用RACE技术对一株肠道病毒3′端进行扩增和序列分析  

Amplification and sequencing analysis of a strain of enterovirus by 3' RACE

作　　者：侯俊[1] 沈宏辉[1] 胡燕[1] 朱雷[1] 王志杰[1] 雷厉[1] 貌盼勇[1] 

机构地区：[1]解放军第三○二医院传染病研究所病毒研究室,北京100039

出　　处：《中华实验和临床病毒学杂志》2006年第4期410-412,共3页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的应用3′RACE技术对一株本室分离肠道病毒基因组3′末端进行扩增,并对其核苷酸序列进行比较分析。方法提取病毒总RNA,以锚定oligdT(17)引物进行逆转录,用特异引物及锚定引物进行3′RACE扩增,将PCR产物进行克隆、测序和序列分析。结果获得了该病毒的3′端核苷酸序列,同源性分析结果显示该肠道病毒的核苷酸序列与肠道病毒76、89、90、91型的同源性最高为90%左右,而与其他型别的肠道病毒的同源性均小于80%;推导的氨基酸同源性与肠道病毒76、89、90、91型均在90%以上。结论用RACE技术对肠道病毒3′端进行扩增及序列分析,证明该病毒属新型肠道病毒类,为进一步研究该病毒的分子生物学特性奠定了基础。Objective The genome of a strain of entero virus isolated by us was amplified by using RACE Technology and the nucleotide sequences compared by using NCBI Blast. Methods The template was prepared by extracting the total RNA from the A549 cells. To generate first strand cDNA from RNA, anchor OligdT（17） priming was used for reverse transcription, then the first strand cDNA was amplified by 3′ RACE using a gene specific primer and the oligo dT-anchor primer. And the PCR products were cloned, positive bacterial clones were randomly selected for sequencing and BLAST analysis. Results The sequence of of the 3′ end had 90 % homology with the EV76, EV89, EV90, EV91 were 90%, but less than 80% with other enterovirus. The amino acid sequence homology was speculated to be more than 90% .Conclusions This virus has proved to be a new type of enterovirus by 3′ RACE technology, which established a basis for understanding the molecular biology of this virus.

关 键 词：肠道病毒属 DNA 序列分析 

分 类 号：R373[医药卫生—病原生物学]