弓形虫体内诱导抗原调节NF-kB信号通路的机制研究

Toxoplasmosis is a global parasitic zoonosis which is caused by Toxoplasma gondii. NF-кB, as a regulatory factor, plays a key role in the active immunity of hosts.The immune systems of hosts recognize the antigens of T. gondii and pass the signals on by the NF-кB pathway. Mice deficient in some NF-кB family members were increased susceptibility to T. gondii, indicating the importance of this pathway in pathogen resistance. Previously, we accomplished the work to immunoscreening the cDNA expression library of T.gondii tachyzoits and obtained 5 in vivo-induced antigens. As preliminary identification turned out some antigens are hypothesized to related with the invasion, reproduction and evasion of T. gondii, whereas the relationship between in vivo-induced antigens of and NF-кB pathway is still unknown. The present study aims to screening and identify the in vivo-induced antigens which can regulate the transcriptional activity of NF-кB, eukaryotic expression plasmid of in vivo-induced antigens of T. gondii with HA tag will be constructed, and the constructed recombinant plasmid will co-transfection into 293T cells with NF-кB-Luc and pRL-TK plasmid, then under the stimulating of TNF or LPS, the activation or suppression of NF-кB patway could be observed. Following the exploration of the ways to regulate NF-кB signal pathway by RNAi , the inhibitors or the mutant of NF-кB family members directing at the corresponding regulatory proteins in this pathway. The host active proteins which can interact with the in vivo-induced antigens of T. gondii would be find out using appropriate methods such as immunoprecipitation, IFAT, western blot or Pull-down. To study deeply on the side of the parasite, the mutant expression vectors of the different sections of the screened antigens of T. gondii will be constructed and co-transfection into 293T cells with NF-кB-Luc and pRL-TK plasmid. The antigen regions of the corresponding antigens which can act on to the NF-кB pathway could be identified by detecting the transcription activity of NF-кB. The biological effects of the stimulated in vivo-induced antigens on the macrophage will be detected. Finally, the interaction mechanisms of the effect of T. gondii antigens on NF-кB signal pathway will be expounded and confirmed further using western blot or real time PCR. The results of the study would not only establish a valuable foundation for the researches of both the molecular pathogenesis and immunemechanism of T. gondii, but also provide the evidences for the molecular candidates for the developments of new medicaments and vaccines against toxoplasmosis.

弓形虫病是弓形虫引起的人畜共患寄生虫病。NF-кB是宿主主动免疫过程中关键性调控因子。宿主免疫系统识别抗原并通过NF-кB传递信息。缺乏某些NF-кB调控元件的小鼠，对弓形虫极为敏感，提示NF-кB信号通路在宿主抵抗弓形虫方面起着重要作用。本课题组前期研究成功筛选得到5种弓形虫体内诱导抗原，这些抗原可能在弓形虫入侵、繁殖和免疫等方面发挥作用，推测体内诱导抗原与NF-кB信号通路之间应有联系。为此，本课题拟利用双荧光素酶检测系统初步筛选可调节NF-кB转录活性的弓形虫体内诱导抗原，并确定其功能区；从NF-кB信号通路接头分子负调控突变体、抑制剂和siRNA三方面确定抗原调节NF-кB信号通路的具体途径；用免疫共沉淀等方法筛选与抗原相互作用的宿主蛋白，并对体内诱导抗原刺激巨噬细胞后的生物学效应进行检测，从而阐明体内诱导抗原作用NF-кB信号通路的机制并为弓形虫致病机理和免疫机理的研究奠定基础。

刚地弓形虫是一种顶复门专性细胞内寄生原虫，生活史复杂，能感染包括人在内的几乎所有温血动物。近年来，一系列弓形虫抗原被发现能够调控宿主NF-κB信号通路，但它们的具体机制尚不明确。本研究首先将RH、Me49、VEG、地方Ⅰ型和地方Ⅱ型弓形虫速殖子感染HEK293T细胞，利用双荧光素酶报告系统检测发现Me49、VEG、地方Ⅰ型和地方Ⅱ型弓形虫能激活NF-κB信号通路，为探索弓形虫是如何调控宿主的NF-κB信号通路，挑选已筛选到的体内诱导抗原基因，克隆并构建真核表达载体，将22种弓形虫抗原真核表达质粒分别转染HEK293T细胞，利用双荧光素酶系统检测发现仅有pCMV-GRA15(Ⅱ)和pCMV-GRA15(Ⅰ)能够激活NF-κB信号通路；利用Western blot验证了Ⅰ型GRA15能够激活NF-κB信号通路。根据生物信息学分析，全长的GRA15存在着两种结构域，跨膜区和核定位信号。因此构建了7个不同长度的GRA15截短真核表达质粒，将它们分别转染HEK293T细胞，利用双荧光素酶报告系统检测到pCMV-GRA15-4、pCMV-GRA15-5、pCMV-GRA15-6和pCMV-GRA15-7均有激活NF-κB信号通路的作用，推测Ⅰ型GRA15的194-518氨基酸区域为激活NF-κB信号通路的结构域。同时利用Realtime‐PCR 检测下游细胞因子，发现GRA15（Ⅰ）对 NF‐κB 信号通路下游细胞因子的影响效果不如 GRA15（Ⅱ）基因显著；在研究GRA15（Ⅰ/Ⅱ）、JK483877和JK483898免疫原性中，免疫活性和保护力实验证实这四个质粒不能够刺激巴贝西小鼠产生体液免疫和细胞免疫，均未表现出对巴贝西小鼠良好的保护力；在研究弓形虫CaM的下调表达对速殖子表型的影响，发现弓形虫CaM的表达下调显著降低速殖子的细胞粘附，入侵和溢出能力 ，但对其胞内繁殖无影响。本研究利用多种技术对弓形虫如何调控宿主NF-κB信号通路进行了探索，不仅加深了对弓形虫与宿主免疫机制的相互作用的理解，更为弓形虫新型药物和疫苗的开发提供了理论基础。