沉默白细胞介素10基因对猪繁殖与呼吸综合征病毒(PRRSV)感染的影响

Effect of porcine interleukin-10 gene silence on infection of pig reproductive and respiratory syndrome virus(PRRSV) in porcine immune cells

为了研究藏猪免疫细胞中IL-10基因表达抑制后对RNA病毒感染及其免疫应答的影响,本实验构建和筛选了抑制猪IL-10基因表达的shRNA干扰分子及其表达载体,并以新型的壳聚糖接枝聚乙二醇和聚乙烯亚胺修饰分子(CS-N-PEI-PEG)通过离子交联法进行分子包装,制备DNA-壳聚糖纳米分子,体外转染藏猪免疫细胞,观察猪繁殖和呼吸障碍综合征病毒(PRRSV)诱导免疫细胞IL-10基因的表达及其增殖感染水平.实验结果表明:与未转染纳米分子或者纳米分子不表达shRNA的空白对照组相比较,shRNA实验组的IL-10基因表达在24～72h内明显抑制(P<0.05),免疫细胞中PRRSV的增殖水平显著下降(P<0.05);同时IL-2、IL-4、IL-6和IFN-γ基因的mRNA水平明显升高(P<0.05).这些提示IL-10的shRNA转染能使免疫细胞的抗感染能力显著增强,甚至可完全抑制PRRSV的复制.

In order to observe the effect of porcine IL-10 gene silence with interference RNA on RNA vi- rus infection, the experiment was conducted to construct and screen novel short hairpin RNA targeted to pig IL-10 gene and their recombinant vectors. The recombinant shRNA silencer plasmid to IL-10 （pS- AS-ILl0） was then packed with chitosan modified with PEG and PEI to prepare nanoparticles by iono- tropic gelation method （CNP）, which contains recombinant plasmid to transcribe a small hairpin RNA （shRNA） for swine IL-10 gene. The ability and effect of CNP-pS-ASqL10 to silence IL-10 gene expres- sion was studied through testing the level of IL-10 mRNA in swine blood immune ceils by quantitative fluorescence real-time RT-PCR, which was cultured in witro and infected by porcine reproductive and re-spiratory syndrome virus （PRRSV）. Meanwhile, the expression of IL-2, IL-4, IL-6 and INF-γ gene were also analyzed in the infected cells by QRT-PCR. The results were found that the small hairpin RNA （shRNA） for swine IL-10 significantly suppressed the expression of IL-10 gene in the infected cells in comparison with that of the control cells （P〈0.05） ; the replication level of PRRSV was significantly lower than that of the control cell （P〈0.05）. Moreover, the mRNA level of IL-2, IL-4, IL-6 and INF- γ gene were markedly higher than those of the control cell （P〈0.05）. These suggest that the silence of pig IL-10 gene expression can increase the level of cytokine genes, and significantly inhibit the replica- tion of PRRSV in swine blood immune cells.