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The Study on the Virulence of Trypanosoma cruzi

克氏锥虫毒力研究

基本信息

批准号：
63570177
负责人：
KANBARA Hiroji
金额：
$ 1.34万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

Although the process of transformation of Trypanosoma cruzi trypomastigote to amastigote seemed to take place spontaneously, it was demonstrated that the process was accelerated by keeping trypomastigotes at low pH that was adjusted by 10 mM citrate-phosphate buffer. The process required certain nutrients and involved the separation of chemical substances which lysed even parasites. The lytic effect was neutralized by bovine albumin, accordingly the transformation was rapidly completed in tissue culture medium at low pH, 4.5-6.0, supplemented with 1% bovine albumin. Low pH adjusted by 10 mM acetate buffer was highly toxic to parasites. The interesting point is that the lytic effect was much stronger in high-virulent clone or strains than in low virulenct. Characterization of the lysin suggested that it might be lipid.The presence of serum in media at pH 7.4 suppressed the transformation, resulting in the long maintenance of the trypomastigote stage. Bovine albumin cloud replace whole serum but the ability to maintain the trypomastigote was less. Comparison of minicircle DNA of the kinetoplast after digestion by various restricted enzymes revealed that the pattern of DNA fragments digested by EcoRl in the high-virulent clone or strains was very distinct from the low-virulent clone or strains, which shared the same pattern. Hybridization test using the probe obtained from the distinct fragment demonstrated presence of the specific sequence to the high-vireulent clone.

虽然克氏锥虫锥鞭毛体转化为无鞭毛体的过程似乎是自发发生的，但事实证明，通过将锥虫鞭毛体保持在低 pH 值并用 10 mM 柠檬酸盐-磷酸盐缓冲液调节，可以加速该过程。这个过程需要一定的营养物质，并涉及化学物质的分离，甚至可以溶解寄生虫。裂解作用被牛白蛋白中和，因此在低pH值4.5-6.0、补充有1%牛白蛋白的组织培养基中转化迅速完成。用 10 mM 醋酸盐缓冲液调节的低 pH 对寄生虫具有剧毒。有趣的一点是，高毒力克隆或菌株的裂解作用比低毒力克隆或菌株强得多。溶素的表征表明它可能是脂质。pH 7.4 的培养基中血清的存在抑制了转化，导致锥鞭毛体阶段得以长期维持。牛白蛋白云替代全血清，但维持锥鞭毛体的能力较差。比较各种限制性酶消化后动质体的小环DNA，发现高毒力克隆或菌株中EcoRI消化的DNA片段的模式与低毒力克隆或菌株有很大不同，低毒力克隆或菌株具有相同的模式。使用从不同片段获得的探针进行的杂交测试证明了高毒力克隆的特定序列的存在。