發(fā)布時(shí)間：2018-03-11 18:41

  本文選題：柔嫩艾美耳球蟲　切入點(diǎn)：Sir2　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目前,抗球蟲藥物仍然是控制雞球蟲病的主要手段,但因其長期廣泛的使用,雞球蟲對幾乎所有已商業(yè)化使用的抗球蟲藥物都產(chǎn)生了抗藥性。盡管雞球蟲病減毒活卵囊疫苗已在一定范圍推廣應(yīng)用,但其帶蟲免疫、仍然具有一定的致病能力且具有潛在環(huán)境散毒和侵蝕的特點(diǎn),決定其不可能完全取代藥物在球蟲病防治上的應(yīng)用。集約化養(yǎng)雞業(yè)生產(chǎn)的可持續(xù)發(fā)展和球蟲病持續(xù)控制亟需新型抗球蟲藥物和疫苗的問世。Sir2作為一種NAD+依賴的組蛋白去乙�；�酶,在染色質(zhì)重塑、衰老、調(diào)節(jié)細(xì)胞代謝生命活動中具有重要作用,醫(yī)學(xué)和模式生物的大量研究結(jié)果揭示其是具有重大潛在價(jià)值的藥物靶標(biāo)。瘧原蟲、弓形蟲等頂復(fù)門原蟲中一般具有兩種Sir2蛋白,SIR2A和SIR2B,且基因組數(shù)據(jù)分析發(fā)現(xiàn)組蛋白修飾和染色質(zhì)占位等表觀遺傳機(jī)理可能是它們發(fā)育過程中基因表達(dá)調(diào)控的主要方式。艾美耳球蟲全基因組數(shù)據(jù)分析表明,球蟲與弓形蟲、瘧原蟲等相似,也具有2種Sir2組蛋白去乙�；�酶,但目前關(guān)于球蟲Sir2的研究尚鮮見報(bào)道。為探討Sir2對球蟲發(fā)育和寄生生活過程中對基因表達(dá)的調(diào)控作用及其作為藥物靶標(biāo)候選分子的意義,我們先期進(jìn)行了柔嫩艾美耳球蟲(Eimeria tenella)Sir2(EtSir2)的基因克隆、表達(dá)及其在不同發(fā)育階段表達(dá)動態(tài)的研究,以為深入研究其藥靶活性、基因表達(dá)調(diào)控活性及其機(jī)理提供實(shí)驗(yàn)基礎(chǔ)。獲得主要結(jié)果如下:1.PCR克隆得到了EtSir2A的全長ORF序列及EtSir2B部分編碼序列,并通過生物信息學(xué)軟件對EtSir2A序列進(jìn)行了初步分析。EtSir2A ORF全長909bp,編碼302氨基酸,其理論等電點(diǎn)為6.04,分子量大小約為43.4kDa,無信號肽序列,表明其是胞內(nèi)定位和發(fā)揮生物學(xué)活性的蛋白質(zhì)。構(gòu)建了pMAL-c2x-EtSir2A原核表達(dá)載體,并將重組質(zhì)粒轉(zhuǎn)化入表達(dá)菌Transetta(DE3)中,經(jīng)由終濃度為1mmol/L的IPTG誘導(dǎo)后,成功獲得了EtSir2A-MBP融合蛋白的可溶性表達(dá)。2.采用實(shí)時(shí)定量PCR檢測并以相對定量法計(jì)算EtSir2A mRNA在E.tenella不同發(fā)育階段轉(zhuǎn)錄量的差值,并選取E.tenellaβ-actin作為內(nèi)參基因。結(jié)果顯示,EtSir2A mRNA在E.tenella不同發(fā)育階段的轉(zhuǎn)錄有明顯差異,未孢子化卵囊階段EtSir2A mRNA轉(zhuǎn)錄量最高,而第二代裂殖子階段轉(zhuǎn)錄量最低。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.7

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本文編號：1599419