高效抗猪瘟病毒shRNA的筛选

Identification of potent antiviral shRNAs against CSFV

为降低RNAi技术的毒副作用,筛选高效抑制猪瘟病毒复制的shRNA序列,根据有关报道构建了荧光报告系统shRNA筛选平台。该平台以9种抑制效率分别为强、中、弱的shRNA筛选载体作为参照,结合荧光报告系统进一步量化shRNA的抑制效率。根据2种已知体外有效抑制猪瘟病毒复制的siRNA-N2、siRNA-NS5B,分别设计得到shRNA-N2、shRNA-NS5B表达载体,继而采用高效、高灵敏度的shRNA筛选平台对这2种shRNA进行了检测,结果显示单拷贝情况下,shRNA-N2抑制靶序列效率较强,shRNA-NS5B抑制效率弱。本试验通过新策略大大提高了shRNA干扰效率检测的灵敏度,为采用shRNA转基因克隆动物进行抗病育种的研究提供了保障。

In order to minimize toxicities induced by RNAi and identify potent antiviral shRNAs against classical swine fever virus, we developed a fluorescent reporter assay that enables identifica tion of potent shRNAs according to relevant reports. We constructed 9 pSENSOR vectors harbo ring preexisting shRNAs of different potency and combined it with the singlevector reporter as say that functionally monitors the interaction of shRNAs with their specific target sites. We con structed two pSENSOR vectors to validate the potency of shRNAs against CSFV based on two preexisting antiCSFV siRNAN2,siRNANSSB. Our results showed that shRNAN2 has a inter mediate potency and shRNANSSB inhibits its target site weakly. In our study, the new method greatly improves the sensitivity of identifying shRNAs,provides the guarantee for research of shR NAtransgenic animal preparation.