恶性疟疾多表位DNA疫苗M.RCAg-1在不同免疫方案中的免疫原性及其免疫机制的研究

发布时间：2018-01-25 03:56

本文关键词： 恶性疟疾 DNA疫苗基因枪活体电穿孔脂质体肌肉注射免疫原性　出处：《中国协和医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 疟疾是目前世界上对人类危害最严重的蚊媒寄生原虫病。全世界每年有5亿人感染疟原虫，约三百万人死于恶性疟疾，其中80％是五岁以下的儿童和孕妇。恶性疟原虫及蚊媒抗药性的不断增加和蔓延，使得疟疾的防治成为世界性难题之一。疟原虫生活周期复杂，抗原具有阶段特异性、虫株特异性和高度变异性等特点，长期以来形成研制抗疟原虫疫苗的技术瓶颈。因此，针对恶性疟原虫不同生活时期的免疫反应特点，选取诱导产生多种免疫反应类型的不同抗原构建多表位疫苗已经成为研究抗疟疫苗的热点。本课题组在前期工作中借鉴了分子育种技术(即DNA改组)的随机重组原理，建立了表位改组技术并构建了多表位基因库，用库免疫血清筛选出高抗原性的阳性克隆，经实验证实其中VR312(后改名为M．RCAg-1)基因克隆及其编码蛋白不仅能在小鼠模型中诱导交叉免疫保护，而且能在家兔模型中诱导抑制抗恶性疟原虫生长的抗体。由于蛋白质疫苗成本昂贵，不适宜在非洲等落后地区推广使用；单一DNA疫苗的免疫原性较低，难以达到预期的免疫反应强度和令人满意的免疫保护效果。因此本研究试图采用不同的免疫策略以观察对DNA疫苗免疫原性的影响，并分析其免疫反应机制，重点观察了基因枪免疫中不同粒径金颗粒对免疫佐剂效应的影响。初步证实将恶性疟疾多表位DNA疫苗M．RCAg-1D通过基因枪皮内免疫小鼠，能够获得高滴度特异性抗体水平，诱生明显的CD4~+T细胞反应，而且在用P．yoelii攻击时能明显保护小鼠延长其存活时间。提示基因枪皮内免疫能够显著提高基因疫苗的免疫原性，这可能与其提高外源基因的转染及表达有关。与此同时，应用活体电穿孔免疫小鼠，也能够获得较高滴度特异性抗体水平和诱生明显的CD4~+T细胞反应，但是不能对P．yoelii产生交叉保护。而脂质体混合基因疫苗M．RCAg-1DDNA后肌肉注射所诱导产生的免疫原性尽管显著高于裸DNA肌肉注射和裸DNA皮下注射，但是诱导产生的抗体不能对P．yoelii产生交叉免疫保护。基因枪免疫时诱导产生的抗体水平受到金粉粒径的影响，其中1.0μm金颗粒作为质粒DNA载体进行基因免疫时，在小鼠体内诱导产生的特异性抗体水平最高。以上工作对DNA疫苗的应用和改进均提供了有价值的实验依据。
[Abstract]:Malaria is the most serious mosquito - borne protozoan disease in the world . More than 500 million people are infected with Plasmodium falciparum every year , about 3 million people die from malignant malaria , and 80 percent of them are children and pregnant women under the age of five . Based on the principle of random recombination of molecular breeding technology ( i.e . , DNA shuffling ) in the previous work , this group established the epitope shuffling technique and constructed a multi - epitope gene pool , screened the positive clones with high antigenicity by using the library immune serum , and confirmed the VR312 ( renamed as M ) . RCAg - 1 ( RCAg - 1 ) gene clone and its encoded protein not only induce cross - immune protection in mouse model , but also induce anti - P . falciparum growth antibody in rabbit model . Because the cost of protein vaccine is expensive , it is not suitable to be popularized in such areas as Africa and so on . The immunogenicity of single DNA vaccine is low , it is difficult to achieve the expected immune response intensity and the satisfactory immune protection effect . Therefore , the present study attempts to adopt different immune strategies to observe the effect of different particle size gold particles on the immune adjuvant effect of DNA vaccine . RCAg - 1D was able to obtain high titer specific antibody level and induce obvious CD4 ~ + T cell reaction . It was suggested that the immunity of gene vaccine could significantly improve the immunogenicity of the gene vaccine , which could enhance the immunogenicity of the gene vaccine . At the same time , it was possible to obtain high titer specific antibody level and induce obvious CD4 ~ + T cell response when challenged with P.yoelii , but it was not possible to cross - protect P.yoelii . The antibody level induced by intramuscular injection after RCAg - 1DDNA was significantly higher than that of naked DNA intramuscular injection and naked DNA injection , but the antibody produced by induction could not cross - immune protection against P.yoelii . The antibody level induced by induction of gene gun was affected by the particle size of gold powder .

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【引证文献】