蓝氏贾第鞭毛虫糖代谢通路的研究

Giardia lamblia infection is pandemic worldwild and causes giardiasis with symptoms of diarrhoea and intestinal upset . Giarida is an anerobic protozoan parasite and important model organism.Giarida gains ATP mainly from Embden-Meyerhof-Parnas glycolysis and may tansfer to amino acid source energy pathway when glucose is limited.The Inorganic pyrophosphate(PPi) is a key material for the production of ATP. Bisphosphonates is an isomer of PPi and can compete with PPi to combine with Pyrophosphate-dependent phosphofructokinase (PPi-PFP) and pyruvate-phosphate dikinase (PPDK) , resulting in the inhibition of glycolysis pathway in Giardia.The work for pre-project, we found PPDK is the key enzemy to calazy the production of pyruvate enven though the pyruvate kinase(PK) also exits.The present study will focus on the changes of the production of energy, the consumption of glucose and amino acid in Giardia trophozoites when being cultured in vitro with or without diimide bisphosphonates or lack of glucose to evolute the alternatiove pathway on the energy metabolish of Giardia protozoan. Two specific hammerhead ribozymes were designed and synthesized refered to the perious study. The ribozymes cDNA and the fragements involved in the construction of recombinate vector were obtained by PCR ampilication. cDNA encoding hammerhead ribozyme flanked with antisense PPDK or PK RNA were cloned into a viral vector Giardia canis virus (GCV) by digestion and ligation, so that the recombinant plasmids (pGCV634/H5/2174 and pGCV634/H3/2174) were constructed. PPKD and PK specific epitope antibodies were synthetised to clear the functions and the locations of PPDK and PK in Giardia sugar metabolic pathway .The results will provide worthy experimental data for interpreting energy metabolism of Giardia protozoan. From the glycolytic pathway ,the enzymes depend on the PPi is significantly different in Giardia from those in the host including PFP and PPDK so this study may also be observed to evalute potential drug for treating Giardia infection.

蓝氏贾第鞭毛虫是引起腹泻的常见病原体，是重要的模式生物，能量代谢依赖糖酵解途径。糖利用受限时是否存在氨基酸源能量通路转换，PPDK和PK的分工如何，是该虫能量代谢和新药靶点研究的重点。双磷酸盐类化合物为贾第虫糖酵解途径中必需物质PPi的异构体，能与PPi依赖酵解关键酶竞争结合，有效阻断糖酵解进程。本项目在明确PPDK是催化丙酮酸生成关键酶基础上，以亚胺二磷酸盐作用于贾第虫滋养体，切断PPDK催化的糖酵解，分析经该磷酸盐作用前、后，以及在糖饥饿培养情况下，糖、氨基酸的利用和能量生成等情况，综合分析探讨贾第虫在糖利用受限时，其能量代谢通路的改变机制；设计PPDK和PK特异性锤头状核酶，构建核酶-贾第虫病毒载体，合成PPDK和PK特异性多表位抗体，明确二者的功能定位和在贾第虫糖代谢通路中的功能分工。本研究可为贾第虫能量代谢的深入研究提供有价值数据资料，也可为抗贾第虫新药筛选提供理论和实验依据。

蓝氏贾第鞭毛虫是引起腹泻的病原体之一，是重要的模式生物，能量代谢依赖糖酵解途径。糖利用受限时是否存在能量通路转换，PPDK和PK的分工如何，是该虫能量代谢和新药靶点研究的重点。双磷酸盐类化合物为贾第虫糖酵解途径中必需物质PPi的异构体，能与PPi依赖酵解关键酶竞争结合，有效阻断糖酵解进程。本项目在明确PPDK是催化丙酮酸生成关键酶基础上，以亚胺二磷酸盐作用于贾第虫滋养体，切断PPDK催化的糖酵解，分析在体外培养情况下,亚胺二磷酸盐作用前后和糖饥饿培养时，虫体能量生成、糖和氨基酸的利用情况，探讨贾第虫在糖利用受限时，能量代谢通路的改变；设计PPDK和PK特异性锤头状核酶，构建核酶-贾第虫病毒载体，合成PPDK和PK特异性多表位抗体，明确PPDK和PK的功能定位和在贾第虫糖代谢通路中的分工。结果表明，在贾第虫滋养体胞浆中存在多聚焦磷酸和钙离子的储存单位，即酸性钙离子体；PPDK和PK的功能定位分析显示，PPDK 主要定位于细胞浆内，为蓝氏贾第鞭毛虫糖酵解途径催化磷酸烯醇式丙酮酸合成和产生ATP的关键酶；PK主要要定位于细胞核，其功能主要与虫体的分裂增殖有关；基于PPDK三维结构的进化分析表明，针对环境和代谢条件的不同，PPDK的结构发生了适应性改变，从而导致功能的进化；同正常培养对照组相比，双膦酸盐类药物的作用明显降低糖的利用（P＜0.05），说明双膦酸盐类药物可有效切断贾第虫糖酵解途径。通过基因组学的分析，贾第虫存在脂肪酸物质的分解代谢酶，且代谢过程中参与乙酰-COA的生成，为脂类代谢参与的能量代谢研究提供线索。本研究为贾第虫能量代谢的深入研究提供有价值数据资料，同时为抗贾第虫新药筛选提供理论和实验依据。