中华鳖IRF4基因克隆及免疫功能研究

干扰素调节因子IRF4基因仅表达于免疫相关细胞,在先天性免疫和特异性免疫中发挥重要作用。本研究根据中华鳖高通量测序预测的IRF4基因序列(GenBank登录号:XM_014581444),设计特异性引物扩增片段验证其编码区;通过荧光定量检测IRF4基因在健康中华鳖中组织表达情况以及脂多糖、聚肌胞苷酸诱导后在外周血淋巴细胞中的表达情况;构建真核表达载体p3xflag-IRF4,检测过表达IRF4基因后细胞对中华鳖虹彩病毒的敏感性,以及对干扰素启动子、NF-κB启动子的活性。试验结果显示,IRF4基因编码区和GenBank中序列完全相同,其推测的氨基酸序列含有1个DNA结合结构域和1个IRF相关结构域;荧光定量结果分析显示,IRF4基因表达于所检测的组织和器官,其中在血液、肝、脾中表达较高,脂多糖和聚肌胞苷酸均能诱导外周血淋巴细胞中IRF4基因的表达;过表达IRF4基因后降低细胞对中华鳖虹彩病毒的敏感性,过表达IRF4基因能显著诱导干扰素启动子活性,抑制NF-κB启动子活性。上述结果为更深入研究中华鳖IRF4参与机体免疫应答提供基础。

Cloning and Immune Function of IRF4 Gene in Soft-shelled Turtle Pelodiscus sinensis

A pair of specific primers was designed to qualify the open reading frame (ORF) of IRF4, limited in immune-related cells, deposited in Genbank (XM_014581444)by high-throughput sequencing to evaluate the immune function of IRF4 in soft-shelled turtle Pelodiscus sinensis . The tissue distribution and induction expression in peripheral blood lymphocytes(PBLs) of IRF4 after treatment with lipopolysaccharide (LPS) or polycytidine monophosphate (PolyI:C) were detected by real-time quantitative PCR to examine the sensitivity of STA cells to soft-shelled turtle iridovirus (STIV), and effects on IFN promoter and NF-κB promoter activity was examined in STA cells transfected with overexpression vector p3xflagIRF4. The results showed that the amplified IRF4 ORF was identical to the sequence deposited in Genbank and the deduced amino acid sequence contains a DBD domain and a IAD domain. Rt-qPCR results indicated that IRF4 was mainly expressed in blood, liver and spleen and both LPS and PolyI:C induced IRF4 mRNA expression level in PBLs. Overexpression IRF4 led to decrease the sensitivity of STA cells to STIV, to activate the IFN promoter activity and inhibit the NF-κB promoter activity. These findings provided a fundation to investigate the mechanism of IRF4 involved in immune response in soft-shelled turtle.