Zhou Fang, Chen Rui-ting, Lu Yan, Liang Shuang, Wang Mei-xian, Miao Yun-gen
Key Laboratory of Animal Virology of Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, People's Republic of China.
Mol Biol Rep. 2014 Dec;41(12):8247-54. doi: 10.1007/s11033-014-3726-0. Epub 2014 Sep 9.
The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to sericulture industry. In this study, we developed a system of RNAi to prevent the BmNPV infection using the piggyBac transposon-derived targeting short hairpin RNA (shRNA) interference. The shRNAs targeting the genes of i.e.-1, lef-1, lef-2 and lef-3 of BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in Bm cells. The highest activity was presented in the shRNA targeting the i.e.-1c of BmNPV, of which the inhibition rate reached 94.5 % in vitro. Further a stable Bm cell line of piggyBac transposon-derived targeting shRNA interference against BmNPV was established, which has a highly efficacious suppression on virus proliferation. These results indicated that the recombinant shRNA expression system was a useful tool for resistance to BmNPV in vitro. The approach by recombinant shRNAs opens a door of RNAi technology as a strategy that offering technically simpler, cheaper, and quicker gene knockdown for promising research and biotechnology application on silkworm lethal diseases.
家蚕核型多角体病毒(BmNPV)是家蚕最具毁灭性的病害之一,给养蚕业造成了主要损失。在本研究中,我们利用源自piggyBac转座子的靶向短发夹RNA(shRNA)干扰技术开发了一种防止BmNPV感染的RNAi系统。设计了靶向BmNPV ie-1、lef-1、lef-2和lef-3基因的shRNAs,并用于抑制BmNPV在Bm细胞内的复制或增殖。靶向BmNPV ie-1c的shRNA活性最高,其体外抑制率达到94.5%。进一步建立了对BmNPV具有piggyBac转座子衍生靶向shRNA干扰的稳定Bm细胞系,该细胞系对病毒增殖具有高效抑制作用。这些结果表明,重组shRNA表达系统是体外抗BmNPV的有用工具。重组shRNAs方法为RNAi技术打开了一扇门,作为一种策略,它为家蚕致死性疾病的有前景的研究和生物技术应用提供了技术上更简单、更便宜、更快的基因敲低方法。