鼻咽癌细胞HNE_2cDNA文库的构建  

Construction of cDNA Library From Human Nasopharyngeal Carcinoma Cell HNE_2

作　　者：舒峻[1] 李官成[1] 何小鹃[1] 李跃辉[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：《中国医师杂志》2003年第4期441-443,共3页Journal of Chinese Physician

摘　　要：目的　快速构建人鼻咽癌细胞株HNE2 的定向cDNA文库。方法　从人鼻咽癌细胞抽提总RNA磁珠法分离mRNA ,以含SfiI酶切位点的oligo(dT)引物合成cDNA第一链 ,利用SMART(switchingmechanismat 5’endofRNAtranscript)技术 ,经LD -PCR合成双链cDNA ,该物经SfiI酶切后分级分离 ,插入片段与λTripIEx2载体连接经体外包装成噬菌体cDNA文库。结果　经鉴定 ,该文库含0 78× 10 6 个重组子 ,重组率 >96% ;扩增文库滴度为 1 0 2× 10 9pfu/ml ,插入片段平均长度为 1 2kb。结论　构建的人鼻咽癌细胞HEN2 cDNA文库质量较好 。Objective To construct a cDNA library from human nasopharyngeal carcinoma cell HNE 2.Methods The total RNA was separated from human NPC(nasopharyngeal carcinoma)cell HNE 2 and the mRNA was isolated from the total RNA by MagneSphere technique,then the first-strand cDNA was synthesized with oligo(dT) primer containing sfiI site while the double-strand cDNA was amplified through LD-PCR(Long-distance PCR) by SMART technique.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 λTripIEx2 vector and then was packaged in vitro.Results The unamplified human NPC cell HNE 2 cDNA library consists of 0 78×10 6 independent clones,and the percentage of recombinant clones was more than 96%.The titer of the amplified cDNA library was 1 02×10 9 pfu/ml and the average insert of the recombinants was 1 2kb.Conclusions The quality of the constructed human NPC cell HNE 2 cDNA library is excellent and helpful to screen NPC specific-antigen.

关 键 词：鼻咽癌 CDNA文库 基因克隆 

分 类 号：R739.63[医药卫生—肿瘤]