抑制低氧诱导因子活性的转录载体pTZU6+1 RNAi系统的构建  

Construction of pTZU6+l RNAi system of inhibiting hypoxia inducible factor-1 activity

作　　者：曾波[1] 姜政[1] 向廷秀[1] 黄爱龙[2] 王丕龙[1] 

机构地区：[1]重庆医科大学附属第一医院消化科,400016 [2]重庆医科大学病毒性肝炎研究所,400010

出　　处：《重庆医学》2006年第14期1283-1285,共3页Chongqing medicine

摘　　要：目的构建抑制低氧诱导因子(HIF-1a)活性的shRNA表达载体,为进一步探讨其对HIF-1a基因表达的抑制作用。方法设计并合成2对HIF-1a编码基因的反向重复序列,中间分别间隔4nt和8nt,分别定向克隆至载体pTZU6+l的U6转录启动子下,构建pshRNA-HIF-1al和pshRNA-HIF-1a2重组质粒。结果构建的pshRNA-HIF-1al和pshRNA-HIF-1a2重组载体经双酶切鉴定及插入基因片段序列分析,成功地将目的基因片段插入到预计位点,并且与实验设计完全一致。结论重组载体的成功构建,为进一步研究pshRNA-HIF-1a对HIF-1a活性的抑制作用和基因功能的分析、肿瘤的治疗等奠定了基础。Objective To construct the plasmid containing short hairpin RNA （shRNA） of DNA of hypoxia inducible factor-1 （HIF-1） in order to suppress HIF-1 activity in human liver cancer. Methods Two pair of oligos for hairpin RNA expression which targeted HIF-1a gene were chemically synthesized and annealed, pTZU6＋1 vector was linearized with Sail and Xbal. Finally, annealed oligos were inserted into the down stream of U6 promoter to construct RNAi plasmid （pshRNA-HIF-1a） respectivily. Resuits Recombinant pshRNA-HIF-1a vector were identified by digestion with Sail and Xbal and confirmed by sequencing analysis with T7 primer. The results demonstrated that oligos had been inserted the expected site of the vector. Furthermore, the insertion sequences were exactly correct, Conclusion pshRNA-HIF-1a systems have been constructed successfully. These will facilitate the study of HIF-1 activity inhibition. Based on the results, we can develop the RNAi synthesis in vivo. This technique not only facilitates a wide range of gene functional analysis, but also offers therapeutic means for treatment of tumors.

关 键 词：pTZU6+1 SHRNA RNAI 低氧诱导因子 hHIF-1a 

分 类 号：Q78[生物学—分子生物学]