犬贾第虫TERT互作蛋白的筛选及验证

发布时间：2018-02-21 01:25

  本文关键词： 犬贾第虫TERT cDNA文库 互作蛋白 酵母双杂交　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：贾第鞭毛虫（Giardia）简称贾第虫，内膜系统非常简单，只有内质网（ER）和次级囊泡（PVs），是一种原始的真核生物，堪称模式生物。贾第虫是一种呈世界性分布的人兽共患原虫，宠物犬、警犬、伴侣动物等都增加了人与贾第虫的接触，犬贾第虫(G.canis)对人的健康构成了严重的威胁，但是由于贾第虫的分子生物学特性及虫体细胞调控机制至今尚不清楚，迄今为止尚无防治贾第虫病的理想办法。因此研究贾第虫细胞和分子生物学，揭示贾第虫细胞分子生物学特性，寻找新的药物作用靶位，已迫在眉睫。端粒酶及其相关蛋白的发现为深入研究细胞分子生物特性及药物疫苗靶位等开辟了新的研究领域。 随着端粒酶研究的深入，hGar1p、hNhp2、hNhp10、PinX1、Hsp90、Hsp23、KIP等端粒酶相关蛋白已被报道，这些蛋白与端粒酶活性密切相关。目前，端粒酶相关蛋白的研究主要集中在哺乳动物，寄生虫端粒酶相关蛋白研究报道很少。本研究以模式生物贾第虫为研究对象，通过酵母双杂交系统筛选犬贾第虫TERT保守结构域—TRBD互作蛋白，并通过pull-down和Co-IP试验验证。犬贾第虫端粒酶的互作蛋白的获得，有助于阐述贾第虫生物学特性及虫体细胞调控机制。贾第虫作为良好的生物模型，其端粒酶互作蛋白的筛选及研究，对真核生物端粒酶、端粒酶逆转录酶及其互作蛋白的研究具有重要意义。 贾第虫酵母双杂交cDNA文库的构建本研究用Trizol法手提犬贾第虫滋养体总RNA，构建了酵母双杂交cDNA文库，文库滴度为2.3×107cfu/ml，文库库容为5.5×109cfu，文库插入片段主要分布在1-2kb，重组克隆阳性率≥85%，文库质量较好，可用于酵母双杂交文库的筛选。 犬贾第虫TERT互作蛋白的筛选构建诱饵质粒pGBK-GcTRBD，经自激活和毒性作用检测，诱饵蛋白无自激活和毒性作用，然后进行文库宿主菌和诱饵菌株的杂交培养，筛选TERT互作蛋白，获得2个候选阳性菌，经测序比对，确定候选捕获基因是复制因子亚基1和二硫键异构酶4。 蛋白相互作用的验证原核表达His-PDI-4蛋白、真核表达GFP-TRBD蛋白，pull-down试验验证两种蛋白相互作用关系，结果表明TRBD结构蛋白和PDI-4蛋白无直接相互作用；构建pGADT7-gPDI-4重组质粒，和诱饵质粒共转化Y187酵母菌，，α-半乳糖苷酶试验检测两种蛋白的相互作用关系，结果表明TRBD结构蛋白和PDI-4蛋白无直接相互作用。 利用原核表达的RFC1、真核表达的TRBD蛋白，进行pull-down试验，结果表明TRBD结构蛋白与RFC1蛋白存在相互作用；构建真核表达质粒pEGFP-C1-GcTRBD、pcDNA3.1-His A-RFC1，共转染293T细胞，Co-IP试验结果表明TRBD结构蛋白与RFC1蛋白在真核细胞内存在相互作用。为下一步研究RFC1蛋白功能及端粒酶相关蛋白的调节机制奠定基础。
[Abstract]:Giardia (Giardia) is a primitive eukaryote, called Giardia, with a very simple intimal system, only the endoplasmic reticulum (ERR) and the secondary vesicle (PVS). Giardia is a worldwide zoonotic protozoa, a pet dog. Police dogs and companion animals have increased the contact between people and giardia. G. canis poses a serious threat to human health. However, the molecular biological characteristics of Giardia lamblia and its somatic and cellular regulation mechanisms are still unclear. So far, there is no ideal method to prevent and cure giardiosis. Therefore, we study the cell and molecular biology of Giardia, reveal the molecular biological characteristics of giardia cells, and find new targets for drug action. The discovery of telomerase and its related proteins has opened up a new research field for the further study of cell molecular biological properties and drug vaccine targets. With the development of telomerase research, telomerase related proteins such as hGar1pHP2hNhp10 PinX1 Hsp90 Hsp23KIP have been reported, and these proteins are closely related to telomerase activity. At present, telomerase related proteins are mainly studied in mammals. There are few studies on telomerase associated proteins of Giardia canis. In this study, TRBD interaction proteins of TERT conserved domain of Giardia canis were screened by yeast two-hybrid system. The results of pull-down and Co-IP tests showed that the acquisition of telomerase interaction proteins of giardia canis was helpful to explain the biological characteristics and somatic regulation mechanism of Giardia canis, which was a good biological model. The screening and study of telomerase interaction proteins are of great significance for the study of telomerase, telomerase reverse transcriptase and their interaction proteins in eukaryotic organisms. Construction of a two-hybrid cDNA library of Giardia japonica in this study the cDNA library of yeast two-hybrid was constructed by using Trizol method in the trophoblast of Giardia canis. The titer of library was 2.3 脳 107cfurml.The library capacity was 5.5 脳 109cfu. the inserted fragment was mainly distributed in 1-2kb, the positive rate of recombinant clone was 鈮

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