导读:本文包含了动力素相似蛋白论文开题报告文献综述及选题提纲参考文献，主要关键词:疟疾,动力素相似蛋白,透射电镜,内吞作用
动力素相似蛋白论文文献综述
周洪昌[1]（2008）在《恶性疟原虫动力素相似蛋白1的功能研究》一文中研究指出疟疾是目前地球上对人类危害最严重的蚊媒寄生原虫病。全世界每年有3至5亿人受到疟原虫感染，二百至叁百余万人死于恶性疟疾，其中80%是五岁以下儿童。疟原虫是疟疾的病原体，寄生于人体的疟原虫共有4种，恶性疟原虫是其中最致命的一种。由于恶性疟原虫对现有药物的抗药性不断出现和蔓延，蚊媒对杀虫剂抗药性的产生等因素影响，加上迄今尚无有效的疟疾疫苗出现，致使对疟疾的控制成为世界性的大难题之一。对疟疾的防治是多种多样的，可以针对不同的发育阶段和传播环节采取不同的措施。目前公认最有发展前景的策略包括：1)能够有效抑制疟原虫的某一特殊生命过程的药物；2)具有预防及治疗作用的疫苗。恶性疟原虫动力素相似蛋白1(Plasmodium falciparum dynamin like protein1)是本实验筛选出的新虫源基因，前期工作证明其功能具有多样性，可能在内吞通路和分泌通路中具有重要的作用，并且是维持恶性疟原虫生存的重要基因。为了更深入地了解其功能，本文尝试使用基因敲除和特异性抑制剂等技术手段，进行进一步的研究。首先，构建了用于全长敲除和C端敲除的双交换敲除载体，经电脉冲转染方法，获得了稳定的转染子。随后利用有限稀释法培养了单克隆。遗传实验证明没有得到预期的全长敲除的恶性疟原虫虫株，但是有发生单交换的虫体存在。C末端敲除的稳定转染子由于时间关系，没有及时单克隆化并进行分析，对C末端敲除后的效果尚待观察。其次，本研究中尝试了P．f dynamin like protein 1蛋白在原核和真核表达系统中全长表达，试用各种诱导条件均未成功。随后改用密码子优化法，将P．fdynamin like protein 1基因密码子全部替换成大肠杆菌(E．coli)偏爱的密码子，实现了P．f dynamin like protein 1全长蛋白的表达，并证实其具有GTPase活性，且不受C末端131个氨基酸缺失的影响。最后，通过使用dynamin特异性抑制剂dynasore，发现dynasore能够有效地抑制P．fdynamin like protein 1的GTPase活性。将dynasore与培养的恶性疟原虫共孵育，能够抑制恶性疟原虫的生长。结合前期研究结果与已经明确dynamin家族的功能，提出了P．f dynamin like protein 1参与疟原虫血红蛋白内吞的假设。通过生物化学方法显示，dynasore的使用可以明显地减少恶性疟原虫体内血红蛋白的累积以及疟色素的形成，并能极大减少示踪剂FITC标记的葡聚糖在恶性疟原虫中的累积，表明dynasore抑制了P．f dynamin like protein 1的GTPase活性，进而影响了恶性疟原虫对血红蛋白的内吞作用；电镜结果显示，dynasore处理的恶性疟原虫体内累积了大量的囊泡样结构，提示dynamin在内吞通路中有重要作用。本研究表明，P．f dynamin like protein 1参与了恶性疟原虫血红蛋白内吞过程，并发挥了重要作用。为揭示恶性疟原虫的内吞机理提供了新的线索，也为将P．f dynamin like protein 1作为抗疟药靶点提供了理论依据。(本文来源于《中国协和医科大学》期刊2008-01-18）
Zhou,Hongchang,Wang,Heng,(Molecular,Parasitology,Laboratory,Department,of,Etiology,Peking,Union,Medical,College,Chinese,Academy,of,Medical,Sciences,Institute,of,Basic,Medical,Sciences[2]（2005）在《使用密码子优化策略高效表达恶性疟原虫动力素相似蛋白1(英文)》一文中研究指出Malaria is by far the most important human parasitic disease, causing disease in 200-300million people and killing l-3million people annually. The two widely used anti malarial drugs, chloroquine(CQ) and sulphadoxine-pyrimethamine (SP) are failing at an accelerating rate in most malaria endemic regions, with consequent increases in malaria-related morbidity and mortality. Increased efforts in anti malarial drug discovery are urgently needed. Dynamins are large GTPase that belong to a protein superfamily. They are involved in many processes, including budding of vesicles, division of organelles, cytokinesis and pathogen resistance. We have identified a dynamin like protein gene by cDNA library screen and our previous study suggested that the P.f dynamin like protein 1 has the potential to be a drug target for malaria therapy. The expression and GTPase activity assay of P.f dynamin like protein 1 is the important step for further drug screen. In this experiment, the Plasmodium falciparum dynamin like protein 1 was highly expressed in E.coli with codon optimization and showed GTPase activity. First, The whole gene was divided into 4 fragments with one unique restriction site at each end. Each sequence was altered to obtain optimized codon usage percentages of E.coli by using DNA works computer program. The oligoes were de nove designed based on TBIO method. Each fragment was obtained with several times of PCR using Pfu and sub-cloned to T-vector and sequenced. Four T-vector- fragment-plasmid and pET30a were digested with appropriate enzyme. Then,the five fragments were gel-purified and ligated with T4 DNA ligase at 16℃over night. The positive clone were detected and sequenced. Histidine-tagged P.f dynamin like protein was expressed in E.coli (BL21DE3). A production of about 100kD was obtained. The whole protein was isolated using Ni2+chelate chromatography. The nature of the GTPase activity of P.f dynamin like protein 1 was determined by using nonradioactive malachite green phosphomolybdate assay. We are going to calculate the Km and Kcat.(本文来源于《2005年寄生虫学国际学术研讨会暨中国动物学会寄生虫专业委员会第十次学术研讨会论文摘要汇编》期刊2005-10-01）
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Malaria is by far the most important human parasitic disease, causing disease in 200-300million people and killing l-3million people annually. The two widely used anti malarial drugs, chloroquine(CQ) and sulphadoxine-pyrimethamine (SP) are failing at an accelerating rate in most malaria endemic regions, with consequent increases in malaria-related morbidity and mortality. Increased efforts in anti malarial drug discovery are urgently needed. Dynamins are large GTPase that belong to a protein superfamily. They are involved in many processes, including budding of vesicles, division of organelles, cytokinesis and pathogen resistance. We have identified a dynamin like protein gene by cDNA library screen and our previous study suggested that the P.f dynamin like protein 1 has the potential to be a drug target for malaria therapy. The expression and GTPase activity assay of P.f dynamin like protein 1 is the important step for further drug screen. In this experiment, the Plasmodium falciparum dynamin like protein 1 was highly expressed in E.coli with codon optimization and showed GTPase activity. First, The whole gene was divided into 4 fragments with one unique restriction site at each end. Each sequence was altered to obtain optimized codon usage percentages of E.coli by using DNA works computer program. The oligoes were de nove designed based on TBIO method. Each fragment was obtained with several times of PCR using Pfu and sub-cloned to T-vector and sequenced. Four T-vector- fragment-plasmid and pET30a were digested with appropriate enzyme. Then,the five fragments were gel-purified and ligated with T4 DNA ligase at 16℃over night. The positive clone were detected and sequenced. Histidine-tagged P.f dynamin like protein was expressed in E.coli (BL21DE3). A production of about 100kD was obtained. The whole protein was isolated using Ni2+chelate chromatography. The nature of the GTPase activity of P.f dynamin like protein 1 was determined by using nonradioactive malachite green phosphomolybdate assay. We are going to calculate the Km and Kcat.
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动力素相似蛋白论文参考文献
[1].周洪昌.恶性疟原虫动力素相似蛋白1的功能研究[D].中国协和医科大学.2008
[2].Zhou,Hongchang,Wang,Heng,(Molecular,Parasitology,Laboratory,Department,of,Etiology,Peking,Union,Medical,College,Chinese,Academy,of,Medical,Sciences,Institute,of,Basic,Medical,Sciences.使用密码子优化策略高效表达恶性疟原虫动力素相似蛋白1(英文)[C].2005年寄生虫学国际学术研讨会暨中国动物学会寄生虫专业委员会第十次学术研讨会论文摘要汇编.2005