Coimbra E S, Gonçalves-da-Costa S C, Costa B L S, Giarola N L L, Rezende-Soares F A, Fessel M R, Ferreira A P, Souza C S F, Abreu-Silva A L, Vasconcelos E G
Departamento de Bioquímica, Microbiologia e Imunologia, ICB, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil.
Parasitology. 2008 Mar;135(3):327-35. doi: 10.1017/S0031182007003927. Epub 2007 Nov 16.
A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.
从无鞭毛体的质膜中通过制备性非变性聚丙烯酰胺凝胶电泳部分纯化了亚马逊利什曼原虫（利什曼原虫属）的ATP二磷酸水解酶同工型。SDS-PAGE之后进行的蛋白质印迹分析，用多克隆抗马铃薯焦磷酸酶抗体显色，鉴定出约58 - 63 kDa的弥散条带，可能是该蛋白的糖基化形式。通过ELISA技术发现，与未感染小鼠相比，无鞭毛体感染小鼠血清中针对马铃薯焦磷酸酶的总IgG抗体水平显著更高，这既证实了寄生虫与植物蛋白之间存在共同表位，也证实了寄生虫ATP二磷酸水解酶的抗原性。通过蛋白质印迹分析，来自无鞭毛体感染的BALB/c小鼠的血清能识别马铃薯焦磷酸酶以及从无鞭毛体中分离出的这种抗原性ATP二磷酸水解酶同工型，这表明它在无鞭毛体阶段也有表达。对无鞭毛体感染小鼠90天的感染监测显示，在感染早期IgG2a抗体有反应性，而IgG2a反应消失以及针对该共同表位的IgG1抗体血清水平升高与实验性利什曼病的进展相关。这是首次观察到亚马逊利什曼原虫ATP二磷酸水解酶同工型的抗原性，以及与马铃薯焦磷酸酶的交叉免疫反应性在感染小鼠中区分利什曼病血清学阶段的能力。
