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目的研究环指蛋白34(RING finger protein 34,RNF34)对天然免疫的调控。方法利用重组PCR方法构建pcDNA3-Flag-RNF34、pcDNA3-Flag-RNF34-ΔFYVE、pcDNA3-Flag-RNF34-ΔCID和pcDNA3-Flag-RNF34-ΔRING质粒,并在HEK293T细胞中瞬时表达;利用双荧光素酶报告基因技术检测RNF34及其3种突变体对仙台病毒(SeV)和N-RIG-Ⅰ激活NF-κB和IFN-β转录活性的影响。结果成功构建了Flag-RNF34及其3种结构域缺失突变体的真核表达质粒;发现在SeV刺激下,RNF34对NF-κB和IFN-β转录活性有明显抑制作用,RNF34-ΔFYVE、RNF34-ΔCID和RNF34-ΔRING与RNF34相比此种抑制作用减弱。同时发现对于N-RIG-Ⅰ激活的NF-κB和IFN-β活性,RNF34及其3种结构域缺失突变体也有相似的抑制作用。结论 RNF34通过负调控RIG-Ⅰ-MAVS信号通路调控天然免疫。
Objective To study the regulation of innate immunity by RING finger protein 34 (RNF34). Methods The pcDNA3-Flag-RNF34, pcDNA3-Flag-RNF34-ΔFYVE, pcDNA3-Flag-RNF34-ΔCID and pcDNA3-Flag-RNF34-ΔRING plasmids were constructed by recombinant PCR and transiently expressed in HEK293T cells. The reporter gene was used to detect the effect of RNF34 and its three mutants on the activation of NF-κB and IFN-β by Sendai virus (SeV) and N-RIG-Ⅰ. RESULTS: Flag-RNF34 and its 3 domain deletion mutants were successfully constructed. RNF34 and RNF34-RNF34 were significantly inhibited by SeV stimulation. RNF34-ΔFYVE, RNF34- This inhibition was attenuated by ΔCID and RNF34-ΔRING compared to RNF34. It was also found that RNF34 and its three domain deletion mutants also showed similar inhibitory effects on N-RIG-I-activated NF-κB and IFN-β. Conclusion RNF34 regulates natural immunity through negative regulation of RIG-Ⅰ-MAVS signaling pathway.