發(fā)布時(shí)間：2018-05-06 15:45

  本文選題：斯氏艾美耳球蟲 + 斯氏艾美耳球蟲病毒��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：兔球蟲病是由艾美耳屬的多種球蟲所引起的嚴(yán)重危害養(yǎng)兔業(yè)的一種寄生性原蟲病。在國(guó)內(nèi)目前已報(bào)道的17種兔球蟲中,斯氏艾美耳球蟲(Eimeria stiedai)的致病力最強(qiáng)且危害最大,寄生于兔膽管上皮細(xì)胞,可引起嚴(yán)重的肝球蟲病。當(dāng)前對(duì)于兔球蟲病的控制主要是依靠化學(xué)藥物,然而隨著球蟲耐藥性和藥物殘留等問(wèn)題的出現(xiàn),使抗球蟲藥物難以從根本上解決球蟲的威脅。究其原因是缺乏對(duì)兔球蟲細(xì)胞和分子生物學(xué)特性的了解。斯氏艾美耳球蟲病毒的發(fā)現(xiàn)為兔球蟲分子生物學(xué)特性的研究提供了新的方向。原蟲病毒是ds RNA病毒(Double-stranded RNA viruses),屬于全病毒科,在許多寄生性原蟲內(nèi)發(fā)現(xiàn)了病毒的存在,例如,賈第蟲、陰道毛滴蟲、利什曼原蟲及艾美耳屬球蟲。研究表明,利什曼原蟲病毒可以通過(guò)激活宿主的TRL3信號(hào)通路,促進(jìn)利什曼原蟲的持久性感染。另外,還有一些研究表明,ds RNA病毒能夠增加原蟲的致病性。此外,病毒RNA介導(dǎo)的轉(zhuǎn)染載體已經(jīng)在藍(lán)氏賈第蟲中實(shí)現(xiàn)了基因表達(dá)調(diào)控和蛋白功能的研究。雖然,病毒樣粒子已在斯氏艾美耳球蟲中發(fā)現(xiàn),但尚無(wú)關(guān)于斯氏艾美耳球蟲病毒的全基因組序列和分類的報(bào)道。本研究首次對(duì)斯氏艾美耳球蟲病毒基因組序列進(jìn)行了克隆、測(cè)序及分析,并構(gòu)建了斯氏艾美耳球蟲病毒轉(zhuǎn)染載體。本研究為了解兔球蟲的分子生物學(xué)特性提供了新思路。斯氏艾美耳球蟲病毒全長(zhǎng)c DNA的克隆及序列分析:根據(jù)已知病毒的保守序列設(shè)計(jì)簡(jiǎn)并引物,通過(guò)RT-PCR及SMART RACE技術(shù)成功獲得了斯氏艾美耳球蟲病毒基因組的全長(zhǎng)序列。測(cè)序結(jié)果顯示,病毒基因組全長(zhǎng)為6219 bp,包含兩個(gè)開放閱讀框,且在兩個(gè)開放閱讀框的交界處存在四個(gè)堿基的轉(zhuǎn)換片段(AUGA)。ORF1長(zhǎng)為2400 bp,編碼病毒衣殼蛋白(799個(gè)氨基酸);ORF2長(zhǎng)為3303 bp,編碼病毒RNA依賴的RNA聚合酶(1100個(gè)氨基酸)。BLASTp分析發(fā)現(xiàn)斯氏艾美耳球蟲病毒與E.tenella RNA virus 1(Et RV1)的氨基酸序列同源性最高。斯氏艾美耳球蟲病毒基因組具有全病毒科的典型特征,可認(rèn)定為全病毒科的一個(gè)新種。斯氏艾美耳球蟲病毒序列的生物信息學(xué)分析:將獲得的Es RV基因組序列和Totiviridae病毒科的病毒序列進(jìn)行多重序列比對(duì)后,構(gòu)建進(jìn)化樹。進(jìn)化樹分析顯示,Es RV、Et RV1及Eb RV1形成一個(gè)獨(dú)立的分支,而與其它的原蟲病毒距離較遠(yuǎn),因此應(yīng)將Eimeriaviruses劃分為Totiviridae家族的一個(gè)新屬。密碼子偏好性分析表明真菌病毒比原蟲病毒具有更高的宿主適應(yīng)性,而原蟲病毒中的GLV和Et RV1的宿主適應(yīng)性遠(yuǎn)超其它原蟲病毒。通過(guò)Cp G島和啟動(dòng)子區(qū)域預(yù)測(cè),顯示在coat和Rd Rp編碼框上游具有獨(dú)立啟動(dòng)子。Rd Rp蛋白結(jié)構(gòu)預(yù)測(cè)顯示,具有palm,fingers和thumb結(jié)構(gòu)域。斯氏艾美耳球蟲不同發(fā)育階段病毒蛋白的表達(dá)研究:從感染斯氏艾美耳球蟲不同時(shí)期的兔肝臟中收集斯氏艾美耳球蟲裂殖子、配子體及未孢子化卵囊,體外進(jìn)行孢子化孵育后,收集斯氏艾美耳球蟲孢子化卵囊,分別提取m RNA和蛋白樣品。對(duì)提取的樣品進(jìn)行RT-PCR和Western blot分析。結(jié)果顯示,Rd Rp蛋白在斯氏艾美耳球蟲的四個(gè)不同發(fā)育階段均可以表達(dá);而coat蛋白僅在斯氏艾美耳球蟲卵囊形成及孢子化階段的蟲體內(nèi)表達(dá)。斯氏艾美耳球蟲病毒載體的構(gòu)建:以斯氏艾美耳球蟲病毒基因組序列為基礎(chǔ),參照相關(guān)病毒載體的構(gòu)建策略,將綠色熒光蛋白(GFP)作為報(bào)告基因替換斯氏艾美耳球蟲病毒部分序列,構(gòu)建斯氏艾美耳球蟲病毒載體。通過(guò)電穿孔技術(shù)將構(gòu)建的斯氏艾美耳球蟲病毒載體轉(zhuǎn)入未孢子化的斯氏艾美耳球蟲卵囊內(nèi)。通過(guò)激光共聚焦掃描、流式細(xì)胞術(shù)和Western blot檢測(cè),研究表明報(bào)告基因GFP在斯氏艾美耳球蟲傳代的第二代和第三代蟲體中均得到表達(dá)。綜上所述,本研究對(duì)了解斯氏艾美耳球蟲病毒的基礎(chǔ)特征及研發(fā)斯氏艾美耳球蟲的基因操作工具,具有重要的意義,同時(shí)斯氏艾美耳球蟲病毒載體的構(gòu)建為研究球蟲細(xì)胞和分子生物學(xué)特性奠定了重要基礎(chǔ)。
[Abstract]:Rabbit coccidiosis is a kind of parasitic protozoon, which is caused by various coccidiosis of Eimeria. In the 17 kinds of rabbit coccidiosis, Eimeria stiedai has the strongest pathogenicity and the greatest harm. It is parasitic on the rabbit bile duct epithelial cells, which can cause serious liver coccidiosis. The control of rabbit coccidiosis mainly depends on chemical drugs. However, with the emergence of the drug resistance and drug residues, the anti coccidian drugs can not solve the threat of coccidiosis fundamentally. The reason is the lack of understanding of the cell and molecular biological characteristics of rabbit coccidiosis. The discovery of Eimeria serieriba virus is the molecular birth of rabbit coccidiosis. The study of physical properties provides a new direction. The protozoa virus is the DS RNA virus (Double-stranded RNA viruses), belonging to the whole virus family, and the presence of the virus in many parasitic protozoa, such as Giardia, Trichomonas vaginalis, Leishmania and Eimeria. Studies have shown that the Leishmania virus can be activated by the host The TRL3 signaling pathway promotes the persistent infection of Leishmania. In addition, some studies have shown that the DS RNA virus can increase the pathogenicity of the protozoa. In addition, the viral RNA mediated transfection vector has already realized gene expression regulation and protein function in Giardia len. Although the virus like particles have been in the eimeriella sp. The whole genome sequence and classification of Eimeria seriba virus were not reported. The genome sequence of Eimeria serieri virus was cloned, sequenced and analyzed for the first time, and the vector of Eimeria serieri virus transfection was constructed for the first time. This study was provided to solve the molecular biological characteristics of the rabbit coccidiosis. New ideas. Cloning and sequence analysis of the full length C DNA of Eimeria seriba virus: a full-length sequence of the genome of Eimeria serieri virus was successfully obtained by RT-PCR and SMART RACE techniques based on the conserved sequence of known viruses. The total length of the virus genome was 6219 BP, including two open reading. Frame, and there are four base conversion fragments (AUGA).ORF1 long 2400 BP at the junction of two open reading frames, encoding viral capsid protein (799 amino acids), ORF2 long 3303 BP, and RNA polymerase (1100 amino acid).BLASTp analysis of RNA dependent RNA dependent RNA polymerase (1100 amino acid).BLASTp analysis to find ammonia of the E.tenella RNA virus 1 (Et) The homology of the base acid sequence is the highest. The Eimeria serieri virus genome has the typical characteristics of the whole family. It can be identified as a new species of the family. The bioinformatics analysis of the sequence of Eimeria serieri virus: the multiple sequence alignment of the Es RV genome and the virus sequence of the Totiviridae virus family. Phylogenetic tree. Phylogenetic tree analysis showed that Es RV, Et RV1 and Eb RV1 formed an independent branch and were far away from other protozoans, so Eimeriaviruses should be divided into a new genus of Totiviridae family. Codon preference analysis showed that fungal viruses have higher host adaptability than protozoon, and protozoan viruses are more adaptable than protozoans. The host adaptability of the GLV and Et RV1 is far beyond the other protozoans. Through the prediction of the Cp G island and the promoter region, the prediction of the.Rd Rp protein structure of the independent promoter in the upstream of the coat and Rd Rp coding frames shows that the expression of the viral proteins in the different developmental stages of Eimeria serieri has been studied. M RNA and protein samples were collected from Eimeria's livers of Eimeria swell, gametophyte and oocyst in vitro, and m RNA and protein samples were extracted respectively. The results showed that the Rd Rp protein was found in the extracted samples by RT-PCR and Western. Four different developmental stages of Eimeria serieri can be expressed, while coat protein is expressed only in the oocyst and sporulation stage of Eimeria esimeria. The construction of Eimeria serieri virus vector is based on the genome sequence of Eimeria esimeria virus and the construction strategy of related virus vectors, will be green. Color fluorescent protein (GFP) is used as a reporter to replace partial sequence of Eimeria serieri virus and construct Eimeria seriba virus vector. Through electroporation, the vector of Eimeria serieri virus is transferred into the oocyst of Eimeria serieri without sporulation. Laser confocal scanning, flow cytometry and Western blo T detection shows that the reporter gene GFP is expressed in the second and third generation of Eimeria serieri. In summary, this study is important for understanding the basic characteristics of Eimeria swell virus and the research and development of the gene operation tool of Eimeria seriba. The construction of the body laid an important foundation for studying the cell and molecular biological characteristics of coccidia.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.7

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