CHARACTERIZATION OF TRYPANOSOMA BRUCEI GP63 PROTEIN

布氏锥虫 GP63 蛋白的表征

• 批准号: 6136273
• 负责人: Douglas J. LaCount
• 金额: $ 3.24万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-10-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6136273
• 关键词: SDS polyacrylamide gel electrophoresis; Trypanosoma brucei rhodesiense; affinity chromatography; complement pathway; confocal scanning microscopy; cytolysis; enzyme activity; epitope mapping; fluorescence microscopy; fungal proteins; gene complementation; gene expression; genetic regulation; host organism interaction; immunoprecipitation; intracellular parasitism; laboratory mouse; laboratory rabbit; metalloendopeptidases; molecular cloning; northern blottings; protein structure function; western blottings

Trypanosoma brucei, the causative agent of African trypanosomiasis, multiplies extracellularly in the bloodstream of its mammalian hosts but is resistant to complement mediated lysis (CML). The objective of this proposed research is to determine the role of T. brucei GP63 (Tb-GP63) genes in resistance to CML. Tb-GP63 is a homolog of leishmania GP63 (L- GP63), a cell surface zinc metalloprotease that is critical for the resistance of leishmania to CML. Two groups of Tb-GP63 genes have been identified A and B. which are approximately 40 % identical and 60 % similar to L- GP63 and to each other. All disulfide bonded cysteines in L-GP63 and nine prolines are conserved in Tb-GP63-A and -B, suggesting that the Tb-GP63 proteins will be structurally similar to L-GP63. To study the role of Tb- GP63 in the resistance of T. brucei to CML Tb-GP63 will expressed in a baculovirus expression system and purified antibodies will be generated against Th-GP63 and cell lines that over-express or that lack Tb-GP63 will be created. With these reagents. the following questions will be addressed: (i) is Tb-Gp63-A is a functional cell surface protease? (ii) is Tb-GP63-B also a functional GP63 homolog? (iii) does Tb-GP63 increase the resistance cells to CML? (iv) is Tb-GP63 is required for growth of T. brucei in mice? and (v) do the Tb-GP63 gene 3' UTRs regulate stage specific expression of Tb-GP63? These studies may identify a new target for anti-trypanosome drugs and will provide insight into the molecular basis for the success of T. brucei as a parasite.

布氏锥虫是非洲锥虫病的病原体，在哺乳动物宿主的血液中细胞外繁殖，但对补体介导的酵解（CML）具有抗性。本研究的目的是确定布鲁氏T. GP63 （Tb-GP63）基因在CML耐药中的作用。Tb-GP63是利什曼原虫GP63 （L- GP63）的同源物，后者是一种细胞表面锌金属蛋白酶，对利什曼原虫对CML的抗性至关重要。已鉴定出两组Tb-GP63基因A和b，它们与L- GP63基因相同约40%，彼此相似约60%。L-GP63中的所有二硫键结合半胱氨酸和9个脯氨酸在Tb-GP63- a和-B中都是保守的，这表明Tb-GP63蛋白在结构上与L-GP63相似。为了研究结核-GP63在布鲁氏t细胞对CML的抗性中的作用，结核-GP63将在杆状病毒表达系统中表达，并产生针对Th-GP63的纯化抗体，并创建过表达或缺乏结核-GP63的细胞系。用这些试剂。以下问题将被解决：(i) Tb-Gp63-A是一个功能性的细胞表面蛋白酶吗？（ii） Tb-GP63-B也是GP63的功能性同源物吗？（iii） Tb-GP63是否会增加细胞对CML的抗性？（iv）结核菌在小鼠体内生长是否需要Tb-GP63 ？(v) Tb-GP63基因3′utr是否调控Tb-GP63的阶段特异性表达？这些研究可能确定抗锥虫药物的新靶点，并将为布鲁氏锥虫作为寄生虫的成功提供分子基础。