【摘要】 为构建稳定表达马NOD样受体热蛋白结构域相关蛋白3(eqNLRP3)的细胞系，并用于eqNLRP3炎性小体激活的研究，本研究采用同源重组的方法将经PCR扩增的eqNLRP3和GFP基因克隆至pLPCX中，构建重组表达质粒pLPCX-Flag-eqNLRP3-GFP，经PCR和测序鉴定正确后与pCGP、pVSV-G共转染HEK293T细胞，48 h后获得携带eqNLRP3的慢病毒。将该慢病毒感染HEK293T细胞，并采用嘌呤霉素筛选携带绿色荧光(GFP+)的单克隆细胞。采用western blot鉴定该细胞中eqNLRP3蛋白的表达、反应原性及细胞遗传稳定性。Western blot结果显示在146.1 ku处出现特异性条带，且传至1、4、7、10代的细胞均出现该条带，表明获得稳定表达eqNLRP3蛋白的HEK293T细胞系HEK293Teq NLRP3(2G1细胞)。采用NLRP3特异性激活剂尼日利亚菌素(Nig)处理2G1细胞1 h；将表达质粒pCDNA3.1-eqASC-HA转染2G1细胞，加入Nig处理1 h，采用激光共聚焦显微镜分别观察上述细胞中e NLRP3的聚化状态，及在2G1细胞中表达eqASC后能否形成ASC斑点(eqNLRP3-eqASC的复合物，即NLRP3炎性小体激活的典型特征)。结果显示，正常2G1细胞中呈绿色荧光的eqNLRP3弥散分布于细胞质中，加入Nig后弥散的eqNLRP3在细胞质中形成典型绿色荧光的点状聚集。转染表达eqASC质粒的2G1细胞中出现的红色荧光(eqASC)主要在细胞质中呈弥散性分布；转染表达eqASC的2G1细胞用Nig处理后形成2.5μm的中间呈绿色荧光四周呈红色荧光的典型ASC斑点。上述结果表明2G1细胞可以响应特异性激活剂并诱导eqNLRP3的聚化，招募ASC形成ASC斑点。将本实验室构建的iGLuc报告系统中的4种表达炎性小体蛋白质粒分别与EIV各蛋白对应质粒共转染HEK293T细胞，24 h后利用该报告系统检测各组细胞中的荧光素酶活性；采用western blot检测各共转染细胞中各EIV蛋白的表达对该系统中4种炎性小体蛋白表达的影响，通过上述两个试验筛选能够激活eqNLRP3炎性小体的EIV蛋白。iGLuc报告系统检测结果显示，仅表达EIV M2蛋白质粒的细胞上清中荧光素酶活性显著高于阳性对照组(P<0.05);western blot结果显示，EIV M2蛋白的表达对4种炎性小体蛋白的表达均无明显影响，表明EIV M2蛋白激活了eqNLRP3炎性小体。将表达EIV M2蛋白及表达eqASC的质粒共转染2G1细胞，24 h后经激光共聚焦显微镜观察以进一步验证上述结果。结果显示，该组细胞中带绿色荧光的eqNLRP3出现点状聚集，并招募细胞质中带红色荧光的eqASC形成了ASC斑点，与i GLuc报告系统筛选结果一致。上述结果表明，EIV感染对HEK293Teq NLRP3细胞系中的eqNLRP3产生了聚化效应，并招募ASC形成ASC斑点。本研究建立了稳定表达eqNLRP3蛋白的HEK293T细胞系，为马属动物病原感染激活NLRP3炎性小体的评价奠定了物质基础。更多还原

【Abstract】 To create a stable cell line expressing equine NOD-like receptor pyrilin domain-associated protein 3(NLRP3) and to study the activation of the eqNLRP3 inflammasome, we cloned PCR-amplified eqNLRP3 and GFP genes into pLPCX by homologous recombination. The recombinant expression plasmid pLPCX-Flag-eqNLRP3-GFP was constructed and correctly identified by PCR and sequencing, and co-transfected with pCGP and pVSV-G into HEK293T cells. Lentivirus carrying eqNLRP3was obtained 48 hours later. The lentivirus was infected with HEK293T cells, and the green fluorescent(GFP+) monoclonal cells were screened by purinomycin screening. The expression, reactivity and genetic stability of eqNLRP3 protein were determined by western blot. Western blot results showed that specific bands were present at 146.1ku, and the bands were present in all the cells transmitted to the 1st, 4th, 7th and 10th generations, indicating that HEK293T cell line HEK293Teq NLRP3(2G1 cells), which statically expressed NLRP3 protein, had been obtained. 2G1 cells were treated with the NLRP3 activator Nigeriectin(Nig) for 1 hour. The expression plasmid pCDNA3.1-eqASC-HA was transfected into 2G1 cells and treated with Nig for 1 hour. The aggregation state of eqNLRP3 in the above cells was observed by confocal laser microscopy, and whether ASC spots(eqNLRP3-eqASC complex,which is typical of NLRP3 inflammasome activation) could be formed after expression of eqASC in 2G1 cells. The results showed that eqNLRP3 with green fluorescence in normal 2G1 cells was dispersed in the cytoplasm, and after the addition of Nig, the dispersed eqNLRP3 formed a typical green fluorescent dot aggregation in the cytoplasm. The red fluorescence(eqASC) in 2G1 cells transfected with eqASC was mainly distributed in the cytoplasm. 2G1 cells transfected with eqASC were treated with Nig to form2.5μm typical ASC spots with green fluorescence in the middle and red fluorescence around. These results indicate that HEK293Teq NLRP3can respond to specific activator stimulation and induce the aggregation of NLRP3, recruiting ASC to form ASC spots. HEK293T cells were transfected with four kinds of inflammasome-expressing protein granules in the i GLuc reporting system constructed in our laboratory and corresponding plasmids of EIV proteins, respectively. The luciferase activity in each group of cells was detected by the reporting system 24 hours later. Western blot was used to detect the effect of the expression of EIV proteins in the cotransfected cells on the expression of four inflammasome proteins in the system, and the EIV proteins that can activate the eqNLRP3 inflammasome were screened through the above two tests. The results of i GLuc reporting system showed that the luciferase activity in the supernub of cells expressing only EIV M2 protein granules was higher than that in positive control group(P<0.05). Western blot results showed that the expression of EIV M2 protein had no significant effect on the expression of the four inflammasome proteins, indicating that EIV M2 protein activated the eqNLRP3 inflammasome. Plasmid expressing EIV M2 protein and eqASC were co-transfected into 2G1 cells and observed by confocal laser microscope 24 hours later to further verify the above results. The results showed that eqNLRP3 with green fluorescence appeared spot-like aggregation in this group of cells, and eqASC with red fluorescence in the cytoplasm was recruited to form ASC spots, which was consistent with the screening results of i GLuc reporting system. These results suggest that EIV infection has a aggregative effect on NLRP3 in HEK293Teq NLRP3cell line and recruits ASCs to form ASC spots. In this study, HEK293T cell line with stable expression of eqNLRP3 protein was established, which laid a material basis for the evaluation of NLRP3 inflammasome activated by pathogen infection in equine animals.更多还原