国家重点基础研究发展计划(973计划)(2014CB744404)
目的 构建pEGFP-N1/IL-37b真核表达载体,并检测其在THP-1细胞中的表达情况。方法 从人PBMCs中提取总RNA,利用RT-qPCR技术扩增出IL-37b基因编码区序列,克隆至pEGFP-N1真核表达载体,将构建的重组质粒pEGFP-N1/IL-37b转染到THP-1细胞中,通过RT-qPCR和Western blot检测IL-37的表达。结果 双酶切及基因测序结果显示IL-37b基因正确插入真核表达载体pEGFP-N1中;RT-qPCR和Western blot结果显示转染THP-1细胞后,IL-37表达水平明显升高(P < 0.01)。结论 成功构建了新型抗炎因子IL-37真核表达载体pEGFP-N1/IL-37b,为进一步研究IL-37功能及与相关疾病的关系奠定基础。
Objective To construct the eukaryotic expression vector pEGFP-N1/IL-37b and analyze the expression of IL-37 gene in THP-1 cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells (PBMCs) and the coding region of IL-37b gene was amplified by RT-qPCR. Then, the gene was cloned into pEGFP-N1 eukaryotic expression vector. After transfected the recombinant plasmid into THP-1 cells, the expression of IL-37 was detected by RT-qPCR and Western blot. Results Double restriction enzyme digestion and gene sequencing showed that IL-37b gene was correctly inserted into the eukaryotic expression vector pEGFP-N1.RT-qPCR and Western blot showed that the IL-37 expression level was increased significantly (P < 0.01) after transfection in THP-1 cells. Conclusions We successfully constructed a novel anti-inflammatory cytokine IL-37 eukaryotic expression vector pEGFP-N1/IL-37b, which lays a foundation for further study on IL-37 functions and its association with related diseases.
姚静,程江,裴雪枫,王静宇,袁明.人IL-37b基因真核表达载体的构建与表达[J].中国实验动物学报,2016,24(3):268~272.
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