我国分离的XJ-160病毒全基因组克隆的构建  

Construction of Full-length cDNA Clone of XJ-160 Virus

作　　者：何海怀[1] 杨益良[1] 付士红[1] 李蕾[1] 周国林[1] 吕新军[1] 梁国栋[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《病毒学报》2001年第2期161-165,共5页Chinese Journal of Virology

基　　金：国家自然科学基金资助项目(批准号 3 9970 0 3 7)

摘　　要：XJ 160 virus was isolated in 1990 from Xinjiang, China. The biological characteristics of XJ 160 virus suggested that it might be a Togavirus. The serological tests showed that XJ 160 virus is a Sindbis like virus. The complete 11626 base nucleotide sequence of XJ 160 has been determined recently. It is necessary to establish a reverse genetic system for rescue of XJ 160 virus from its cDNA clone. We describe here the constructon of full length cDNA clone of XJ 160 virus. The total RNA were extracted from BHK cells infected with XJ 160 virus. Specific primers were used to amplify five fragments covering the whole genome of XJ 160 virus. All of these five fragments have the restriction enzyme sites at both termini to be assembled into the full length cDNA clone. The five PCR fragments were cloned into pGEM T vector. The clones with SP6 inserted direction were selected for subsequent manipulation. After a series of plasmid manipulation, the full length cDNA clone of XJ 160 virus was assembled into pBluescript vector. The restriction enzyme assay and sequencing analysis demonstrated that the whole genome of XJ 160 virus was correctly assembled into pBluescript vector, with added SP6 promoter in 5′ terminus and poly A in its 3′ terminus.XJ 160 virus was isolated in 1990 from Xinjiang, China. The biological characteristics of XJ 160 virus suggested that it might be a Togavirus. The serological tests showed that XJ 160 virus is a Sindbis like virus. The complete 11626 base nucleotide sequence of XJ 160 has been determined recently. It is necessary to establish a reverse genetic system for rescue of XJ 160 virus from its cDNA clone. We describe here the constructon of full length cDNA clone of XJ 160 virus. The total RNA were extracted from BHK cells infected with XJ 160 virus. Specific primers were used to amplify five fragments covering the whole genome of XJ 160 virus. All of these five fragments have the restriction enzyme sites at both termini to be assembled into the full length cDNA clone. The five PCR fragments were cloned into pGEM T vector. The clones with SP6 inserted direction were selected for subsequent manipulation. After a series of plasmid manipulation, the full length cDNA clone of XJ 160 virus was assembled into pBluescript vector. The restriction enzyme assay and sequencing analysis demonstrated that the whole genome of XJ 160 virus was correctly assembled into pBluescript vector, with added SP6 promoter in 5′ terminus and poly A in its 3′ terminus.

关 键 词：XJ-160病毒 辛德毕斯病毒 全基因克隆 

分 类 号：R373.3[医药卫生—病原生物学]