体外TLR3配体增强鸭坦布苏病毒灭活苗效果的免疫机制研究

Study on the Immune Mechanism of Effects of Inactivated Vaccine of Duck Tembusu Virus Enhanced by TLR3 Ligand in Vitro

为了阐明TLR3配体对鸭坦布苏病毒(DTMUV)灭活苗的免疫增强作用,以TLR3配体poly(I:C)和灭活DTMUV作用雏鸭外周血单个核细胞(PMBC),通过体外检测TLR3相关信号蛋白和细胞因子转录及表达水平,明确TLR3配体发挥免疫增强作用的主要信号通路。制备雏鸭PMBC,选择TLR3配体poly(I:C)与灭活DTMUV的不同组和与雏鸭PBMC相互作用,通过SYBR GreenⅠ荧光定量PCR和间接ELISA方法对细胞因子(IL-6)和干扰素(α-IFN、β-IFN、γ-IFN)的mRNA转录水平和蛋白表达水平进行测定,结果显示各细胞因子和干扰素的mRNA转录水平和蛋白表达均升高。通过SYBR GreenⅠ荧光定量PCR对TLR3相关信号蛋白(TLR3、TRIF、MyD88、NF-κB)的mRNA转录水平测定,结果显示TLR3、TRIF的mRNA转录水平升高显著,MyD88的mRNA转录水平升高不明显。抑制NF-κB的试验组,细胞因子和干扰素mRNA的转录水平显著降低,可见,NF-κB对poly(I:C)+灭活DTMUV组细胞因子和干扰素的产生发挥重要的调节作用。TLR3配体poly(I:C)通过与TLR3结合,激活了TRIF-NF-kB通路,从而引起细胞因子和Ⅰ型干扰素的产生。

In order to clarify the immune enhancement effect of DTMUV inactivated vaccine enhanced by TLR3 ligand,TLR3 ligand poly(I:C)and inactivated DTMUV were used to act on duck peripheral blood mononuclear cells(PMBC).The transcription and expression levels of signaling proteins and cytokines about TLR3 were detected in vitro to identify the main signaling pathway in which TLR3 ligand plays a signifi cant role.Firstly,PMBC was prepared from ducklings.TLR3 ligand poly(I:C)and inactivated DTMUV were selected to interact with duckling PBMC,and the mRNA transcription and protein expression levels of IL-6,α-IFN,β-IFN andγ-IFN in each group were measured by SYBR GreenⅠQuantitative real-time PCR and indirect ELISA.The results showed that the mRNA transcription and protein expression levels of the cytokine and interferons were increased.The mRNA transcription levels of TLR3,TRIF,MyD88 and NF-κB were determined by SYBR GreenⅠQuantitative real-time PCR.The results showed that the mRNA transcriptionlevels of TLR3 and TRIF were signifi cantly increased,while the mRNA transcription levels of MyD88 were not signifi cantly increased.NF-κB played an important role in regulating the production of cytokines and interferon in the poly(I:C)+inactivated DTMUV group.So TLR3 ligand poly(I:C)can activate the TRIF-NF-KB pathway by binding to TLR3 to cause the production of cytokines and type I interferon.