用基因打靶技术构建恶性疟原虫MSP1转基因鼠疟模型及其应用

发布时间：2018-09-06 08:33

【摘要】：疟疾是世界范围流行的严重威胁人类健康的寄生虫病。在四种人体疟原虫中,恶性疟原虫(Plasmodium falciparum,Pf)是引起严重疟疾病症乃至死亡的主要病原体。据WHO估计,每年患病人数超过2亿,死亡200—300万,大多数为5岁以下的儿童。由于疟原虫抗药性以及蚊媒对杀虫剂抗性的产生和扩散,使疟疾防治面临严重困难。因此,研制有效的疟疾疫苗已成为疟疾防治的重要途径。疟原虫裂殖子表面蛋白1(Merozoite Surface Protein 1,MSP1)是一种红细胞内期原虫表达、分布于裂殖子表面的糖蛋白。该分子是诱导机体产生保护性免疫的主要抗原之一,尤其是其C末端的19KDa(MSP1-19)片段是其保护性免疫功能域。该片段的单抗和抗血清在体外能有效抑制疟原虫的生长。本实验室构建的恶性疟红内期疫苗PfCP-2.9含有该片段的组分,目前该疫苗已进入临床试验。目前疟疾疫苗研究遇到的最大困难之一就是缺乏廉价有效的动物模型。尽管南美洲夜猴能感染恶性疟原虫,可作疫苗评价的动物模型,但夜猴并非是恶性疟原虫的天然宿主,且来源非常有限,价格昂贵。近年来疟原虫转基因技术已日趋成熟,并已广泛应用于研究疟原虫蛋白结构和功能,与宿主相互作用和基因表达调控等。根据等位替换的实验研究表明,疟原虫不同种间一些重要的功能蛋白,例如TRAP、MSP1,虽然序列上差异很大,但在功能上却是保守的,经同源替换后疟原虫仍然能完成生活史。依据这个特点,本研究以鼠伯氏疟原虫(Plasmudium berghei,Pb)为实验对象,通过疟原虫的基因打靶技术用PfMSP1-19替换内源性的PbMSP1-19,建立疟原虫的基因转染技术并产生表达PfMSP1-19转基因的Pb。由于此转基因伯氏疟原虫含有PfMSP1-19组分,因此用此转基因鼠疟原虫来评价PfCP-2.9疫苗体内的免疫保护效果。应用专业软件设计引物,从Pb和Pf的基因组PCR扩增目的基因片段。在Pb中获得来自伯氏疟原虫裂殖子表面蛋白1(PbMSP-1)的1.4Kb片段和PbMSP-13’UTR的500bp片段;在Pf中获得PfMSP-1 C末端19KDa的330bp片段。将PbMSP-1的1.4Kb与Pf的330bp的片段连接形成PbfMSP1融合基因。将PbfMSP1
[Abstract]:Malaria is a worldwide epidemic of parasitic diseases that pose a serious threat to human health. Among the four human malaria parasites, Plasmodium falciparum (Plasmodium falciparum,Pf) is the leading cause of severe malaria and even death. According to WHO estimates, more than 200 million people are sick and 2-3 million die each year, most of them children under 5 years of age. Malaria control is difficult because of the resistance of malaria parasite and the emergence and spread of mosquito vector resistance to insecticides. Therefore, the development of effective malaria vaccine has become an important way of malaria control. Plasmodium parasite merozoite surface protein (1 (Merozoite Surface Protein 1 MSP1) is a glycoprotein expressed in erythrocyte parasites and distributed on the surface of merozoites. This molecule is one of the major antigens that induce the body to produce protective immunity, especially the 19KDa (MSP1-19) fragment at its C-terminal is its protective immune functional domain. The McAb and antiserum of this fragment can effectively inhibit the growth of Plasmodium falciparum in vitro. PfCP-2.9, an internal falciparum malaria vaccine constructed in our laboratory, contains the component of this fragment, and the vaccine has been put into clinical trial. One of the biggest difficulties in malaria vaccine research is the lack of cheap and effective animal models. Although South American night monkeys can infect Plasmodium falciparum as an animal model for vaccine evaluation, night monkeys are not natural hosts of Plasmodium falciparum. In recent years, Plasmodium transgenic technology has become more and more mature, and has been widely used to study the structure and function of Plasmodium protein, interaction with host, gene expression regulation and so on. According to the experimental studies of allelic substitution, some important functional proteins in different species of Plasmodium, such as TRAP,MSP1, are conservative in function, although they differ greatly in sequence. After homologous replacement, Plasmodium can still complete its life cycle. According to this characteristic, this study took Plasmodium berghei (Plasmudium berghei,Pb) as experimental object, established the gene transfection technology of Plasmodium falciparum with PfMSP1-19 instead of endogenous PbMSP1-19, and produced the Pb. expressing PfMSP1-19 transgenic through the gene targeting technology of Plasmodium falciparum. Because the transgenic Plasmodium berghei contains PfMSP1-19 component, this transgenic mouse Plasmodium falciparum is used to evaluate the immune protection effect of PfCP-2.9 vaccine in vivo. Primers were designed to amplify the target gene fragments from the genomic PCR of Pb and Pf. 1.4Kb fragment and 500bp fragment of PbMSP-13'UTR from merozoite surface protein 1 (PbMSP-1) of Plasmodium berghei were obtained from Pb and 330bp fragment of PfMSP-1 C-terminal 19KDa from Pf. The PbfMSP1 fusion gene was formed by ligating the 1.4Kb of PbMSP-1 with the fragment of 330bp of Pf. PbfMSP1
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R-332

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