【作者】 周变华；

【导师】 薛飞群；

【作者基本信息】 中国农业科学院 ， 基础兽医学， 2010， 博士

【摘要】 鸡球虫病是由一种或几种鸡艾美球虫寄生于鸡的肠上皮细胞,引起的以肠道病变为主的细胞内寄生虫病,给养禽业带来了巨大的经济损失。目前抗球虫主要依靠药物预防,针对耐药性的产生,迫切需要抗球虫新药的研发。本研究通过人工接种鸡E. tenella孢子化卵囊,分别建立感染组(接种卵囊+正常饲料)和地克珠利组(接种卵囊+ 1 mg/kg地克珠利饲料)球虫感染模型,地克珠利添加时间为接种后第96 h到120 h。以E. tenella第二代裂殖子为研究对象,采用透射电镜观察地克珠利对E. tenella第二代裂殖子超微结构的影响;采用流式细胞术(FCM)测定E. tenella第二代裂殖子凋亡率及线粒体膜电位,在细胞水平上研究地克珠利对E. tenella第二代裂殖子超微结构损伤的机制;采取抑制性消减杂交技术(SSH)和cDNA微阵列技术,从转录基因组水平研究地克珠利抗E. tenella第二代裂殖子作用机理。最终为阐明地克珠利抗鸡E. tenella作用机制及新型抗球虫药物的靶点的筛选提供理论依据。结果如下:1动物模型的建立及E. tenella第二代裂殖子的提取本试验以罗曼优质黄羽蛋公雏口服感染E. tenella孢子化卵囊,复制E. tenella正常感染和地克珠利作用动物模型。在接种后第120 h,通过酶消化、离心沉淀、红细胞裂解和Percoll密度梯度离心等方法的联合应用提取获得纯净的E. tenella第二代裂殖子,扫描电镜观察鉴定获得大量纯净的第二代裂殖子。2地克珠利对E. tenella第二代裂殖子超微结构的影响地克珠利对E. tenella第二代裂殖子超微结构产生了明显的损伤。在透射电镜下,感染组E. tenella第二代裂殖子细胞核双层核膜结构完整,染色质均匀,附着在核膜内侧面,胞质内线粒体丰富。地克珠利组E. tenella第二代裂殖子细胞核染色质明显减少、发生异染色质化,凝集成大的黑色团块、与核膜分离并发生趋边化,线粒体脊模糊、断裂,胞质中出现了大量的空泡,出现了类似于多细胞生物的凋亡现象。3地克珠利对E. tenella第二代裂殖子线粒体膜电位和凋亡率的影响地克珠利对E. tenella第二代裂殖子的线粒体膜电位和凋亡率产生了显著影响。与感染组相比,地克珠利组正常膜电位线粒体的数量降低了97.58% (P < 0.01),散失膜电位线粒体的数量提高了45.04 %( P < 0.01),坏死线粒体的数量提高了266.67% (P < 0.01)。E. tenella第二代裂殖子的早期凋亡率提高了180.75% (P < 0.01),晚期凋亡率提高了86.82% (P < 0.05),与E. tenella第二代裂殖子膜电位的变化呈正相关。以地克珠利组E. tenella第二代裂殖子为实验组,感染组第二代裂殖子为驱动组,构建了消减cDNA文库(T1-H);以感染组第二代裂殖子为实验组,地克珠利组第二代裂殖子为驱动组,构建了消减cDNA文库(T2-H)。经PCR鉴定,文库的重组率都为98%,插入片段都在500bp左右。结合具有高通量筛选能力的cDAN微阵列技术,对差异克隆进行了杂交鉴定。cDNA微阵列杂交分析后共获得881个差异表达基因克隆,其中从T1-H cDNA文库中获得532个,T2-H cDNA文库中获得268个。实时荧光定量PCR验证芯片杂交结果表明与芯片筛选克隆差异上下调趋势相同,程度相似,证明芯片数据可靠。选择251个差异表达基因克隆进行测序,结果获得了229条有效的ESTs序列,采用DNAStar软件进行拼接,最终的拼接序列共计23条Contigs,涉及到182条ESTs序列,32条Singletons序列。Blast X同源性比较分析发现差异基因编码蛋白同源蛋白主要包括:E. tenella微线蛋白、E. tenella表面抗原、E. tenella肌动蛋白解聚因子、E. acervulina热激蛋白90,T. gondii激活蛋白激酶C受体、T. annulata甘油醛-3-磷酸脱氢酶等。这些蛋白参与虫体和宿主细胞间的识别、黏附、入侵以及寄生虫的代谢、信号传导等过程。本研究鉴定获得的差异表达基因为探讨地克珠利的抗球虫分子作用机理的研究奠定的理论基础。5荧光定量PCR检测差异表达基因荧光定量PCR测定表明地克珠利降低了E. tenella第二代裂殖子EtMIC1的65.63% (P < 0.01)、EtMIC2的64.12% (P < 0.01)、EtMIC3的56.82% (P < 0.05)、EtMIC4的73.48% (P < 0.01)、EtMIC5的78.17% (P < 0.05)以及ADF的63.86%(P < 0.01)的mRNA表达量,提示地克珠利可能是通过抑制裂殖子入侵相关基因的表达,抑制入侵复合体的组装,进而干扰虫体入侵宿主细胞,同时药物处理后E. tenella第二代裂殖子的数量的显著降低及感染鸡盲肠病理组织结构的改善也进一步支持我们的推论。6差异基因全长cDNA的克隆、表达和分析根据筛选获得差异表达基因的ESTs序列,利用RACE技术扩增了EtRACK基因的全长cDNA,利用电子克隆的方法扩增了G3PDH基因的全长cDNA。构建了pET-28a-mz-ADF和pET-28a-mz-EtRACK表达质粒,并在E. coli BL21(DE3)中成功诱导表达,采用镍亲和层析法对融合蛋白进行了纯化,制备得到相应的抗体,这将为后续进行球虫发育阶段性表达检测、蛋白表达的定位、蛋白蛋白相互作用及蛋白生物学功能的研究奠定了基础。综上结论,地克珠利对E. tenella第二代裂殖子产生了显著的影响,可能通过以下途径发挥抗E. tenella第二代裂殖子的作用,从而影响虫体的正常发育:(1)降低虫体细胞线粒体膜电位和诱导虫体细胞凋亡;(2)影响虫体骨架相关蛋白的形成,从而使虫体散失生命能力;(3)抑制虫体与宿主细胞间的正常识别;(4)抑制虫体入侵宿主细胞的马达复合物的组装和形成;(5)抑制虫体的信号传导。有关地克珠利抗球虫的分子作用机制还需要进一步的研究和验证。更多还原

【Abstract】 Protozoan parasites of the genus Eimeria which infect chickens develop in the intestine cause great economic losses in the poultry industry. At present, coccidiosis is still mainly controlled by the use of chemotherapeutic agents; thus, new drugs are urgently needed due to the rising problem of drug-resistant strains of Eimeria. In this study, to evaluated the potential mechanism of diclazuril action on the second-generation merozoites of E. tenella, two treatment conditions were employed: (1) chickens were challenged with E. tenella oocysts and received no diclazuril treatment, as an Infected group or (2) chickens were challenged with E. tenella oocysts and administered a regular diet containing 1 mg/kg diclazuril continuously for 24 h, from the 96th h to 120th h after inoculation, as a Diclazuril group. Chickens were inoculated by oral gavage with an 8×104 oocysts per chicken suspended in 1ml of distilled water. Ultrastructural changes were monitored by transmission electron microscopy (TEM). Apoptosis and mitochondrial transmembrane potential were determined by flow cytometry (FCM). Differential expressed genes in second-generation merozoites induced by diclazuril were isolated and analyzed using suppression subtractive hybridization and microarray technologies. The findings in the present study help to elucidate the complex mechanism underlying diclazuril effects on coccidiosis at the molecular level. The merozoite organelles and its genome would appear to represent valuable targets for development of new therapeutic drugs. The results are as the followings:1 Establishment of animal moded infected by E. tenella and preparation of second-generation merozoitesFine Chinese Yellow Broiler male chickens were inoculated by oral gavage with E. tenella sporulated oocysts. By this way, the coccidiosis animal model had been reproduced successfully. Purified second-generation merozoites were obtained from infected chicken caecal tissue at the 120th h after inoculation by a combination of enzymatic digestion, centrifugation, erythrocytes disruption and percoll density gradient centrifugation. Scanning electron microscopy (SEM) examination showed a high degree of purity of the merozoites preparation.2 Observation of ultrastructural changes in second-generation merozoites of E. tenella induced by TEMIn the Infected group, considerable numbers of viable merozoites showed normal features under TEM. However, in the Diclazuril group, merozoites that were characterised by obvious apoptotic features were observed. Apoptotic merozoites had morphological changes including reduced amounts of chromatin, which was condensed and compressed against the nuclear envelope and aggregated into large dark, compact masses and cytoplasm association with abundant vacuolisation. These findings which have not been reported previously lead to new drug targets for chemotherapeutic intervention in this parasite. 3 Determination of apoptosis and mitochondrial transmembrane potential in second-generation merozoites of E. tenella by FCMApoptosis and mitochondrial transmembrane potential in second-generation merozoites of E. tenella were determined using double staining method by FCM. The results showed that Rh123+PI- in the Diclazuril group was decreased by 97.58% (P < 0.01), compared with the Infected group. Rh123-PI- and Rh123-PI+ in the Diclazuril group were increased by 45.04 % (P < 0.01) and 266.67% (P < 0.01), respectively, in comparison with the Infected group. With the changes of mitochondrial membrane potential, diclazuril treatment also significantly increased early apoptosis by 180.75% (P < 0.01) and late apoptosis by 86.82% (P < 0.05). A positive correlation was suggested between the collapse of mitochondrial transmembrane potential and apoptosis. The mitochondrial dysfunction induced by diclazuril in E. tenella was most likely associated with the mitochondrial dependent apoptosis pathway.4 Isolation and analysis of differentially expressed genes in second-generation merozoites of E. tenella using SSH and cDNA microarrayIn the forward-subtracted library, the cDNA from the Diclazuril group was used as the“tester group”; and the cDNA from the Infected group was used as the“tester group”in the reverse-subtracted one. PCR amplification showed that the recombinant clones were 98% both in the two subtractive library, and the inserts were about 500bp. Rapid and high throughput screening of differentially genes in SSH subtracted libraries were used by cDNA microarray. The microarray hybridization results showed 881 differentially expression genes were obtained in all and 532 clones from the T1-H cDNA library and 268 from the T1-H cDNA library. Further QRT-PCR test on the chip hybridization results showed that clone differences is the same degree of similarity. A total of 229 ESTs from 251 differentially clones sequence were group into 55 contigs that contain 23 Contigs and 32 Singletons. All contigs then were submitted to Blast P for homologous searching with the nucleotide database in GeneBank. The homologous proteins including E. tenella microneme proteins, E. tenella surface antigens, E. tenella actin-depolymerizing factor (ADF), E. acervulina heat shock protein 90, T. gondii receptor for activated C kinase, T. annulata glyceraldehyde-3-phosphate dehydrogenase, etc.. These coding proteins identified in this study are related closely to the parasite discern, attach, motility, host-cell invasion, metabolism, signal transmission and so on. The results showed that diclazuril may be involved in the regulation of key molecules essential to parasite invasion and development in the clinical action of anticoccidiosis. These differential genes identified in this study can be acted as attractive target for control coccidiosis, remains to be investigated.5 Analysis of differential expression genes by QRT-PCRQRT-PCR analysis showed that diclazruil treatment resulted in downregulation of EtMICs and ADF genes of second-generation merozoites in E. tenella: EtMIC1 by 65.63% (P < 0.01), EtMIC2 by 64.12% (P < 0.01), EtMIC3 by 56.82% (P < 0.05), EtMIC4 by 73.48% (P < 0.01), EtMIC5 by 78.17% (P < 0.05) and ADF by 63.86% (P < 0.01). This indicated that downregulation of invasion-related EtMICs and ADF genes by diclazuril had interfered with the formation of biological complexes acting as important role during the course of invasion. As the same, the attenuation of damage to caecal mucosa induced by merozoite infection under SEM and the 65.13% (P < 0.01) decrease in merozoite number in the Diclazuril group also support our speculation.6 Cloning, expression and analysis of differential genesBased on the ESTs of differentially expressed genes, E. tenella receptor for activated C kinase (EtRACK) full-length cDNA was amplified using the RACE technology. G3PDH sequence was obtains by the electronic clone method. Recombinant expression plasmids pET-28a-mz-ADF and pET-28a-EtRACK were constructed successfully, and expressed in E. coli BL21(DE3) cell with the IPTG induction, respectively. The fusion protein purified using nickel-ion metal chelating column have been finished preparing the antibody and this pave a road to the study of protein expression in developmental stage, location of protein expression, protein-protein interaction and protein biological function, etc..In conclusion, the effects of diclazuril treatment on the normal development of second-generation merozoites in E. tenella are as the followings: First, decreased the mitochondrial transmembrane potential and induced apoptosis. Second, affect the skeleton-related protein and lost the living capacity. Third, interfere with the normal reorganization between parasite and hose cell. Forth, inhibit the gliding motor complex overhauling and formation. Fifth, shutoff the signal transduction of the parasite. However, the molecular mechanism of diclazuril action on the anticocidiosis will need further investigation and verification.更多还原