斯氏艾美耳球虫dsRNA病毒全基因组序列测定及其转染载体的构建

发布时间：2018-05-06 15:45

本文选题：斯氏艾美耳球虫 + 斯氏艾美耳球虫病毒　；参考：《吉林大学》2016年博士论文

【摘要】：兔球虫病是由艾美耳属的多种球虫所引起的严重危害养兔业的一种寄生性原虫病。在国内目前已报道的17种兔球虫中,斯氏艾美耳球虫(Eimeria stiedai)的致病力最强且危害最大,寄生于兔胆管上皮细胞,可引起严重的肝球虫病。当前对于兔球虫病的控制主要是依靠化学药物,然而随着球虫耐药性和药物残留等问题的出现,使抗球虫药物难以从根本上解决球虫的威胁。究其原因是缺乏对兔球虫细胞和分子生物学特性的了解。斯氏艾美耳球虫病毒的发现为兔球虫分子生物学特性的研究提供了新的方向。原虫病毒是ds RNA病毒(Double-stranded RNA viruses),属于全病毒科,在许多寄生性原虫内发现了病毒的存在,例如,贾第虫、阴道毛滴虫、利什曼原虫及艾美耳属球虫。研究表明,利什曼原虫病毒可以通过激活宿主的TRL3信号通路,促进利什曼原虫的持久性感染。另外,还有一些研究表明,ds RNA病毒能够增加原虫的致病性。此外,病毒RNA介导的转染载体已经在蓝氏贾第虫中实现了基因表达调控和蛋白功能的研究。虽然,病毒样粒子已在斯氏艾美耳球虫中发现,但尚无关于斯氏艾美耳球虫病毒的全基因组序列和分类的报道。本研究首次对斯氏艾美耳球虫病毒基因组序列进行了克隆、测序及分析,并构建了斯氏艾美耳球虫病毒转染载体。本研究为了解兔球虫的分子生物学特性提供了新思路。斯氏艾美耳球虫病毒全长c DNA的克隆及序列分析:根据已知病毒的保守序列设计简并引物,通过RT-PCR及SMART RACE技术成功获得了斯氏艾美耳球虫病毒基因组的全长序列。测序结果显示,病毒基因组全长为6219 bp,包含两个开放阅读框,且在两个开放阅读框的交界处存在四个碱基的转换片段(AUGA)。ORF1长为2400 bp,编码病毒衣壳蛋白(799个氨基酸);ORF2长为3303 bp,编码病毒RNA依赖的RNA聚合酶(1100个氨基酸)。BLASTp分析发现斯氏艾美耳球虫病毒与E.tenella RNA virus 1(Et RV1)的氨基酸序列同源性最高。斯氏艾美耳球虫病毒基因组具有全病毒科的典型特征,可认定为全病毒科的一个新种。斯氏艾美耳球虫病毒序列的生物信息学分析:将获得的Es RV基因组序列和Totiviridae病毒科的病毒序列进行多重序列比对后,构建进化树。进化树分析显示,Es RV、Et RV1及Eb RV1形成一个独立的分支,而与其它的原虫病毒距离较远,因此应将Eimeriaviruses划分为Totiviridae家族的一个新属。密码子偏好性分析表明真菌病毒比原虫病毒具有更高的宿主适应性,而原虫病毒中的GLV和Et RV1的宿主适应性远超其它原虫病毒。通过Cp G岛和启动子区域预测,显示在coat和Rd Rp编码框上游具有独立启动子。Rd Rp蛋白结构预测显示,具有palm,fingers和thumb结构域。斯氏艾美耳球虫不同发育阶段病毒蛋白的表达研究:从感染斯氏艾美耳球虫不同时期的兔肝脏中收集斯氏艾美耳球虫裂殖子、配子体及未孢子化卵囊,体外进行孢子化孵育后,收集斯氏艾美耳球虫孢子化卵囊,分别提取m RNA和蛋白样品。对提取的样品进行RT-PCR和Western blot分析。结果显示,Rd Rp蛋白在斯氏艾美耳球虫的四个不同发育阶段均可以表达;而coat蛋白仅在斯氏艾美耳球虫卵囊形成及孢子化阶段的虫体内表达。斯氏艾美耳球虫病毒载体的构建:以斯氏艾美耳球虫病毒基因组序列为基础,参照相关病毒载体的构建策略,将绿色荧光蛋白(GFP)作为报告基因替换斯氏艾美耳球虫病毒部分序列,构建斯氏艾美耳球虫病毒载体。通过电穿孔技术将构建的斯氏艾美耳球虫病毒载体转入未孢子化的斯氏艾美耳球虫卵囊内。通过激光共聚焦扫描、流式细胞术和Western blot检测,研究表明报告基因GFP在斯氏艾美耳球虫传代的第二代和第三代虫体中均得到表达。综上所述,本研究对了解斯氏艾美耳球虫病毒的基础特征及研发斯氏艾美耳球虫的基因操作工具,具有重要的意义,同时斯氏艾美耳球虫病毒载体的构建为研究球虫细胞和分子生物学特性奠定了重要基础。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.7

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