大黄鱼白细胞介素2的转录调控及功能研究

Interleukin-2 (IL-2) is a cytokine playing important roles in promoting T cell proliferation and cell differentiation, stimulating B cell proliferation and antibody secretion, as well as enhancing the killing activity of natural killer cells. While currently little is known about the function of fish IL-2. In preliminary study, the large yellow croaker (Larimichthys crocea) IL-2 gene (LycIL-2) and its 5’ promoter sequence have been cloned. Analysis on LycIL-2 expression modulation after immune stimulation and promoter activity were performed. Recombinant IL-2 protein was expressed and purified in Phichia pastoris. In this research, we will conduct the regulational analysis on lycIL-2 promoter activity by transcription factor AP1, NF-κB and NFAT, so as to clarify the transcriptional regulation mechanism of LycIL-2. On the other hand, the function of LycIL-2 on promoting the in vivo and in vitro T cell proliferation, Th1 and Th2 cell differentiation and its effect on the Th1/Th2 cell mediated immune response will be investigated, thus revealing the function of LycIL-2 on promoting the T cell proliferation, Th1 and Th2 cell differentiation and in immune response regulation. These results will not only help to reveal the function and the transcriptional regulation mechanism of fish IL-2, but also push forward the application of LycIL-2 as immune adjuvant in the disease prevention of large yellow croaker.

哺乳类白细胞介素2（IL-2）是调控宿主免疫应答的重要细胞因子，其主要功能是促进T细胞增殖与分化，刺激B细胞增殖与抗体分泌，增强自然杀伤细胞的杀伤活性。目前对于鱼类IL-2的功能仍知之甚少。前期我们克隆了大黄鱼IL-2基因，研究了其表达调控模式和启动子活性，并获得了重组大黄鱼IL-2蛋白。本项目拟在此基础上研究AP1、NF-κB和NFAT等转录因子对大黄鱼IL-2启动子活性的调控作用以探明大黄鱼IL-2的转录调控机制；通过大黄鱼IL-2在T细胞增殖和Th1、Th2细胞分化中的作用，对PI-3K、MAPK和JAK-STAT信号通路的激活作用，及对Th1、Th2细胞介导免疫学效应的影响等研究，揭示大黄鱼IL-2在促进T细胞增殖和Th1、Th2细胞分化，以及调控免疫应答中的功能。研究结果对于认识鱼类IL-2的功能及其转录调控机制具有重要理论意义，也为将其作为免疫佐剂应用于大黄鱼病害防治提供依据。

在前期克隆鉴定大黄鱼白细胞介素2（LcIL-2）启动子的基础上，证实了PHA、Con-A和T细胞活化剂CI、PMA和CI+PMA等刺激Jurkat T细胞后可显著提高LcIL-2的启动子活性，而LPS和Poly(I:C)刺激后启动子活性无明显变化。大黄鱼转录因子NFAT和NF-κB的LcRelA亚基、Lcc-Rel亚基的过表达可显著增强LcIL-2启动子的活性（P < 0.01），而AP1的Lcc-Jun亚基过表达则对LcIL-2启动子的活性无明显影响（P > 0.05）。LcIL-2启动子上NFAT或NF-κB结合位点核心序列的突变可显著降低相应转录因子对LcIL-2启动子活性的增强作用。这些结果表明，T细胞有丝分裂原和T细胞活化剂可以诱导LcIL-2的表达，且其表达受转录因子NFAT和NF-κB调控。.在毕赤酵母中重组表达并获得了纯化的重组LcIL-2蛋白（rLcIL-2），rLcIL-2可在大黄鱼活体内或在离体培养的头肾淋巴细胞中显著促进ZAP70+ T细胞的增殖，同时诱导Th1、Th2发育分化相关基因及IL-2下游关键转录因子STAT5等的表达上调，诱导Th17发育分化相关基因的表达下调，说明LcIL-2可参与调节T细胞增殖并促进CD4+ T细胞向Th1、Th2型细胞分化，抑制CD4+ T细胞向Th17型细胞分化。此外，LcIL-2还可显著促进大黄鱼IgM+ B细胞增殖，刺激血清中针对弧菌亚单位疫苗DLD特异性IgM抗体的产生，同时提高DLD对大黄鱼的攻毒保护率至76%，这说明LcIL-2在调控体液免疫应答方面也发挥重要作用。.研究结果在SCI收录期刊发表论文11篇，申请国家发明专利1项。另有一篇文章已被英文期刊 The Journal of Immunology接收。.培养硕士研究生5人，其中3人已通过论文答辩获得硕士学位，2人在读；培养博士研究生2人，1人已通过论文答辩获得博士学位，另1人拟于2022年6月进行论文答辩。