AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expres...AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.展开更多
1977年3月,在巴塞尔生物:中心(那次 John 作了一个关于质粒克隆载体发展的学术报告)首次相见时,我们俩就确信,寻找一个能高效克隆大型 DNA 片段(】10kb)的全新的方法,是基因技术能否广泛应用的重要一环。当时,John 需要(这个需要就是发...1977年3月,在巴塞尔生物:中心(那次 John 作了一个关于质粒克隆载体发展的学术报告)首次相见时,我们俩就确信,寻找一个能高效克隆大型 DNA 片段(】10kb)的全新的方法,是基因技术能否广泛应用的重要一环。当时,John 需要(这个需要就是发明的原动力)从细菌克隆出青霉素酰基转移酶基因。这用传统的载体是难以办到的(那时象 EcoRI、SmaI、HindⅢ和 Sau3A 这些限制性酶只能由自己制备)。John展开更多
Using plant mini-Ti cosmid pEND4K, the cosmid genomic library of Sesbania rostratawas constructed. Sizes of plant DNA inserts in clones were 25- 33 kb. The restrictionmapping showed that different recombinant involved...Using plant mini-Ti cosmid pEND4K, the cosmid genomic library of Sesbania rostratawas constructed. Sizes of plant DNA inserts in clones were 25- 33 kb. The restrictionmapping showed that different recombinant involved various types of plant DNA insert. 4clones containing leghemoglobin gene sequence of S. rostrata were obtained by in situ hy-bridization of colonies. The cloning of leghemoglobin gene sequence has been confirmedby plasmid DNA dot hybridization and Southern blot hybridization.展开更多
基金the National Natural Science Foundation of China,No.39500129
文摘AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.
文摘一个从cosmid分子克隆库中筛选特别基因顺序的遗传学方法——体内同源重组(invlvo homologous recombination)法。即使探针DNA与分子克隆库中带有与探针同源顺序的克隆发生体内重组,然后以遗传学方法进行筛选。cosmid分子克隆库构建在rec宿主细胞内,经体内包装(in vivo Packaging)成λ噬菌体颗粒,把该噬菌体颗粒转入带有探针DNA的rec^+细胞内,探针是已被克隆在与cosmid载体没有同源顺序的质粒(如PUC8或PUC9)内的。经过一段时间(1—3小时),待重组发生后,把cosmid进行体内包装。此时探针DNA连同质粒已整合入cosmid基因组内,因此它带有原为两个载体所分别带有的双重抗性——Amp^r(氨苄青霉素,PUC8或PUC9)和Kan^r(卡那霉素,cosmid)。这种双重抗性菌落可在含有这2种抗菌素的培养平皿上选出,该重组cosmid借助于λ切除酶的作用将已被整合的探针质粒重新切除,再经体内包装后,该cosmid被还原并纯化,然后可用一含有Xgal的培皿识别和选出。本文用此法以有关DNA探针从cosmid分子克隆库中分离得到含有与小鼠t复合体连锁的基因组顺序的克隆,并对该克隆作了物理图谱分析。
文摘1977年3月,在巴塞尔生物:中心(那次 John 作了一个关于质粒克隆载体发展的学术报告)首次相见时,我们俩就确信,寻找一个能高效克隆大型 DNA 片段(】10kb)的全新的方法,是基因技术能否广泛应用的重要一环。当时,John 需要(这个需要就是发明的原动力)从细菌克隆出青霉素酰基转移酶基因。这用传统的载体是难以办到的(那时象 EcoRI、SmaI、HindⅢ和 Sau3A 这些限制性酶只能由自己制备)。John
基金Project supported by the National Natural Science Foundation of China.
文摘Using plant mini-Ti cosmid pEND4K, the cosmid genomic library of Sesbania rostratawas constructed. Sizes of plant DNA inserts in clones were 25- 33 kb. The restrictionmapping showed that different recombinant involved various types of plant DNA insert. 4clones containing leghemoglobin gene sequence of S. rostrata were obtained by in situ hy-bridization of colonies. The cloning of leghemoglobin gene sequence has been confirmedby plasmid DNA dot hybridization and Southern blot hybridization.
