spots for proteins from both promastigotes and ax- enic amastigotes. The dominant antigens from promastigotes that reacted with patient serum were elongation factor 1 and heat shock protein 70, and the dominant antigens from axenic amastigotes that reacted with patient serum were 13-tubulin, elongation factor 1, phosphoglycan beta 1,3 galactosyltransferase, and phosphoglycan beta 1,2 arabinosyltransferase. Conclusion Immunoproteomic analysis of human visceral leishmaniasis will facilitate the development of vaccine antigens and new serodiagnostic antigens.