pSIREN-HIF-1α/shRNA表达载体的构建与鉴定  

Construction and Identification of pSIREN-HIF-1α/shRNA Expression Vector

作　　者：赵留芳[1] 张书萍[1] 李晓江[1] 任艳鑫[1] 张康[1] 喻博[1] 马静[1] 隋军[1] 

机构地区：[1]昆明医科大学第三附属医院/云南省肿瘤医院头颈外科,云南昆明650118

出　　处：《昆明医科大学学报》2013年第10期58-61,72,共5页Journal of Kunming Medical University

基　　金：云南省卫生科技计划项目(2010NS085;2011WS0068)

摘　　要：目的构建pSIREN-HIF-1α/shRNA重组质粒并测序鉴定,为下一步探索鼻咽癌基因治疗的RNA干扰(RNA interference,RNA)i途径奠定基础.方法根据基因库HIF-1αcDNA序列(NM_001530/NM_181054)以及Reynolds设计原则,设计并合成两端有酶切位点的60个碱基的寡核苷酸链,退火成互补双链后将退火产物与RNAi-Ready pSIREN-RetroQ-ZsGreen载体链接后,热转化至大肠杆菌JM109菌株中,提取质粒进行1%琼脂糖凝胶电泳鉴定,测序分析.结果 PCR扩增片段出现约470 bp大小的目的基因条带,与预期结果相符;凝胶电泳证实重组载体构建成功,插入片段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-HIF-1α/shRNA,为下一步用脂质体包装并转染鼻咽癌细胞的研究奠定基础.Objective To construct and identity pSIREN-HIF-1α/shRNA expression vector in order to make foundation of gene therapy for further exploration of RNA interference to nasopharyngeal darcinoma. Methods According to HIF-1αcDNA gene sequence in the gene bank （NM_001530/NM_181054）, a pair of 60 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-RetroQ-ZsGreen.Transfected into JM109, the recombinants were finally sequenced and identified by 1%agarose gel electrophoresis. Results The size of the target gene fragment amplified by PCR was 470 bp and in accordance with the expected result.pSIREN-HIF-1α was successfully constructed and identitfied by 1%agarose gel electrophoresis.Sequence analysis of inserted fragment revealed the same sequence as synthesized shRNA Oligonucleotides. Conclusion pSIREN-HIF-1α /shRNA expression vector has been successfully constructed, and can make the foundation of research using liposome packaging transfectiing nasopharyngeal darcinoma cell for the next step .

关 键 词：RNA干扰 鼻咽癌 质粒 HIF-1Α 短发卡RNA 

分 类 号：R739.6[医药卫生—肿瘤]