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目的构建丁酸钠诱导McF-7细胞凋亡过程的反义cDNA文库,以分离丁酸钠抗肿瘤效应基因。方法收集经2．5mmol/L丁酸钠处理0、12、18、24、36、48、72h的人乳腺癌MCF-7细胞,提取poly(A)+RNA,逆转录合成cDNA第一链和第二链,双链cDNA修平末端后连上EcoRⅠ/HindⅢ接头,分别用EcoRⅠ/HindⅢ酶切消化后,反向连上载体pcDNA 3．1,连接产物转化大肠杆菌得一反义cDNA文库,随机挑取40个克隆子进行酶切鉴定。结果构建的反义cDNA文库含1×10~6重组子,重组效率为90%,插入子平均长度为1．5kb。结论构建的文库容量较大,质量较高,为进一步分离丁酸钠抗肿瘤效应基因奠定了基础。
Objective To construct an antisense cDNA library of sodium butyrate-induced apoptosis in McF-7 cells and to isolate the sodium butyrate antitumor effect gene. Methods Human breast cancer MCF-7 cells were harvested at 0, 12, 18, 24, 36, 48 and 72 h after treatment with 2.5 mmol / L sodium butyrate and poly (A) In the second strand, the double-stranded cDNA was trimmed to the EcoR I / HindIII linker and digested with EcoR I / Hind III, respectively, and then ligated with the vector pcDNA 3.1 in the reverse direction to obtain an antisense cDNA library. 40 clones were picked for restriction enzyme digestion. Results The constructed antisense cDNA library contained 1 × 10 ~ 6 recombinants with a recombination efficiency of 90% and an average insert length of 1.5 kb. Conclusion The constructed library has high capacity and high quality, which lays the foundation for further separation of sodium butyrate antitumor effect genes.