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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 ,有利于进一步筛选鼻咽癌特异性抗原基因
Objective To rapidly construct a directional cDNA library of human nasopharyngeal carcinoma cell line HNE2. Methods Total RNA was isolated from human nasopharyngeal carcinoma cells by magnetic beads method. The first strand of cDNA was synthesized by oligo (dT) primer containing SfiI restriction sites. The cDNA was amplified by semi-quantitative reverse transcription-polymerase chain reaction (LD-PCR) using SMART (switchingmechanismat 5’endofRNAtranscript) The double-stranded cDNA was synthesized and the product was fractionated by SfiI digestion. The inserted fragment was ligated to λTripIEx2 vector and packaged into a phage cDNA library by in vitro packaging. Results The library contained 0 78 × 10 6 recombinants and the recombination rate was> 96%. The titer of the amplified library was 102 × 10 9 pfu / ml, and the average length of the insert was 12 kb. Conclusion The constructed HEN2 cDNA library of human nasopharyngeal carcinoma cells is of good quality and is favorable for further screening of nasopharyngeal carcinoma specific antigen genes