贾第虫TERT对保守假定蛋白CHP的钓取及互作鉴定

发布时间：2018-04-21 23:36

本文选题：贾第虫TERT + 保守假定蛋白　；参考：《东北农业大学》2017年硕士论文

【摘要】：十二指肠贾第鞭毛虫(Giardia duodenalis),简称贾第虫,是一种结构简单的真核模式生物,被世界卫生组织列为重要的人兽共患寄生虫。该寄生虫流行区域广泛,寄生宿主种类多样,近年来对公共卫生构成了严重威胁。贾第虫病在世界范围的不定期流行或爆发,给许多发展中和发达国家带来了极大的经济损失。贾第虫生活史虽然简单,只包含滋养体和包囊两个阶段,然而有关贾第虫分子生物学特征、细胞调控机制和致病机理的研究都还处于探索阶段。此外,进行贾第虫蛋白质组学研究,寻找新的药物靶点蛋白是当前贾第虫病防控研究待解决的重要科学问题之一。端粒(Telomere)是位于真核生物染色体末端的一小段核酸重复序列,其复制的过程由于端粒酶的插入而终止,从而导致细胞自身的程序性死亡。在众多影响端粒机制的结构中,最为关键和重要的是端粒酶,因此对其相关蛋白的研究是解释端粒对细胞作用机制的重要内容。近年来,有关寄生虫端粒的研究正不断兴起,目前已报到过利氏曼原虫中端粒酶相关蛋白对其生长和抗氧化应激的研究、布氏锥虫端粒同源物与C/D sno RNA家族和甲基转移酶的结合研究等。贾第虫为最古老的真核生物之一,对其端粒相关机制的研究尤为关键,目前关于贾第虫的端粒抑制剂四聚体链、端粒酶相关蛋白二硫键异构酶和复制因子亚基已有过相关研究。为更深入研究影响端粒酶的相关蛋白,本试验以贾第虫滋养体为研究对象,利用酵母双杂交(Yeast two hybrid,YTH)Gal 4系统针对贾第虫端粒酶逆转录酶(telomerase reverse transcriptase,TERT)中RNA结合结构域互作的蛋白质进行了筛选,使用GST pull-down技术和免疫共沉淀(Co-immunoprecipitation,Co-IP)技术对筛选的结果进行了验证。采用Trizol法提取的贾第虫滋养体阶段总RNA,经反转录后得到cDNA。并以此为模板构建了诱饵质粒p GBKT7-Gl_TRBD,转染Y187酵母菌后形成的诱饵蛋白经检测未发生毒性作用和自激活现象。利用已构建的贾第虫cDNA文库进行贾第虫TERT互作蛋白质的钓取,通过酵母双杂交Gal 4系统最终筛选出两种与TERT互作的蛋白质,经BLAST比对分别确认为CHP(登录号为XM_001707200.1,全长846 bp)和VSP(登录号为XM_001705026.1,全长2280 bp)。由于VSP蛋白的高变性和不稳定性,本研究选取CHP蛋白进行GST pull-down和免疫共沉淀试验并验证其相互作用。根据蛋白的疏水性分析,使用293T细胞真核表达Gl_TRBD基因,使用p GEX4T-1载体原核表达筛选的CHP蛋白。通过谷胱甘肽亲和树脂与诱饵蛋白TRBD结合,成功捕获了目的蛋白CHP,使用Western Blot技术验证了CHP与TERT间的相互作用。通过两种蛋白在293细胞中共转染,并分别以各自的标签抗体进行抗原抗体特异性检测,使用Western Blot技术验证了两蛋白在细胞内依然可以产生相互作用。本论文得出的结果为研究贾第虫CHP蛋白功能及端粒酶相关蛋白的调节机制提供参考依据,为研究贾第虫乃至整个真核生物的端粒、端粒酶及其相关蛋白的作用原理;寻找防治贾第虫病的药物新靶点提供试验依据,为最终推动关于细胞衰老、死亡、癌变等相关机制的深入研究奠定基础。
[Abstract]:Giardia duodenum (Giardia duodenalis), abbreviated as Giardia, is a simple eukaryotic model organism, which is listed as an important zoonotic parasite by the WHO. The parasite has a wide range of epidemic areas and a variety of parasitic hosts. It has been a serious threat to public health in recent years. Irregular epidemic or outbreak has brought great economic losses to many developing and developed countries. Although the life history of Giardia is simple, it contains only two stages of trophoblast and cyst. However, the study of the molecular biological characteristics of Giardia, the mechanism of cell regulation and the pathogenesis of Giardia is still at the exploratory stage. The search for new drug target proteins is one of the important scientific problems to be solved in the prevention and control of giardiasis. The telomere (Telomere) is a small sequence of nucleic acid repeats at the end of eukaryotic chromosomes. The process of its replication is terminated by the insertion of telomerase, resulting in the programmed cell death of the cells. Telomerase is the most important and important factor affecting telomere mechanisms. Therefore, the study of its related proteins is an important part of the mechanism of telomere action. In recent years, the study of telomere has been rising. At present, it is reported that telomerase related proteins in the parasite of leielhitin have been responsible for its growth and antioxidation. Exciting studies, the binding of telomere homologs of Trypanosoma brucellus to the C/D SnO RNA family and methyltransferase. Giardia is one of the oldest eukaryotes. It is particularly critical for its telomere related mechanisms. At present, the telomere inhibitor four polymer chain, telomerase related protein two sulphur bond isomerase and replicator subunits of Giardia are present. In order to further study the related proteins related to telomerase, this experiment uses the Giardia trophoblastic as the research object and uses yeast two hybrid (Yeast two hybrid, YTH) Gal 4 system to sieve the protein of RNA binding domain of Giardia telomerase reverse transcriptase (telomerase reverse transcriptase, TERT). GST pull-down technology and Co-immunoprecipitation (Co-IP) technology were used to verify the results of the screening. The total RNA in the Giardia trophoblastic phase extracted by Trizol was obtained by reverse transcription, and cDNA. was obtained after reverse transcription, and the decoy plasmid P GBKT7-Gl_TRBD was constructed, and the bait protein formed after the transfection of Y187 yeast was used as a template. No toxicity and self activation were detected. Using the established Giardia cDNA library to catch the TERT interaction protein of Giardia, two kinds of proteins interacting with TERT were screened by yeast two hybrid Gal 4 system, and CHP (login number XM_001707200.1, total length 846 BP) and VSP (login number X) were identified by BLAST comparison. M_001705026.1, the total length of 2280 BP). Due to the high denaturation and instability of VSP protein, this study selected CHP protein to carry out GST pull-down and immunoprecipitation test and verify its interaction. According to the hydrophobicity analysis of the protein, the Gl_TRBD gene was expressed by 293T cells, and the CHP protein screened by the P GEX4T-1 carrier was used. Cystamine affinity resin combined with bait protein TRBD, successfully captured the target protein CHP. The interaction between CHP and TERT was verified by Western Blot. Two proteins were transfected in 293 cells, and the antigen antibody specificity was detected with their respective label antibodies, and the two protein was verified by Western Blot technique. The results of this paper provide a reference for the study of the function of Giardia CHP and the regulation mechanism of telomerase related proteins, and to study the telomere, telomerase and related proteins of Giardia and even the whole eukaryotes, and to find a new drug target for the prevention and control of giardiasis. The basis of this study will lay a foundation for further research on the mechanisms of cell senescence, death and carcinogenesis.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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