二烯丙基二硫诱导HT-29细胞差异表达cDNA文库的构建

目的:构建二烯丙基二硫(diallyl disulfide,DADS)诱导人结肠癌HT-29细胞差异表达cDNA文库,以期寻找、克隆DADS特异性作用靶点基因。方法:用120μmol/L的DADS处理人结肠癌HT-29细胞,从DADS处理组及对照组HT-29细胞中提取poly A+RNA,应用高效、灵敏的抑制性消减杂交(SSH)技术构建DADS诱导人结肠癌细胞差异表达cDNA消减文库,再转染大肠杆菌进行文库扩增。结果:成功构建具有高消减效率的DADS诱导人结肠癌细胞差异表达cDNA文库,文库扩增得到了500个克隆,随机挑取100个制备质粒,酶切分析均得到150~1 300 bp大小插入片段,这些片段可能就是载有高度特异性的目的片段。结论:建立了DADS诱导人结肠癌细胞差异表达cDNA文库,为进一步寻找、克隆DADS特异性作用靶点基因奠定了基础。

Construction and identification for differential expression cDNA subtractive library in HT-29 cell lines induced by DADS

Objective: To construct differential expression cDNA subtractive library of human colon carcinoma HT-29 cell lines induced by DADS(diallyl disulfide,DADS),to find and cloning DADS specific target gene.Methods: Human colon carcinoma HT-29 cell was processed with 120μmol/L DADS,Poly A+ RNA were isolated from colon carcinoma HT-29 cells treated for trial group and control group respectively.Differential expression cDNA subtractive library was constructed by using the suppression subtractive hybridization(SSH),then used it to transfect E.coli strain JM109,the library was amplified with the transfected E.coli strain JM109.Results: Library of human colon carcinoma HT-29 cells induced by DADS subtractive library with high subtractive efficiency was set up successfully.The amplified library contained 500 clones.100 clones were selected randomly to make plasmid,with PCR ampification showed that all plasmids in the clones contained 150～1300 bp insert sequences;which might contain high specific sequences of target.Conclusion: The cDNA subtractive library of human colon carcinoma HT-29 cells is constructed,which lays a foundation for searching and cloning specific target gene of DADS.