本文選題：弓形蟲 + GRA1��； 參考：《福建醫(yī)科大學》2010年博士論文

【摘要】： 弓形蟲疫苗研制經(jīng)歷了全蟲疫苗、蟲體特異組分疫苗、基因工程疫苗、DNA疫苗4個發(fā)展過程。但到目前為至,僅有的疫苗是一種活的速殖子S48的減毒株,作為用于羊弓形蟲病的商品化疫苗,并沒有可用于人的弓形蟲疫苗。越來越多的證據(jù)表明弓形蟲生活期特異的免疫只能誘導局限于該期的保護,較多研究認為多抗原DNA疫苗提供較好的抗弓形蟲病保護并且優(yōu)于單一抗原DNA疫苗。組合多種弓形蟲抗原基因的DNA疫苗成為一種主要的研究策略。免疫佐劑的加入可能顯著增強原有組合疫苗的免疫效果。當前已有被研究應(yīng)用為弓形蟲DNA疫苗的候選抗原基因主要有:弓形蟲膜表面抗原(SAG)、致密顆�？乖�(GRA)、棒狀體蛋白(ROP)、微線體蛋白等。在這個思路下,我們構(gòu)建了包括候選抗原基因GRA1、SAG1、ROP2、AMA1的DNA疫苗,并加入佐劑IL-12的質(zhì)粒載體,以期取得較好的小鼠受弓形蟲感染時的保護效果和免疫效應(yīng)。 一、弓形蟲GRA1、SAG1、ROP2、AMA1雞尾酒DNA疫苗的構(gòu)建和鑒定 根據(jù)本課題組先期的研究結(jié)果,弓形蟲DNA疫苗候選基因GRA1和SAG1具有部分的抗弓形蟲感染保護作用和免疫效應(yīng)。他人有關(guān)研究認為候選基因ROP2及AMA1也具有這種作用。本研究成功構(gòu)建了用于組合弓形蟲雞尾酒DNA疫苗的重組質(zhì)粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1。成功構(gòu)建用于檢測蛋白表達的質(zhì)粒pVAX1-ROP2His、pVAX1-AMA1His。 將pVAX1-ROP2His、pVAX1-AMA1His轉(zhuǎn)染巨噬細胞后,用Western Blot方法檢測到了蛋白的表達。pVAX1-GRA1、pVAX1-SAG1的蛋白表達能力先期本課題組已應(yīng)用免疫細胞化學染色方法檢測驗證,本次所構(gòu)建pVAX1-GRA1、pVAX1-SAG1序列與先期相應(yīng)重組質(zhì)粒序列完全一致。將美國Alexander Rakhmilevich博士惠贈的pNGVL3-mIL12重組質(zhì)粒轉(zhuǎn)染巨噬細胞后,用ELISA方法檢測到了蛋白的表達。 二、重組質(zhì)粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1、pNGVL3-mIL12的免疫效果 不同的鼠系對于弓形蟲的敏感性不同,C3H、BALB/c、C57BL/6等小鼠是常用于弓形蟲DNA疫苗研究的鼠系,C3H/He (H-2k)是一種對弓形蟲感染中等易感的鼠系。本研究選用C3H/He (H-2k)小鼠作為免疫效果觀察動物,期望能夠觀察到一種較長期的免疫保護存活的效果。 早期的弓形蟲DNA疫苗研究中往往使用較大劑量的弓形蟲進行免疫后小鼠受攻擊保護效果的評價,較多的研究使用104及更高劑量的弓形蟲RH株速殖子,有研究認為104的劑量較高。這些研究往往都較難以觀察到存活時間較長的數(shù)據(jù),即可以計算存活率的數(shù)據(jù)。近期來,弓形蟲疫苗研究時,弓形蟲的感染攻擊劑量已從最早期的105降至104、500直至最新報道的使用50個弓形蟲RH株滋養(yǎng)體,最初的延長生存時間變成了存活率指標。 Quan Liu等只用了50個弓形蟲RH株的滋養(yǎng)體攻擊BALB/c小鼠,BALB/c小鼠又是較C3H小鼠對弓形蟲的抵抗力更強的鼠系,我們的研究使用500個弓形蟲RH株速殖子的攻擊劑量,本研究結(jié)果C3H小鼠受保護的存活率不如最近期的Quan Liu等的研究,可能弓形蟲攻擊劑量仍然較高。 本次研究構(gòu)建的DNA疫苗pVAX1 - GRA1 + SAG1 + ROP2 + AMA1實驗組在C3H小鼠中取得了較好的保護效果,其受弓形蟲攻擊時生存時間較較少基因pVAX1-ROP2+AMA1組生存時間延長。該種保護又由于IL-12免疫佐劑的應(yīng)用得到了提高,以弓形蟲RH株攻擊時觀察到了生存時間的較大延長,該實驗組10只小鼠受弓形蟲攻擊時觀察到了1只小鼠存活達30天以上。 經(jīng)雞尾酒DNA疫苗免疫小鼠血清抗弓形蟲(TLA)IgG高于對照組,加免疫佐劑pNGVL3-mIL12組檢測到IL-12水平的顯著升高,實驗組pVAX1- ROP2+AMA1的IFN-γ水平高于pVAX1對照組,更多基因組又較少基因組的IFN-γ水平升高,IL-12佐劑遞次誘導了更高IFN-γ水平。實驗組與對照組之間的IL-4水平?jīng)]有顯著差異。實驗組均較pVAX1對照組淋巴細胞增殖反應(yīng)更高。pVAX1-GRA1+SAG1+ROP2+AMA1+pNGVL3-mIL12基因免疫組CD4+/CD8+淋巴細胞比值降低。說明該雞尾酒DNA疫苗誘導了C3H小鼠的特異性免疫應(yīng)答。 結(jié)果表明,該種雞尾酒DNA疫苗具有良好的免疫保護性,復合免疫的方式增強了免疫效果,這種效果可因免疫佐劑的使用而得到遞次提高。
[Abstract]:The development of Toxoplasma gondii vaccine has experienced 4 development processes: whole insect vaccine, worm specific vaccine, gene engineering vaccine, and DNA vaccine. But to present, the only vaccine is a live tachyzoite S48 strain, as a commercialized vaccine for sheep toxoplasmosis, and is not available for human Toxoplasma vaccine. More and more evidence table The specific immunization of Toxoplasma can only be induced in this period, and many studies suggest that the DNA vaccine provides better protection against toxoplasmosis and is superior to the single antigen DNA vaccine. The DNA vaccine combining multiple Toxoplasma antigen genes is a major research strategy. The addition of immuno adjuvant may be significantly enhanced. The immune effect of the original combined vaccine. The candidate antigens of Toxoplasma DNA vaccine have been used as candidate antigens mainly: Toxoplasma membrane surface antigen (SAG), compact particle antigen (GRA), rod body protein (ROP), and micro body protein. In this way, we have constructed DNA vaccines including candidate antigen genes, GRA1, SAG1, ROP2, AMA1. Adjuvant plasmid IL-12 was added in order to obtain better protective effect and immune effect in mice infected with Toxoplasma gondii.
Construction and identification of DNA vaccine against Toxoplasma gondii GRA1, SAG1, ROP2 and AMA1 Cocktail
According to the research results of the research group, the candidate gene GRA1 and SAG1 of the DNA vaccine of Toxoplasma gondii have some protective and immune effects on the infection of Toxoplasma gondii. Other studies suggest that the candidate gene ROP2 and AMA1 also have this effect. The recombinant plasmid pVAX1-GRA1 for combining Toxoplasma cocktail DNA vaccine is successfully constructed in this study. PVAX1-SAG1, pVAX1-ROP2 and pVAX1-AMA1. successfully constructed plasmid pVAX1-ROP2His, pVAX1-AMA1His. for detecting protein expression.
After transfection of pVAX1-ROP2His and pVAX1-AMA1His to macrophages, the expression of.PVAX1-GRA1 was detected by Western Blot method. The first phase of the protein expression of pVAX1-SAG1 was tested by immunocytochemical staining, and the sequence of pVAX1-GRA1, pVAX1-SAG1 sequence and corresponding recombinant plasmid was completely constructed. After transfection of pNGVL3-mIL12 recombinant plasmid, which was donated by Dr. Alexander Rakhmilevich, the expression of protein was detected by ELISA.
Two, the immune effect of recombinant plasmid pVAX1-GRA1, pVAX1-SAG1, pVAX1-ROP2, pVAX1-AMA1 and pNGVL3-mIL12.
Different mice are sensitive to Toxoplasma, C3H, BALB/c, C57BL/6 and other mice are commonly used in the mice of Toxoplasma DNA vaccine. C3H/He (H-2k) is a moderately susceptible mouse strain of Toxoplasma gondii. This study selects C3H/He (H-2k) mice as an immune observation animal, hoping to observe a longer immune protection. Protect the survival effect.
The early Toxoplasma gondii DNA vaccine is often used to evaluate the effect of an aggressive dose of Toxoplasma gondii in immunized mice. More studies use 104 and higher doses of Toxoplasma gondii RH tachyonus. Studies suggest that the dose of 104 is higher. These studies are often difficult to observe data with longer survival time. In the recent study of the Toxoplasma vaccine, the dose of Toxoplasma gondii has fallen from 105 to 104500 in the earliest period of study until the latest report of the use of 50 Toxoplasma RH trophoblastic plants, and the initial prolongation of survival became a survival index.
Quan Liu and other 50 toxoplasmosis RH strains attacked BALB/c mice and BALB/c mice were more resistant to Toxoplasma than C3H mice. Our study used the attack dose of 500 Toxoplasma gondii strain tachyonus. The results of this study showed that the survival rate of C3H mice was not as good as the most recent Quan Liu. The attack dose of the parasite is still high.
The experimental group of DNA vaccine pVAX1 - GRA1 + SAG1 + ROP2 + AMA1 obtained a better protective effect in the C3H mice. The survival time of the vaccine was longer than that of the less gene pVAX1-ROP2+AMA1 group when the Toxoplasma was attacked. The protection was also improved by the application of the IL-12 immune adjuvant and the time view of the RH strain of Toxoplasma gondii. A larger prolongation of survival time was observed. When the 10 mice were attacked by Toxoplasma gondii, 1 mice survived for more than 30 days.
The serum anti Toxoplasma gondii (TLA) IgG of mice immunized with DNA vaccine was higher than that of the control group, and the IL-12 level of the immune adjuvant pNGVL3-mIL12 group was significantly increased. The IFN- y level of pVAX1- ROP2+AMA1 in the experimental group was higher than that of the pVAX1 control group. The level of IFN- gamma in the more genome and the less genome increased, and the IL-12 adjuvant induced the higher IFN- gamma water. There was no significant difference in the level of IL-4 between the experimental group and the control group. Compared with the pVAX1 control group, the lymphocyte proliferation reaction was higher in the.PVAX1-GRA1+SAG1+ROP2+AMA1+pNGVL3-mIL12 gene immunization group than in the pVAX1 control group, indicating that the DNA vaccine of the cocktail induced the specific immune response of the C3H mice.
The results showed that the DNA vaccine of this kind of cocktail had good immune protection, and the immune effect was enhanced by compound immunization. This effect could be improved by the use of immuno adjuvant.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392

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本文編號：1976273