CpG免疫刺激序列增強(qiáng)弓形蟲速殖子復(fù)合基因疫苗免疫保護(hù)作用的研究
發(fā)布時(shí)間：2018-11-02 17:11
【摘要】： 剛地弓形蟲(Toxoplasma gondii)是一種專性細(xì)胞內(nèi)寄生的機(jī)會(huì)性致病原蟲,其生活史及致病機(jī)理復(fù)雜,人類對(duì)弓形蟲普遍易感,孕婦感染弓形蟲后可導(dǎo)致胎兒先天性弓形蟲病,嚴(yán)重可致畸胎,死胎,在AIDS,惡性腫瘤等免疫力低下患者中,弓形蟲作為一種機(jī)會(huì)性感染因子,是引發(fā)致死的病因之一。目前尚無有效治療弓形蟲病的藥物。因此,弓形蟲病的預(yù)防尤為重要。 對(duì)弓形蟲疫苗的研究經(jīng)歷了死疫苗、減毒活疫苗、基因工程疫苗以及DNA疫苗,DNA疫苗可同時(shí)誘導(dǎo)細(xì)胞免疫和體液免疫,但其誘導(dǎo)免疫反應(yīng)的效率相對(duì)較低,尤其對(duì)于大型動(dòng)物種系和人類。為提高DNA疫苗誘導(dǎo)的免疫反應(yīng)的強(qiáng)度,構(gòu)建基因佐劑成為一種替代方法。免疫刺激序列為非甲基化的短核苷酸重復(fù)序列,即CpG基序,是目前廣被接受的一種基因佐劑,CpG基序在細(xì)菌,病毒的DNA中普遍存在,研究表明,人工合成的非甲基化CpG基序同樣能通過Toll樣受體9直接活化B細(xì)胞,誘導(dǎo)其增殖并抑制其凋亡,并能促進(jìn)免疫球蛋白和IL-6, IL-10, IL-12等細(xì)胞因子分泌。在寄生蟲等感染性疾病中,CpG序列可在動(dòng)物體內(nèi)激發(fā)強(qiáng)烈的Th1型相關(guān)反應(yīng),在寄生蟲病預(yù)防的研究中,CpG的應(yīng)用也不乏成功的例子。由于質(zhì)粒DNA分為兩個(gè)明顯的功能單位,一個(gè)是轉(zhuǎn)錄單元指導(dǎo)抗原表達(dá),一個(gè)是DNA骨架中的佐劑單元(CpG基序),誘導(dǎo)免疫反應(yīng)激活。在質(zhì)粒DNA骨架中適量增加CpG基序數(shù)量,可增強(qiáng)DNA疫苗的免疫效應(yīng)。 SAG1抗原作為弓形蟲速殖子期特異的主要表面抗原之一,具有高度的免疫原性和免疫保護(hù)性,是弓形蟲感染免疫診斷和疫苗開發(fā)的的主要候選抗原。SAG1蛋白分布于弓形蟲速殖子表膜、速殖子內(nèi)以及納蟲泡的管狀結(jié)構(gòu)中,約占弓形蟲體總蛋白的3-5%,卻可抑制患者血清中抗體活性的50%,是誘導(dǎo)宿主免疫應(yīng)答的主要靶抗原。ROP2蛋白是弓形蟲棒狀體分泌的一種蛋白,主要協(xié)助蟲體入侵宿主細(xì)胞,在弓形蟲生活史的速殖子期、緩殖子期和子孢子期中均有表達(dá),具有高度的保守性和免疫原性。SAG1和ROP2在蟲體入侵宿主細(xì)胞的過程中有相互促進(jìn)作用,其相應(yīng)的抗體可有效拮抗弓形蟲的感染。 本研究根據(jù)已報(bào)道對(duì)小鼠有免疫刺激作用的免疫刺激序列,設(shè)計(jì)并合成一段含三對(duì)免疫刺激序列的核苷酸,作為內(nèi)源性佐劑插入質(zhì)粒pcDNA3.1中,構(gòu)建質(zhì)粒pcDNA3.1/GpG作為DNA疫苗載體,再與弓形蟲抗原基因片段SAG1-ROP2相連,以重組質(zhì)粒肌肉注射免疫小鼠,觀察基因疫苗與佐劑的免疫效果。結(jié)果表明,增加CpG基序的基因疫苗的免疫效果優(yōu)于對(duì)照組,且以細(xì)胞免疫為主。CpG基序作為內(nèi)源性佐劑可增強(qiáng)免疫效應(yīng)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：2306424
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
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