【作者】 王艳华；

【导师】 殷宏； 张德林；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2011， 博士

【摘要】 弓形虫是一种专性细胞内寄生原虫,具有广泛的宿主群并在世界范围内流行,引起人兽共患寄生虫病。在畜牧业方面,怀孕动物尤其是牛和羊会出现流产或死胎,猪会大批死亡,给畜牧业造成巨大经济损失。因此建立特异、快速、灵敏的弓形虫病诊断方法和研制有效预防弓形虫病的疫苗成为迫切需要解决的问题。由于弓形虫抗原成分较复杂,实验表明,单一抗原疫苗免疫效果或诊断试剂检测效果均不理想,研制多表位疫苗和诊断试剂必然是一个新的发展策略。本研究在弓形虫代谢分泌抗原和多表位抗原免疫保护研究基础之上,对主要抗原的线性B细胞表位进行了鉴定,为制备弓形虫B细胞表位抗原奠定基础。主要研究结果如下：1)代谢分泌抗原(ESA)较其它可溶性抗原和包囊抗原能更好的诱导细胞免疫反应,是研制抗弓形虫疫苗的一种非常好的候选抗原。本研究评估了ESA与弗氏佐剂乳化疫苗对猪的免疫保护效果,通过对细胞免疫和体液免疫水平、血液中荷虫量、猪临床症状、病理剖解及组织变化等一些重要参数的评估,表明ESA加弗氏佐剂乳化疫苗诱导猪产生的细胞和体液免疫反应能抵抗急性弓形虫感染。2)本研究中,我们首次探讨一个线性B细胞表位(来自表面蛋白1(SAG1)保守区域)和两个T细胞表位(来自致密颗粒蛋白1(GRA1)和致密颗粒蛋白4(GRA4))通过GG间隔序列串联,然后连接到赖氨酸以多表位肽(MAP)形式免疫小鼠所引发的免疫反应。结果,MAP结构+弗氏佐剂不仅诱导BALB/c和昆明小鼠产生高水平特异性抗体,而且还诱导小鼠发生强烈的细胞免疫反应,用致死剂量的弓形虫GJS强毒株(1×103)攻击免疫鼠,小鼠的存活时间比对照组的明显延长。数据证明多表位肽疫苗可作为一种弓形虫候选疫苗值得进一步进行研究。3)本研究利用B细胞表位分析软件预测和肽扫描技术两种方法对弓形虫两个主要抗原SAG1和GRA1的优势线性B细胞表位进行筛选。对于SAG1鉴定到2个新的高度保守的线性B细胞抗原表位91PTLAYSPNRQICPAGTTSSCTSKAVTLSSL120和151PVTTQTFVVGCIKGDDAQSCMVTVTVQARA180;对于GRA1鉴定了3个新的高度保守的线性B细胞抗原表位134 YSEVGNVNMEEVIDTMKSMQ153、164NKGETVEEAIEDVAQAEGLN183和214LEKDKQQLKDDIGFLTGERE233),所鉴定到的表位与弓形虫感染猪血清反应原性良好,有研发诊断试剂和表位疫苗的价值。4)本研究以91PTLAYSPNRQICPAGTTSSCTSKAVTLSSL120和164NKGETVEEAIEDVAQAEGLN183两条合成肽作为抗原建立了一种检测猪弓形虫抗体的肽ELISA(Pep-ELISA)方法。敏感性和特异性分别为88.06%和97.87%。Pep-ELISA敏感性或许可通过增加其它抗原表位(源于同种蛋白或其他蛋白)得到进一步提高,因此基于合成肽ELISA可能会成为弓形虫病诊断的重要工具之一。更多还原

【Abstract】 Toxoplasma gondii is an obligate intracellular parasite capable of infecting a variety of animals and causing toxoplasmosis. Infections of T. gondii in livestock have economic importance due to abortions, stillbirth and neonatal losses, especially in sheep and goats, and even death in pigs. It is essential to establish a rapid, highly specific and accurate method for diagnosing T. gondii infection and to develop effective vaccine against toxoplasmosis.Several vaccination trials have confirmed that diagnostic method and vaccine using single antigen are not satisfactory because of the complexity of the parasite life cycle. The diagnostic method and vaccine based on multi-epitope peptides have become an attractive strategy. In this study, based on the immune protection study of excreted/secreted antigens (ESA) and multi-epitope peptide, the linear B cell antigenic epitopes derived from major protein were identified. The results of this study lay a foundation for further study of diagnostic method and vaccine for toxoplasmosis. The main results are summarized as following:1) The ESA is able to stimulate a better cell-mediated immune response as compared to soluble or cysts antigen. Therefore, this antigen is a good candidate for development of immunizing agents against T. gondii infection. In the present study, we have evaluated the immunogenicity of EAS antigens of the the Gansu Jingtai strain (GJS) of T. gondii after administration in the pigs. The important parameters (cellular and humoral immunity, the amount of parasites in blood, the clinical symptoms, pathological changes and pathological anatomy) in immunization experiments were evaluated. The response elicited by ESA was capable of protecting against acute T. gondii infection in pigs.2) For the first time, we investigated murine immune responses to one linear B-cell epitope (derived from conserved regions of SAG1) when conjugated to two other defined T-cell epitopes (from conserved regions of GRA1 and GRA4) situated in tandem through the GG spacer sequence, with the latter positioned adjacent to a polylysine core. Immunization of BALB/c and Kunming mice with the MAP construct in Freund’s adjuvant induced not only a humoral immune response but also a cellular response. After challenge with lethal doses of T. gondii (1×103), vaccinated mice had increased survival time in comparison with unvaccinated controls. Our data demonstrated that a MAP construct could trigger strong humoral and cellular responses against T. gondii, and that the MAP is a vaccine candidate worth of further study.3) The B cell epitopes of SAG 1 and GRA1 were screened and identified by prediction of linear B cell epitopes and pepscan technique. The two new highly conserved linear B cell epitopes: 91PTLAYSPNRQICPAGTTSSCTSKAVTLSSL120 and 151PVTTQTFVVGCIKGDDAQSCMVTVTVQARA180 derived from SAG1 were identified successfully. The three new highly conserved linear B cell epitopes: 134YSEVGNVNMEEVIDTMKSMQ153,164NKGETVEEAIEDVAQAEGLN183 and 214LEKDKQQLKDDIGFLTGERE233 derived from GRA1 were identified successfully. A good reactivity was monitored with sera from pig infected with T. gondii. The results of this study may aid in the design of an epitope vaccine against T. gondii and the development of diagnostic reagents for toxoplasmosis.4) In this study, we developed a Pep- ELISA using peptides, 91PTLAYSPNRQICPAGTTSSCTSKAVTLSSL120 and 164NKGETVEEAIEDVAQAEGLN183, that can detect antibody against T. gondii. The sensitivity and specificity were 88.06% and 97.87% respectively. It is noteworthy that the diagnostic sensitivity of the Pep-ELISA may be improved by the inclusion of other peptides, originating from the same or other proteins. The Pep-ELISA described here may constitute an important tool for the diagnosis of toxoplasmosis.更多还原