本文選題：賈第蟲TERT + 保守假定蛋白��； 參考：《東北農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：十二指腸賈第鞭毛蟲(Giardia duodenalis),簡稱賈第蟲,是一種結(jié)構(gòu)簡單的真核模式生物,被世界衛(wèi)生組織列為重要的人獸共患寄生蟲。該寄生蟲流行區(qū)域廣泛,寄生宿主種類多樣,近年來對公共衛(wèi)生構(gòu)成了嚴(yán)重威脅。賈第蟲病在世界范圍的不定期流行或爆發(fā),給許多發(fā)展中和發(fā)達(dá)國家?guī)砹藰O大的經(jīng)濟(jì)損失。賈第蟲生活史雖然簡單,只包含滋養(yǎng)體和包囊兩個階段,然而有關(guān)賈第蟲分子生物學(xué)特征、細(xì)胞調(diào)控機(jī)制和致病機(jī)理的研究都還處于探索階段。此外,進(jìn)行賈第蟲蛋白質(zhì)組學(xué)研究,尋找新的藥物靶點蛋白是當(dāng)前賈第蟲病防控研究待解決的重要科學(xué)問題之一。端粒(Telomere)是位于真核生物染色體末端的一小段核酸重復(fù)序列,其復(fù)制的過程由于端粒酶的插入而終止,從而導(dǎo)致細(xì)胞自身的程序性死亡。在眾多影響端粒機(jī)制的結(jié)構(gòu)中,最為關(guān)鍵和重要的是端粒酶,因此對其相關(guān)蛋白的研究是解釋端粒對細(xì)胞作用機(jī)制的重要內(nèi)容。近年來,有關(guān)寄生蟲端粒的研究正不斷興起,目前已報到過利氏曼原蟲中端粒酶相關(guān)蛋白對其生長和抗氧化應(yīng)激的研究、布氏錐蟲端粒同源物與C/D sno RNA家族和甲基轉(zhuǎn)移酶的結(jié)合研究等。賈第蟲為最古老的真核生物之一,對其端粒相關(guān)機(jī)制的研究尤為關(guān)鍵,目前關(guān)于賈第蟲的端粒抑制劑四聚體鏈、端粒酶相關(guān)蛋白二硫鍵異構(gòu)酶和復(fù)制因子亞基已有過相關(guān)研究。為更深入研究影響端粒酶的相關(guān)蛋白,本試驗以賈第蟲滋養(yǎng)體為研究對象,利用酵母雙雜交(Yeast two hybrid,YTH)Gal 4系統(tǒng)針對賈第蟲端粒酶逆轉(zhuǎn)錄酶(telomerase reverse transcriptase,TERT)中RNA結(jié)合結(jié)構(gòu)域互作的蛋白質(zhì)進(jìn)行了篩選,使用GST pull-down技術(shù)和免疫共沉淀(Co-immunoprecipitation,Co-IP)技術(shù)對篩選的結(jié)果進(jìn)行了驗證。采用Trizol法提取的賈第蟲滋養(yǎng)體階段總RNA,經(jīng)反轉(zhuǎn)錄后得到cDNA。并以此為模板構(gòu)建了誘餌質(zhì)粒p GBKT7-Gl_TRBD,轉(zhuǎn)染Y187酵母菌后形成的誘餌蛋白經(jīng)檢測未發(fā)生毒性作用和自激活現(xiàn)象。利用已構(gòu)建的賈第蟲cDNA文庫進(jìn)行賈第蟲TERT互作蛋白質(zhì)的釣取,通過酵母雙雜交Gal 4系統(tǒng)最終篩選出兩種與TERT互作的蛋白質(zhì),經(jīng)BLAST比對分別確認(rèn)為CHP(登錄號為XM_001707200.1,全長846 bp)和VSP(登錄號為XM_001705026.1,全長2280 bp)。由于VSP蛋白的高變性和不穩(wěn)定性,本研究選取CHP蛋白進(jìn)行GST pull-down和免疫共沉淀試驗并驗證其相互作用。根據(jù)蛋白的疏水性分析,使用293T細(xì)胞真核表達(dá)Gl_TRBD基因,使用p GEX4T-1載體原核表達(dá)篩選的CHP蛋白。通過谷胱甘肽親和樹脂與誘餌蛋白TRBD結(jié)合,成功捕獲了目的蛋白CHP,使用Western Blot技術(shù)驗證了CHP與TERT間的相互作用。通過兩種蛋白在293細(xì)胞中共轉(zhuǎn)染,并分別以各自的標(biāo)簽抗體進(jìn)行抗原抗體特異性檢測,使用Western Blot技術(shù)驗證了兩蛋白在細(xì)胞內(nèi)依然可以產(chǎn)生相互作用。本論文得出的結(jié)果為研究賈第蟲CHP蛋白功能及端粒酶相關(guān)蛋白的調(diào)節(jié)機(jī)制提供參考依據(jù),為研究賈第蟲乃至整個真核生物的端粒、端粒酶及其相關(guān)蛋白的作用原理;尋找防治賈第蟲病的藥物新靶點提供試驗依據(jù),為最終推動關(guān)于細(xì)胞衰老、死亡、癌變等相關(guān)機(jī)制的深入研究奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.7

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