【摘要】：環(huán)形泰勒蟲是一種蜱傳血液原蟲,能引起牛的熱帶泰勒蟲病。環(huán)形泰勒蟲和小泰勒蟲裂殖體感染的牛淋巴細(xì)胞在體外表現(xiàn)具有無(wú)限增值(永生化)的能力,即其具有轉(zhuǎn)化宿主淋巴細(xì)胞的特性,因此被稱為可轉(zhuǎn)化淋巴細(xì)胞泰勒蟲。泰勒屬的寄生蟲是截止目前已發(fā)現(xiàn)的可使哺乳動(dòng)物細(xì)胞發(fā)生永生化的唯一一類真核生物,為寄生蟲與宿主相互作用研究提供了理想模型。截至目前,人們已經(jīng)揭示了可轉(zhuǎn)化淋巴細(xì)胞泰勒蟲參與調(diào)控宿主細(xì)胞的部分機(jī)制,涉及到的分子有Tash AT(Theileria annulata macroschizont-specific AT hook-containing protein)、SVSP(subtelomere-encodedvariable secreted protein)等,并證明與NF-κB、ISG15、TGF-β、PI-3K、E2F等分子及凋亡調(diào)控路徑有關(guān)。目前,人們已經(jīng)證明了小泰勒蟲Tp SCOP(T.parva schizont-derived cytoskeleton-bindingprotein)蛋白能夠激活宿主細(xì)胞的NF-κB通路,進(jìn)而實(shí)現(xiàn)對(duì)宿主細(xì)胞的調(diào)控。由于環(huán)形泰勒蟲Ta SDP(T.annulata schizont-derived protein)蛋白與已知的的小泰勒蟲Tp SCOP蛋白為同源蛋白。本研究對(duì)Ta SDP基因進(jìn)行了原核表達(dá),利用了原核表達(dá)獲得的重組蛋白制備了兔源多克隆抗體并對(duì)該蛋白在環(huán)形泰勒蟲感染細(xì)胞上進(jìn)行了定位,同時(shí)運(yùn)用雙熒光素酶報(bào)告基因系統(tǒng)就該蛋白對(duì)HEK293T細(xì)胞NF-κB信號(hào)通路的影響做了初步的探索,確定了電穿孔轉(zhuǎn)染環(huán)形泰勒蟲感染細(xì)胞的最佳電轉(zhuǎn)緩沖液和電壓,為研究環(huán)形泰勒蟲Ta SDP蛋白在轉(zhuǎn)化的宿主細(xì)胞中的作用奠定基礎(chǔ)。主要研究結(jié)果如下:1.Ta SDP基因的表達(dá)及Ta SDP蛋白多克隆抗體的制備。利用PCR技術(shù)從環(huán)形泰勒蟲c DNA中擴(kuò)增Ta SDP基因,構(gòu)建原核表達(dá)載體進(jìn)行蛋白的表達(dá),r Ta SDP蛋白以包涵體的形式存在;將純化后的融合蛋白免疫家兔,制備多克隆抗體。制備的抗體不僅可與重組表達(dá)的His-Ta SDP發(fā)生反應(yīng),而且可以識(shí)別天然的蟲體蛋白,為開展其在細(xì)胞調(diào)控方面的研究準(zhǔn)備了材料。2.Ta SDP蛋白在環(huán)形泰勒蟲感染細(xì)胞中的定位。為探尋Ta SDP蛋白在環(huán)形泰勒蟲感染細(xì)胞內(nèi)的位置,以純化的抗r Ta SDP蛋白免疫血清為一抗,運(yùn)用間接免疫熒光實(shí)驗(yàn),并借助于激光共聚焦顯微鏡對(duì)該蛋白進(jìn)行細(xì)胞定位。結(jié)果表明,Ta SDP蛋白定位于環(huán)形泰勒蟲裂殖體上。該蛋白可以作為一個(gè)標(biāo)記分子用于后續(xù)裂殖體相關(guān)的研究。3.Ta SDP蛋白對(duì)HEK293T細(xì)胞內(nèi)NF-κB轉(zhuǎn)錄活性的影響。為確定環(huán)形泰勒蟲Ta SDP蛋白對(duì)HEK293T細(xì)胞內(nèi)NF-κB轉(zhuǎn)錄活性的影響,我們運(yùn)用了雙熒光素酶報(bào)告基因系統(tǒng)。結(jié)果表明:Ta SDP蛋白對(duì)HEK293T細(xì)胞內(nèi)NF-κB通路具有顯著激活作用,活性升高大約1.7倍。實(shí)驗(yàn)中對(duì)環(huán)形泰勒蟲感染細(xì)胞的C5細(xì)胞系的電轉(zhuǎn)染條件進(jìn)行了篩選,確定了最佳的電轉(zhuǎn)緩沖液及相應(yīng)的最適電壓。為將雙熒光素酶報(bào)告基因系統(tǒng)用于環(huán)形泰勒蟲感染細(xì)胞的研究奠定了基礎(chǔ)。
[Abstract]:Taylor ring is a tick-borne blood protozoa that causes tropical Taylor's disease in cattle. The bovine lymphocytes infected by Taylor's ring and small Taylor's lobes have the ability of infinite increment (immortalization) in vitro, that is, they have the characteristics of transforming host lymphocytes, so they are called transforming lymphocyte Taylor's. The parasites of the genus Taylor are the only eukaryotes found to immortalize mammalian cells, which provide an ideal model for the study of the interaction between parasites and hosts. Up to now, it has been revealed that Taylor's can participate in the regulation of host cells, involving Tash AT (Theileria annulata macroschizont-specific AT hook-containing protein), SVSP (subtelomere-encodedvariable secreted protein) and other molecules, and NF- 魏 B, ISG15 TGF- 尾, PI-3K,E2F and other molecules and apoptosis regulation pathway related to the role of TGF- 尾, PI-3K,E2F and so on. At present, it has been proved that the Tp SCOP (T.parva schizont-derived cytoskeleton-bindingprotein) protein can activate the NF- 魏 B pathway of host cells and thus regulate the host cells. Because the Ta SDP (T.annulata schizont-derived protein) protein is homologous to the known Tp SCOP protein. In this study, Ta SDP gene was expressed in prokaryotic cells and rabbit polyclonal antibody was prepared by using the recombinant protein expressed in prokaryotic expression. The effects of the protein on the NF- 魏 B signaling pathway in HEK293T cells were studied by using double luciferase reporter gene system. The optimal electroporation buffer and voltage were determined. The results provide a basis for the study of the role of Ta SDP protein in transformed host cells. The main results are as follows: expression of 1.Ta SDP gene and preparation of polyclonal antibody against Ta SDP protein. The Ta SDP gene was amplified from c DNA of Taylor's ring by PCR technique, and the prokaryotic expression vector was constructed to express, r Ta SDP protein in the form of inclusion body. The purified fusion protein was immunized with rabbit to prepare polyclonal antibody. The prepared antibody can not only react with recombinant His-Ta SDP, but also recognize natural insect proteins, which provides a material for the study of cell regulation. The localization of 2.Ta SDP protein in the cells infected with Taylor's ring. In order to find out the position of Ta SDP protein in the cells infected with Taylor's ring, the purified anti-r Ta SDP protein immunized serum was used as the first antibody. The cell localization of the protein was carried out by means of indirect immunofluorescence assay and laser confocal microscopy. The results showed that the, Ta SDP protein was located on the merozoite of Taylor's ring. This protein can be used as a marker molecule for further studies on splitosome. The effect of 3.Ta SDP protein on the transcriptional activity of NF- 魏 B in HEK293T cells. In order to determine the effect of Ta SDP on the transcriptional activity of NF- 魏 B in HEK293T cells, a double luciferase reporter gene system was used. The results showed that: Ta SDP protein significantly activated the NF- 魏 B pathway in HEK293T cells, and the activity increased about 1.7 times. In the experiment, the electrotransfection conditions of C5 cell line infected by Taylor's ring were screened, and the optimal electrotransposable buffer and the corresponding optimum voltage were determined. The results laid a foundation for the application of double luciferase reporter gene system in the study of Taylor ring infection cells.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7

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