【作者】 方瑞；

【导师】 赵俊龙；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2010， 博士

【摘要】 刚地弓形虫(Toxoplasma gondii)广泛存在于世界各地,寄生于人和200多种脊椎动物的有核细胞内而引起人兽共患弓形虫病(Toxoplasmosis)。该病严重影响人类优生优育与畜牧业的健康发展。目前弓形虫病的防治主要依靠药物防治,但药物防治无法从根本上清除宿主体内的弓形虫,仅仅是将弓形虫从快速繁殖的速殖子转变为缓慢繁殖的缓殖子,一旦宿主的免疫功能出现障碍,弓形虫将从缓殖子再度活化为速殖子。因此研制安全高效的新型疫苗来预防和控制弓形虫病成为各国研究者关注的焦点。但由于弓形虫的生活史复杂,影响免疫的因素多,目前弓形虫病的免疫预防研究进展不大。本研究对弓形虫病两种新型疫苗进行了探索,构建了“自杀性DNA疫苗”pSCA-MIC3和重组杆状病毒疫苗Ac-V-MIC3、Ac-V-SAG1,并在实验鼠模型BALB/c上对构建的候选疫苗进行了免疫效力研究,主要研究结果如下：1.弓形虫“自杀性DNA疫苗”pSCA-MIC3的构建及免疫原性研究DNA疫苗具有高效、稳定、易构建等优点被研究者认为是最有潜力的新型疫苗,但DNA疫苗免疫后长期存在于宿主体内,一旦整合进宿主染色体可能会引起宿主基因的改变,另外宿主体内可能会产生针对该DNA疫苗的抗体,因此存在很大的安全隐患。基于甲病毒复制子的自杀性DNA疫苗继承了DNA疫苗的上述优点,但它能随宿主细胞的凋亡自我凋亡,同时具有免疫增强的作用,因此被认为是一种优良的新型疫苗载体。本研究从弓形虫全基因组中利用PCR扩增弓形虫MIC3全基因,并克隆到“自杀性DNA疫苗”质粒载体pSAGl和常规DNA疫苗载体pcDNA3.1+中,获得“自杀性DNA疫苗”pSCA-MIC3和常规DNA疫苗pcDNA-MIC3。间接免疫荧光实验和RT-PCR检测结果表明pSCA-MIC3和pcDNA-MIC3均能够介导弓形虫MIC3基因在BHK-21细胞中有效表达。将该疫苗与常规DNA疫苗pcDNA-MIC3分别以50ug和100ug免疫六周龄BALB/c鼠,试验结果表明,在首疫后两周pSCA-MIC3、pcDNA-MIC3均能利用ELISA检测到微弱的抗弓形虫MIC3抗体,第四周,第六周加强免疫后两组的抗体水平继续升高,但pSCA-MIC3、pcDNA-MIC3两组之间抗体水平差异不显著,PBS和空载体对照组均检测不到MIC3抗体的存在。细胞免疫水平检测表明pSCA-MIC3、pcDNA-MIC3免疫组的BALB/c鼠诱导的淋巴细胞增殖、IFN-y的表达均显著高于PBS和空载体对照组,但pSCA-MIC3、pcDNA-MIC3两组之间细胞免疫水平差异不显著。使用弓形虫RH株攻击时二者均能显著延长BALB/c鼠的存活时间。以上研究结果表明“自杀性DNA疫苗”pSCA-MIC3具有良好的免疫原性,同时其随宿主细胞自我凋亡的特性导致它比常规的DNA疫苗有着更高的安全性,是一种极具潜力的弓形虫候选疫苗。2.弓形虫重组杆状病毒疫苗Ac-V-MIC3、Ac-V-SAG1的构建及免疫原性的研究在合适的启动子驱动下,杆状病毒可以在多种哺乳动物细胞中表达外源基因,但自身并不感染哺乳动物细胞。但杆状病毒介导外源基因表达的能力在一定程度上受到血清补体系统的抑制,从而影响杆状病毒疫苗的应用。水泡性口炎病毒G蛋白的引入,可以消除杆状病毒对补体的敏感性。因此利用哺乳动物极早期启动子CMV驱动的,水泡性口炎病毒G蛋白修饰的杆状病毒载体pFast-VSV-G-CMV (Ac-V)成为一种优良的疫苗载体。本研究分别将弓形虫的MIC3、SAG1基因插入杆状病毒载体pFast-VSV-G-CMV构建了重组杆状病毒Ac-V-MIC3、Ac-V-SAG1.间接免疫荧光实验表明重组杆状病毒Ac-V-MIC3、Ac-V-SAG1均能高效转导哺乳动物PK-15细胞。将重组杆状病毒Ac-V-MIC3、Ac-V-SAG1分别以不同的剂量(1010pfu/mL,109pfu/mL,108pfu/mL)免疫BALB/c鼠,试验结果表明,在首免后第三周,重组病毒Ac-V-MIC3、Ac-V-SAG1免疫组和相应的DNA疫苗对应组均可检测到特异的抗弓形虫ELISA抗体,二次免疫之后,重组病毒Ac-V-MIC3、Ac-V-SAG1免疫组ELISA平均抗体水平随之升高,均显著的高于其它对照组。重组杆状病毒Ac-V-MIC3、Ac-V-SAG1免疫组的细胞免疫水平同样显著高于对照组,重组病毒1010pfu/mL免疫组表现出最高的体液和细胞免疫水平,显著高于108pfu/mL免疫组和DNA疫苗对照组。使用弓形虫RH株攻击时重组病毒1010pfu/mL免疫组BALB/c鼠存活时间最长,显著高于108pfu/mL免疫组和DNA疫苗对照组。上述结果表明重组杆状病毒Ac-V-MIC3、Ac-V-SAG1是一种具有良好发展前景的弓形虫新型候选疫苗。更多还原

【Abstract】 Toxoplasma gondii is an obligate intracellular protozoan parasite. As a significant human and animal pathogen, it was estimated to infect about one-third of the world’s human population and all warm-blooded mammals which was frequently associated with congenital infection and abortion (Montoya & Liesenfeld,2004; Tenter et al.,2000). Therefore, T. gondii is of veterinary and medical importance. So far, drugs can not clean T. gondii from host. Thus, development of an effective and safe vaccine for controlling T. gondii infection in those animals is an important goal for scientists worldwide. However, due to T. gondii multiplicity immunity, the progress of new vaccines against T. gondii maintains is slowly.Therefore, in the present study, we explored the new vaccine design against T. gondii, and constructed "suicidal DNA vaccine" pSCA-MIC3 and the recombinant baculovirus vaccine Ac-V-MIC3, Ac-V-SAG1. Furthermore, the immune efficacy of the candidate vaccines was investigated in BALB/c mice. The research works were as following:1. Construction and immune effect of Toxoplasma gondii suicidal DNA vaccine pSCA-MIC3Among new kinds of vaccines, DNA vaccines become a focus as such vaccines have been shown to elicit potent, long-lasting humoral and cell-mediated immunity and low cost in manufacture,storage, and administration. But conventional DNA vaccines raise certain concerns such as potential integration into the host genome and cell transformation as well as their potency since they do not have the intrinsic ability to amplify in vivo as viral vaccines do. More recently, "suicidal DNA vaccine vector pSCA1", based on the replicon of alphaviruses, has been developed. Besides the advantages of conventional DNA vaccines, it has some prominent advantages. For example, it could break immunological tolerance by activating innate antiviral pathways, catalyzes cytoplasmic self-amplification of recombinant RNA,and high-level expression of the heterologous antigens.Furthermore,when a suicidal DNA vaccine is transfected into cells, it leads eventually to apoptosis of the transfected cells, which is particularly important in alleviating the concerns of potential integration and celltransformation generated by the use of conventional DNAvaccines. All these advantages indicate that suicidal DNA vaccine is a good delivery vehicle and could be used to replace conventional DNA vaccines.In this study, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferationresponse and IFN-y induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P< 0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P< 0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P> 0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis.2. Construction and immune effect of recombinant baculovirus vaccine Ac-V-MIC3、Ac-V-SAG1Although AcMNPV are failing to replicate in vertebrate cells, it does express aline genes that are dependent on the strength of the promoter used to drive transcription of the foreign gene. Follwing these findings, baculovirus have emerged as a vector with great potential for gene transfer in mammalian cells. Furthermore, it has been reported that a pseudotype baculovirus displaying the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope can extend the host range and increase the transduction efficiency in mammalian cells.so it was considered as an excellent expressing vector.In this study, the value of Bac-VSV-G in delivering T. gondii antigen was investigated. T. gondii MIC3、SAG1 gene was cloned into Bac-VSV-G, and recombinant baculovirus Ac-V-MIC3、Ac-V-SAG1 were obtained. Indirect immunofluorescence test showed Ac-V-MIC3、Ac-V-SAG1 were efficiently transduced and expressed in pig kidney cells. Then BALB/c mice were immunized with Ac-V-MIC3、Ac-V-SAG1 at different doses (1010pfu/mL,109pfu/mL,108pfu/mL/mouse) and challenged with T. gondii RH strain tachyzoites after immunization. The levels of specific T. gondii antibody, IFN-y, IL-4, IL-10 expression and release, and the survival rate of treated mice were evaluated. Compared with the mice immunized with DNA vaccine (pcDNA/SAG1、pcDNA/MIC3) encoding the same gene, Ac-V-MIC3、Ac-V-SAG1 induced higher levels of specific T. gondii antibody and IFN-y expression and the survival rate of mice with Ac-V-MIC3、Ac-V-SAG1 were significantly improved. These results indicated that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop new generation of vaccines against T. gondii infection.更多还原