CAJ | 학술논문

【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。
【목적】탐토응용다단발잡RNA（ shRNA）천련침묵구제역병독RNA복제적가행성。【방법】침대구제역병독（Foot and mouth disease， FMDV）비결구단백기인3B화3D보수구역설계합성3개shRNA적천련편단，병분별용3개불동서렬적계동자인도，성공구건료3shRNA천련적만병독RNAi재체。이용만병독3질립포장계통공전우293T세포포장성만병독과립。이용포장적만병독처리세포급유서，병접충FMDV，관찰FMDV억제정황。【결과화결론】결과현시，만병독처리BHK-21획득적전기인세포중검측shRNA표체；통과O형FMDV접충발현전기인세포대구제역병독적복제유명현적억제，기중재접충후24 h병독고패량부위보통세포적1/3；O형FMDV독주접충우항구제역만병독재체예처리과적3～5일령유서，재5 LD50적도하전부유서균존활，재20 LD50적도하존활솔야제고50％。구건적만병독개도다shRNA천련표체항구제역재체능제고BHK-21세포화유서대구제역병독적저항력，유효지피면료5 LD50적도내유서적사망현상，구유저항구제역병독독성적량호성능。
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 chemi-cally synthesized according to the conservative area in 3B and 3D regions of FMDV;induced by three dif-ferent 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 trans-genic 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 .