稳定表达人TLR5及NF-κB响应报告分子的HEK-293细胞系的建立

发布时间：2018-07-08 12:36

本文选题：人Toll样受体- + 核因子-κB　；参考：《国际药学研究杂志》2017年05期

【摘要】：目的构建人Toll样受体-5(hTLR5)和NF-κB信号通路响应的荧光素酶(luciferase)-绿色荧光蛋白(GFP)报告分子的慢病毒质粒,获得稳定表达hTLR5及NF-κB响应报告分子的HEK-293细胞系。方法通过PCR及全基因合成方法获得hTLR5和5×NF-κB结合位点序列,分别构建pWSLV-hTLR5-puro和pWSLV-5×NF-κB-nanluc-GFP慢病毒质粒;将2个慢病毒质粒分别与包装质粒共转染HEK-293T细胞进行慢病毒包装,将获得的慢病毒上清经浓缩后共感染HEK-293细胞,经嘌呤霉素筛选和流式细胞分选后得到表达hTLR5及NF-κB响应报告分子的HEK-293细胞。经传代后,采用Western印迹法和免疫荧光法鉴定hTLR5表达情况,荧光显微镜下观察并采用荧光素酶定量法检测不同浓度细菌鞭毛蛋白(flagellin)对NF-κB信号通路的激活情况。结果质粒酶切鉴定及DNA测序结果表明插入目的片段与预期相符,重组慢病毒质粒构建正确;HEK293-N-T细胞高表达hTRL5,且hTLR5能正确定位到细胞膜表面;鞭毛蛋白能显著激活HEK293-N-T细胞的NF-κB信号通路,且具有剂量依赖关系。结论正确构建了hTLR5和NF-κB信号通路响应的荧光素酶-GFP报告分子的慢病毒质粒,并成功建立了稳定表达hTLR5及NF-κB响应报告分子的HEK-293细胞系。
[Abstract]:Objective to construct the lentiviral plasmid of human Toll-like receptor 5 (hTLR5) and NF- 魏 B signal pathway responsive luciferase (luciferase) green fluorescent protein (GFP) reporter molecules and to obtain HEK-293 cell lines stably expressing hTLR5 and NF- 魏 B response reporter molecules. Methods pWSLV-hTLR5-puro and pWSLV-5 脳 NF- 魏 B-nanluc-GFP lentivirus plasmids were constructed by polymerase chain reaction (PCR) and complete gene synthesis, and the two lentivirus plasmids were cotransfected into HEK-293T cells for lentiviral packaging. The purified lentivirus supernatant was coinfected with HEK-293 cells. After purine mycin screening and flow cytometry sorting, HEK-293 cells expressing hTLR5 and NF- 魏 B responsive reporter molecules were obtained. After passage, the expression of hTLR5 was identified by Western blotting and immunofluorescence. The activation of NF- 魏 B signaling pathway by different concentrations of bacterial flagellin (flagellin) was detected by fluorescence microscope and luciferase quantitative method. Results the results of restriction endonuclease digestion and DNA sequencing showed that the inserted target fragment was in accordance with the expectation. The recombinant lentivirus plasmid was constructed correctly to express hTRL5 on HEK293-N-T cells, and hTLR5 could be correctly located on the surface of cell membrane. Flagellin can activate NF- 魏 B signaling pathway in HEK293-N-T cells in a dose-dependent manner. Conclusion Lentivirus plasmids of luciferase -GFP reporter molecules responding to hTLR5 and NF- 魏 B signaling pathway were constructed correctly, and HEK-293 cells stably expressing hTLR5 and NF- 魏 B response reporter molecules were successfully established.
【作者单位】：北京药物化学研究所;
【基金】：国家“重大新药创制”科技重大专项资助项目(2009ZX09103-384)
【分类号】：R392

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