AalDV-3介导的miRNA海绵载体构建及其在埃及伊蚊体内外的抑制效果测试

Construction of AalDV-3 Mediated mirRNA Sponge Delivery System and Its Effect on Cell and Larva of Aedes aegypti

miRNA不仅广泛参与了蚊生长、发育的调控,还参与了媒介与病原体的相互作用,因此miRNAs可能作为蚊虫防制与蚊媒病原体控制的靶点,但目前尚无有效的转导系统。为研究白纹伊蚊浓核病毒-3(AalDV-3)是否可以作为有效的载体,我们利用内含子内表达miRNA海绵的策略,以埃及伊蚊内源性miR-210为靶基因,构建了重组病毒AalDV-anti-210。使用跨内含子引物通过RT-PCR验证了内含子可在蚊体内外的剪接效果,通过qPCR方法对埃及伊蚊Aag2细胞与埃及伊蚊幼虫的感染后的miR-210的表达水平进行检测。结果证实,含有miRNA海绵的内含子可以有效的蚊体内外剪接,重组病毒可以在蚊体内外高效的抑制靶miR-210的表达水平。该结果为探索AalDV-3在蚊虫基因功能研究及生物杀虫剂方面的潜在价值提供了理论依据。

microRNAs（miRNAs）not only play the key roles in regulation of the growth and development of mosquito,but also has an important function in interaction between the pathogen and vector.So,miRNAs can be used as the molecular target for development of an alternative method for mosquito and mosquito-borne disease control.However,an effective delivery system is still need.Mosquito densovirus have the potential for vector control as transducing agents to express foreign toxins or small interfering RNAs molecules in vitro and in vivo.In this study,we report the development of a recombinant Aedes albopictus densovirus-3（AalDV-3）miRNA sponge expression system,using an intronic miRNA sponge expression strategy.To test the inhibition effect of recombinant virus medicated miRNA sponge on endogenous miR-210,Aedes aegypti Aag2 cell and 1st-2nd larvae were infected,respectively.The splicing of the intronic miRNA sponge expression constructs in vitro and in vivo were tested by RT-PCR with intron span primers,and the relatively expression level of miR-210 was confirmed by qPCR.As results,AalDV-3can be used to decrease endogenous miRNAs by generating an antisense sponge in vitro and in vivo,which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of densovirus for vector control.