本文選題：弓形蟲 + ROP18基因　； 參考：《山東大學(xué)》2012年碩士論文

【摘要】：弓形蟲是一種專性細(xì)胞內(nèi)寄生機(jī)會(huì)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(dòng)物。人類對(duì)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(dǎo)致畸胎、死胎。弓形蟲病也是最常見的機(jī)會(huì)性寄生蟲病,該病成為免疫功能嚴(yán)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(hù)性研究已逐漸引起了重視,并認(rèn)為疫苗接種可能是預(yù)防弓形蟲病的最佳策略。 大量的動(dòng)物和人體實(shí)驗(yàn)表明,DNA疫苗不僅可以預(yù)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(jì)的方法。由于弓形蟲生活史較復(fù)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(guān)弓形蟲疫苗的過程中,發(fā)展多種抗原組合、針對(duì)不同生活史發(fā)育階段的復(fù)合多價(jià)疫苗是研究弓形蟲疫苗過程中的一個(gè)發(fā)展方向與共識(shí),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(xì)胞的信號(hào)傳導(dǎo),阻止宿主細(xì)胞凋亡,改變感染細(xì)胞的基因表達(dá),介導(dǎo)受感染細(xì)胞出現(xiàn)病理損傷。在最新研究中,科學(xué)家們發(fā)現(xiàn)當(dāng)弓形蟲進(jìn)入宿主細(xì)胞時(shí),會(huì)生成一層保護(hù)膜包裹自身,使自己雖受細(xì)胞環(huán)境影響而不被殺死,ROP18的作用則是讓宿主細(xì)胞某些蛋白失效而無法破壞這層保護(hù)膜。 微線體蛋白2(microneme protein2, MIC2)屬于血小管反應(yīng)蛋白相關(guān)匿名蛋白家族,是蟲體入侵宿主細(xì)胞過程中一個(gè)必需的入侵因子,并在弓形蟲連接到宿主細(xì)胞的過程中發(fā)揮重要作用。然而,至今還未有研究對(duì)ROP18、MIC2作為抗弓形蟲病疫苗的潛力進(jìn)行評(píng)估和檢測。 本文利用一新型真核表達(dá)載體pBudCE4.1,此含有人巨細(xì)胞病毒(CMV)高效啟動(dòng)子、增強(qiáng)子序列,是一種外源基因在哺乳動(dòng)物內(nèi)高效表達(dá)的良好載體。該載體的特點(diǎn)是含有兩側(cè)分別有一些可插入的酶切位點(diǎn),而且兩側(cè)分別都有各自的啟動(dòng)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(dá)兩種蛋白,以檢測蛋白在小鼠體內(nèi)誘導(dǎo)的免疫原性和安全性,并通過動(dòng)物攻擊實(shí)驗(yàn),評(píng)價(jià)其免疫保護(hù)性。結(jié)果表明ROP18-MIC2雙基因疫苗的效果顯著優(yōu)于ROP18或者M(jìn)IC2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(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】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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

，

本文編號(hào)：1886460