慢病毒介导多短发卡 RNA 串联载体构建与体内外抗口蹄疫效果评估
Construction of lentivirus-mediated multi-shRNAs vector and evaluation of anti-FMDV in vitro and vivo
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摘要: 【目的】探讨应用多短发卡 RNA（shRNA） 串联沉默口蹄疫病毒RNA复制的可行性.【方法】针对口蹄疫病毒 （Foot and mouth disease, FMDV） 非结构蛋白基因 3B 和 3D 保守区域设计合成3个 shRNA 的串联片段，并分别用3个不同序列的启动子引导，成功构建了3shRNA串联的慢病毒RNAi载体.利用慢病毒3质粒包装系统共转于293T细胞包装成慢病毒颗粒.利用包装的慢病毒处理细胞及乳鼠，并接种 FMDV，观察 FMDV 抑制情况.【结果和结论】结果显示，慢病毒处理 BHK-21 获得的转基因细胞中检测 shRNA 表达；通过 O 型 FMDV 接种发现转基因细胞对口蹄疫病毒的复制有明显的抑制，其中在接种后 24 h 病毒拷贝量仅为普通细胞的 1/3；O 型 FMDV 毒株接种于抗口蹄疫慢病毒载体预处理过的 3~5 日龄乳鼠，在 5 LD50滴度下全部乳鼠均存活，在 20 LD50 滴度下存活率也提高50%.构建的慢病毒介导多 shRNA 串联表达抗口蹄疫载体能提高 BHK-21 细胞和乳鼠对口蹄疫病毒的抵抗力，有效地避免了 5 LD50 滴度内乳鼠的死亡现象，具有抵抗口蹄疫病毒毒性的良好性能.Abstract: 【Objective】 The study was conducted to investigate the inhibit replication of foot-and-mouth disease virus (FMDV) by multi-shRNAs expression.【Method】 Three shRNAs were designed and chemically synthesized according to the conservative area in 3B and 3D regions of FMDV; induced by three different promoters respectively, they were constructed into a multi-shRNAs expressing lentiviral plasmid. The anti-FMDV multi-shRNAs expressing lentiviral particles were packaged by co-transfecting the three plasmid lentivirus packaging system into 293T cells. Infected FMDV into lentivirus-treated cells and suckling mice, inhibitions of FMDV were observed.【Result and conclusion】 Results showed that transgenic BHK-21 cells were obtained by infecting lentivirus. The expression of shRNAs in transgenic cells was detected by stem-loop RT-PCR. Inoculated with FMDV type O, the transgenic cells were proven to have an obvious inhibition to FMDV replication, which could reduce virus growth by three folds (24 h post-infection). After infection of FMDV type O strain into 3-5 days suckling mice, no mouse mortality was observed under 5 LD50 titer, and survival time of the dead mice extended compared with negative control under 20 LD50 titer. Based on the above results, it can be concluded that the anti-FMDV multi-shRNAs expressing lentiviral vector can improve FMDV resistance of BHK-21 cells and suckling mice.
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Keywords:
- foot-and-mouth disease virus /
- multi-shRNAs /
- lentiviral vector /
- anti-virus
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