家蚕核型多角体病毒lef-11基因RNA干扰策略的优化

Optimization of RNA interference strategy for lef-11 gene of Bombyx mori nucleopolyhedrovirus

摘要:

[目的]家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus，BmNPV）lef-11基因作为杆状病毒高度保守的晚期表达因子之一，对病毒晚期基因的表达极其重要。因此对lef-11基因进行有效RNA干扰将提高宿主细胞对BmNPV的抗性。[方法]本文基于经典的sh-RNA-loop骨架及家蚕内源性的miRNA骨架，构建以BmNPV lef-11基因为靶标的干扰载体pIZ-shRNA1、pIZ-shRNA2和pIZ-DsRed-amiR279；分别构建基于A4、IE1、IE1-295、IE2、IE2-339、hr3A4和hsp70启动子驱动的干扰载体，用以评价不同启动子驱动的干扰载体的抗病毒效果，并对干扰载体进行优化。[结果]首先，本文通过比较miRNA-based和shRNA-based RNAi载体对同一靶基因同一位点的干扰效率，发现pIZ-DsRed-amiR279对BmNPV lef11基因的干扰效率超过90%，远优于shRNA-based RNAi载体的干扰效果；其次，通过对干扰载体进行优化，发现IE1启动子的效果最优，由其驱动的pIZ-DsRed-milef11干扰载体也是本研究中的最优干扰载体，对病毒的增殖抑制效果最为明显。[结论]对目的基因的干扰效果并非启动子的活性越高、miRNA积累得越多就越好，只有综合考虑干扰片段的选择、启动子的活性以及靶基因自身的功能等多方面的因素，才能提高干扰效率，达到干扰目的。

Abstract:

[Objective] Gene lef-11 is one of the high conservative late expression factors in Bombyx mori nucleopolyhedrovirus (BmNPV), and contributes to virus replication and proliferation. The effective RNA interference will increase the resistance of host cells to BmNPV. [Methods] Choosing two classical skeleton sh-RNA-loop and miRNA skeleton based on silkworm endogenous, we constructed targeted-lef-11 interference vectors: pIZ-shRNA1, pIZ-shRNA2 and pIZ-DsRed-amiR279 and optimized these vectors via different promoters. [Results] First, comparing the interference efficiency of shRNA-based and miRNA-based RNAi vectors on the same site, we found the interference efficiency of pIZ-DsRed-amiR279 on target gene was more than 90%, better than that of RNAi shRNA-based and could be applied for follow-up experiments. Second, we evaluated the antiviral effect of different interference vectors driven by A4, IE1, IE1-295, IE2, IE2-339, hr3A4 and hsp70 promoters and found that the most active promoter had less interference effect on the target gene and IE1 was the optimal screening seven promoters, which demonstrated that the accumulation of pre-miRNA was not contribute to interference. [Conclusion] The results showed that the interference effect on the target gene depended on kinds of factors, such as the interference skeleton, the activity of promoter and the function of target gene.