E. maxima Th1细胞因子抑制相关抗原的确定及其抑制作用机制的研究

It has been reported that the infection of chicken coccidian could inhibit the production of Th1 cytokines. In our previous research, vaccination with some sub expression library of E. maxima inhibited the mRNA of Th1 cytokines. Antigen components of chicken coccidian are complex and many immune protective antigens have been reported, however, the immunosuppression associated antigens are indeterminate and the immune suppression mechanism is also unclear. In order to determine Th1 cytokine inhibition associated antigen of E. maxima and their inhibitory mechanisms, in this study, the expression immunization library of E. maxima will be constructed. The constructed library will be divided into several sub-libraries to immunize chickens and the specific transcription factors and cytokines of Th1 and Th2 cells will be detected to determine Th1 cytokine inhibition associated antigen gens. The inhibition of cytokines induced by the inhibitory antigen will be tested in vivo and in vitro. The recombinant protein of the inhibitory antigen, mitogen and chicken peripheral blood lymphocytes will be co-cultured to determine inhibitory antigen-binding molecules. The changes of key molecules in the cytokine transcriptional signal transduction pathway, regulatory pathway and mitotic signal pathway will be detected to determine the inhibited signal transduction pathway of cytokines production induced by mitogen. The inhibitory effects on cytokine secretion and signaling pathway in macrophages will be also be detected. These results are of great significance for clarifying the immunosurppressive mechanism of chicken coccidiosis and also play an important role in the immune prevention of chicken coccidiosis.

研究表明，鸡球虫的感染可以引起Th1细胞因子的抑制，我们期前研究发现某些E. maxima亚文库能够抑制Th1细胞因子的转录。鸡球虫抗原成分复杂，免疫保护性抗原的报道已经很多，但免疫抑制相关抗原未见报道，其免疫抑制机制也不清楚。为确定E. maxima Th1细胞因子抑制相关抗原及其抑制机制，本研究拟构建E. maxima 表达免疫文库，分组免疫鸡，检测Th细胞特异性转录因子和细胞因子的表达情况，确定Th1细胞因子抑制相关抗原基因。分别从体内和体外验证抑制性抗原对Th1细胞因子的抑制作用。将重组抑制性抗原和有丝分裂原共刺激鸡外周血淋巴细胞，鉴定淋巴细胞抑制抗原结合分子；检测细胞因子转录信号传导通路、调控通路关键分子的变化，确定抑制相关抗原抑制有丝分裂原诱导细胞因子的信号传导通路；检测抑制抗原对巨噬细胞细胞因子分泌及其信号通路的影响。对于阐明鸡球虫的免疫抑制机制及免疫预防具有重要意义。

鸡球虫可以通过抑制Th1细胞因子的表达，从而逃避宿主免疫系统的攻击。然而，鸡球虫抗原成分复杂，具体的Th1细胞因子抑制性抗原是什么？其作用机制如何？这些问题都不清楚。为此，本研究以E. maxima为研究对象，通过表达文库免疫技术，筛选到了EmSAG, EmHPSP-1, EmHPSP-2 和EmHPSP-3 4个能够抑制Th1 类细胞因子表达的抗原基因。Western blot分析发现这4个抗原的重组蛋白能被E. maxima感染鸡血清所识别，免疫荧光技术发现这4个分子均在E. maxima子孢子阶段表达。体内验证实验发现这4个抗原无论是以其真核表达质粒的形式（pVAX-EmSAG、pVAX-EmHPSP-1、pVAX-EmHPSP-2和pVAX-EmHPSP-3）或者重组蛋白的形式注射鸡体，鸡血清中Th1类细胞因子IL-2、IFN-γ 的表达显著下降。在体外验证实验中也发现这4个抗原的重组蛋白能够显著抑制鸡外周血淋巴细胞Th1类细胞因子IL-2和IFN-γ 的表达。免疫荧光方法发现这4种Th1类细胞因子抑制性抗原均能与鸡外周血淋巴细胞结合。经酵母双杂交筛选、GST pull-down和RNA干扰验证，证明EmHPSP-1是通过与其在淋巴细胞上的结合分子POLR2F结合，从而抑制淋巴细胞Th1类细胞因子的表达。用POLR2F抗血清对鸡进行被动免疫，发现POLR2F抗血清对E. maxima感染有一定的干扰作用。检测了E. maxima Th1类细胞因子抑制性抗原对鸡Th1和Th2淋巴细胞分化信号通路的影响，发现这4个抑制性抗原可能通过抑制STAT1、STAT4，促进STAT6来抑制Th1类细胞分化信号通路。检测了E. maxima Th1类细胞因子抑制性抗原对先天免疫细胞功能的影响，发现其能够显著下调鸡巨噬细胞和树突状细胞IL-12的mRNA水平和蛋白表达水平，并且还可以抑制TLR-2、TLR-4、TLR-21的mRNA基因表达，ERK1/2为4种抑制性抗原在鸡巨噬细胞中主要作用的信号通路靶标。最后，动物免疫攻虫试验结果显示这4个抑制性抗原对E. maxima感染有一定的抵抗作用。综上，本研究明确了鸡球虫Th1 细胞因子抑制性抗原的种类及其作用机制，对于阐明鸡球虫的免疫逃避机制具有重要意义，对于鸡球虫和其他细胞内原虫的免疫预防也具有重要的借鉴意义。