靶向端粒酶调节相关基因TRAP siRNA表达载体的构建

Construction of Expressing Vector of siRNA Targeting Telomeras Regulation-associated Protein (TRAP) Gene

目的构建靶向端粒酶调节相关基因TRAP的siRNA表达载体。方法根据siRNA设计原则,设计并化学合成2段编码短发夹RNA序列、靶向端粒酶调节相关基因TRAP的寡核苷酸,各64个碱基,退火,克隆到线性化的pSilencerTM21u6neo质粒U6启动子下游重组构建RNAi质粒。结果重组质粒pSilencerTM21u6neoTRAP经过插入片段基因序列分析,结果表明64个碱基成功插入到预定位子,并且序列完全一致。结论成功构建靶向TRAP基因的siRNA表达载体,为进一步研究TRAP在调控hTERT的表达、降低端粒酶活性和肿瘤的基因治疗方面的作用做了基础。

Objective: To construct expression vector of siRNA targeting telomeras regulation-associated protein gene. Methods: Each 64 base-pair oligonucleotide for hairpin RNA expression which targeted telomeras regulation-associated protein gene was designed and chemical synthesized. After annealing, oligonucleotides were inserted into the downstream of U6 promoter of pSilencer^TM 2.1-u6 neo. Result: Recombinant pSilencer^TM 2.1-u6 neo-TRAP vector was confirmed by sequencing analysis with M13F primer,the results demonstrated that 64bp had been inserted the expected site. Furthermore, the inserted sequence was exactly correct. Conclusion:pSilencer^TM 2.1-u6 neo-TRAP has been constructed successfully.This will be a base for the study of its function in regulating hTERT, to reduce telomerase activity and in gene treatment of tumor. [