人细胞表面黏附分子P选择素启动子报告基因的构建及鉴定

发布时间：2018-05-14 20:11

本文选题：细胞黏附分子类 + P选择素　；参考：《南方医科大学学报》2016年03期

【摘要】：目的构建人细胞表面黏附分子P选择素(p-selectin)基因启动子荧光素酶报告基因载体pGL3-pselectin-promoter,检测其转录活性,并应用于筛选药物对其转录活性的影响。方法根据UCSC软件查找的人基因组DNA的p-selectin启动子序列并设计两端引物,扩增人基因组DNA中的p-selectin启动子。用限制性内切酶KpnⅠ和XhoⅠ双酶切质粒pGL3-Basic和p-selectin启动子后,将p-selectin基因启动子插入到pGL3-basic报告基因载体上。重组质粒命名为pGL3-pselectin-promoter。将其与内参质粒p RL-SV40瞬时共转染293F细胞,检测双荧光素酶活性。对不同启动子片段长度的p-selectin报告基因进行双荧光素酶的检测。以炎症因子和药物分组刺激转染了报告基因质粒的293F细胞并检测双荧光素酶活性。结果成功构建p-selectin基因启动子荧光素酶报告基因载体pGL3-pselectin-promoter,质粒酶切及测序结果完全正确。瞬时共转染pGL3-pselectin-promoter/p RL-SV40组荧光素酶活性为0.8573±0.4703,高于转染pGL3-Basic/p RL-SV40组的荧光素酶活性值0.03955±0.05894。pGL3-1826 bp相比较于pGL3-1092 bp组和pGL3-3738 bp组具有最强的转录活性。炎症因子LPS和TNF-α和药物As2O3均具有上调pGL3-pselectin-promoter转录活性的作用。结论 pGL3-pselectin-promoter在293F细胞中能被转录激活,并验证了炎症因子对其转录表达的作用,并为药物筛选与评价提供解决方案。
[Abstract]:Objective to construct a human cell surface adhesion molecule P selectin (P-selectin) promoter luciferase reporter gene vector pGL3-pselectin-promoter, to detect its transcriptional activity, and to screen the effect of the drug on its transcriptional activity. Methods the P-selectin promoter sequence of human genome DNA was searched according to the UCSC software and the two primers were designed. The P-selectin promoter in the human genome DNA was amplified. The P-selectin gene promoter was inserted into the pGL3-basic reporter gene carrier with the restrictive endonuclease Kpn I and Xho I double enzyme pGL3-Basic and P-selectin promoter, and the recombinant plasmid was named pGL3-pselectin-promoter. to co transfect 293F with the P RL-SV40 of the internal reference plasmid. The activity of double luciferase was detected. The P-selectin reporter gene of different promoter fragments was detected by double luciferase. 293F cells of the reporter gene plasmid were transfected with inflammatory factors and drug groups, and the activity of double luciferase was detected. The results successfully constructed the P-selectin gene promoter luciferase reporter gene. The results of plasmid digestion and sequencing were completely correct. The luciferase activity of the transient co transfected pGL3-pselectin-promoter/p RL-SV40 group was 0.8573 + 0.4703, which was higher than that of the transfected pGL3-Basic/p RL-SV40 group and the luciferase activity of 0.03955 + 0.05894.pGL3-1826 BP in the pGL3-1092 BP group and pGL3-3738 BP group. Transcriptional activity. Both inflammatory factors LPS and TNF- alpha and drug As2O3 all have the role of up regulation of pGL3-pselectin-promoter transcriptional activity. Conclusion pGL3-pselectin-promoter can be transcriptional activated in 293F cells, and the effect of inflammatory factors on its transcriptional expression is verified, and the solution for screening and evaluation of drugs is provided.

【作者单位】：南方医科大学病理生理学教研室//广东省医学休克微循环重点实验室;
【基金】：国家自然科学基金(81272095) 大学生创新创业训练计划项目(201512121268)~~
【分类号】：R459.7

【相似文献】