CpG免疫刺激序列增強弓形蟲速殖子復合基因疫苗免疫保護作用的研究
發(fā)布時間：2018-11-02 17:11
【摘要】： 剛地弓形蟲(Toxoplasma gondii)是一種專性細胞內(nèi)寄生的機會性致病原蟲,其生活史及致病機理復雜,人類對弓形蟲普遍易感,孕婦感染弓形蟲后可導致胎兒先天性弓形蟲病,嚴重可致畸胎,死胎,在AIDS,惡性腫瘤等免疫力低下患者中,弓形蟲作為一種機會性感染因子,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,弓形蟲病的預防尤為重要。 對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疫苗可同時誘導細胞免疫和體液免疫,但其誘導免疫反應的效率相對較低,尤其對于大型動物種系和人類。為提高DNA疫苗誘導的免疫反應的強度,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細胞,誘導其增殖并抑制其凋亡,并能促進免疫球蛋白和IL-6, IL-10, IL-12等細胞因子分泌。在寄生蟲等感染性疾病中,CpG序列可在動物體內(nèi)激發(fā)強烈的Th1型相關(guān)反應,在寄生蟲病預防的研究中,CpG的應用也不乏成功的例子。由于質(zhì)粒DNA分為兩個明顯的功能單位,一個是轉(zhuǎn)錄單元指導抗原表達,一個是DNA骨架中的佐劑單元(CpG基序),誘導免疫反應激活。在質(zhì)粒DNA骨架中適量增加CpG基序數(shù)量,可增強DNA疫苗的免疫效應。 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%,是誘導宿主免疫應答的主要靶抗原。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,具有高度的保守性和免疫原性。SAG1和ROP2在蟲體入侵宿主細胞的過程中有相互促進作用,其相應的抗體可有效拮抗弓形蟲的感染。 本研究根據(jù)已報道對小鼠有免疫刺激作用的免疫刺激序列,設(shè)計并合成一段含三對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)于對照組,且以細胞免疫為主。CpG基序作為內(nèi)源性佐劑可增強免疫效應。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前10條
1 郭桐生,馮強,郭剛,解慶華,鄒全明;CpG基序?qū)τ拈T螺桿菌疫苗的免疫佐劑效應實驗研究[J];第三軍醫(yī)大學學報;2004年03期
2 曾政,鄧小紅,于三科;CpG-DNA的功能及應用前景[J];動物醫(yī)學進展;2004年04期
3 盧汀;劉倩;喬代蓉;;免疫佐劑作用機制及CpG ODN作為新型佐劑的研究[J];生命科學研究;2006年S1期
4 李娜;孫志偉;俞煒源;;CpG免疫刺激DNA序列及其在疫苗佐劑中的應用[J];生物技術(shù)通訊;2008年04期
5 李文波,姚志強,周永興;CpG DNA的免疫學活性在疾病防治中的應用[J];細胞與分子免疫學雜志;2000年04期
6 張正姬;CpG DNA在寄生蟲感染免疫中的作用[J];中國血吸蟲病防治雜志;2003年06期
7 孫玉紅,朱朝敏,謝堯;含CpG基序的寡脫氧核苷酸聯(lián)合乙型肝炎表面抗原免疫轉(zhuǎn)基因小鼠的實驗研究[J];中華肝臟病雜志;2005年01期
8 李俊華;吳少庭;翁亞彪;;弓形蟲疫苗及其保護性研究現(xiàn)狀[J];中國病原生物學雜志;2006年01期
9 楊翠萍;萬紅嬌;蔡長春;;弓形蟲疫苗研究的現(xiàn)狀與展望[J];中國病原生物學雜志;2006年03期
10 包安裕;王惠玲;蔣明森;;弓形蟲與宿主行為——微妙的關(guān)系[J];中國病原生物學雜志;2007年05期
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