应用抑制消减杂交和cDNA芯片技术筛选流行性感冒病毒感染相关基因  

Combining Suppression Subtractive Hybridization(SSH)and cDNA Microarray for Rapid Identification of Differentially Expressed Genes From Influenza Virus Infected Host Cells

作　　者：陈忠斌[1] 杨静[1] 王华[1] 孙偶军[1] 王升启[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《病毒学报》2003年第1期21-26,共6页Chinese Journal of Virology

基　　金：国家自然科学基金项目 (项目号 :30 0 0 0 14 6 )

摘　　要：病毒基因组有限的编码能力和以病毒蛋白为靶的抗病毒药物易出现耐药性 ,使从病毒感染宿主筛选病毒感染相关生物大分子作为抗病毒药靶和诊断标志物成为新的研究方向。为了筛选流行性感冒 (流感 )病毒感染相关基因 ,采用抑制消减杂交 (suppressionsubtractivehybridization ,SSH)技术 ,以流感病毒A/鲁防 / 93 - 9(H3N2 )感染的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。从文库中随机挑取约 80 0个克隆 ,PCR扩增其中插入片段 ,经纯化、紫外定量后 ,用基因芯片自动点样仪点在氨基片上 ,制备cDNA芯片。将流感病毒感染的MDCK细胞和正常MDCK细胞的总RNA分别用Cy3、Cy5反转录荧光标记后 ,与cDNA芯片杂交 ,用芯片扫描仪扫描获得芯片杂交信号 ,经阳性对照校正和归一化处理后 ,以如下条件作为判定基因差异表达的标准 ;(a)Cy3与Cy5的信号比值大于 1 5 (正常细胞用Cy5标记 )或小于 0 6 7(正常细胞用Cy3标记 ) ;(b)Cy3和Cy5信号值之一必须大于 10 0 0。经cDNA芯片筛选获得了 18个流感病毒感染特异性克隆 ,经测序和生物信息学分析发现均为流感病毒感染相关新基因EST。流感病毒感染相关基因cDNA片段的获得 ,为新型病毒药靶诊断标志物发现和功能研究提供了基础。Current antiviral drugs mainly target to viral proteins,such as reverse transcriptase and proteinase.However,virus protein-targeted drugs usually bring alout resistance.On the other hand,the number of targets for virus is limited due to the small coding capacity of viral genome.So,an alternative strategy for antiviral drug target research is to screen and validate the host-derived genes and/or proteins that are essential for the virus to complete its replication.To find the potential cellular antiviral targets which are the 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-MCDK cells as tester and the mock-infected MDCK cells as driver.Then cDNA microarray containing 768 cDNA fragments and control genes was used to further identify the differentially expressed genes in cDNA library.It showed that the length of inserts in subtractive cDNA library were between 250～1000bp.18 differentially expressed clones were selected by cDNA microarray to be novel ESTs as no functional clues were associated with them by bioinformatic analysis.These novel ESTs differentially expressed in the course of influenza virus infection would provide a good foundation for further finding of host-derived antiviral target and/or diagnostic biomarker,and also using for dissection of the molecular pathogenesis of influenza virus infection.

关 键 词：流行性感冒病毒 抑制消减杂交 CDNA芯片 病毒感染基因组学 药靶 

分 类 号：R373.13[医药卫生—病原生物学]