【作者】 韩颖丽；

【导师】 苏钟；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2011， 硕士

【摘要】 疟疾是由疟原虫引起的,是最严重的传染病之一,每年导致100万人死亡,目前109个国家正遭受疟疾感染的危险。疟疾是由按蚊传播,随着叮咬人体而将疟原虫注入人体血液循环中,它可以导致低血糖症、发热、贫血等临床症状。目前有四种人疟和四种鼠疟,其中夏氏疟原虫是研究恶性疟原虫最佳的小鼠模型。目前研发的用于抵抗疟疾的疫苗种类有很多,亚单位疫苗是研究最多的。它可以引起很强的体液免疫,但细胞免疫较弱,不能引起持久的免疫保护力。亚单位疫苗需要佐剂的辅助来增强免疫反应。MSP1是最重要的亚单位候选疫苗,可以引起一定的保护力。它经一系列切割最后只剩C端19kDa经GPI连接在虫表面并进入红细胞中。有前人报道发现从疟原虫体外培养的上清中有某些物质可以与疟疾感染的病人的阳性血清发生作用。因此本实验我们想研究MSP1和疟原虫培养上清共同免疫的效果。首先利用大肠杆菌表达系统获得夏氏疟原虫AS株MSP1蛋白C端11kDa;其次体外短期培养夏氏疟原虫获得代谢分泌物(ES)。将MSP1与代谢分泌物共同免疫小鼠,检测感染率、ELISA检测抗体滴度;免疫小鼠的脾细胞培养,研究细胞因子的分泌和细胞激活状态。实验发现:(a)MSP1以ES为佐剂免疫可以有效抑制血虫水平;产生较高的抗体水平,主要有IgG1、IgG2c和IgG2b;(b)激活了树突状细胞,使CD40、CD86分子表达升高,MHC-II表达下降;(c)激活了CD4+T细胞,使其表面分子CD62L表达下降;(d)诱导淋巴细胞的增殖,产生较高的IFN-γ、IL-12和IL-10。本研究第一次将代谢分泌物作为新型佐剂。我们的研究结果揭示了抗疟疾免疫诱导的复杂机制,并可能对疟疾疫苗的研发具有重要意义。更多还原

【Abstract】 Malaria caused by infection with Plasmodium parasite is one of the most serious infectious diseases, which causes 1 million deaths every year. At present, more than 109 countries of the world are suffering from the risk of malaria infection. Malaria is transmitted by Anopheles, which bite human body and inject the parasites into blood circulation. Malaria infection causes severe clinical symptoms including anemia, hypoglycemia and fever. There are four species of Plasmodium that infect human. The rodent malaria parasite, Plasmodium chabaudi, is one of the model to study the Plasmodium falciparum infection.Many vaccines against malaria are currently developed, among which subunit vaccines are most studied. The subunit vaccines can induce strong humoral immune response, but the cell-mediated immunity is weak and the protection is not long lasting. Adjuvants are often needed to help subunit vaccines to strengthen immune responses. Merozoite surface protein1 (MSP1) is a leading candidate vaccine antigen and has been shown to confer certain protection. MSP1 undergoes a series of proteolytic cleavages and finally the C-terminal GPI-anchored MSP119 remains on the parasite surface as it enters the RBC. It was reported that the supernatants from Plasmodium culture medium are recognized by the sera from malaria-infected adults.In this study, we intend to determine the coimmune effects of P. chabaudi culture supernatant and MSP1. We first expressed the 11 kDa fragment of MSP1 C-terminal domain of Plasmodium chabaudi AS in Escherichia coli expression system. A protocol of short-term in vitro culture of Plasmodium chabaudi was used to acquire the excretory and secretary products (ES). After infecting mouse with MSP1 plus ES, we monitored the parasitemia and measured antibody titers using ELISA assay. In addition, spleen cells of the immunized mice were cultured to study the cytokine secretion and immune cell activation. We observed that: (a) immunization with MSP1 using ES as adjuvant effectively inhibited the parasitemia and induced higher levels of IgG1, IgG2c and IgG2b antibody titers; (b) CD11c+DC cells were activated, which showed up-regulated expression of CD40,CD86,but down-regulated expression of MHC-II; (c) the CD4+T cells were activated and CD62L were consequently down-regulated, and (d) splenocytes proliferated more vigorously and produced high levels of IFN-γ,IL-12 and IL-10. We report here first time that the excretory and secretary products can be used as a new immune adjuvant. Our results reveal the complex mechanisms of induction of anti-malarial immunity and may have important implication in development of malaria vaccines.更多还原