CpG免疫刺激序列增强弓形虫速殖子复合基因疫苗免疫保护作用的研究

发布时间：2018-11-02 17:11

【摘要】： 刚地弓形虫(Toxoplasma gondii)是一种专性细胞内寄生的机会性致病原虫,其生活史及致病机理复杂,人类对弓形虫普遍易感,孕妇感染弓形虫后可导致胎儿先天性弓形虫病,严重可致畸胎,死胎,在AIDS,恶性肿瘤等免疫力低下患者中,弓形虫作为一种机会性感染因子,是引发致死的病因之一。目前尚无有效治疗弓形虫病的药物。因此,弓形虫病的预防尤为重要。对弓形虫疫苗的研究经历了死疫苗、减毒活疫苗、基因工程疫苗以及DNA疫苗,DNA疫苗可同时诱导细胞免疫和体液免疫,但其诱导免疫反应的效率相对较低,尤其对于大型动物种系和人类。为提高DNA疫苗诱导的免疫反应的强度,构建基因佐剂成为一种替代方法。免疫刺激序列为非甲基化的短核苷酸重复序列,即CpG基序,是目前广被接受的一种基因佐剂,CpG基序在细菌,病毒的DNA中普遍存在,研究表明,人工合成的非甲基化CpG基序同样能通过Toll样受体9直接活化B细胞,诱导其增殖并抑制其凋亡,并能促进免疫球蛋白和IL-6, IL-10, IL-12等细胞因子分泌。在寄生虫等感染性疾病中,CpG序列可在动物体内激发强烈的Th1型相关反应,在寄生虫病预防的研究中,CpG的应用也不乏成功的例子。由于质粒DNA分为两个明显的功能单位,一个是转录单元指导抗原表达,一个是DNA骨架中的佐剂单元(CpG基序),诱导免疫反应激活。在质粒DNA骨架中适量增加CpG基序数量,可增强DNA疫苗的免疫效应。 SAG1抗原作为弓形虫速殖子期特异的主要表面抗原之一,具有高度的免疫原性和免疫保护性,是弓形虫感染免疫诊断和疫苗开发的的主要候选抗原。SAG1蛋白分布于弓形虫速殖子表膜、速殖子内以及纳虫泡的管状结构中,约占弓形虫体总蛋白的3-5%,却可抑制患者血清中抗体活性的50%,是诱导宿主免疫应答的主要靶抗原。ROP2蛋白是弓形虫棒状体分泌的一种蛋白,主要协助虫体入侵宿主细胞,在弓形虫生活史的速殖子期、缓殖子期和子孢子期中均有表达,具有高度的保守性和免疫原性。SAG1和ROP2在虫体入侵宿主细胞的过程中有相互促进作用,其相应的抗体可有效拮抗弓形虫的感染。本研究根据已报道对小鼠有免疫刺激作用的免疫刺激序列,设计并合成一段含三对免疫刺激序列的核苷酸,作为内源性佐剂插入质粒pcDNA3.1中,构建质粒pcDNA3.1/GpG作为DNA疫苗载体,再与弓形虫抗原基因片段SAG1-ROP2相连,以重组质粒肌肉注射免疫小鼠,观察基因疫苗与佐剂的免疫效果。结果表明,增加CpG基序的基因疫苗的免疫效果优于对照组,且以细胞免疫为主。CpG基序作为内源性佐剂可增强免疫效应。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a kind of opportunistic protozoa parasitic in specific cells. Its life history and pathogenic mechanism are complex. Human beings are generally susceptible to Toxoplasma gondii. Pregnant women infected with Toxoplasma gondii can lead to congenital toxoplasmosis of the fetus. Toxoplasma gondii, as an opportunistic infection factor, is one of the causes of death in patients with severe teratogenesis, stillbirth and low immunity, such as malignant tumor of AIDS,. There is no effective drug for Toxoplasma gondii. Therefore, the prevention of toxoplasmosis is particularly important. The research on Toxoplasma gondii vaccine has experienced death vaccine, live attenuated vaccine, genetic engineering vaccine and DNA vaccine. DNA vaccine can induce both cellular and humoral immunity, but the efficiency of inducing immune response is relatively low. Especially for large animal species and humans. In order to enhance the intensity of immune response induced by DNA vaccine, the construction of gene adjuvant has become an alternative method. The immunostimulatory sequence is a non-methylated short nucleotide repeat sequence (CpG motif), which is a widely accepted gene adjuvant. CpG motifs are ubiquitous in the DNA of bacteria and viruses. The synthetic unmethylated CpG motifs can also directly activate B cells through Toll like receptor 9, induce their proliferation and inhibit their apoptosis, and promote the secretion of cytokines such as immunoglobulin and IL-6, IL-10, IL-12. In infectious diseases such as parasites, CpG sequences can stimulate strong Th1 related responses in animals, and the application of CpG in the study of parasitic disease prevention is also successful. The plasmid DNA is divided into two distinct functional units, one is transcriptional unit to guide antigen expression, the other is the adjuvant unit (CpG motif) in the framework of DNA, which induces the activation of immune response. Increasing the number of CpG motifs in plasmid DNA skeleton can enhance the immune effect of DNA vaccine. As one of the major surface antigens of Toxoplasma gondii Tachyzoites, SAG1 antigen has high immunogenicity and immunogenicity. SAG1 protein distributes in Toxoplasma gondii Tachyzoites epidermis, tachyzoites and the tubular structure of nadidia vesicles, and accounts for about 3-5% of Toxoplasma gondii body total protein, which is the main candidate antigen for Toxoplasma gondii infection diagnosis and vaccine development. ROP2 protein is a kind of protein secreted by Toxoplasma gondii rodlike body, which mainly assists the parasite invading host cells, during the tachyzoite stage of Toxoplasma gondii's life cycle. Both the bradyzoite stage and the sporozoite stage were expressed with high conserved and immunogenicity. SAG1 and ROP2 promoted each other during the invasion of host cells, and their corresponding antibodies could effectively antagonize the infection of Toxoplasma gondii (Toxoplasma gondii). In this study, a nucleotide containing three pairs of immunostimulatory sequences was designed and synthesized according to the reported immunostimulatory sequence in mice, which was inserted into plasmid pcDNA3.1 as an endogenous adjuvant. The plasmid pcDNA3.1/GpG was constructed as DNA vaccine vector and then connected with Toxoplasma gondii antigen gene fragment SAG1-ROP2. Mice were immunized with recombinant plasmid intramuscularly to observe the immune effect of gene vaccine and adjuvant. The results showed that the immune effect of the gene vaccine which increased CpG motif was better than that of the control group, and the cellular immunity was dominant. The CpG motif as an endogenous adjuvant could enhance the immune effect.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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