本文選題：弓形蟲 + ROP18基因�。� 參考：《山東大學》2012年碩士論文

【摘要】：弓形蟲是一種專性細胞內(nèi)寄生機會致病原蟲,分布廣泛,可感染包括人類在內(nèi)的大部分哺乳動物。人類對弓形蟲普遍易感,孕婦感染后可通過胎盤垂直傳染給胎兒,導致畸胎、死胎。弓形蟲病也是最常見的機會性寄生蟲病,該病成為免疫功能嚴重低下患者(如HIV/AIDS、惡性腫瘤患者)的主要死亡原因。迄今尚無治療弓形蟲病的特效藥物,弓形蟲疫苗和免疫保護性研究已逐漸引起了重視,并認為疫苗接種可能是預防弓形蟲病的最佳策略。 大量的動物和人體實驗表明,DNA疫苗不僅可以預防,還可以治療病原生物的感染。在弓形蟲感染和包囊形成的防治過程中,疫苗是一種潛在高效經(jīng)濟的方法。由于弓形蟲生活史較復雜,抗原成分具有發(fā)育階段的特異性或差異性,因此在研究有關弓形蟲疫苗的過程中,發(fā)展多種抗原組合、針對不同生活史發(fā)育階段的復合多價疫苗是研究弓形蟲疫苗過程中的一個發(fā)展方向與共識,這有助于克服單一抗原成分作為候選疫苗的不足。 棒狀體蛋白在蟲體入侵和納蟲空泡的形成和修飾過程中起著重要作用,是重要的入侵和毒力作用因子。棒狀體蛋白18(rhoptry protein18, ROP18)可以改變受感染的宿主細胞的信號傳導,阻止宿主細胞凋亡,改變感染細胞的基因表達,介導受感染細胞出現(xiàn)病理損傷。在最新研究中,科學家們發(fā)現(xiàn)當弓形蟲進入宿主細胞時,會生成一層保護膜包裹自身,使自己雖受細胞環(huán)境影響而不被殺死,ROP18的作用則是讓宿主細胞某些蛋白失效而無法破壞這層保護膜。 微線體蛋白2(microneme protein2, MIC2)屬于血小管反應蛋白相關匿名蛋白家族,是蟲體入侵宿主細胞過程中一個必需的入侵因子,并在弓形蟲連接到宿主細胞的過程中發(fā)揮重要作用。然而,至今還未有研究對ROP18、MIC2作為抗弓形蟲病疫苗的潛力進行評估和檢測。 本文利用一新型真核表達載體pBudCE4.1,此含有人巨細胞病毒(CMV)高效啟動子、增強子序列,是一種外源基因在哺乳動物內(nèi)高效表達的良好載體。該載體的特點是含有兩側(cè)分別有一些可插入的酶切位點,而且兩側(cè)分別都有各自的啟動子。將ROP18與MIC2構(gòu)建在此載體上,并作為DNA疫苗直接注射免疫小鼠來表達兩種蛋白,以檢測蛋白在小鼠體內(nèi)誘導的免疫原性和安全性,并通過動物攻擊實驗,評價其免疫保護性。結(jié)果表明ROP18-MIC2雙基因疫苗的效果顯著優(yōu)于ROP18或者MIC2單基因疫苗。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a parasitic opportunistic protozoa, which can infect most mammals, including humans. Human is susceptible to Toxoplasma gondii, pregnant women can be infected through the placenta vertical transmission to the fetus, resulting in teratogenesis, stillbirth. Toxoplasma gondii is also the most common opportunistic parasitic disease, which is the leading cause of death in patients with severe hypoimmunity (e.g. HIV / AIDS, malignant tumor). Up to now, there is no special drug to treat Toxoplasma gondii. Toxoplasma gondii vaccine and immune protection have been paid more and more attention, and it is considered that vaccination may be the best strategy to prevent Toxoplasma gondii from Toxoplasma gondii. A large number of animal and human experiments have shown that DNA vaccine can not only prevent, but also treat the infection of pathogenic organisms. Vaccine is a potentially efficient and economical method in the prevention and treatment of Toxoplasma gondii infection and cyst formation. Because the life history of Toxoplasma gondii is complicated and the antigen components have the specificity or difference of development stage, many kinds of antigen combinations have been developed in the research of Toxoplasma gondii vaccine. Multivalent vaccine for different stages of life cycle development is a development direction and consensus in the research of Toxoplasma gondii vaccine, which is helpful to overcome the shortage of single antigen as candidate vaccine. Rodlike proteins play an important role in the formation and modification of vacuoles and play an important role in invading and virulence. 18(rhoptry protein 18 (ROP18) can change the signal transduction of infected host cells, block the apoptosis of host cells, change the gene expression of infected cells, and mediate the pathological damage of infected cells. In a new study, scientists found that when Toxoplasma gondii enters the host cell, it forms a protective film that wraps itself. The effect of not killing ROP18, though affected by the cellular environment, is to invalidate certain proteins in the host cell and fail to destroy the protective membrane. Microline protein 2(microneme protein 2 (MIC2) belongs to the blood tubule reaction protein-associated anonymous protein family. It is an essential invading factor during the invasion of host cells and plays an important role in the process of connecting Toxoplasma gondii to host cells. However, no research has been done to evaluate and test the potential of ROP18 MIC2 as a vaccine against toxoplasmosis. In this paper, a new eukaryotic expression vector pBudCE4.1, which contains human cytomegalovirus (CMV) promoter and enhancer sequence, is a good vector for high expression of exogenous gene in mammals. The characteristic of the vector is that there are some insertable endonuclease sites on both sides of the vector, and each side has its own promoter. ROP18 and MIC2 were constructed on this vector and immunized mice with DNA vaccine directly to express the two proteins to detect the immunogenicity and safety of the proteins induced in mice and to evaluate their immune protection by animal attack experiments. The results showed that the effect of ROP18-MIC2 double gene vaccine was better than that of ROP18 or MIC2 single gene vaccine.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

【參考文獻】

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1 張鳳珍;李景峰;;弓形蟲疫苗研究進展[J];畜牧獸醫(yī)科技信息;2010年07期

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本文編號：1886460