斑马鱼il-11b基因5′端启动子序列及体外活性分析

Sequence analysis of 5′ upstream promoter of il-11b gene in zebrafish and construction of reporter gene vectors

白细胞介素11(interleukin-11,IL-11)是白细胞介素家族的重要成员,其在伤口愈合、癌细胞迁移及免疫调控等过程中起到关键作用。为了深入研究IL-11的转录调控机制,通过生物信息学分析,找出斑马鱼(Danio rerio)il-11b基因的3000 bp启动子序列,经缺失处理获得2908 bp、2000 bp、1000 bp和500 bp的片段,进行PCR扩增并与pGL3-enhancer载体重组构建pGL3-il-11b-promoter-Enhancer报告基因质粒,之后转染HEK293T细胞,再用双荧光素酶报告基因检测系统检验其转录活性。结果显示:il-11b启动子序列上存在3个潜在的启动子区和7个CpG岛,潜在的转录因子结合位点包括Sp1、CACCC-box、Egr-1、ICSBP、c-Jun、COUP、GR、RAP1、NF-kappaB、REV-ErbAalpha、ER、RAR-alpha1、RXR-beta、SRF、Oct-1、Oct-2、Pit-1a、GCN4、HNF-1、TBP、C/EBPalpha、HNF-1C、Oct-2.1、USF等。成功扩增出il-11b不同片段启动子,重组质粒双酶切检测条带大小一致,测序比对正确,双荧光素酶检测pGL3-il-11b、pGL3-il-11b-Δ1、pGL3-il-11b-Δ2、pGL3-il-11b-Δ3、pGL3-il-11b-Δ4、pGL3-il-11b-Δ5报告基因质粒的活性分别是pGL3-enhancer对照组的1.94、14.92、5.33、9.11、0.75、1.32倍。该结果为研究细胞因子参与的免疫相关信号调控提供了有力的研究工具。

As one of the important members of interleukin, interleukin-11(IL-11) plays a pivotal role in many biological processes, including wound healing, cancer cells migrating, immunity regulating, and so on. To study the transcription regulatory mechanism of il-11 b, the 3 000 bp promoter of an interleukin-11 b( il-11 b)in zebrafish(Danio rerio) was found through bioinformatics analysis, and we obtained 2 908 bp, 2 000 bp, 1 000 bp and 500 bp different fragments after deletion processing. These promoter sequences were amplified through PCR method and reconstructed into pGL3-enhancer empty plasmid to form reporter gene vectors. We used dual-luciferase reporter assay system to detect the activity of these promoters in HEK293 T cell line. Bioinformatics results indicated there are about 3 promoter regions and 7 CpG islands in the sequence of il-11 b promoter, and 60 binding sites including Adf-1, Krox-24, Sp1, CACCC-box, Egr-1, ICSBP, c-Jun, COUP, GR, RAP1, NF-kappaB, REV-Erbalpha, ER, RAR-alpha1, RXR-beta, SRF, Oct-1, Oct-2, Pit-1 a, GCN4, HNF-1, TBP, C/EBPalpha, HNF-1 C, Oct-2.1, USF, etc. The fragment sizes of all recombinant plasmids were consistent by double enzyme digestion, and the sequences were correct. Compared to control group, the results of luciferase showed the activity of pGL3-il-11 b, pGL3-il-11 b-Δ1, pGL3-il-11 b-Δ2, pGL3-il-11 b-Δ3, pGL3-il-11 b-Δ4, pGL3-il-11 b-Δ5 were about 1.94, 14.92, 5.33, 9.11, 0.75, 1.32 folds, respectively. These results may provide a tool to investigate the immune signals in which cytokines are involved.