稳定表达人TLR5及NF-κB响应报告分子的HEK-293细胞系的建立

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.%Objective To construct the lentivirus plasmids of human Toll like receptor-5(TLR5)and nuclear factor(NF)-κB signaling pathway responsive luciferase and green fluorescent protein(GFP)reporters,and establish a HEK-293 cell line stably expressing human TLR5 and NF-κB responsive reporters. Methods The TLR5 and 5×NF-κB-binding sites genes were cloned to lenti?viral expressing plasmid pWSLV-puro and pWSLV-nanluc-GFP,respectively. The recombinant plasmids,pWSLV-hTLR5-puro and pWSLV-5×NF-κB-nanluc-GFP were respectively transfected with packaging plasmid into HEK293T cells in order to package lentivirus. The lentivirus was used to coinfect HEK-293 cells,and the positive clones stably expressing TLR5 and NF-κB responsive reporter (HEK293-N-T)were generated using colony-purification in present of puromycin and fluorescence activated cell sorting. The cells ex?pressing TLR5 were confirmed by Western blot and immunofluorescence. Additionally,the cells expressing NF-κB responsive reporter were confirmed by fluorescence microscopy and luciferase quantitative assay. Results The results of recombinant plasmids digestion identification and the sequencing of cDNA showed that the sequences inserted into the plasmid and the expected sequences of hTLR 5 and 5×NF-κB-binding sites genes were completely consistent. It was confirmed that the hTLR5 and 5×NF-κB-binding sites genes were correctly inserted into the vectors,and that the recombinant plasmids,pWSLV-hTLR5-puro and pWSLV-5 × NF-κB-nanluc-GFP, were successfully constructed. Additionally,Western blot and immunofluorescence analysis showed that TLR5 were stably expressed in a high level in the HEK293-N-T cells and located in the cell membranes. Moreover,the results of fluorescence microscopy and lucif?erase quantitative assay indicated that the NF-κB responsive reporter could reflect the activation of NF-κB signaling in a dose-depen?dent manner. Conclusion The HEK-293 cell line stably expressing human TLR5 and NF-κB responsive reporter has been successful?ly established and the HEK293-N-T cells can be used for the screening and evaluation of drugs target to TLR5.