人鼻咽癌组织定向cDNA文库的构建  

Construction of directional cDNA library from human NPC tissue

作　　者：舒峻[1] 李官成[1] 何小鹃[1] 

机构地区：[1]中南大学湘雅医学院肿瘤研究所免疫生物室,湖南长沙410078

出　　处：《中国耳鼻咽喉颅底外科杂志》2004年第4期200-203,共4页Chinese Journal of Otorhinolaryngology-skull Base Surgery

摘　　要：目的　快速构建成人鼻咽癌组织cDNA文库。方法　从人鼻咽癌组织分离总RNA ,以含有sfiIB酶切位点的oligo(dT)引物合成第一链cDNA ,以含sfiIA酶切位点的SMART寡核苷酸为引物经LD PCR扩增双链cDNA ,双链cDNA经sfiI(IA&IB)酶切 ,以CHROMASPIN 4 0 0柱分级分离cDNA ,收集符合需要的cDNA片段并纯化 ,随后将之与λTriplEx2载体连接经体外包装成噬菌体cDNA文库。 结果　经检测共获得 3.6 4× 10 6个重组子 ,重组率 >94 % ,文库经扩增后滴度为 3.8× 10 9pfu/ml。结论　用SMART方法构建的鼻咽癌组织cDNA文库质量较高 。Objective To quickly construct a directional cDNA library from human nasopharyngeal carcinoma tissue. Methods The total RNA was separated from human NPC (nasopharyngeal carcinoma) tissue, then the first-strand cDNA was synthesized with oligo (dT) primer containing SfiI-digested sites before the double-strand cDNA was amplified through LD-PCR (Long-distance PCR) by SMART technology. The double-strand cDNA was digested by SfiI (IA & IB) restriction enzyme before cDNA size fractionation, the double-strand cDNA fractionated was ligated into the λ TripIEx2 vector and packaged in vitro. Results The unamplified human NPC tissue cDNA library consists of (3.64)×10 6 independent clones with recombinant clones more than 94%. The titer of the amplified cDNA library is (3.8)×10 9 pfu/ml and the average size of the recombinants insert (1.2 kb). Conclusion The quality of the constructed human NPC tissue cDNA library is excellent and helpful to screen NPC specific-antigen.

关 键 词：CDNA文库 定向克隆 鼻咽肿瘤/遗传学 

分 类 号：R739.63[医药卫生—肿瘤]