本文關鍵詞： 犬賈第蟲TERT cDNA文庫 互作蛋白 酵母雙雜交　出處：《吉林大學》2015年碩士論文　論文類型：學位論文

【摘要】：賈第鞭毛蟲（Giardia）簡稱賈第蟲，內膜系統(tǒng)非常簡單，只有內質網（ER）和次級囊泡（PVs），是一種原始的真核生物，堪稱模式生物。賈第蟲是一種呈世界性分布的人獸共患原蟲，寵物犬、警犬、伴侶動物等都增加了人與賈第蟲的接觸，犬賈第蟲(G.canis)對人的健康構成了嚴重的威脅，但是由于賈第蟲的分子生物學特性及蟲體細胞調控機制至今尚不清楚，迄今為止尚無防治賈第蟲病的理想辦法。因此研究賈第蟲細胞和分子生物學，揭示賈第蟲細胞分子生物學特性，尋找新的藥物作用靶位，已迫在眉睫。端粒酶及其相關蛋白的發(fā)現為深入研究細胞分子生物特性及藥物疫苗靶位等開辟了新的研究領域。 隨著端粒酶研究的深入，hGar1p、hNhp2、hNhp10、PinX1、Hsp90、Hsp23、KIP等端粒酶相關蛋白已被報道，這些蛋白與端粒酶活性密切相關。目前，端粒酶相關蛋白的研究主要集中在哺乳動物，寄生蟲端粒酶相關蛋白研究報道很少。本研究以模式生物賈第蟲為研究對象，通過酵母雙雜交系統(tǒng)篩選犬賈第蟲TERT保守結構域—TRBD互作蛋白，并通過pull-down和Co-IP試驗驗證。犬賈第蟲端粒酶的互作蛋白的獲得，有助于闡述賈第蟲生物學特性及蟲體細胞調控機制。賈第蟲作為良好的生物模型，其端粒酶互作蛋白的篩選及研究，對真核生物端粒酶、端粒酶逆轉錄酶及其互作蛋白的研究具有重要意義。 賈第蟲酵母雙雜交cDNA文庫的構建本研究用Trizol法手提犬賈第蟲滋養(yǎng)體總RNA，構建了酵母雙雜交cDNA文庫，文庫滴度為2.3×107cfu/ml，文庫庫容為5.5×109cfu，文庫插入片段主要分布在1-2kb，重組克隆陽性率≥85%，文庫質量較好，可用于酵母雙雜交文庫的篩選。 犬賈第蟲TERT互作蛋白的篩選構建誘餌質粒pGBK-GcTRBD，經自激活和毒性作用檢測，誘餌蛋白無自激活和毒性作用，然后進行文庫宿主菌和誘餌菌株的雜交培養(yǎng)，篩選TERT互作蛋白，獲得2個候選陽性菌，經測序比對，確定候選捕獲基因是復制因子亞基1和二硫鍵異構酶4。 蛋白相互作用的驗證原核表達His-PDI-4蛋白、真核表達GFP-TRBD蛋白，pull-down試驗驗證兩種蛋白相互作用關系，結果表明TRBD結構蛋白和PDI-4蛋白無直接相互作用；構建pGADT7-gPDI-4重組質粒，和誘餌質粒共轉化Y187酵母菌，，α-半乳糖苷酶試驗檢測兩種蛋白的相互作用關系，結果表明TRBD結構蛋白和PDI-4蛋白無直接相互作用。 利用原核表達的RFC1、真核表達的TRBD蛋白，進行pull-down試驗，結果表明TRBD結構蛋白與RFC1蛋白存在相互作用；構建真核表達質粒pEGFP-C1-GcTRBD、pcDNA3.1-His A-RFC1，共轉染293T細胞，Co-IP試驗結果表明TRBD結構蛋白與RFC1蛋白在真核細胞內存在相互作用。為下一步研究RFC1蛋白功能及端粒酶相關蛋白的調節(jié)機制奠定基礎。
[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

本文編號：1520599