团头鲂白细胞介素6基因启动子报告载体的构建及活性分析

Reporter plasmids construction of Megalobrama amblycephala interleukin 6 gene promoter and analysis of their bioactivity

为了解鱼类白细胞介素6(IL-6)基因的转录调控原理及其免疫作用机制,构建5个团头鲂il-6基因启动子的荧光素酶报告质粒(PGL3-IL-6P),以PGL3-basic质粒作为阴性对照,将它们分别与内参质粒pRL-TK共转染进鲤EPC细胞,并用100ng/mL的LPS诱导转染过重组载体的细胞,24h后检测荧光素酶的活性。结果显示:与阴性对照组相比,质粒转染组PGL3-IL-6P-0、PGL3-IL-6P-1、PGL3-IL-6P-2、PGL3-IL-6P-3与PGL3-IL-6P-4的荧光素酶活性均明显升高,其中PGL3-IL-6P-4组的荧光素酶活性最高,表明该启动子缺失体(-379至+34)包含il-6的核心启动子。用LPS刺激转染过重组载体的细胞后发现,与未经LPS刺激的对照组相比,仅PGL3-IL-6P-4组的活性明显增强,而其余缺失体的活性则没有显著变化,这进一步表明PGL3-IL-6P-4缺失体包含核心启动子区,并推测该核心启动子区域存在的潜在转录因子NF-κB与C/EBPβ等可能是响应LPS刺激的重要作用元件。

Interleukin-6(IL-6)is a multifunctional cytokine,playing important roles in immune defense.To better understand the transcriptional principle and immune mechanism of fish il-6,five reporter plasmids of the Megalobrama amblycephala il-6promoter were constructed,co-transfected with the internal control plasmid pRL-TK into carp EPC cell,induced by LPS,and the luciferase activity was then detected after 24 h.The results showed that compared with the negative control PGL3-basic group,the luciferase activity of the PGL3-IL-6P-0,PGL3-IL-6P-1,PGL3-IL-6P-2,PGL3-IL-6P-3and PGL3-IL-6P-4groups were increased,and the PGL3-IL-6P-4group showed the highest luciferase level,suggesting that the promoter deletion(-379 to +34)contains the core promoter region of the il-6promoter.After stimulation by 100ng/mL LPS,the luciferase activity of the PGL3-IL-6P-4group was significantly activated,while that of the other groups did not change significantly,further indicating that PGL3-IL-6P-4indeed contains the core promoter region and the potential transcription factor NF-κB and C/EBPβin this promoter region may play primary roles in response to LPS induction.