乳酸脱氢酶在地克珠利抗柔嫩艾美耳球虫中作用研究

The diclazuril can inhibit effectively the the anaerobic stage of Eimeria tenella (schizogony and gametogony), however, little effect was found to the aerobic stage (sporogony). Previous studies had shown that the mRNA transcriptional level of E. tenella lactate dehydrogenase (EtLDH) was only observed in the intracellular stage (the schizont and meronts stage). The mRNA level of EtLDH was found to be significantly higher in the diclazuril-resistant strain in comparison with the drug-sensitive strain. Diclazuril had no effect on the diclazuril-resistant strain of Eimeria, but it can kill effectively the drug-sensitive strain, suggested that LDH plays an important role in the mechanism of diclazuril against E. tenalla..Lactate dehydrogenase, the last enzyme of the anaerobic glycolytic pathway, can catalyze NADH to deoxygenate the pyruvate into lactic acid while releasing of the NAD+. This NAD+ was utilized by glyceraldehyde 3-phosphate, the next round of the glycolytic cycle was proceeded and the ATP was provided to the parasites. Therefore, we speculated that the mechanism of diclazuril against E. tenalla was: the activity of E. tenella LDH was inhibited by diclazuril, which could not catalyze NADH to deoxygenate the pyruvate into lactic acid. The NAD+ was not generated and no NAD+ could be utilized by the glyceraldehyde 3-phosphate which caused the stop of next round of glycolysis pathway. The ATP could not be produced and the energy supply was ended which lead to the parasites dead. .The purpose of this project is to investigate the potential role of lactic dehydrogenase in the mechanism of diclazuril against E. tenalla with the following works: 1)Comparison the differences of EtLDH gene and its target proteins between the E. tenella drug-sensitive strain and diclazuril- resistant strain; 2)Effect of diclazuril on the recombinant EtLDH from the drug-sensitive strain and diclazuril- resistant strain; 3)Effect of diclazuril on LDH, NADH and NAD+ from the anaerobic stage of E. tenella (schizogony and gametogony); 4)EtLDH gene was transfected into yeast cells to verify the relationship between the EtLDH and diclazuril; 5) Flow cytometry was used to study the effect of NAD+ and ATP on the invasion capability of sporozoites and merozoites after treated with diclazuril.. Theses studies not only could help to understand the role of lactic dehydrogenase in the mechanism of diclazuril against E. tenalla, but also to learn more about the biological function of EtLDH. These would have important theoretical significance and practical value to clarify the anti-coccidial molecular mechanisms of diclazuril and discover new anticoccidial-drug targets.

地克珠利主要抑制柔嫩艾美耳球虫厌氧阶段虫体，但机制还不清楚。乳酸脱氢酶（LDH）是无氧糖酵解途径中末端酶，与球虫的生存密切相关。前期研究发现，柔嫩艾美耳球虫LDH仅在厌氧阶段虫体中高效表达，在地克珠利耐药株中表达量明显上调。我们推测地克珠利是抑制了球虫LDH酶活性，无法再生NAD+，糖酵解途径被终止，能量供给受阻而引发球虫发生死亡。本项目以柔嫩艾美耳球虫敏感株和地克珠利耐药株为研究对象，通过研究两个虫株的LDH基因及其靶标蛋白特性差异；药物对重组EtLDH酶活性作用，以及对需氧与厌氧阶段虫体LDH、NADH和NAD+影响的差异；将球虫LDH转染至酵母细胞以验证LDH与地克珠利之间关系；用流式细胞仪技术检测NAD+、ATP对药物处理后球虫子孢子和裂殖子入侵活力的影响，以探讨EtLDH在地克珠利抗柔嫩艾美耳球虫作用中扮演的角色。结果对阐明地克珠利抗球虫的分子机制及筛选新的药物靶标具有重要意义。

地克珠利主要抑制柔嫩艾美耳球虫厌氧阶段虫体，但机制还不清楚。为了阐明乳酸脱氢酶（LDH）在地克珠利抗柔嫩艾美耳球虫作用中扮演的角色，本项目对柔嫩艾美耳球虫LDH（EtLDH）的特性进行了研究，发现第二代裂殖子中EtLDH的mRNA转录水平和蛋白质翻译水平均明显高于其他发育阶段虫体，表明EtLDH在球虫糖酵解途径中发挥重要功能。比较了柔嫩艾美耳球虫敏感株与地克珠利耐药株之间 LDH特性的差异，发现二者LDH的氨基酸序列仅有1个碱基的差异，且该位点不在酶功能结构域内；敏感株孢子化卵囊、孢子化卵囊、子孢子和第二代裂殖子等4个不同发育阶段中LDH 的mRNA转录水平均明显高于耐药株中相应阶段的；耐药株第二代裂殖子中LDH的蛋白翻译水平和孢子化卵囊中的酶催化活力均明显低于相应的敏感株；说明长期的药物诱导使EtLDH的基因特性及酶学特性发生了改变。研究了地克珠利对球虫孢子生殖过程中LDH特性的影响，发现地克珠利能明显抑制卵囊的孢子化；能显著增强EtLDH还原反应的催化活力，但明显抑制氧化反应的催化活力；明显下调EtLDH的mRNA转录和蛋白翻译水平；说明地克珠利抑制卵囊孢子化的分子机理可能与抑制糖酵解途径中LDH的基因表达及酶活力有关。研究了地克珠利对球虫裂殖生殖过程中LDH特性的影响，发现地克珠利能显著抑制EtLDH氧化反应和还原反应的催化活力、mRNA转录和蛋白翻译水平，且使EtLDH在裂殖子中的分布范围缩小，表达量明显下降；推测EtLDH是地克珠利的一种潜在药物靶标。研究了NAD+、ATP对地克珠利处理后球虫子孢子入侵活力的影响，发现补给NAD+、ATP能显著地降低地克珠利抑制球虫子孢子的能力。利用酵母双杂交技术筛选了EtLDH潜在互作的裂殖子蛋白，获得了27个潜在的互作蛋白；对其中的EtSIR2和EtSTK蛋白的基因进行克隆、表达，利用免疫共沉淀技术和BiFC技术验证了其与EtLDH的关系，结果发现二者与EtLDH均不发生互作关系。对EtSIR2和EtSTK特性的进行了研究，发现二者均具有较好了抗原性，具有一定的抗球虫效果。这些结果对阐明地克珠利抗球虫的分子机制及筛选新的药物靶标具有重要意义。