捻转血矛线虫诱导山羊Th9型免疫反应特点及其刺激性抗原鉴定

Th9 immunity was a new subtype of immune response. Research in humans and mice indicated that Th9 play important roles in promoting effective immunity against tumor and some parasites infection. However, there were no literatures on Th9 immunity of goats. We do not know the Th9 responses in goat induced by Haemonchus contortus. In this project, how the parasite induces Th9 immunity in goat would be studied both in vivo and in vitro experiments. The dynamics of Th9 cells, Interleukin 9 (IL-9), target cells would be analyzed after infection with H. contortus or incubated with proteins extracted from this parasite. To explore the mechanisms in modulating Th9 immunity by H. contortus, the signal transduction pathways in promoting interleukin-9 production and Th9 cell differentiation would be studied. Signal transducer and activator of transcription (STAT), nuclear factor of activated T cells (NFAT), transforming growth factor-β/SMAD, and nuclear factor-κB (NF-κB) signaling would be quantitated by Western Blot and qRT-PCR. The antibodies and RNAis target STAT1, STAT5, STAT6, GATA binding protein 3 (GATA3), Forkhead box protein 3 (FOXP3), SMAD, NFAT, Interferon Response Factor 4 (IRF4), PU.1 and NF-κB would be used to inhabit the functions of relative pathways. To prove the immune responses and protective effects induced by Th9 immunity, Th9 cells would be transformed to blank goats, followed by challenge with L3. To select the parasite proteins that binding to goat Th9 cells, technologies as flow cytometry, co-immunoprecipitation and mass spectrum would be applied. After which, the genes encoding these proteins would be cloned and expressed. The recombinant proteins would be incubated with goat peripheral blood mononuclear cell (PBMC), and the ability in stimulating Th9 cell differentiation and interleukin 9 (IL-9) secreting would be studied. Then the protective effects induced by these Th9 cell stimulating antigens would be studied by animal experiments. This project would be helpful to understand the mechanisms of parasite in modulating the host immunity. The antigens with ability to stimulate Th9 immunity in goat would be the vaccine candidates against this parasite.

Th9型免疫反应是新近发现的一种免疫类型，在人和小鼠等动物过敏性疾病发生、对抗某些肿瘤和寄生虫感染中发挥重要作用。然而，尚无山羊Th9型免疫反应的报道，山羊感染捻转血矛线虫后宿主Th9免疫反应的特点、作用机制等问题还不清楚。本项目拟通过体内和体外实验，研究捻转血矛线虫感染和虫体抗原刺激引起山羊Th9细胞分化增殖、IL-9产生、其他亚类T细胞和肥大细胞等靶细胞活化作用，研究其对Th9免疫相关信号通路的调节作用，研究Th9细胞被动转移对新宿主相关免疫反应的影响和免疫保护作用；用流式细胞分选、免疫共沉淀、质谱等技术筛选与Th9细胞相结合的虫体分子，对相关分子进行基因克隆和表达，鉴定具有Th9免疫刺激作用的抗原，研究这些抗原的免疫保护性。该项目可为深入研究捻转血矛线虫调节宿主免疫应答机制、探索虫体-宿主间相互作用提供新的数据和支撑；发掘Th9细胞刺激性抗原可为该病的免疫预防提供新的思路和候选分子。

已有研究表明Th9免疫在抗寄生虫感染中发挥重要作用，但捻转血矛线虫能否诱导山羊产生Th9型免疫反应还不清楚。本项目通过体外和体内试验，研究了虫体抗原和虫体感染后山羊Th9免疫反应的动态变化，首次证明了捻转血矛线虫能够诱导山羊产生Th9型免疫应答。本项目研究了虫体排泄分泌蛋白（ESP）对山羊Th9免疫相关信号通路中TGF-β1、TGF-βRI、TGF-αRII、Smad3、Smad4、Smad7、PU.1和IRF-4等关键基因的调节作用，发现捻转血矛线虫主要通过ESP蛋白调节山羊STAT6、PU.1、NF-κB等信号通路从而诱导宿主Th9免疫。本研究共筛选到218种能与山羊Th9细胞相结合的虫体蛋白，鉴定出HcDR、HcCBG、HcL6、HcS1和HcGATA等为山羊Th9细胞刺激分子。用HcDR、HcS1等Th9刺激分子的重蛋白免疫山羊能够产生较好的免疫保护效果。该项目研究结果为深入了解捻转血矛线虫与宿主之间的相互作用及其分子机制提供了新的依据，为捻转血矛线虫病疫苗研究提供了新的候选抗原和研究思路，兼具理论意义和应用价值。