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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 major antigenic proteins recognized by the serums of visceral leishmaniasis in China. Methods The Leishmania donovani promastigotes were cultured and transformed into amastigotes in vitro. The total protein was extracted. The 2-DE Western blot was performed by 2-DE electrophoresis and the serum was taken as the primary antibody against visceral leishmaniasis. The corresponding antigen proteins were identified by MALDI-TOF / TOF tandem mass spectrometry. Results Equivalent amounts of Leishmania donovani promastigotes and amastigote proteins showed approximately 700 protein spots by 2-DE electrophoresis, and the major amastigote antigen recognized by visceral leishman’s serum was β-microtubules Protein, phosphoglycerate β-1,3-galactosyltransferase, PA7 phosphoglycerate β-1,3-arabinosyltransferase and elongation factor 1, identified as the main promastigote antigen protein heat shock protein 70 and extension Factor 1 and so on. Conclusion Serum of human leishmaniasis can recognize a variety of Leishmania donovani proteins, which is good for screening anti-visceral leishmaniasis vaccine antigens and new serological diagnostic antigens.