斑节对虾Toll9受体基因免疫功能的研究与表达分析

发布时间：2018-02-08 20:51

本文关键词： 斑节对虾 Toll受体蛋白细胞免疫模型 TLR信号通路微生物刺激先天性免疫　出处：《南方水产科学》2017年05期 　论文类型：期刊论文

【摘要】：Toll受体蛋白(Toll receptors)是一类重要的模式识别受体,在无脊椎动物先天性免疫系统中发挥重要作用。文章对斑节对虾(Penaeus monodon)新型Toll9受体基因(Pm Toll9)进行了研究:以人源胚胎肾细胞(HEK293T)成功构建体外细胞免疫模型,通过免疫印迹方法证实Pm Toll9重组真核蛋白可在HEK293T中成功表达。双荧光素酶报告系统检测发现在200 ng转染浓度处Pm Toll9对NF-κB报告基因的激活效果显著。q RT-PCR数据证明PmToll9成功激活HEK293T细胞Toll-like receptor(TLR)信号通路,促进通路下游髓样分化因子(My D88)、肿瘤坏死因子(TNF-α)和白介素(IL-10)的上调表达,同时酶联免疫吸附测定结果证明Pm Toll9可促进TNF-α蛋白表达水平显著上调。斑节对虾体内细菌刺激实验结果显示无乳链球菌(Streptococcus agalactiae)可激活Pm Toll9在肝胰腺、肠、淋巴和鳃中的表达,哈维氏弧菌(Vibrio harveyi)可显著抑制Pm Toll9在肝胰腺中的表达。提示无乳链球菌可通过Pm Toll9激活Toll信号通路,引起机体免疫防御反应,而哈维氏弧菌对此过程具有一定的抑制作用。
[Abstract]:Toll receptor protein Toll receptor is an important type of pattern recognition receptor. In this paper, the novel Toll9 receptor gene Pm Toll9 of Penaeus Monodon (Penaeus Monodon) was studied. Human embryonic kidney cells (HEK293T) were used to successfully construct a cell immune model in vitro. The recombinant eukaryotic protein of PmToll9 was successfully expressed in HEK293T by Western blot. The results of double luciferase report system showed that the activation effect of PmToll9 on NF- 魏 B reporter gene was significant at 200ng transfection concentration. Q RT-PCR data proved PmToll9. Toll-like receptor TLR signaling pathway was successfully activated in HEK293T cells. To promote the up-regulation of the expression of myeloid differentiation factor (my D88A), tumor necrosis factor (TNF- 伪) and interleukin-10 (IL-10) downstream of the pathway. The results of enzyme-linked immunosorbent assay (Elisa) showed that pm Toll9 could significantly up-regulate the expression of TNF- 伪 protein, and Streptococcus agalactiae could activate the expression of pm Toll9 in hepatopancreas, intestine, lymph nodes and gills. Vibrio harveyi) could significantly inhibit the expression of pm Toll9 in hepatopancreas, suggesting that Streptococcus actinomycetes can activate Toll signaling pathway through pm Toll9 and induce immune defense response, but Vibrio harveyi can inhibit this process to some extent.
【作者单位】：中国水产科学研究院南海水产研究所农业部南海渔业资源开发利用重点实验室;上海海洋大学水产与生命学院;
【基金】：国家自然科学基金项目(31202019) 广州市珠江科技新星专项(201506010054) 现代农业(虾)产业体系建设专项资金(CARS-47) 广东省科技创新青年拔尖人才项目(2015TQ01N583)
【分类号】：S945.47

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