人胚胎干细胞培养及其cDNA文库的构建  

Construction and characterization of a cDNA library from human embryonic stem cells

作　　者：周素芳[1] Angela M Eastham Peter L Stern 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,广西南宁530021 [2]Cancer Research UK Immunology Group,Paterson Institute for Cancer Research,Christie Hospital NHS Trust,Manchester,UK.

出　　处：《中国现代医学杂志》2007年第17期2049-2053,共5页China Journal of Modern Medicine

基　　金：广西自然科学基金项目(桂科自0728121)

摘　　要：目的构建人胚胎干细胞cDNA文库,为筛查疾病抗原基因奠定基础。方法培养和收集人胚胎干细胞克隆,提取胚胎干细胞总RNA,分离纯化mRNA,利用mRNA作模板反转录合成双链cDNA,双链DNA经pfu-DNA聚合酶将链末端补平,与EcoRⅠ接头连接,XhoⅠ酶切消化产生粘端。用Sepharose CL2B柱分离纯化及除去小分子cDNA片段,与Uni-ZAP XR噬菌体连接,体外转化XL1-blue MRF'菌,建成cDNA文库并计算重组效率。对cDNA文库进行扩增,测定扩增文库的滴度。阳性重组子用EcoRⅠ+XhoⅠ双酶切鉴定插入片段的大小。结果构建成含1.2×106重组子的人胚胎干细胞cDNA文库,重组子插入外源DNA片段长度不小于1000kb。结论已成功构建人胚胎干细胞cDNA文库,适合用于筛选目的基因克隆。[Objective] To construct a eDNA library of human embryonic stem （hES） cell for screening, cloning and studying new genes. [Methods] Thomson-derived human embryonic stem cells were cultured and isolated. Total RNA was extracted from hES cell and mRNA was purified using mRNA oligo （dT） magnetic particle purification technique. The first and second strand eDNA was synthesized by reverse transcriptase and DNA polymerase Ⅰ respectively. The ds-cDNA termini was blunted with pfu DNA polymerase. The blunted cDNAs were added EcoR Ⅰ adaptor and then digested by Xho Ⅰ. The eDNA fragments longer than 0.5 kb were collected by Sepharose CL2B chromatography and ligated to Uni-ZAP XR expression vector. The ligated products were packaged in vitro and the lambda phage particles infected the host strains XL1-BlueMRF＇ to produce an unamplified library. The library was amplified and tittered. The size of eDNA fragment was evaluated by EcoR Ⅰ and Xho Ⅰ restriction endonuclease digestion. [Results] The efficiency of the primary library was 1.2 ×10^6 recombinant and the amplified library was 1.0×10^9 pfu/mL. The percentage of positive recombinant clones was 98.5%. The length of the inserted eDNA fragment was over 1 kb. [Conclusion] The quality of the constructed hES eDNA library is excellent and helpful to screen new genes.

关 键 词：人胚胎干细胞 CDNA文库 分子克隆 

分 类 号：R383.24[医药卫生—医学寄生虫学]