第一篇：柔嫩艾美耳球虫2-甲基柠檬酸合成酶的酶促动力学分析第二篇：鸡球虫基因组DNA甲基化修饰的检测

发布时间：2018-10-29 13:02

【摘要】：基因组数据分析发现,鸡球虫具有真菌样Ⅰ型2-MCC途径催化酶的编码基因。抑制或阻断2-MCC途径有可能成为抗球虫药物的新药靶,对开发新型抗球虫药物、建立球虫病防治的新策略具有重要意义。本文在基因组数据分析的基础上,通过克隆和原核表达柔嫩艾美耳球虫2-MCC途径相关酶的基因,并对柔嫩艾美耳球虫中的2-MCC途径关键酶2-甲基柠檬酸合成酶进行了体外酶活力测定和动力学分析,为进一步研究鸡球虫2-MCC途径及其相关酶的功能以及寻找新的药靶和开发新型抗球虫药物奠定了基础。本文的主要工作包括：1.通过RT-PCR的方法,成功克隆获得了编码EtMS. EtACN. EtCS. EtACS的基因的全长ORF序列以及EtMD和EtML编码基因的部分序列,证实了2-MCC途径在鸡球虫中的存在。2.鸡球虫基因组数据生物信息学分析发现,编码EtPrpE的基因序列与编码EtACS基因序列完全一致。根据在部分细菌中已报道的研究结果推测,在鸡球虫中,ACS可能同时具有PrpE功能。3. EtMS. EtACN. EtCS和EtACS分别在大肠杆菌表达系统中成功获得表达,为进一步研究其功能奠定了基础。4.获得了具有活性的重组表达蛋白MBP-EtMS,并对其进行了酶促动力学分析。证实EtMS不仅能利用丙酰辅酶A为底物还能以乙酰辅酶A为底物,进一步说明鸡球虫中的2-MCC途径与细菌中的2-MCC途径类似,可能也具有TCA循环的旁路功能。但其抑制物的筛选和抑制动力学分析有待进一步深入研究。DNA甲基化是在DNA甲基转移酶(DNA Methyltransferases, DNMTs)催化作用下,利用S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)提供甲基,在CpG二核苷酸中胞嘧啶嘧啶环的5号碳原子上加上甲基的共价修饰过程(5-methylcytosine,5-mC)。基因组DNA甲基化作为一种重要的表观遗传修饰系统,在调控基因表达、细胞分化以及其它各种生命过程中起到了非常重要的作用。本研究通过检测4种鸡球虫基因组DNA甲基化水平,还检测了刚地弓形虫、新孢子虫和3种隐孢子基因组DNA甲基化水平。另外,我们还对鸡球虫不同发育阶段的基因组DNA甲基化水平进行了检测。通过DNA甲基转移酶抑制物5-azacytidine处理子孢子和子孢子总蛋白提取物DNA甲基转移酶活性检测来验证鸡球虫体内是否具有有活性的DNA甲基转移酶,同时我们还克隆表达了柔嫩艾美耳球虫DNMT2编码基因,并对其进行了酶活力测定。通过体外培养模型,对DNA甲基化水平与鸡球虫入侵细胞的关系进行了初步研究。本文的主要结论是：1.证实了5-mC在顶复门原虫中广泛存在,但水平较低。2.5-mC水平在鸡球虫不同发育阶段差异显著,子孢子阶段明显高于其它4个阶段,说明基因组DNA甲基化在鸡球虫发育转化中可能发挥着重要作用。3.柔嫩艾美耳球虫子孢子基因组DNA甲基化水平能被DNA甲基转移酶抑制物5-azacytidine抑制,而且在柔嫩艾美耳球虫子孢子总蛋白提取物中能检测到DNA甲基转移酶样活性,说明鸡球虫体内存在可能的DNA甲基转移酶样机制。4. EtDNMT2可能不具有DNA甲基转移酶活性。5.体外试验证实了抑制子孢子基因组DNA甲基化水平能促进其入侵细胞。
[Abstract]:Genome data analysis revealed that the chicken coccidia has a fungal-like type I 2-MCC pathway catalytic enzyme coding gene. Inhibition or blocking of 2-MCC pathway may be a new drug target for anti-coccidial drugs. It is of great significance to develop new anti-coccidial drugs and to establish new strategies for the prevention and cure of coccidia. On the basis of genomic data analysis, the gene of Eimeria tenella 2-MCC pathway related enzyme was cloned and prokaryotic expression, and the enzyme activity determination and kinetic analysis were carried out on 2-MCC pathway key enzyme 2-methylcitric acid synthase in Eimeria tenella. In order to further study the function of 2-MCC pathway and its related enzymes in chicken coccidia and to find new drug targets and develop new anti-coccidial drugs. The main work of this paper includes: 1. The EMS was successfully cloned by RT-PCR. EtOH. EtCS. The full-length ORF sequence of EtACS and the partial sequence of EtMD and EtML coding genes confirmed the presence of 2-MCC pathways in Cocciella. Bioinformatics analysis of the genomic data of Eimeria tenella found that the gene sequence encoding EtPrpE was completely consistent with the sequence of EACS gene. According to the results reported in some bacteria, ACS may have the PrpE function at the same time in coccidia. EtMS. EtOH. EtCS and EtACS were successfully expressed in E. coli expression system, which laid the foundation for further study of its function. The recombinant expression protein A-EtMS with activity was obtained, and the enzymatic kinetic analysis was carried out. It was confirmed that EtMS was not only able to use propylated coenzyme A as a substrate but also NADA as a substrate, and further explained that the 2-MCC pathway in chicken coccidia was similar to the 2-MCC pathway in bacteria, possibly also with the bypass function of TCA cycle. However, the screening and inhibition kinetic analysis of its inhibitor still need to be studied further. DNA methylation is a covalent modification process (5-xylenol, 5-mC) which uses S-methionogen (SAM) to provide methyl in the catalysis of DNA methyltransferase (DNMTs). Genomic DNA methylation plays an important role in regulating gene expression, cell differentiation and other life processes as an important epigenetic modification system. In this study, the DNA methylation level of 4 species of chicken coccidia was detected, and the DNA methylation levels of Toxoplasma, neospora and three cryptosporidium were also detected. In addition, we also tested genomic DNA methylation level at different developmental stages of chicken coccidia. DNA methyltransferase activity detection of the total protein extract of sporozoites and sporozoites is processed by DNA methyltransferase inhibitor 5-azacytidine to verify whether there is active DNA methyltransferase in the chicken coccidian body, and meanwhile, the DNMT2 coding gene of Eimeria tenella is also cloned, The enzyme activity assay was carried out. Through in vitro culture model, the relationship between DNA methylation level and chicken coccidia invasion cells was studied. The main conclusions of this paper are: 1. It was confirmed that 5-mC was widely existed in the protozoa, but the level was lower. The levels of 5-mC were significantly different in different developmental stages of coccidia, and the sporozospore stage was significantly higher than that of the other 4 stages. The genomic DNA methylation might play an important role in the development and transformation of chicken coccidia. The DNA methylation level of the insect spores genomic DNA of EAMU can be inhibited by the DNA methyltransferase inhibitor 5-azacytidine, and the DNA methyltransferase-like activity can be detected in the total protein extract of the tender Eamey ball worm spore total protein. It is shown that possible DNA methyltransferase-like mechanism exists in chicken coccidia. EtDNMT2 may not have DNA methyltransferase activity. 5. In vitro tests demonstrated that inhibition of the DNA methylation level of sporozoite genomic DNA could promote its invasion of cells.
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：S852.7

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第一篇：柔嫩艾美耳球虫2-甲基柠檬酸合成酶的酶促动力学分析 第二篇：鸡球虫基因组DNA甲基化修饰的检测

第一篇：柔嫩艾美耳球虫2-甲基柠檬酸合成酶的酶促动力学分析第二篇：鸡球虫基因组DNA甲基化修饰的检测