【摘要】 为研究猪德尔塔冠状病毒（PDCoV）感染能否诱导宿主细胞产生IL-8，本研究将PDCoV分别感染猪小肠上皮细胞（IPEC）和ST细胞，感染后6 h、12 h和24 h收集细胞，通过荧光定量PCR方法检测IL-8 mRNA转录水平。构建表达PDCoV M蛋白的真核表达质粒pCAGGS-M，经测序鉴定无误后转染HEK 293T细胞，采用间接免疫荧光试验（IFA）和western blot方法检测PDCoV M蛋白的表达。将pCAGGS-M质粒转染IPEC细胞，采用荧光定量PCR方法检测PDCoV M蛋白对IL-8转录的影响。为进一步评价PDCoV M蛋白对IL-8转录水平的影响，将不同浓度pCAGGS-M质粒（10 ng、100 ng和200 ng）、IL-8启动子质粒和pRL-TK质粒共转染HEK 293T细胞，采用双荧光素酶活性分析试验评价PDCoV M蛋白对IL-8转录活性的影响。荧光定量PCR结果显示，与未感染组相比，PDCoV感染能够显著上调细胞中IL-8 mRNA转录水平，其中PDCoV感染IPEC细胞6 h、12 h和24 h后IL-8 mRNA分别上调3倍、37倍和173倍；PDCoV感染ST细胞6 h、12 h和24 h后IL-8 mRNA分别上调20倍、19 000倍和48 000倍。IFA和western blot结果显示PDCoV M蛋白正确表达，与空载体质粒转染组相比，转染表达PDCoV M蛋白的质粒能够上调IPEC细胞IL-8转录水平约4.3倍。双荧光素酶活性分析试验结果显示，与对照组相比，转染10 ng、100 ng和200 ng pCAGGS-M质粒能够分别诱导细胞中IL-8转录活性上调1.9倍、3.8倍和5.7倍；表明PDCoV M蛋白能够上调宿主细胞IL-8的转录活性。综上所述，PDCoV感染能够显著上调IPEC和ST细胞中IL-8的转录水平，其中PDCoV M蛋白参与诱导细胞IL-8的产生，本研究为深入了解PDCoV致病机制提供参考依据。更多还原

【Abstract】 In order to investigate whether PDCoV infection can induce the production of IL-8, IPEC and ST cells were infected with PDCoV, respectively, and the transcription level of IL-8 mRNA was detected by real-time quantitative PCR at 6, 12and 24 hpi. Constructed PDCoV M eukaryotic expression plasmid named pCAGGS-M and the protein expression was verified in HEK 293T cells by immunofluorescence and western blot experiments. Besides, the IPEC cells were transfected with PDCoV M protein, and the effect of PDCoV M protein on IL-8 transcription was detected by real-time PCR. In order to further evaluate the effect of PDCoV M on IL-8 transcription level, HEK 293T cells were co-transfected with different concentrations of PDCoV M protein plasmid(10 ng, 100 ng and 200 ng), IL-8 promoter plasmid and pRL-TK plasmid and the effect of PDCoV M protein on IL-8 transcription activity was evaluated by luciferase assay. The results showed that compared with the uninfected cells, the transcription level of IL-8 mRNA was significantly up-regulated after PDCoV infection. In IPEC cells, IL-8 mRNA level was upregulated by 3 times, 37 times and 173 times at 6, 12 and 24 hpi. In ST cells, IL-8 mRNA level was up-regulated by 20 times,19000 times and 48000 times at 6, 12 and 24 hpi. Immunofluorescence and western blot experiments showed that PDCoV M was successfully expressed. Compared with the empty vector plasmid transfection group, PDCoV M significantly up-regulated the IL-8transcription level of IPEC cells about 4.3 times. In addition, the results of luciferase assay analysis showed that compared with the control group, the transcription activity of IL-8 was increased by 1.9 times, 3.8 times and 5.7 times by transfecting 10ng, 100ng and200ng pCAGGS-M plasmids. These results indicated that PDCoV M protein could up-regulate IL-8 transcriptional activity. In conclusion, PDCoV infection can significantly up-regulate the transcription level of IL-8 in IPEC and ST cells, and PDCoV M protein is involved in inducing the production of IL-8 in cells. This study provides a theoretical basis for in-depth understanding of the pathogenesis of PDCoV.更多还原