弓形虫鸡尾酒DNA疫苗的免疫保护作用

发布时间：2018-06-04 06:01

本文选题：弓形虫 + GRA1　；参考：《福建医科大学》2010年博士论文

【摘要】： 弓形虫疫苗研制经历了全虫疫苗、虫体特异组分疫苗、基因工程疫苗、DNA疫苗4个发展过程。但到目前为至,仅有的疫苗是一种活的速殖子S48的减毒株,作为用于羊弓形虫病的商品化疫苗,并没有可用于人的弓形虫疫苗。越来越多的证据表明弓形虫生活期特异的免疫只能诱导局限于该期的保护,较多研究认为多抗原DNA疫苗提供较好的抗弓形虫病保护并且优于单一抗原DNA疫苗。组合多种弓形虫抗原基因的DNA疫苗成为一种主要的研究策略。免疫佐剂的加入可能显著增强原有组合疫苗的免疫效果。当前已有被研究应用为弓形虫DNA疫苗的候选抗原基因主要有:弓形虫膜表面抗原(SAG)、致密颗粒抗原(GRA)、棒状体蛋白(ROP)、微线体蛋白等。在这个思路下,我们构建了包括候选抗原基因GRA1、SAG1、ROP2、AMA1的DNA疫苗,并加入佐剂IL-12的质粒载体,以期取得较好的小鼠受弓形虫感染时的保护效果和免疫效应。一、弓形虫GRA1、SAG1、ROP2、AMA1鸡尾酒DNA疫苗的构建和鉴定根据本课题组先期的研究结果,弓形虫DNA疫苗候选基因GRA1和SAG1具有部分的抗弓形虫感染保护作用和免疫效应。他人有关研究认为候选基因ROP2及AMA1也具有这种作用。本研究成功构建了用于组合弓形虫鸡尾酒DNA疫苗的重组质粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1。成功构建用于检测蛋白表达的质粒pVAX1-ROP2His、pVAX1-AMA1His。将pVAX1-ROP2His、pVAX1-AMA1His转染巨噬细胞后,用Western Blot方法检测到了蛋白的表达。pVAX1-GRA1、pVAX1-SAG1的蛋白表达能力先期本课题组已应用免疫细胞化学染色方法检测验证,本次所构建pVAX1-GRA1、pVAX1-SAG1序列与先期相应重组质粒序列完全一致。将美国Alexander Rakhmilevich博士惠赠的pNGVL3-mIL12重组质粒转染巨噬细胞后,用ELISA方法检测到了蛋白的表达。二、重组质粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1、pNGVL3-mIL12的免疫效果不同的鼠系对于弓形虫的敏感性不同,C3H、BALB/c、C57BL/6等小鼠是常用于弓形虫DNA疫苗研究的鼠系,C3H/He (H-2k)是一种对弓形虫感染中等易感的鼠系。本研究选用C3H/He (H-2k)小鼠作为免疫效果观察动物,期望能够观察到一种较长期的免疫保护存活的效果。早期的弓形虫DNA疫苗研究中往往使用较大剂量的弓形虫进行免疫后小鼠受攻击保护效果的评价,较多的研究使用104及更高剂量的弓形虫RH株速殖子,有研究认为104的剂量较高。这些研究往往都较难以观察到存活时间较长的数据,即可以计算存活率的数据。近期来,弓形虫疫苗研究时,弓形虫的感染攻击剂量已从最早期的105降至104、500直至最新报道的使用50个弓形虫RH株滋养体,最初的延长生存时间变成了存活率指标。 Quan Liu等只用了50个弓形虫RH株的滋养体攻击BALB/c小鼠,BALB/c小鼠又是较C3H小鼠对弓形虫的抵抗力更强的鼠系,我们的研究使用500个弓形虫RH株速殖子的攻击剂量,本研究结果C3H小鼠受保护的存活率不如最近期的Quan Liu等的研究,可能弓形虫攻击剂量仍然较高。本次研究构建的DNA疫苗pVAX1 - GRA1 + SAG1 + ROP2 + AMA1实验组在C3H小鼠中取得了较好的保护效果,其受弓形虫攻击时生存时间较较少基因pVAX1-ROP2+AMA1组生存时间延长。该种保护又由于IL-12免疫佐剂的应用得到了提高,以弓形虫RH株攻击时观察到了生存时间的较大延长,该实验组10只小鼠受弓形虫攻击时观察到了1只小鼠存活达30天以上。经鸡尾酒DNA疫苗免疫小鼠血清抗弓形虫(TLA)IgG高于对照组,加免疫佐剂pNGVL3-mIL12组检测到IL-12水平的显著升高,实验组pVAX1- ROP2+AMA1的IFN-γ水平高于pVAX1对照组,更多基因组又较少基因组的IFN-γ水平升高,IL-12佐剂递次诱导了更高IFN-γ水平。实验组与对照组之间的IL-4水平没有显著差异。实验组均较pVAX1对照组淋巴细胞增殖反应更高。pVAX1-GRA1+SAG1+ROP2+AMA1+pNGVL3-mIL12基因免疫组CD4+/CD8+淋巴细胞比值降低。说明该鸡尾酒DNA疫苗诱导了C3H小鼠的特异性免疫应答。结果表明,该种鸡尾酒DNA疫苗具有良好的免疫保护性,复合免疫的方式增强了免疫效果,这种效果可因免疫佐剂的使用而得到递次提高。
[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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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