乳鼠成骨细胞酵母双杂交cDNA文库构建  

Construction of cDNA library of suckling mouse osteoblasts using yeast two-hybrid system

作　　者：林振[1] 江建明[1] 袁亮[1] 冯瑞强[1] 付兆宗[1] 孟越[1] 杨德鸿[1] 

机构地区：[1]南方医科大学南方医院,脊柱骨科,广东广州510515

出　　处：《中国骨质疏松杂志》2013年第9期930-933,共4页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金资助(30973061)

摘　　要：目的构建乳鼠成骨细胞的酵母双杂交cDNA文库。方法用TRIzol法提取乳鼠成骨细胞总RNA，按照SMART cDNA Library Construction Kit （CLONTECH）说明书反转录合成双链cDNA，Spin Column去除短片段，然后用同源重组的方法将双链cDNA和PGADT7-rec载体共转化到酵母细胞Y187中，建立文库并计算文库滴度。结果提取的总RNA的A260／A280为1．99，琼脂糖凝胶电泳示28SrRNA、18SrRNA2条带，且28S与18S带亮度比值约为2。酵母文库库容为1．68×10^7，重组率为100％。插入片段PCR检测提示大小分布为1．5-4．0kb，平均长度约为3．0kb。结论乳鼠成骨细胞酵母双杂交cDNA文库构建成功。Objective To construct a yeast two-hybrid cDNA library from suckling mouse osteoblasts. Methods Total RNA of suckling mouse osteoblasts was isolated using TRIzol method. The double-strand cDNA was synthesized according to the instructions （SMART cDNA Library Construction Kit） , and short clips were removed using Spin Column. Then the PGADT7-ree vector and the amplified double-strand cDNA were co-transfeeted into Saceharomyceseerevisiae Y187 cells using homologous recombination mediated approach. After the construction of cDNA library, the titer of the library was counted. Results The ratio of A260 to A280 of the extracted RNA was 1.99. Two bands （28SrRNA, 18S rRNA） exhibited in agar gel eleetrophoresis, and the ratio of 28S rRNA to 18S rRNA of the light density was about 2. A total of 1.68 ×10^7 recombinants were obtained from the cDNA library, and the percentage of recombinant was 100%. The inserted fragment size of recombinants ranged from 1.5 kb to 4.0kb, detecting using PCR method. The average length of the inserted cDNA fragment was 3.0 kb. Conclusion A cDNA library of suckling mouse osteoblasts using yeast two-hybrid system has been constructed successfully.

关 键 词：成骨细胞 CDNA文库 酵母双杂交 

分 类 号：R681[医药卫生—骨科学]