伯氏疟原虫酸性钙体的分离纯化及其蛋白质组学研究
发布时间：2018-09-19 16:06
【摘要】：研究背景及目的： 疟疾仍是严重危害人类健康的‘超级杀手’,这种传染病每年夺去大约100万人的性命,导致4亿人感染。在热带和亚热带地区,疟疾是常见疾病也是严重的公共卫生问题,统计数字显示,每年全球有几亿人感染疟疾,虽然大多数患者能够治愈,可是仍有一百万人死亡,其中以儿童占了多数。世界卫生组织对疟疾给予密切的关注,遏制疟疾已成为全球奋斗的目标。我国也提出到2015年大部分地区消除疟疾,到2020年全国实现消除疟疾的目标[3]。有效的药物是治疗疟疾的主要方法。然而,疟原虫对抗疟药耐药性和蚊虫对杀虫剂的抗药性已成为实现全球疟疾控制所面临的最主要威胁。以青蒿素为基础的联合疗法(ACT)是目前治疗恶性疟疾这一最致命的疾病形式最强有力的武器,其疗效能达到90%以上,但在柬埔寨和泰国边境地区近期已确认出现青蒿素耐药性。鉴于目前医学界使用的所有治疗疟疾药物都出现耐药性现象,因此开发抗疟疾新药不但至关重要而且面临着急迫性。 寻找有效的药物作用靶标是研制抗疟新药的基础。寄生虫为了适应不同的外界环境和宿主环境,在长期进化过程中,其体内许多特殊的细胞器被保留下来,而这类细胞器在宿主体内不存在,是寄生虫特有的。因此,通过对这些细胞器的结构和功能的深入研究,就有可能发现针对这些寄生虫的特异靶标。近期研究发现并命名的一个新细胞器—酸性钙体(Acidocalcisome, Ac)就是其中之一。酸性钙体一种近期发现的细胞器,大量研究表明其参与了寄生虫的生长代谢、侵袭宿主、产生毒力等过程,是一种潜在的抗疟靶点。 酸性钙体是由美国伊利诺依大学Dr. Docampo Roberto教授的研究团队首先在锥虫体内发现并命名。随后又在利什曼原虫,顶复门原虫：弓形虫、疟原虫、艾美原虫,绿藻、霉菌、细菌及人类血小板中均发现有该类细胞器的存在。该细胞器与过去报道的“颗粒”(granules)相似,其共同特点是内含大量钙和多聚磷酸盐。虽然这些“颗粒”早在1904年就有文献报道,但直到最近二十年才对其结构和功能进行了系统研究,并命名为酸性钙体。许多具有酸性钙体的寄生原虫或细菌,皆为致病病原体,而酸性钙体的特点在于其焦磷酸酶(pyrophosphatase)活性,由于哺乳类细胞缺乏此酶,如果能针对此酶研发药物以阻断其活性,便有可能杀死具有此类胞器的致病病原体。因此,开展对酸性钙体的深入研究具有重要应用价值。 对酸性钙体的研究中,以锥虫和弓形虫的研究最为深入。研究发现,酸性钙体的膜上有许多泵：如钙泵(Ca2+-ATPase)、质子泵(Vacuolar-H+-ATPase, Vacuolar-H+-pryophosphatase)、钠/氢泵(Na+/H+exchanger)、钙/氢泵(Ca2+/H+exchanger)和水蛋白通道(Aquaporin)、离子通道等。这些成分对寄生虫的毒性、代谢和入侵宿主具有非常重要的作用。目前对疟原虫酸性钙体研究的诸多热点领域如起源与进化、与其它相似细胞器的关系多种酶的基因序列比对及系统发生关系等方面,都需要以蛋白质组学数据提供理论基础,因此,对酸性钙体进行蛋白质组学研究意义重大。 疟原虫中也已证实有酸性钙体的存在,但对其研究远不如锥虫和弓形虫深入,其具体的结构、生物学功能和代谢特点尚未十分清楚。由于恶性疟原虫在体外培养存在一些问题：新鲜血液来源受限,寄生虫在体外培养生长状态不稳定,难以扩大繁殖等,这些问题限制了恶性疟原虫的研究运用。而啮齿类寄生虫伯氏疟原虫与恶性疟原虫在形态结构、基因组成、生理特点、生长周期等方面非常相似,因此,本研究把伯氏疟原虫作为研究疟疾酸性钙体的生物模型。 研究方法与结果： 首先将伯氏疟原虫腹腔注射昆明小鼠内使其大量繁殖,待小鼠发病后摘眼球采血取虫。利用60%的percoll分离液分离得到感染大滋养体和裂殖体的红细胞,皂素裂解红细胞,反复洗涤得到较纯的伯氏疟原虫疟原虫。虫体经碳化硅机械研磨后充分裂解,用buffer A反复清洗以排除大量疟色素对实验的干扰。接着根据大量文献报道及反复摸索实验条件,确定伯氏疟原虫酸性钙体分离纯化的最优碘克沙醇不连续密度梯度梯度为15%,20%,25%,30%,34%,38,利用这个最优梯度分离纯化得到沉淀。同时我们也对percoll分离液分离得到感染大滋养体和裂殖体的红细胞进行固定、包埋、切片处理,制配良好的细胞切片,在电压为80kv的透射电子显微镜(FEI TecnaiTM G2Spirit transmission electron microscope)下观察,同时直接观察碘克沙醇密度梯度分离得到的沉淀物,并利用Norvar薄窗探测器对样品网格进行x-ray元素分析。在电镜下分别可见类似包涵体的空泡、大量大小不等的圆形致密颗粒状,X射线能谱分析结果显示该颗粒富含钙、磷、镁等化学元素。 通过蛋白质组学方法挖掘出新的酸性钙体蛋白是本研究的重要方面,对深入研究酸性钙体功能特点具有重大意义。因此,我们将碘克沙醇密度梯度分离得到样品进行裂解,浓缩蛋白后进行SDS电泳,最后割胶做LC-MS/MS检测。利用Mascot2.3.02软件在建立的数据库中搜索与得到的肽段匹配的蛋白,最后得到一系列的蛋白质鉴定信息。对这些蛋白质进行数据统计,共得到125804个谱图,1183个鉴定的谱图,472个鉴定肽段,369个鉴定蛋白质。接着对鉴定蛋白质进行生物信息学分析,主要包括GO注释、COG分析、passway分析。在GO注释分析结果中,分子功能分析结果可见鉴定蛋白质主要具有催化活性(41.05%)和结合活性(42.47%)；亚细胞定位结果显示,鉴定蛋白质分布范围较集中,主要在细胞器(23.83%)及膜(23.83%)上；生物学进程分析可见,鉴定蛋白质主要参与了代谢通路(27.85%)、细胞过程(29.84%)、信号转导(2.69%)等途径。将组学鉴定到的蛋白质和COG数据库进行比对,预测这些蛋白质可能的功能并统计,结果显示能量生成与转化,脂质转运与代谢,无机离子转运与代谢在功能分类中所占比例较高。接着,我们参考大量的文献报道结果、多种亚细胞定位预测软件结果及GO注释结果,对鉴定蛋白质的亚细胞定位分布进行了统计,做出较准确的鉴定蛋白质亚细胞定位图。最后综合GO分析,COG分析及亚细胞定位分布结果,我们初步预测18个蛋白质定位于伯氏疟原虫酸性钙体。 为验证前面实验初步预测的18个蛋白是否确实定位于酸性钙体,我们将采用免疫荧光方法来验证,但制备酸性钙体的标志性蛋白-液泡型质子泵焦磷酸酶单克隆抗体是首要任务。焦磷酸酶为膜蛋白,含有较多跨膜域,难以利用原核表达系统获得抗原来制备抗体,所以我们选择合成抗原表位肽的方式来生产免疫原。首先我们用TMHMM对伯氏疟原虫焦磷酸酶进行跨膜域的预测,发现了15个跨膜域,对抗原肽段的选择需要避开这些跨膜区。同时用IEDB软件做B细胞表位分析,,红线表示临界值0.350,评分高于红线的为可能具有抗原性的表位,共预测6个抗原表位,接着将伯氏疟原虫、恶性疟原虫、弓形虫三者V-PPase氨基酸序列同源性比对结果,最终选定一段保守的序列为目标抗原肽----TKAADCGADLSGKNEYGIPEDDDIM。因其分子较小,不具备好的抗原性,所以人工合成肽段后再与BSA偶联制成抗原,免疫BALB/c小鼠制备单抗。免疫小鼠后,采用经典的单克隆抗体制备方法制备抗焦磷酸酶单抗,运用间接ELISA的方法对杂交瘤细胞株进行阳性筛选,最后共构建了9株单抗,其中3D3、4E7、1H5杂交瘤株呈强阳性,将其注射小鼠腹腔产生腹水。经Western-blot鉴定腹水,发现4E7株能与弓形虫、恶性疟原虫、伯氏疟原虫的80KD左右大小蛋白都有相互作用。 结论： 本实验成功建立了碘克沙醇不连续密度梯度分离纯化伯氏疟原虫酸性钙体的方法,并分离获得伯氏疟原虫的酸性钙体。对分离获得的伯氏疟原虫酸性钙体进行蛋白质组学分析,鉴定获得369个蛋白质,系统生物信息学分析初步预测有18个蛋白质可能定位于伯氏疟原虫酸性钙体。成功制备了酸性钙体靶标蛋白-焦磷酸酶的单抗,从而为其它酸性钙体相关蛋白的亚细胞定位及进一步的功能鉴定提供了有效的工具 我们的实验结果为疟原虫的相关基础研究及探索新的抗疟药物靶点奠定基础。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R382.31
本文编号：2250628
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R382.31
【参考文献】
相关期刊论文 前2条
1 车河龙;林栋;;疟疾的防控现状及进展[J];热带医学杂志;2010年02期
2 张坤炎,周洁娴,黄健康,林博华,吴振宏,阮彩文,黄祺林;间日疟原虫、恶性疟原虫和伯氏疟原虫各期的提纯和分离[J];中国寄生虫学与寄生虫病杂志;1993年04期
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