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采用一步法快速从培养的Ｋ５６２细胞中抽提、纯化ｍＲＮＡ，逆转录成ｃＤＮＡ分子后，通过在ｃＤＮＡ分子两端加ＥｃｏＲＩ适配子、Ｔ４多核苷酸激酶作用下磷酸化、ＮｏｔＩ内切酶消化、过ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ柱等步骤，与λＥｘＣｅｌｌ／ＮｏｔＩ／ＥｃｏＲＩ／ＣＩＰ载体连接，然后用包装蛋白在体外包装连接产物，感染Ｅ·ＣｏｌｉＮＭ５２２以构建ｃＤＮＡ文库。经检测：该文库含有１．０×１０６个重组子，ｃＤＮＡ分子长度在０．４～８．５ｋ６范围，扩增后的滴度达１．２×１０１０ｐｆｕ／ｍｌ，完全达到筛选低表达基因的要求，为从Ｋ５６２细胞中克隆新的锌指蛋白ｃＤＮＡ基因奠定了基础。并对构建ｃＤ－ＮＡ文库的关至和应用进行了讨论
One-step rapid extraction and purification of mRNA from cultured K562 cells followed by reverse transcription into cDNA molecules, followed by the addition of EcoRI aptamers at both ends of the cDNA molecule, phosphorylation by T4 polynucleotide kinase, digestion with NotI endonuclease After passing through a Sepharose CL-4B column, etc., it was ligated with the λExCell/NotI/EcoRI/CIP vector, and then packaging products were packaged in vitro with packaging proteins to infect E.coliNM522 to construct a cDNA library. After detection, the library contains 1.0×106 recombinants, the length of cDNA molecules is in the range of 0.4 to 8.5 k6, and the titer after amplification reaches 1.2×10 10 pfu/ml, which fully achieves the screening of low-expressed genes. The requirements laid the foundation for the cloning of a new zinc finger protein cDNA gene from K562 cells. And discussed the construction and application of cD-NA library