【摘要】 为从多发性骨髓瘤细胞中克隆与鉴定恶性相关基因并试图阐明其发病分子机制,建立了一种改进的快速表达克隆法,简单、快捷、直接从多发性骨髓瘤细胞株ARH 77cDNA文库中克隆恶性相关基因.其特点是常用的表达克隆法与经典的DNA介导的NIH 3T3转染实验相结合,直接从cDNA表达文库中克隆恶性相关基因.首先建立高质量的cDNA表达文库,将cDNA表达文库分成几个基因池,转染NIH 3T3细胞;将转化活性最强的基因池再分成几个亚基因池,转染NIH 3T3细胞;将转化活性最强的亚基因池再分成次级亚基因池,直到获得有明显转化活性的单个cDNA克隆.该技术亦可用于从其他肿瘤细胞中克隆恶性相关基因.更多还原

【Abstract】 The cloning and identification of a tumorassociated gene from human multiple myeloma (MM) is one of the most important issues to elucidate its molecular mechanisms and to establish a novel strategy against it.Since MM develops as a sporadic disease,linkage analysis is not available for tumorassociated gene cloning.We report an expression screening technique to clone the tumorassociated genes from human MM and this method can be used for the cloning of tumorassociated gene from other malignant cells.mRNA was isolated from human MM cell line ARH77 and then cDNA was synthesized,which was linked to a kind of special adaptors and amplified by polymerase chain reaction (PCR).The library was constructed by ligating cDNA with eukaryote expression vector pcDNA3.1(+) through EcoRⅠ sticky ends,and then the competent cells DH5α was transformed.The total clones of cDNA library were transferred in situ to nylon membrane,which was divided into eight equals (A～H) to set up gene pool and cultured in LB medium.Corresponding mixed plasmids of A～H gene pools were extracted,and the NIH/3T3 cells were transfected by calcium phosphateDNA coprecipitation.Counted the foci,and chose A gene pool which had more foci than others for next screening.After several cycles,two clones were gained,A6512 and A6217,whichhad significant transforming ability.DNA sequencing and homologous comparison were performed.These sequences were not the known oncogenes and transforming genes that cloned so far.Comparison with other oncogene cloning techniques,this method is simple and efficient,and can get the cDNA fragment of transforming gene from cDNA library directly.更多还原