大肠癌细胞HRT-18cDNA定向文库的构建

Construction of cDNA library from human colorectal cancer cell HRT-18

目的 :快速构建人大肠癌细胞HRT 1 8的定向cDNA文库。方法 :从人大肠癌细胞HRT 1 8中抽提总RNA后磁珠法分离mRNA ,以含sfiⅠ酶切位点的oligo(dT)引物合成cDNA的第一链 ,利用SMART(switchingmech anismat 5′endofRNAtranscript)技术 ,经LD PCR合成双链cDNA ,该产物经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 6 .5× 1 0 6个重组子 ,重组率 >96 % ,扩增文库滴度为 1 .1× 1 0 9pfu/ml,插入片段平均长度为 1 .1kb。结论 :构建的人大肠癌细胞HRT 1 8文库质量好 。

Objective To construct a cDNA library from human colorectal cancer cell HRT 18. Methods The total RNA was extracted from human CRC(colorectal cancer )cell HRT 18 and the mRNA was isolated from the total RNA by MagneSphere technique,and then the first strand cDNA was synthesized with oligo(dT)primer containing sfiⅠsite while the double strand cDNA was amplified through LD PCR (long distance PCR) by SMART technique. The double strand cDNA was digested by sfi Ⅰ(ⅠA&ⅠB)restriction enzyme before cDNA size fractionation, and the double strand cDNA fractionated was ligated into the λTripIEX2 vector and then was packaged into λ phage in vitro. Results The unamplified human CRC cell HRT 18 cDNA library consisted of 6.5×10 6 independent clones, recombinants clones was more than 96%.The titer of the amplified cDNA library was 1.1×10 9 pfu/ml and the average insert of the recombinants was 1.1 kb.Conclusion The quality of the constructed cDNA library from human CRC cell HRT 18 is excellent and it is helpful to screen CRC specific antigen.