斑节对虾Toll9受体基因免疫功能的研究与表达分析

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。

Immune function and expression of Toll9 receptor gene from Penaeus monodon

Toll receptors are important pattern recognition receptors (PRRs) and play a vital role in the immune system of inverte brates.This paper investigates a new type of Toll receptor from Penaeus monodon (PmToll9).By constructing an in vitro cellular immune model in HEK293T cells,we found that PmToll9 could express a recombinant eukaryotic protein in HEK293T cells successfully with western blot technology.The dual-luciferase reporter system shows that PmToll9 could activate NF-κB reporter gene significantly when it was transfected with 200 ng DNA in HEK293T cells.The qRT-PCR data reaveal that PmToll9 can activate TLR signaling path way of HEK293T cells and promote high expression level of the downstream factors (MyD88,TNF-α and IL-10).Meanwhile,ELISA results confirm that PmToll9 could increase TNF-α protein level.The in vivo bacteria stimulation in P.mondon shows that Streptococcus agalactiae could activate the mRNA expression of PmToll9 in hepatopancreas,intestine,lymph and gill,while Vibrio harveyi could inhibit the mRNA expression of PmToll9 in hepatopancreas.It is revealed that PmToll9 could be activated and inhibited by S.agalactiae and V.harveyi via Toll signaling pathway to induce the immune defenses,respectively.