疟原虫QSOX生物学功能及其传播阻断潜能的研究

Malaria is one of the parasitic diseases which severely impairs humanity health. Transmission blocking vaccine (TBV) plays an essential role in malaria elimination, especially Plasmodium vivax malaria. The discovery of TBV candidate and clarify its functions in the malaria parasite development stage are prerequisite for TBV studies. The enzyme family Quiescin Sulfhydryl Oxidase (QSOX), which catalyzes generate disulfide bonds into unfolded reduced proteins, is vital for proteins to play a physiological activity. Nonetheless, the existence and functions of QSOX in Plasmodium are still unknown. Based on the significance of TBV control the spread of Plasmodium and the current research situation of Plasmodium QSOX, in our previous study, we identified a QSOX member in Plasmodium. The expression of QSOX was only detected on the surface in sexual stages and antisera against this recombinant QSOX antigen possessed strong transmission blocking activity. We therefore postulate that Plasmodium QSOX may be a potential candidate antigen for TBV. The project intends to use parasite transfection technology to knock out qsox gene in Plasmodium berghei ANKA model and study the functions during parasite development. By expression of QSOX recombinant protein, we will further clarify the characteristic of the enzyme activity, identify the enzyme inhibitor and detail analysis its effect on parasite development. Further, we will also obtain antisera and monoclonal antibody, systematic evaluation of the transmission blocking activities both in Plasmodium berghei and Plasmodium vivax model. This study will reveal the parasite developmental mechanism of QSOX in sexual stage, and expect to provide a new theoretical and experimental basis for the development of an effective Plasmodium vivax malaria vaccine.

疟疾是严重威胁人类健康的感染性寄生虫病。传播阻断疫苗（TBV）对控制疟疾特别是间日疟的流行意义重大，而候选抗原的发掘及其在疟原虫发育阶段功能的阐明是TBV研发的关键。静息巯基氧化酶（QSOX）是催化生物体二硫键正确形成、促进蛋白质发挥生理活性的关键酶类，但迄今为止疟原虫QSOX的生物学特点及功能尚未阐明。我们在前期研究中筛选确定了疟原虫QSOX，阐明了其在疟原虫有性生殖阶段表面表达，并初步证明了其抗血清的传播阻断能力，提示QSOX具有成为TBV候选抗原的潜能。本项目拟对疟原虫QSOX的生物学功能进行系统研究，利用基因重组技术揭示QSOX对疟原虫生长发育的影响；通过重组蛋白的表达明确酶活性特点，并确定酶抑制剂对疟原虫发育的影响；制备免疫血清与单克隆抗体，通过鼠疟模型与间日疟膜饲实验系统验证QSOX的传播阻断活性。以期寻求间日疟原虫防控的新靶位，为研发新型、高效的TBV抗原提供理论与实验依据。

疟疾是严重威胁人类健康的感染性寄生虫病。传播阻断疫苗（TBV）对控制疟疾特别是间日疟的流行意义重大，而候选抗原的发掘及其在疟原虫发育阶段功能的阐明是TBV研发的关键。静息巯基氧化酶（QSOX）是催化生物体二硫键正确形成、促进蛋白质发挥生理活性的关键酶类，但迄今为止疟原虫QSOX的生物学特点及功能尚未阐明。本项目通过生物信息学分析，证实了疟原虫的QSOX；应用基因打靶技术，敲除了pbqsox基因，构建了基因敲除型疟原虫（Δpbqsox），发现pbqsox缺失后，主要影响疟原虫雄配子出丝与动合子的成熟，pbqsox的缺失也导致了动合子表面蛋白硫醇基团的显著剩余，表明QSOX可能在维持表面蛋白二硫键的完整性方面发挥作用，这可能是动合子发育所必需的。接着，我们通过原核表达系统表达并纯化了伯氏疟原虫QSOX重组蛋白（rPbQSOX），明确了PbQSOX的酶活性特点，并确定了ebselen可明显抑制PbQSOX的酶活性，同时证明了ebselen可影响伯氏疟原虫的有性生殖阶段的发育，提示提示ebselen可能会成为控制疟疾传播的潜在药物。rPbQSOX免疫小鼠后制备了免疫血清与单克隆抗体，并构建了PbQSOX HA-标签型疟原虫，应用三者共同确定了PbQSOX主要表达于雌雄配子体、活化的雌雄配子、合子、成熟的动合子表面，这种蛋白表达特点符合传播阻断疫苗的设计原则。接着，我们通过体外动合子形成率抑制实验与体内蚊饲实验，共同证明了抗rPbQSOX抗体具有一定的传播阻断能力。为了进一步验证抗间日疟原虫QSOX（PvQSOX ）抗体的传播阻断能力，我们表达了PvQSOX重组蛋白（rPvQSOX），并应用基因打靶技术获得表达PvQSOX蛋白的pbqsox基因敲除型伯氏疟原虫虫株（Pv-Tr-PbQSOX）。应用抗rPvQSOX免疫血清与Pv-Tr-PbQSOX疟原虫，明确了PvQSOX同样在疟原虫雌雄配子体、活化的雌雄配子、合子与成熟的动合子表面表达；膜饲实验显示，rPvQSOX免疫血清可明显阻断间日疟原虫卵囊的形成，具有明显的传播阻断能力。总之，我们应用鼠疟和人疟模型，共同证明了抗QSOX抗体具有一定的传播阻断能力，QSOX可能是潜在的疟原虫TBV候选抗原。