目的：构建pSIREN-survivin/shRNA重组质粒并鉴定, 为探索瘢痕疙瘩基因治疗的RNA干扰（RNAi）途径奠定基础。 方法：根据基因库survivin cDNA序列及Reynolds设计原则, 设计并合成两端有酶切位点的65个碱基的寡核苷酸链, 退火成互补双链后用T4DNA酶克隆至线性化的RNAi-Ready pSIREN-DNR-DsRed-Express质粒中; 转化大肠杆菌DH-5a菌株, 提取质粒行酶切鉴定, 测序分析。 结果：PCR扩增片断出现465 bp大小的目的基因条带与预期结果相符; 双酶切见约65 bp目的基因条带; 插入片断测序结果与合成的寡核苷酸序列一致。 结论：成功构建重组质粒pSIREN-survivin/shRNA, 为下一步用脂质体转染瘢痕或肿瘤细胞的研究奠定基础。
Objective：To construct and identify a recombinant survivin-shRNA expression vector, for making a foundation of keloid gene therapy via RNA interference. Methods：According to survivin cDNA gene sequence in the gene bank, a pair of 65 nt oligonucleotides each containing the sites of restriction endonuclease at both ends were designed and synthesized by Reynolds design principles. Oligonucleotides were annealed and ligated with linedrized RNAi-Ready pSIREN-DNR-DsRed-Express by T4DNA ligase. The recombinants were transfected into Escherichia coli strains DH-5a. Finally, it was sequenced and identified by restriction endonuclease digestion. Results：The size of the target gene fragment amplified by PCR was 465 bp and in accordance with the expected results. Double digested section showed a target gene band of about 65 bp identitfied by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesized shRNA oligonucleotides. Conclusions：A survivin-shRNA expression vector has been successfully constructed, which can be as the groundwork for future study of liposome mediated transfection into scar or tumor cells.
马静, 赵留芳, 刘流, 李晓江, 杨洁, 孙瑞梅, 隋军. pSIREN-survivin/shRNA表达载体的构建与鉴定[J].中国普通外科杂志,2011,20(5):507-511.复制