应用抑制性消减杂交技术筛选流感病毒感染宿主应答基因

从宿主系统寻找病毒感染特异性相关的生物大分子是研究病毒药物靶标和诊断标志物的新方向 .为了筛选宿主细胞中流感病毒感染特异性基因 ,采用抑制性消减杂交技术 (SSH) ,以流感病毒A 鲁防 93 9(H3N2 )感染MDCK细胞及正常MDCK细胞为材料 ,构建病毒感染特异性差减cDNA文库 ,PCR法扩增鉴定其中插入片段大小 .从差减文库中随机挑取 10 0个克隆进行测序 ,用生物信息学方法对其同源性和基因功能进行分析和预测 .结果显示 ,成功构建了流感病毒感染特异性差减cDNA文库 ,文库中cDNA片段长度在 2 5 0～ 10 0 0bp之间 .从文库中随机选取 10 0个克隆测序 ,获得了 95个有效序列 ,经blast同源性分析发现 ,大部分基因为参与宿主细胞能量代谢和蛋白质生物合成过程中的基因 ;其中 19个为无任何功能线索的新基因片段 .流感病毒感染特异性差减cDNA文库的建立和筛选出病毒感染应答候选新基因cDNA片段 ,为发现新型流感病毒药靶和诊断标志物以及病毒感染机制研究打下基础

Screening and Identification of Differentially Expressed Genes from Influenza Virus Infected-Host Cells by Suppression Subtractive Hybridization

An alternative strategy for antiviral drug finding is to inhibit the function of host protein that are essential for the virus to complete its replication. To find the potential cellular anti-viral targets which were differentially expressed genes during influenza virus infection, subtractive cDNA library was constructed by suppression subtractive hybridization(SSH) with influenza virus A/Lufang/93-9(H3N2)infected- MDCK cells as tester and mock-infected MDCK cells as driver. 100 clones were randomly selected for further DNA sequencing, blast homology analysis and function prediction. It was showed that the inserts in subtractive cDNA library were between 250—1 000 bp. Most of the subtractive genes differentially expressed during Influenza virus infection were related to energy process and protein synthesis pathway. Meanwhile, 19 clones were found to be novel EST as no functional clues were associated with them by bioinformatic analysis. The novel ESTs differentially expressed in the course of influenza virus infection would make a good foundation for further finding host-derived antiviral target and diagnostic biomarker, and also probing the molecular pathogenesis of influenza virus infection.