微隐孢子虫中含铁相关蛋白的表达、纯化与功能研究

【作者】 雷铖；

【导师】 谭相石；

【作者基本信息】 复旦大学 ， 无机化学， 2010， 硕士

【摘要】 微小隐孢子虫(Category B agent)是一种会导致人类和动物急性腹泻的寄生虫。如今在美国只有一种用于治疗对感染了隐孢子虫病艾滋病患者的药物(i.e., nitazoxanide),由于对其抗药性的担心,新药的开发迫在眉睫。本研究者尝试表达的铁氧还蛋白,正是处于微小隐孢子虫的线粒体样细胞器中具有硫铁簇结构的一种功能蛋白(Ferredoxin)、硫铁簇结构在细胞中是普遍存在且必不可少的组成部分,参与各种细胞功能的运作。近30多年对植物性铁氧还蛋白和脊椎动物中的铁氧还蛋白的结构和功能研究都有了一定的基础。在本文中,首次成功表达与纯化CpmtFd和CpmtFNR蛋白,同时对它们进行了表征与生化性质研究。EPR,UV,HPLC和EPR研究表明这两个纯化蛋白分别具有[2Fe-S]簇和FAD辅基,且具有生物功能。CpmtFNR在细胞色素c参与下可以将电子从NADPH传递给CpmtFd。顶复门生物的mtFd和mtFNR从进化角度分析,是与人和其他动物的mtFd和mtFNR存在较大差异的一类。因此,它们可能在将来作为潜在的药物靶点帮助我们攻克隐孢子虫病和其它顶复门寄生虫疾病。更多还原

【Abstract】 Cryptosporidium parvum (Category B agent) is a protozoan parasite that can cause severe watery diarrhea in humans and animals. Currently, only a single drug (i.e., nita-zoxanide [NTZ]) has been approved for treating cryptosporidiosis in immunocompe-tent (but not immunocompromized) patients in the United States. We have successfully expressed recombinant mitochondrial-type ferredoxin (mtFd) and ferredoxin:NADP+ reductase (mtFNR) from Cryptosporidium parvum and characterized their biochemical features for the first time. Both CpmtFd and CpmtFNR were obtained and purified as holo-proteins, in which the correct assembly of [2Fe-2S] cluster in Fd and that of FAD in FNR were confirmed and characterized by UV/Vis and EPR. These proteins were fully functional and CpmtFNR was capable of transferring electrons from NADPH to CpmtFd in a cytochrome c-coupled assay that followed a typical Michaelis-Menten ki-netics. Apicomplexan mtFd and mtFNR proteins were evolutionarily divergent from their counterparts in humans and animals, and could be explored to be acted as poten-tial drug targets in Cryptosporidium and other apicomplexans.更多还原