摘要
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eDNA第一链和第二链，双链cDNA修平末端后连上EeoRⅠ／HindⅢ接头。分别用EcoR Ⅰ／Hind Ⅲ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 anti-sense cDNA library for isolation of essential genes with anti-tumor effects on apoptosis induced by sodium butyrate. Methods Total RNA was extracted from MCF-7 cells treated with 2.5 mmol/L sodium butyrate for 0, 12, 18, 24, 36, 48, 72 h. Poly（A） ＋ RNA was purified by oligo（dT） cellulose. First-strand and second-strand cDNA were synthesized in sequence. The EcoR Ⅰ/Hind Ⅲ linkers were ligated to the blunted eDNA termini, eDNA was ligated into pcDNA3.1 in an anti-sense orientation. The ligation mixture was transformed into E. coli DH10B and independent clones were produced. 40 clones were picked for preliminary identification by EcoR Ⅰ/HindⅢ digestion. Results An anti-sense cDNA library was constructed containing 1 × 10^6 independent clones. The restriction enzyme digestion demonstrated that inserts of different lengths were cloned into the vectors. The recombinant efficiency was 90 %. The mean length of the inserts was about 1.5 kb. Conclusion An anti-sense cDNAlibrary with high quantity and quality was successfully constructed, which was the basis for screening the essential genes with anti-tumor effects on apoptosis induced by sodium butyrate.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第7期807-809,共3页
Chinese Journal of Experimental Surgery
基金
国家重点基础研究发展计划资助项目(2002CB513100)
国家自然科学基金(30371657)
