利用Aag2细胞过表达体系筛选埃及伊蚊对DENV2易感相关免疫基因

SCREENING THE RELATED IMMUNE GENES SUSCEPTIBLE TO DENV2 IN AEDES AEGYPTI WITH AAG2 CELL OVEREXPRESSION SYSTEM

埃及伊蚊Aedes aegypti是登革病毒的主要传播媒介,埃及伊蚊感染登革Ⅱ型病毒(dengue virus typeⅡ,DENV2)后产生先天免疫反应的相关研究,有助于进一步理解埃及伊蚊感染DENV2的过程和机制。本研究通过埃及伊蚊和Aag2细胞感染DENV2进行转录组分析,与未染毒的蚊虫和细胞进行对比,筛选出12个表达变化较大的基因,之后分别构建含有这12个基因的PSL1180polyUBdsRED质粒载体,并使用细胞转染技术在Aag2细胞中建立目标基因的瞬时转染过表达模型,观察了目标基因过表达后细胞上清中DENV2的复制水平。结果显示,12个基因中羟基类固醇脱氢酶样蛋白、蜕皮激素20单加氧酶、3β-羟基类固醇脱氢酶3个基因过表达后细胞上清中病毒拷贝数出现了具有统计学意义的降低,表明这3个基因的表达可能影响DENV2在Aag2细胞中的复制。这3个基因的相关蛋白都涉及到20-羟基脱皮酮的合成,这提示20-羟基脱皮酮可能在蚊虫感染DENV2时介导了重要的免疫调节途径,为进一步研究蚊虫与虫媒病毒的互作机制提供了新的思路。

Aedes aegypti is the main vector of dengue fever.Studies on the innate immune response caused by dengue virus typeⅡ(DENV2)infection in Aedes aegypti contribute much to further understanding the process and mechanism of DENV2 infection in Ae.aegypti.In this study,transcriptome analysis of Ae.aegypti and Aag2 cells infected with DENV2 had revealed 12 genes with expressions dramatically regulated when compared with noninfected cell line and mosquitoes individuals.And then,the recombined plasmid vectors,PSL1180 polyUBdsRED,were constructed to load one of the 12 target genes respectively,and then transiently transfected into Aag2 cells to establish overexpression model for each gene.The viral load of DENV2 were detected and compared before and after the overexpression of target genes.As a results,3 of the 12 genes were shown overexpressed,including hydroxysteroid dehydrogenase-like protein 2,ecdysone 20-monooxygenase and 3 beta-hydroxysteroid dehydrogenase.The significant decrease of DENV2 copy number in host cell supernatant had been recorded,which indicated that the 3 genes’overexpression may have affected the replication of DENV2 in Aag2 cells.The proteins coded by the 3 genes are all found to be involved in the synthesis of 20-hydroxydesqualone,which prompt that 20-hydroxydesqualone may have mediated an important immunomodulatory pathway in mosquitoes infected with DENV2,that provides new ideas for further study the interaction mechanism of mosquito and arbovirus.