鸡胚成纤维细胞cDNA真核表达文库的构建及鉴定

Construction and identification of a cDNA eukaryotic expression library of chicken embryo-fibroblasts

为构建鸡胚成纤维细胞cDNA真核表达文库并对文库质量进行鉴定,从鸡胚成纤维细胞中直接提取mRNA,用紫外分光光度计测定含量。经电泳确定mRNA的质量,反转录合成第一、二链cDNA,经苯酚氯仿抽提后与EcoRⅠ接头连接,经NotⅠ酶切后用spin column除去小于500 bp的cDNA片段,与经EcoRⅠ和NotⅠ双酶切的真核表达质粒连接,连接产物电转化感受态细胞,测定文库的容量大小并通过colony PCR鉴定插入cDNA片段的大小。构建成含8.8×105个重组子的鸡胚成纤维细胞cDNA文库,重组子中平均插入外源片段长度大于1.0 kb的约占85.7%。从结果看构建的文库合格,适合用于筛选目的cDNA克隆。

Purpose： Construction and identification of a cDNA eukaryotic expression library of chicken embryo-fibroblasts （CEF）. Method： The mRNA was extracted from CEF and the quality identified using an ultraviolet spectrophotometer and form aldehyde-agarose gel electrophoresis. The cDNA was then made and extracted by phenol and chloroform. Extracted cDNA was ligated with the EcoR Ⅰ adaptor and then digested with Not Ⅰ . Fragments smaller than 500 bp were removed by a spin column and those above ligated into the eukaryotic expression plasmid pAP3neo which was digested by EcoR Ⅰ and Not Ⅰ. Competent cells were transformed with the ligated product by electroporation. The size of the cDNA library and that of inserts were also identified. Result： A cDNA eukaryotic expression library of CEF was constructed from 8.8× 10^5 original bacterial colonies that contains recombinant plasmids with 85.7 % inserts larger than 1.0 kb. Conclusion： The cDNA library is probably sufficient for cloning of fuli length cDNAs.