【摘要】：研究背景及目的： 瘧疾仍是嚴(yán)重危害人類(lèi)健康的‘超級(jí)殺手’,這種傳染病每年奪去大約100萬(wàn)人的性命,導(dǎo)致4億人感染。在熱帶和亞熱帶地區(qū),瘧疾是常見(jiàn)疾病也是嚴(yán)重的公共衛(wèi)生問(wèn)題,統(tǒng)計(jì)數(shù)字顯示,每年全球有幾億人感染瘧疾,雖然大多數(shù)患者能夠治愈,可是仍有一百萬(wàn)人死亡,其中以?xún)和剂硕鄶?shù)。世界衛(wèi)生組織對(duì)瘧疾給予密切的關(guān)注,遏制瘧疾已成為全球奮斗的目標(biāo)。我國(guó)也提出到2015年大部分地區(qū)消除瘧疾,到2020年全國(guó)實(shí)現(xiàn)消除瘧疾的目標(biāo)[3]。有效的藥物是治療瘧疾的主要方法。然而,瘧原蟲(chóng)對(duì)抗瘧藥耐藥性和蚊蟲(chóng)對(duì)殺蟲(chóng)劑的抗藥性已成為實(shí)現(xiàn)全球瘧疾控制所面臨的最主要威脅。以青蒿素為基礎(chǔ)的聯(lián)合療法(ACT)是目前治療惡性瘧疾這一最致命的疾病形式最強(qiáng)有力的武器,其療效能達(dá)到90%以上,但在柬埔寨和泰國(guó)邊境地區(qū)近期已確認(rèn)出現(xiàn)青蒿素耐藥性。鑒于目前醫(yī)學(xué)界使用的所有治療瘧疾藥物都出現(xiàn)耐藥性現(xiàn)象,因此開(kāi)發(fā)抗瘧疾新藥不但至關(guān)重要而且面臨著急迫性。 尋找有效的藥物作用靶標(biāo)是研制抗瘧新藥的基礎(chǔ)。寄生蟲(chóng)為了適應(yīng)不同的外界環(huán)境和宿主環(huán)境,在長(zhǎng)期進(jìn)化過(guò)程中,其體內(nèi)許多特殊的細(xì)胞器被保留下來(lái),而這類(lèi)細(xì)胞器在宿主體內(nèi)不存在,是寄生蟲(chóng)特有的。因此,通過(guò)對(duì)這些細(xì)胞器的結(jié)構(gòu)和功能的深入研究,就有可能發(fā)現(xiàn)針對(duì)這些寄生蟲(chóng)的特異靶標(biāo)。近期研究發(fā)現(xiàn)并命名的一個(gè)新細(xì)胞器—酸性鈣體(Acidocalcisome, Ac)就是其中之一。酸性鈣體一種近期發(fā)現(xiàn)的細(xì)胞器,大量研究表明其參與了寄生蟲(chóng)的生長(zhǎng)代謝、侵襲宿主、產(chǎn)生毒力等過(guò)程,是一種潛在的抗瘧靶點(diǎn)。 酸性鈣體是由美國(guó)伊利諾依大學(xué)Dr. Docampo Roberto教授的研究團(tuán)隊(duì)首先在錐蟲(chóng)體內(nèi)發(fā)現(xiàn)并命名。隨后又在利什曼原蟲(chóng),頂復(fù)門(mén)原蟲(chóng)：弓形蟲(chóng)、瘧原蟲(chóng)、艾美原蟲(chóng),綠藻、霉菌、細(xì)菌及人類(lèi)血小板中均發(fā)現(xiàn)有該類(lèi)細(xì)胞器的存在。該細(xì)胞器與過(guò)去報(bào)道的“顆�！�(granules)相似,其共同特點(diǎn)是內(nèi)含大量鈣和多聚磷酸鹽。雖然這些“顆�！痹缭�1904年就有文獻(xiàn)報(bào)道,但直到最近二十年才對(duì)其結(jié)構(gòu)和功能進(jìn)行了系統(tǒng)研究,并命名為酸性鈣體。許多具有酸性鈣體的寄生原蟲(chóng)或細(xì)菌,皆為致病病原體,而酸性鈣體的特點(diǎn)在于其焦磷酸酶(pyrophosphatase)活性,由于哺乳類(lèi)細(xì)胞缺乏此酶,如果能針對(duì)此酶研發(fā)藥物以阻斷其活性,便有可能殺死具有此類(lèi)胞器的致病病原體。因此,開(kāi)展對(duì)酸性鈣體的深入研究具有重要應(yīng)用價(jià)值。 對(duì)酸性鈣體的研究中,以錐蟲(chóng)和弓形蟲(chóng)的研究最為深入。研究發(fā)現(xiàn),酸性鈣體的膜上有許多泵：如鈣泵(Ca2+-ATPase)、質(zhì)子泵(Vacuolar-H+-ATPase, Vacuolar-H+-pryophosphatase)、鈉/氫泵(Na+/H+exchanger)、鈣/氫泵(Ca2+/H+exchanger)和水蛋白通道(Aquaporin)、離子通道等。這些成分對(duì)寄生蟲(chóng)的毒性、代謝和入侵宿主具有非常重要的作用。目前對(duì)瘧原蟲(chóng)酸性鈣體研究的諸多熱點(diǎn)領(lǐng)域如起源與進(jìn)化、與其它相似細(xì)胞器的關(guān)系多種酶的基因序列比對(duì)及系統(tǒng)發(fā)生關(guān)系等方面,都需要以蛋白質(zhì)組學(xué)數(shù)據(jù)提供理論基礎(chǔ),因此,對(duì)酸性鈣體進(jìn)行蛋白質(zhì)組學(xué)研究意義重大。 瘧原蟲(chóng)中也已證實(shí)有酸性鈣體的存在,但對(duì)其研究遠(yuǎn)不如錐蟲(chóng)和弓形蟲(chóng)深入,其具體的結(jié)構(gòu)、生物學(xué)功能和代謝特點(diǎn)尚未十分清楚。由于惡性瘧原蟲(chóng)在體外培養(yǎng)存在一些問(wèn)題：新鮮血液來(lái)源受限,寄生蟲(chóng)在體外培養(yǎng)生長(zhǎng)狀態(tài)不穩(wěn)定,難以擴(kuò)大繁殖等,這些問(wèn)題限制了惡性瘧原蟲(chóng)的研究運(yùn)用。而嚙齒類(lèi)寄生蟲(chóng)伯氏瘧原蟲(chóng)與惡性瘧原蟲(chóng)在形態(tài)結(jié)構(gòu)、基因組成、生理特點(diǎn)、生長(zhǎng)周期等方面非常相似,因此,本研究把伯氏瘧原蟲(chóng)作為研究瘧疾酸性鈣體的生物模型。 研究方法與結(jié)果： 首先將伯氏瘧原蟲(chóng)腹腔注射昆明小鼠內(nèi)使其大量繁殖,待小鼠發(fā)病后摘眼球采血取蟲(chóng)。利用60%的percoll分離液分離得到感染大滋養(yǎng)體和裂殖體的紅細(xì)胞,皂素裂解紅細(xì)胞,反復(fù)洗滌得到較純的伯氏瘧原蟲(chóng)瘧原蟲(chóng)。蟲(chóng)體經(jīng)碳化硅機(jī)械研磨后充分裂解,用buffer A反復(fù)清洗以排除大量瘧色素對(duì)實(shí)驗(yàn)的干擾。接著根據(jù)大量文獻(xiàn)報(bào)道及反復(fù)摸索實(shí)驗(yàn)條件,確定伯氏瘧原蟲(chóng)酸性鈣體分離純化的最優(yōu)碘克沙醇不連續(xù)密度梯度梯度為15%,20%,25%,30%,34%,38,利用這個(gè)最優(yōu)梯度分離純化得到沉淀。同時(shí)我們也對(duì)percoll分離液分離得到感染大滋養(yǎng)體和裂殖體的紅細(xì)胞進(jìn)行固定、包埋、切片處理,制配良好的細(xì)胞切片,在電壓為80kv的透射電子顯微鏡(FEI TecnaiTM G2Spirit transmission electron microscope)下觀(guān)察,同時(shí)直接觀(guān)察碘克沙醇密度梯度分離得到的沉淀物,并利用Norvar薄窗探測(cè)器對(duì)樣品網(wǎng)格進(jìn)行x-ray元素分析。在電鏡下分別可見(jiàn)類(lèi)似包涵體的空泡、大量大小不等的圓形致密顆粒狀,X射線(xiàn)能譜分析結(jié)果顯示該顆粒富含鈣、磷、鎂等化學(xué)元素。 通過(guò)蛋白質(zhì)組學(xué)方法挖掘出新的酸性鈣體蛋白是本研究的重要方面,對(duì)深入研究酸性鈣體功能特點(diǎn)具有重大意義。因此,我們將碘克沙醇密度梯度分離得到樣品進(jìn)行裂解,濃縮蛋白后進(jìn)行SDS電泳,最后割膠做LC-MS/MS檢測(cè)。利用Mascot2.3.02軟件在建立的數(shù)據(jù)庫(kù)中搜索與得到的肽段匹配的蛋白,最后得到一系列的蛋白質(zhì)鑒定信息。對(duì)這些蛋白質(zhì)進(jìn)行數(shù)據(jù)統(tǒng)計(jì),共得到125804個(gè)譜圖,1183個(gè)鑒定的譜圖,472個(gè)鑒定肽段,369個(gè)鑒定蛋白質(zhì)。接著對(duì)鑒定蛋白質(zhì)進(jìn)行生物信息學(xué)分析,主要包括GO注釋、COG分析、passway分析。在GO注釋分析結(jié)果中,分子功能分析結(jié)果可見(jiàn)鑒定蛋白質(zhì)主要具有催化活性(41.05%)和結(jié)合活性(42.47%)；亞細(xì)胞定位結(jié)果顯示,鑒定蛋白質(zhì)分布范圍較集中,主要在細(xì)胞器(23.83%)及膜(23.83%)上；生物學(xué)進(jìn)程分析可見(jiàn),鑒定蛋白質(zhì)主要參與了代謝通路(27.85%)、細(xì)胞過(guò)程(29.84%)、信號(hào)轉(zhuǎn)導(dǎo)(2.69%)等途徑。將組學(xué)鑒定到的蛋白質(zhì)和COG數(shù)據(jù)庫(kù)進(jìn)行比對(duì),預(yù)測(cè)這些蛋白質(zhì)可能的功能并統(tǒng)計(jì),結(jié)果顯示能量生成與轉(zhuǎn)化,脂質(zhì)轉(zhuǎn)運(yùn)與代謝,無(wú)機(jī)離子轉(zhuǎn)運(yùn)與代謝在功能分類(lèi)中所占比例較高。接著,我們參考大量的文獻(xiàn)報(bào)道結(jié)果、多種亞細(xì)胞定位預(yù)測(cè)軟件結(jié)果及GO注釋結(jié)果,對(duì)鑒定蛋白質(zhì)的亞細(xì)胞定位分布進(jìn)行了統(tǒng)計(jì),做出較準(zhǔn)確的鑒定蛋白質(zhì)亞細(xì)胞定位圖。最后綜合GO分析,COG分析及亞細(xì)胞定位分布結(jié)果,我們初步預(yù)測(cè)18個(gè)蛋白質(zhì)定位于伯氏瘧原蟲(chóng)酸性鈣體。 為驗(yàn)證前面實(shí)驗(yàn)初步預(yù)測(cè)的18個(gè)蛋白是否確實(shí)定位于酸性鈣體,我們將采用免疫熒光方法來(lái)驗(yàn)證,但制備酸性鈣體的標(biāo)志性蛋白-液泡型質(zhì)子泵焦磷酸酶單克隆抗體是首要任務(wù)。焦磷酸酶為膜蛋白,含有較多跨膜域,難以利用原核表達(dá)系統(tǒng)獲得抗原來(lái)制備抗體,所以我們選擇合成抗原表位肽的方式來(lái)生產(chǎn)免疫原。首先我們用TMHMM對(duì)伯氏瘧原蟲(chóng)焦磷酸酶進(jìn)行跨膜域的預(yù)測(cè),發(fā)現(xiàn)了15個(gè)跨膜域,對(duì)抗原肽段的選擇需要避開(kāi)這些跨膜區(qū)。同時(shí)用IEDB軟件做B細(xì)胞表位分析,,紅線(xiàn)表示臨界值0.350,評(píng)分高于紅線(xiàn)的為可能具有抗原性的表位,共預(yù)測(cè)6個(gè)抗原表位,接著將伯氏瘧原蟲(chóng)、惡性瘧原蟲(chóng)、弓形蟲(chóng)三者V-PPase氨基酸序列同源性比對(duì)結(jié)果,最終選定一段保守的序列為目標(biāo)抗原肽----TKAADCGADLSGKNEYGIPEDDDIM。因其分子較小,不具備好的抗原性,所以人工合成肽段后再與BSA偶聯(lián)制成抗原,免疫BALB/c小鼠制備單抗。免疫小鼠后,采用經(jīng)典的單克隆抗體制備方法制備抗焦磷酸酶單抗,運(yùn)用間接ELISA的方法對(duì)雜交瘤細(xì)胞株進(jìn)行陽(yáng)性篩選,最后共構(gòu)建了9株單抗,其中3D3、4E7、1H5雜交瘤株呈強(qiáng)陽(yáng)性,將其注射小鼠腹腔產(chǎn)生腹水。經(jīng)Western-blot鑒定腹水,發(fā)現(xiàn)4E7株能與弓形蟲(chóng)、惡性瘧原蟲(chóng)、伯氏瘧原蟲(chóng)的80KD左右大小蛋白都有相互作用。 結(jié)論： 本實(shí)驗(yàn)成功建立了碘克沙醇不連續(xù)密度梯度分離純化伯氏瘧原蟲(chóng)酸性鈣體的方法,并分離獲得伯氏瘧原蟲(chóng)的酸性鈣體。對(duì)分離獲得的伯氏瘧原蟲(chóng)酸性鈣體進(jìn)行蛋白質(zhì)組學(xué)分析,鑒定獲得369個(gè)蛋白質(zhì),系統(tǒng)生物信息學(xué)分析初步預(yù)測(cè)有18個(gè)蛋白質(zhì)可能定位于伯氏瘧原蟲(chóng)酸性鈣體。成功制備了酸性鈣體靶標(biāo)蛋白-焦磷酸酶的單抗,從而為其它酸性鈣體相關(guān)蛋白的亞細(xì)胞定位及進(jìn)一步的功能鑒定提供了有效的工具 我們的實(shí)驗(yàn)結(jié)果為瘧原蟲(chóng)的相關(guān)基礎(chǔ)研究及探索新的抗瘧藥物靶點(diǎn)奠定基礎(chǔ)。
[Abstract]:Background and purpose:
Malaria remains a'superkiller'of serious human health hazards, killing about a million people a year and causing 400 million infections. In tropical and subtropical regions, malaria is a common disease and a serious public health problem. Statistics show that hundreds of millions of people worldwide are infected with malaria every year, although most patients can be treated. The World Health Organization pays close attention to malaria and curbing malaria has become a global goal. China has also proposed to eliminate malaria in most areas by 2015 and to achieve the goal of eliminating malaria nationwide by 2020. However, malaria resistance to antimalarials and mosquito resistance to insecticides have become the major threats to global malaria control. Artemisinin-based combination therapy (ACT) is the most powerful weapon currently available for the treatment of falciparum malaria, the deadliest form of disease, with efficacy reaching more than 90%, but in Cambodia Artemisinin resistance has recently been confirmed along the border with Thailand. The development of new antimalarial drugs is not only critical but also urgent in view of the fact that all the drugs currently used to treat malaria are resistant.
In order to adapt to different external and host environments, parasites retain many special organelles in their long-term evolution, which do not exist in the host and are unique to the parasite. A new organelle named Acidocalcisome (Ac) is one of them. Acidocalcisome (Ac), a recently discovered organelle, is involved in the growth and metabolism of parasites, invasion of hosts, and production. The process of virulence is a potential antimalarial target.
Acidic calcitoids were first found and named in trypanosomes by Professor Dr. Docampo Roberto of the University of Illinois. They were then found in Leishmania, Phytoplasma: Toxoplasma gondii, Plasmodium, Eimeria, green algae, molds, bacteria and human platelets. Although these granules were reported as early as 1904, their structure and function were not systematically studied until the last 20 years, and they were named acidic calcitoids. Many parasites or bacteria with acidic calcitoids were caused by them. The characteristic of acidic calcium is its pyrophosphatase activity. Because of the lack of this enzyme in mammalian cells, it is possible to kill pathogens with this kind of organelles if drugs are developed to block its activity.
Trypanosomes and Toxoplasma gondii are the most in-depth studies of acidic calcium bodies. Studies have found that the membranes of acidic calcium bodies contain many pumps: calcium pump (Ca2 + - ATPase), proton pump (Vacuolar - H + - ATPase, Vacuolar - H + - pryophosphatase), sodium / hydrogen pump (Na + / H + exchanger), calcium / hydrogen pump (Ca2 + / H + exchanger) and water protein channel (Aquaporin), iontophoresis. These components play an important role in parasite toxicity, metabolism and host invasion. Proteomic data are needed to provide information on the origin and evolution of acid calcium bodies of Plasmodium, gene sequence alignment of enzymes related to other similar organelles, and phylogenetic relationships of various enzymes. Therefore, it is of great significance to carry out proteomic research on calcium acid bodies.
The existence of acidic calcium bodies in Plasmodium falciparum has been confirmed, but the study of acidic calcium bodies is far less thorough than that of Trypanosoma and Toxoplasma gondii. The specific structure, biological functions and metabolic characteristics of acidic calcium bodies are not very clear. However, the rodent parasite Plasmodium berghei is very similar to Plasmodium falciparum in morphological structure, genome composition, physiological characteristics, growth cycle and so on. Therefore, Plasmodium berghei is used as a biological model for studying acid calcareous malaria.
Research methods and results:
Plasmodium berghei was injected intraperitoneally into Kunming mice to propagate in large numbers, and then the eyeballs of mice were harvested for blood collection. The erythrocytes infected with macrotrophozoites and merozoites were isolated by 60% percoll. The erythrocytes were lysed by saponin and then washed repeatedly to obtain the pure Plasmodium berghei. Then, according to a large number of literature reports and repeated exploration of experimental conditions, the optimal density gradient of iodoxanol for isolation and purification of acidic calcium bodies of Plasmodium berghei was determined to be 15%, 20%, 25%, 30%, 34%, 38. The optimal gradient was used to isolate and purify the acidic calcium bodies of Plasmodium berghei. At the same time, we also fixed, embedded, sliced Erythrocytes Isolated from the Percoll solution and prepared good cell slices. The cells were observed under the transmission electron microscope (FEI TecnaiTM G2 Spirit transmission electron microscope) at a voltage of 80 kV and iodoxanol density was observed directly. The precipitates obtained by degree gradient separation were analyzed by X-ray element analysis using Norvar thin window detector. Under the electron microscope, the inclusion bodies were found to be similar to vacuoles, with a large number of round and dense particles. The X-ray energy spectrum analysis showed that the particles were rich in calcium, phosphorus, magnesium and other chemical elements.
It is an important aspect of this study to dig out new acidic calcitonin by proteomics method, which is of great significance to further study the functional characteristics of acidic calcitonin. Therefore, we separated the samples by density gradient of iodoxanol, then decomposed them, concentrated them by SDS electrophoresis, and finally tapped them for LC-MS/MS detection. Mascot 2.3.02 software was used. A series of protein identification information were obtained after searching for the protein matched with the peptide segments in the database. A total of 125 804 spectra, 1183 identification spectra, 472 identification peptides and 369 identification proteins were obtained by statistical analysis of these proteins. GO annotation, COG analysis and passway analysis were included. The results of GO annotation analysis showed that the identified proteins had catalytic activity (41.05%) and binding activity (42.47%). Subcellular localization showed that the identified proteins were mainly distributed in organelles (23.83%) and membranes (23.83%). The identified proteins were mainly involved in metabolic pathways (27.85%), cellular processes (29.84%) and signal transduction (2.69%). Secondly, according to a large number of literature reports, a variety of subcellular localization prediction software and GO annotation results, the subcellular localization distribution of identified proteins was statistically analyzed, and a more accurate subcellular localization map of identified proteins was made. Finally, GO analysis, COG analysis and subcellular localization were synthesized. Based on the results, we predicted that 18 proteins were located in Plasmodium bergi.
In order to verify whether the 18 proteins predicted in the previous experiment were positioned in acidic calcium bodies, we will use immunofluorescence method to verify, but the preparation of vacuolar proton-pumped pyrophosphate monoclonal antibody, the marker protein of acidic calcium bodies, is the primary task. Firstly, we used TMHMM to predict the transmembrane domain of P. berghei pyrophosphatase, and found 15 transmembrane domains. The selection of antigen peptides needs to avoid these transmembrane domains. Six antigenic epitopes were predicted with a critical value of 0.350 and a score higher than the red line. Then the homology of V-PPase amino acid sequences of Plasmodium berghei, Plasmodium falciparum and Toxoplasma gondii was compared. Finally, a conserved sequence was selected as the target antigenic peptide, TKAADCGADLS GKNEYGIPEDDIM. After immunizing BALB/c mice, anti-pyrophosphatase monoclonal antibodies were prepared by classical monoclonal antibody preparation methods. Hybridoma cell lines were screened by indirect ELISA method. Finally, nine monoclonal antibodies were constructed. Hybridoma strains 3D3,4E7,1H5 were strongly positive and injected into the abdominal cavity of mice to produce ascites. Western blot analysis showed that 4E7 strains could interact with about 80KD proteins of Toxoplasma gondii, Plasmodium falciparum and Plasmodium berghei.
Conclusion:
A discontinuous density gradient method for isolation and purification of acidic calcium bodies from Plasmodium berghei was successfully established. The acidic calcium bodies of Plasmodium berghei were isolated and purified. Proteomic analysis of the isolated acidic calcium bodies showed that 369 proteins were identified and 18 proteins were preliminarily predicted by systematic bioinformatics analysis. These proteins may be localized in acidic calcium bodies of Plasmodium berghei, and monoclonal antibodies against pyrophosphatase, the target protein of acidic calcium bodies, have been successfully prepared, thus providing an effective tool for subcellular localization and further functional identification of other acidic calcium bodies related proteins.
Our results lay the foundation for the basic research of malaria parasites and the exploration of new antimalarial targets.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R382.31

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本文編號(hào)：2250628