人肺癌细胞cDNA表达文库的构建

Construction of cDNA Expression Library from Lung Cancer Cells

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聚合酶的共同信息处理上合成双链ｃＤＮＡ，经Ｔ４ＤＮＡ聚合使其末端平齐化后，连接到经ＥｃｏＲＩ酶切后消平的质粒表达载体中，电击转化Ｅ．ｃｏｌｉ ＤＨ５α。

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.