NLRP3炎症复合体的体外构建

Reconstruction of the NLRP3 inflammasome in vitro

为体外构建NLRP3炎症复合体,利用PCR方法扩增目的基因NLRP3、IL-1β、ASC和Pro-Caspase-1,分别连接到相应的载体中,构建重组真核表达质粒p3XFLAG-NLRP3、pEGFP-C1-ASC、pEGFP-C1-IL-1β、PCI-CASP-HA,利用Lipofectamine2000试剂转染到HEK293T细胞后,采用Western blot方法验证各蛋白的表达情况;通过质粒共转染体外构建NLRP3炎症复合体,采用ELISA方法检测IL-1β生成情况。结果显示,3XFLAG-NLRP3、pEGFP-C1-ASC、pEGFP-C1-IL-1β、PCI-CASP-HA重组质粒均被成功构建,各蛋白于HEK293T细胞内均被成功表达;质粒共转染后ELISA检测到高水平的IL-1β生成,表明NLRP3炎症复合体体外构建成功。NLRP3炎症复合体在体外的成功构建,为进一步研究其激活机制奠定了基础。

In order to reconstruct NLRP3 inflammasome in vitro,the coding sequences of NLRP3,IL-1β,ASC and Pro-Caspase1 were amplified by PCR,and these PCR products were inserted into corresponding plasmids to get the following recombinant eukaryotic expression vectors:p3 XFLAG-NLRP3,pEGFP-C1-ASC,pEGFP-C1-IL-1β and PCI-CASP-HA.The expression of the four recombinant vectors in HEK293 T cells was confirmed by Western blot.NLRP3 inflammasome was reconstructed in HEK293 T cells by co-transfection of the four recombinant vectors and the production of IL-1β was detected by ELISA.The results showed that recombinant vectors including p3 XFLAG-NLRP3,pEGFP-C1-ASC,pEGFP-C1-IL-1β and PCI-CASP-HA were constructed and each protein was successfully expressed in HEK293 T cells.Significant production of IL-1β was detected by ELISA after co-transfection,which indicated that the NLRP3 inflammasome was successfully reconstructed in vitro.Therefore,the NLRP3 inflammasome is reconstructed in vitro,which establishes foundation to further reveal the mechanism of NLRP3 inflammasome activation.