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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T_4DNA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9.0×10~5,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.
Using magnetic beads to directly isolate mRNA from lung cancer cell line A549. The first strand of cDNA was synthesized using random hexamers as primers under the action of M-MLV reverse transcriptase, and the double strand was synthesized by RNaseH and DNA polymerase. The strand cDNA was blunt-ended by T_4 DNA polymerase and then ligated into EcoRI digested plasmid expression vector and transformed into E. coli DH5α by electroporation. The library was about 9.0×10~5 in size, and the recombinant was positive. The rate reached 85% and the length of the inserted cDNA was between 0.5 and 4 kb. Using this library cDNA as a template, the death protease CPP32 gene was first obtained from lung cancer cells by PCR. This cDNA library was constructed to screen and regulate lung cancer cells. The other genes of death lay the foundation.