柔嫩艾美耳球虫组蛋白去乙酰化酶Sir2克隆表达及表达动态分析

发布时间：2018-03-11 18:41

本文选题：柔嫩艾美耳球虫　切入点：Sir2　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：目前,抗球虫药物仍然是控制鸡球虫病的主要手段,但因其长期广泛的使用,鸡球虫对几乎所有已商业化使用的抗球虫药物都产生了抗药性。尽管鸡球虫病减毒活卵囊疫苗已在一定范围推广应用,但其带虫免疫、仍然具有一定的致病能力且具有潜在环境散毒和侵蚀的特点,决定其不可能完全取代药物在球虫病防治上的应用。集约化养鸡业生产的可持续发展和球虫病持续控制亟需新型抗球虫药物和疫苗的问世。Sir2作为一种NAD+依赖的组蛋白去乙酰化酶,在染色质重塑、衰老、调节细胞代谢生命活动中具有重要作用,医学和模式生物的大量研究结果揭示其是具有重大潜在价值的药物靶标。疟原虫、弓形虫等顶复门原虫中一般具有两种Sir2蛋白,SIR2A和SIR2B,且基因组数据分析发现组蛋白修饰和染色质占位等表观遗传机理可能是它们发育过程中基因表达调控的主要方式。艾美耳球虫全基因组数据分析表明,球虫与弓形虫、疟原虫等相似,也具有2种Sir2组蛋白去乙酰化酶,但目前关于球虫Sir2的研究尚鲜见报道。为探讨Sir2对球虫发育和寄生生活过程中对基因表达的调控作用及其作为药物靶标候选分子的意义,我们先期进行了柔嫩艾美耳球虫(Eimeria tenella)Sir2(EtSir2)的基因克隆、表达及其在不同发育阶段表达动态的研究,以为深入研究其药靶活性、基因表达调控活性及其机理提供实验基础。获得主要结果如下:1.PCR克隆得到了EtSir2A的全长ORF序列及EtSir2B部分编码序列,并通过生物信息学软件对EtSir2A序列进行了初步分析。EtSir2A ORF全长909bp,编码302氨基酸,其理论等电点为6.04,分子量大小约为43.4kDa,无信号肽序列,表明其是胞内定位和发挥生物学活性的蛋白质。构建了pMAL-c2x-EtSir2A原核表达载体,并将重组质粒转化入表达菌Transetta(DE3)中,经由终浓度为1mmol/L的IPTG诱导后,成功获得了EtSir2A-MBP融合蛋白的可溶性表达。2.采用实时定量PCR检测并以相对定量法计算EtSir2A mRNA在E.tenella不同发育阶段转录量的差值,并选取E.tenellaβ-actin作为内参基因。结果显示,EtSir2A mRNA在E.tenella不同发育阶段的转录有明显差异,未孢子化卵囊阶段EtSir2A mRNA转录量最高,而第二代裂殖子阶段转录量最低。
[Abstract]:At present, anti-coccidiosis drugs are still the main means to control chicken coccidiosis, but due to their long-term widespread use, Chicken coccidiosis is resistant to almost all commercially available anti-coccidiosis drugs. Although the attenuated live oocyst vaccine against coccidiosis has been popularized to a certain extent, it is immune to the disease. It still has some pathogenicity and potential environmental toxic and erosive characteristics, The sustainable development of intensive chicken production and the sustainable control of coccidiosis are in urgent need of the advent of new anti-coccidiosis drugs and vaccines. Sir2 as a NAD dependent group. Protein deacetylase, It plays an important role in chromatin remodeling, senescence, and regulation of cell metabolic life, and many medical and model organisms have revealed that it is a potentially significant drug target. There are two kinds of Sir2 proteins SIR2A and SIR2Bin Toxoplasma gondii, and genomic data analysis shows that histone modification and chromatin occupation are the main mechanisms of gene expression regulation in the development of Toxoplasma gondii. Analysis of the whole genome data of Eimeria japonica showed that, Similar to Toxoplasma gondii and Plasmodium, coccidia also has two kinds of Sir2 histone deacetylase. However, there are few reports on the study of coccidia Sir2. In order to investigate the role of Sir2 in the regulation of gene expression during the development and parasitic life of coccidia and its significance as a candidate molecule for drug targeting, We have studied the gene cloning of Eimeria tenellaus Sir2EtSir2 (Eimeria tenellaus) and its expression dynamics at different developmental stages in order to study the target activity of Eimeria tenellaus. The main results are as follows: 1. The full-length ORF sequence and EtSir2B partial coding sequence of EtSir2A were obtained. The EtSir2A sequence was preliminarily analyzed by bioinformatics software. The total length of EtSir2A ORF was 909 BP, encoding 302 amino acids. The theoretical isoelectric point was 6.04, the molecular weight was about 43.4 kDa, and the unsignaled peptide sequence was obtained. The prokaryotic expression vector of pMAL-c2x-EtSir2A was constructed, and the recombinant plasmid was transformed into the expression strain TransettasettaDE3. The recombinant plasmid was induced by IPTG with a final concentration of 1 mmol 路L ~ (-1) 路L ~ (-1) 路mol ~ (-1) 路L ~ (-1) 路mol ~ (-1) 路L ~ (-1) 路L ~ (-1) IPTG. The soluble expression of EtSir2A-MBP fusion protein was successfully obtained. Real-time quantitative PCR detection and relative quantitative analysis were used to calculate the difference of transcription quantity of EtSir2A mRNA in E. tenella at different developmental stages. E.tenella 尾 -actin was selected as the internal reference gene. The results showed that the transcription of EtSir2A mRNA in different developmental stages of E. tenella was significantly different, the EtSir2A mRNA transcription was the highest in the unspore oocyst stage and the lowest in the second generation merozoite stage.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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