靶向O型口蹄疫病毒shRNA转基因猪体细胞抗病毒活性研究

Anti-viral activity of cells from foot-and-mouth disease virus shRNA transgenic pig

为研究靶向O型口蹄疫病毒(FMDV)VP1基因shRNA转基因猪体细胞的抗病毒活性,本实验在成功培育转基因克隆猪的基础上,通过分离与培养转基因猪体细胞,对其shRNA进行PCR和Southern blot检测,并将FMDV感染体细胞中,通过细胞病变(CPE)、间接免疫荧光试验(IFA)和实时荧光定量PCR(Real-time PCR)分析转基因猪体细胞抗FMDV活性。结果表明,转基因猪体细胞基因组DNA中携带有靶向FMDV VP1基因的shRNA基因片段。与非转基因猪体细胞相比,接种FMDV转基因猪体细胞其出现CPE的时间延迟,细胞内病毒含量显著降低,细胞感染病毒36 h时,对细胞中FMDV VP1基因抑制效率为53.6%。表明该靶向FMDV shRNA转基因克隆猪体细胞在体外具有良好的抗病毒活性。本研究为进一步在体内评价转基因动物的抗病毒活性奠定了基础。

To investigate the antiviral activity of cells trom O type foot-and-mouth disease virus （FMDV） VP1 shRNA transgenic pig, the porcine somatic cells was isolated from the transgenic cloned pigs and cultured in vivo for the shRNA identification by PCR and southern blot, and the detection of FMDV replication inoculated in the cells by cytopathic effect （CPE） observation, indirect immunofluorescence assay and real-time PCR. The results indicated that shRNA was stably integrated into genomic DNA of transgenic pig, and showed the anti-virus activities by delaying the presence of CPE, and significantly decreasing the virus VP1 mRNA expression by 53.6% than non-transferred transgenic pig at 36 hours post infection of FMDV, which provided the evidences that integrated shRNA in somatic cells of transgenic pig possessed antiviral activity in vivo. This study laid a foundation for further evaluation of the antiviral activity of transgenic animal in vivo.