人脑胶质瘤组织全长cDNA文库的快速构建与鉴定

Rapid construction and identification of full-length cDNA library of human glioma tissues

目的采用SMART(switching mechanism at 5' end of RNA transcript)技术,快速构建高质量的人脑胶质瘤组织全长cDNA文库。方法提取多份人脑胶质瘤组织标本的总RNA,混合后处理得到纯化的mRNA,利用CDSⅢ/3'PCR引物(含SfiⅠB酶切位点)反转录,用SMARTⅣOligo(dT)(含SfiⅠA酶切位点)作为mRNA5'端延伸出去的模板,合成多出一段SMARTⅣOligo(dT)互补序列的cDNA第一链,进而以此序列为引物合成全长的双链cDNA。双链cDNA经SfiⅠ(A&B)酶切后克隆入经SfiⅠ酶切的λTriplEx2载体,再经噬菌体包装蛋白包装成为cDNA文库。结果获得2.4×106个重组子,重组率达100%,文库扩增后,滴度达4.5×109 pfu/ml,插入cDNA平均长度为1.2kb。结论成功构建高质量的人脑胶质瘤组织全长cDNA文库,为进一步筛选、克隆人脑胶质瘤抑癌基因及特异性表达基因奠定基础。

Objective To explore a method for rapid construction of a full-length cDNA library of human glioma tissues using switching mechanism at 5' end of RNA transcript (SMART). Methods The total RNA was extracted from several samples of human glioma tissues and the mRNA was subsequently separated. Multiple mRNA samples were mixed to be used as the template for the first-strand cDNA synthesis. The CDSⅢ/3' PCR primer (containning SfiⅠB site) was used in the first-strand reaction, and the SMART ⅣOligo(dT) (containning SfiⅠA site) served as the short, extended template at the 5' end of the mRNA. With the above two primers, the primer-extension step generated full-length double-strand cDNA, which was digested by SfiⅠrestriction enzyme and ligated to the SfiⅠ(A&B)-digested λTriplEx2 vector. The ligated vector was then packaged by lambda packaging extract for the final construction of the cDNA library. Results The unamplified human glioma cDNA library consisted of 2.4×10 6 independent clones with a recombination rate of 100%. The titer of the amplified cDNA library was 4.5×10 9 pfu/ml, and the average exogenous inserts of the recombinants was 1.2 kb in length. Conclusion A high-quality full-length cDNA library of human gliomas was constructed successfully, which may facilitate further study of the screening and cloning of new tumor suppressor genes and tissue-specific genes of human glioma.