【摘要】： 弓形蟲是一種分布廣泛的、專性細胞內(nèi)寄生機會致病原蟲,可感染人及多種動物的有核細胞。弓形蟲感染人體后可侵犯多種臟器和組織并可通過母嬰垂直傳播而造成胎兒畸形、神經(jīng)系統(tǒng)發(fā)育障礙、脈絡膜視網(wǎng)膜炎等引起流產(chǎn)、早產(chǎn)甚至死胎。對免疫功能低下者如艾滋病患者是致死主要病因。此外,弓形蟲病也給畜牧業(yè)生產(chǎn)造成嚴重的經(jīng)濟損失。因此,為了有效的防治弓形蟲,疫苗的研制已成為預防及治療弓形蟲病的發(fā)展方向。 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。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。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。SAG2抗原為速殖子膜表面的另一重要抗原,與弓形蟲入侵宿主細胞有密切的關(guān)系,到目前為止,對弓形蟲SAG_2蛋白的功能研究較少,SAG_2蛋白可以使弓形蟲固定于有核細胞的表面,并輔助弓形蟲進入有核細胞,介導弓形蟲速殖子的入侵過程。 同時,為攻克核酸疫苗免疫效力低下的難題,我們選用小鼠白介素12(mIL-12)的真核表達質(zhì)粒作為基因佐劑,以增強弓形蟲復合抗原基因SAG1-ROP2-SAG2的免疫效果。在弓形蟲感染早期IL-12可強烈刺激NK細胞和T細胞分泌IFN-γ,繼之激活巨噬細胞提高其殺傷弓形蟲活力,也能促進CD4~+T細胞向Th1細胞分化,并增強NK細胞和CD8~+T細胞殺傷功能。同時,IL-12對促進IFN-γ的持續(xù)產(chǎn)生也起到重要作用。因此IL-12的表達在弓形蟲感染的急慢性期均起到重要作用。 本研究選用弓形蟲復合抗原基因SAG1-ROP2-SAG2及基因佐劑mIL-12通過肌注方式免疫小鼠,觀察并研究其免疫保護性。結(jié)果表明復合基因疫苗的免疫效果明顯優(yōu)于單基因疫苗SAG2及雙基因疫苗SAG1-ROP2,并且IL-12可以對該復合基因疫苗起到很好的免疫增強作用。在弓形蟲研究領(lǐng)域,國內(nèi)外尚無此項研究報道。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a widely distributed opportunistic protozoa that infects human and many animals. Toxoplasma gondii infection can invade various organs and tissues and cause fetal malformation, nervous system dysplasia, choroidal retinitis, abortion, premature delivery and even stillbirth through vertical transmission from mother to child. For people with low immune function such as AIDS is the main cause of death. In addition, toxoplasmosis also causes serious economic loss to animal husbandry production. Therefore, in order to effectively control Toxoplasma gondii, the development of vaccine has become the development direction of prevention and treatment of toxoplasmosis. In the course of studying Toxoplasma gondii vaccine, the development of multiple antigen combinations and the development of multiple multivalent vaccines at different stages of life cycle development is a development direction and consensus in the research of Toxoplasma gondii vaccine. SAG1 antigen, one of the major surface antigens specific to Toxoplasma gondii tachyzoites, has a high degree of immunogenicity and protection. ROP2 protein, the main candidate antigen for Toxoplasma gondii infection diagnosis and vaccine development, is a protein secreted by Toxoplasma gondii corybodies, which mainly assists the parasites to invade host cells during the tachyzoite stage of Toxoplasma gondii's life cycle. Both bradyzoites and sporozoites are expressed. SAG2 antigen is another important antigen on the surface of Toxoplasma gondii and has a close relationship with the invasion of host cells by Toxoplasma gondii so far. The function of SAG_2 protein of Toxoplasma gondii was studied. The SAG-2 protein could immobilize Toxoplasma gondii on the surface of nucleated cells, and assist Toxoplasma gondii to enter into nucleated cells and mediate the invasion of Toxoplasma Toxoplasma tachyzoites. In order to overcome the problem of low immune efficiency of nucleic acid vaccine, the eukaryotic expression plasmid of mouse interleukin 12 (mIL-12) was selected as gene adjuvant to enhance the immune effect of Toxoplasma gondii complex antigen gene SAG1-ROP2-SAG2. During the early stage of Toxoplasma gondii infection, IL-12 stimulated the secretion of IFN- 緯 by NK cells and T cells, and then activated macrophages to enhance the cytotoxicity of Toxoplasma gondii. It also promoted the differentiation of CD4T cells into Th1 cells and enhanced the killing function of NK cells and CD8T cells. At the same time, IL-12 also plays an important role in promoting the sustained production of IFN- 緯. Therefore, the expression of IL-12 plays an important role in the acute and chronic stage of Toxoplasma gondii infection. In this study, Toxoplasma gondii complex antigen gene SAG1-ROP2-SAG2 and gene adjuvant mIL-12 were used to immunize mice by intramuscular injection. The results showed that the immune effect of the composite gene vaccine was significantly better than that of the single gene vaccine SAG2 and the double gene vaccine SAG1-ROP2.The IL-12 could play a good role in enhancing the immunity of the compound gene vaccine. In the field of Toxoplasma gondii, there is no such research report at home and abroad.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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