摘要
目的 :以酵母穿梭表达质粒 pGADT7 Rec为载体 ,采用CLONTECHSMART(switchingmechanismat 5′endofRNAtranscript)技术 ,在酵母细胞中构建HL 6 0细胞的酵母双杂交cDNA文库。方法 :从体外培养的HL 6 0细胞中提取总RNA ,利用经修饰的CDSⅢ引物合成cDNA第一链 ,在链的末端可自动延伸CCC ,在反应体系中加入SMARTⅢTMOligo作为cDNA第二链合成的引物 ,进行长距离 (LD) PCR合成双链cDNA ,后者经CLONTECHCHROMASPIN +TE 4 0 0 0柱纯化后 ,与线性质粒pGADT7 Rec共转化感受态酵母菌AH10 9,二者在酵母细胞中利用酵母细胞内具有较高的同源重组酶活性 ,进行同源重组成环行的有复制活性的文库质粒 ,在缺亮氨酸 (LEU)培养板上筛选出所有克隆 ,即为人HL 6 0细胞的酵母双杂交cDNA文库。结果 :共获得 1.0 3× 10 6转化子 ,文库扩增后 ,插入cDNA长度在 0 .1kb～ 1.5kb之间。结论 :在酵母细胞中利用CLONTECHSMART技术成功构建了HL 6 0细胞的酵母双杂交cDNA文库。
Objective:To construct yeast two-hybrid cDNA library of HL-60 cells in yeast cells.Methods:Total RNA was extract from HL-60 cells.The first chain of cDNA was synthesized by using the modified CDS Ⅲ as the primer and its terminal was automatically extended by CCC.the second chain was produced by adding SMART Ⅲ TM Oligo,and then ds cDNA was synthesized by Long Distance-PCR.After purified by using CLONTECH CHROMA SPIN+TE-4000 column,the PCR products as well as the linearized plasmid pGADT7-Rec were co-transformed into the competent yeast AH109.They were recombinated by yeast homologous recombinase in the yeast cells and became the active cyclic plasmid.The transformed yeasts grew in the SD/Leu-plates.All the growing clones were harvested and then constituted the cDNA library.Results:1.03×10 6 recombinants were obtained from the cDNA library.The amplified PCR fragments were between 0.1 kb～1.5 kb in size.Conclusion:The yeast two-hybrid cDNA library of HL-60 cells was successfully constructed by CLONTECH SMART method in yeast cells.
出处
《广东医学院学报》
2004年第1期1-3,共3页
Journal of Guangdong Medical College
基金
广东省自然科学基金 (0 1 1 766)
教育部科学技术研究重点项目 (0 30 96)
广东医学院标志性成果扶持项目 (XK0 0 0 2 )
