用磁珠从肺癌细胞株A549中直接分离mRNA.以随机六聚体为引物,在M-MLV反转录酶作用下,合成cDNA第一链,在RNaseH和DNA聚合酶的共同作用下合成双链cDNA,经T4DNA聚合酶使其末端平齐化后,连接到经EcoRI酶切后消平的质粒表达载体中,电击转化E.coliDH5α.该文库大小约为9.0×105,重组子阳性率达85%,插入的cDNA长度约在0.5～4kb之间.以该文库cDNA为模板,应用PCR方法,首次从肺癌细胞中获得了死亡蛋白酶CPP32基因.该cDNA文库的构建为筛选调节肺癌细胞死亡的其它基因打下了基础.

Messenger RNA was isolated directly from lung cancer cell line A549 using magnetic particles. First strand synthesis from mRNA was driven by M-MLV(Moloney Murine Leukemia Virus) reverse transcriptase and random hexam-eric primer, followed by second strand synthesis using RNase H and DNA polymerase I After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA was cloned into the plasmid expression vector digested with EcoRI and followed by removing cohesive end. The number of independent clones of the resulting cDNA library was about 9.0 x 105. The estimated percentage of colonies with inserts was about 85 % . The insert size ranges from 0.5 kb ~ 4 kb. The CPP32 gene coding for death protease was obtained by PCR with the cDNA library from lung cancer cells as a template for the first time. Construction of the cDNA library laid a foundation for screening other genes regulating death of lung cancer cells.