弓形蟲SAG1-MIC8復(fù)合基因疫苗對小鼠的免疫保護性研究
發(fā)布時間：2019-02-19 17:30
【摘要】： 剛地弓形蟲(Toxoplasma gondii)是一種以貓科動物為終宿主的腸道球蟲,呈世界性分布,其中間宿主范圍較廣,可感染包括人類在內(nèi)的多種動物,引起人獸共患的弓形蟲病。一般情況下,弓形蟲感染終宿主或中間宿主感染后往往不產(chǎn)生明顯的臨床癥狀,但是在宿主免疫功能低下時,可致嚴重后果。慢性弓形蟲病患者或者免疫抑制(如器官移植后長期應(yīng)用免疫抑制劑)、免疫缺陷患者(如AIDS)合并感染弓形蟲往往可見肺炎、心肌炎、脈絡(luò)膜視網(wǎng)膜炎以及多系統(tǒng)臟器衰竭的發(fā)生;孕婦孕期感染弓形蟲可導(dǎo)致流產(chǎn)、早產(chǎn)、畸胎、死胎等。由于在臨床上弓形蟲病的癥狀表現(xiàn)多樣,給診斷治療及預(yù)防等帶來許多的不便,且臨床上尚無十分理想的治療藥物,因此尋找有效的預(yù)防與治療措施,研制安全有效的疫苗顯得極為迫切。 DNA疫苗又稱核酸疫苗,是繼死疫苗、減毒或滅活疫苗、體外分離或基因工程制備的亞單位疫苗之后的又一類新型疫苗。大量的實驗結(jié)果表明,DNA疫苗不僅具有預(yù)防疾病的作用,同時還具有治療的作用。但是弓形蟲生活史較復(fù)雜,抗原成分多,且具有發(fā)育階段的特異性或差異性,因此成功的疫苗應(yīng)針對多個特異性抗原,所產(chǎn)生的保護性免疫力需針對生活史中一個以上的感染階段。動物和人體實驗也表明,僅靠單種基因疫苗誘導(dǎo)的免疫效果并不理想,而復(fù)合基因疫苗誘導(dǎo)的各項免疫指標顯著優(yōu)于單價疫苗,有望成為未來基因疫苗中的研究方向。在本實驗中,我們選用了SAG1和MIC8兩種候選基因以構(gòu)建復(fù)合DNA疫苗。 SAG1(main surface antigen 1)是弓形蟲速殖子期特異的主要表面抗原之一,能刺激機體產(chǎn)生IgG、IgM、IgA等高效價的抗體以及細胞因子IFN-γ等殺死蟲體,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。除此之外,它還與粘附和穿入宿主細胞等功能有關(guān)。MIC8(microneme protein 8)是一個單基因拷貝序列,無內(nèi)含子,它所編碼的蛋白是MIC3蛋白的護送蛋白,在速殖子期和緩殖子期都有表達,可幫助MIC3蛋白到達微線體并分泌出胞。最新研究顯示,當MIC8蛋白不存在的時候,蟲體不能與宿主細胞形成接觸融合區(qū)域,使得侵入宿主細胞過程受阻,這種由MIC8參與形成的細胞融合區(qū)域非常重要,不可被其他的微線體蛋白來替代,從而表明MIC8是一種新穎的,功能獨特的蛋白。另外,MIC8蛋白參與的信號級聯(lián)放大反應(yīng),可致棒狀體蛋白的分泌。 本研究成功的將SAG1和MIC8兩個基因構(gòu)建在同一個表達載體pcDNA3.1上,通過肌注的方式免疫C57BL/6J小鼠,觀察復(fù)合基因疫苗的免疫效果。結(jié)果表明SAG1-MIC8復(fù)合基因疫苗的效果顯著優(yōu)于SAG1或者MIC8單基因疫苗。此外,在小鼠體內(nèi)沒有檢測到高濃度的細胞因子白介素4(IL-4),且各組間濃度差別無統(tǒng)計學意義,因而不能確定是否有Th2型免疫反應(yīng)參與了免疫應(yīng)答。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) (Toxoplasma gondii) is a kind of intestinal coccidiosis with cat family as its final host. It has a wide range of intermediate hosts and can infect a variety of animals, including human beings, and cause zoonotic toxoplasmosis (Toxoplasma gondii). In general, Toxoplasma gondii infection with terminal host or intermediate host infection often does not produce obvious clinical symptoms, but when host immune function is low, it can lead to serious consequences. Patients with chronic toxoplasmosis or immunosuppression (such as long-term use of immunosuppressive agents after organ transplantation), immunodeficient patients (such as AIDS) with Toxoplasma gondii often have pneumonia, myocarditis, Choroidal retinitis and multiple system organ failure; Pregnant women infected with Toxoplasma gondii can lead to abortion, premature delivery, teratogenesis, stillbirth, etc. Because the symptoms of Toxoplasma gondii are various in clinic, it brings many inconveniences to diagnosis, treatment and prevention, and there is no ideal therapeutic drug in clinic, so we seek effective prevention and treatment measures. It is urgent to develop a safe and effective vaccine. DNA vaccine, also called nucleic acid vaccine, is a new type of vaccine after dead vaccine, attenuated or inactivated vaccine, subunit vaccine prepared by isolation or genetic engineering in vitro. A large number of experimental results show that DNA vaccine not only can prevent disease, but also has therapeutic effect. However, the life history of Toxoplasma gondii is more complicated, with many antigen components, and has the specificity or difference of development stage, so the successful vaccine should be aimed at many specific antigens. The protective immunity generated should be targeted at more than one stage of infection in the life cycle. Animal and human experiments also show that the immune effect induced by single gene vaccine is not satisfactory, and the immune indexes induced by compound gene vaccine are significantly better than that of monovalent vaccine, which is expected to become the research direction of gene vaccine in the future. In this study, we selected two candidate genes, SAG1 and MIC8, to construct compound DNA vaccine. SAG1 (main surface antigen 1) is one of the major surface antigens specific to Toxoplasma gondii tachyzoites. It can stimulate the production of high titer antibodies, such as IgG,IgM,IgA, and the cytokine IFN- 緯 to kill the parasites, so it has high immunogenicity and immunity protection. Toxoplasma gondii infection is the main candidate antigen for immune diagnosis and vaccine development. In addition, it is related to the function of adhesion and penetration into host cells. MIC8 (microneme protein 8) is a single gene copy sequence without introns. It encodes an escort protein of MIC3 protein, which is expressed in both tachyzoites and bradyzoites. It can help the MIC3 protein to reach the microline and secrete the cells. New research shows that when MIC8 protein does not exist, the parasite can't form contact fusion area with host cells, which prevents the invading of host cells. This kind of cell fusion region which is formed by MIC8 is very important. MIC8 is a novel and functional protein that cannot be replaced by other microsomal proteins. In addition, MIC8 protein involved in signal cascade amplification reaction, can induce the secretion of rodlike proteins. In this study, SAG1 and MIC8 genes were successfully constructed on the same expression vector pcDNA3.1, and the immunized C57BL/6J mice were immunized by intramuscular injection to observe the immune effect of the composite gene vaccine. The results showed that the effect of SAG1-MIC8 compound gene vaccine was better than that of SAG1 or MIC8 single gene vaccine. In addition, no high concentration of cytokine interleukin-4 (IL-4) was detected in mice, and there was no significant difference in the concentrations among the groups. Therefore, it was not possible to determine whether the Th2 type immune response was involved in the immune response.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.1
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) (Toxoplasma gondii) is a kind of intestinal coccidiosis with cat family as its final host. It has a wide range of intermediate hosts and can infect a variety of animals, including human beings, and cause zoonotic toxoplasmosis (Toxoplasma gondii). In general, Toxoplasma gondii infection with terminal host or intermediate host infection often does not produce obvious clinical symptoms, but when host immune function is low, it can lead to serious consequences. Patients with chronic toxoplasmosis or immunosuppression (such as long-term use of immunosuppressive agents after organ transplantation), immunodeficient patients (such as AIDS) with Toxoplasma gondii often have pneumonia, myocarditis, Choroidal retinitis and multiple system organ failure; Pregnant women infected with Toxoplasma gondii can lead to abortion, premature delivery, teratogenesis, stillbirth, etc. Because the symptoms of Toxoplasma gondii are various in clinic, it brings many inconveniences to diagnosis, treatment and prevention, and there is no ideal therapeutic drug in clinic, so we seek effective prevention and treatment measures. It is urgent to develop a safe and effective vaccine. DNA vaccine, also called nucleic acid vaccine, is a new type of vaccine after dead vaccine, attenuated or inactivated vaccine, subunit vaccine prepared by isolation or genetic engineering in vitro. A large number of experimental results show that DNA vaccine not only can prevent disease, but also has therapeutic effect. However, the life history of Toxoplasma gondii is more complicated, with many antigen components, and has the specificity or difference of development stage, so the successful vaccine should be aimed at many specific antigens. The protective immunity generated should be targeted at more than one stage of infection in the life cycle. Animal and human experiments also show that the immune effect induced by single gene vaccine is not satisfactory, and the immune indexes induced by compound gene vaccine are significantly better than that of monovalent vaccine, which is expected to become the research direction of gene vaccine in the future. In this study, we selected two candidate genes, SAG1 and MIC8, to construct compound DNA vaccine. SAG1 (main surface antigen 1) is one of the major surface antigens specific to Toxoplasma gondii tachyzoites. It can stimulate the production of high titer antibodies, such as IgG,IgM,IgA, and the cytokine IFN- 緯 to kill the parasites, so it has high immunogenicity and immunity protection. Toxoplasma gondii infection is the main candidate antigen for immune diagnosis and vaccine development. In addition, it is related to the function of adhesion and penetration into host cells. MIC8 (microneme protein 8) is a single gene copy sequence without introns. It encodes an escort protein of MIC3 protein, which is expressed in both tachyzoites and bradyzoites. It can help the MIC3 protein to reach the microline and secrete the cells. New research shows that when MIC8 protein does not exist, the parasite can't form contact fusion area with host cells, which prevents the invading of host cells. This kind of cell fusion region which is formed by MIC8 is very important. MIC8 is a novel and functional protein that cannot be replaced by other microsomal proteins. In addition, MIC8 protein involved in signal cascade amplification reaction, can induce the secretion of rodlike proteins. In this study, SAG1 and MIC8 genes were successfully constructed on the same expression vector pcDNA3.1, and the immunized C57BL/6J mice were immunized by intramuscular injection to observe the immune effect of the composite gene vaccine. The results showed that the effect of SAG1-MIC8 compound gene vaccine was better than that of SAG1 or MIC8 single gene vaccine. In addition, no high concentration of cytokine interleukin-4 (IL-4) was detected in mice, and there was no significant difference in the concentrations among the groups. Therefore, it was not possible to determine whether the Th2 type immune response was involved in the immune response.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.1
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