针对MAGE-1的pSUPERRNAi系统的构建

Construction of pSUPER RNAi system of inhibiting MAGE-1 activity.

目的构建抑制MAGE-1的siRNA表达载体。方法化学合成2对编码短发夹RNA序列的、靶向MAGE-1基因的寡核苷酸链,各64个碱基,退火,克隆到经BglⅠ、HindⅢ双酶切的pSUPER载体上,重组构建RNAi质粒。通过双酶切鉴定及基因片段序列分析验证构建效果。结果重组构建的pSUPER-MAGE-1载体经双酶切电泳分析及插入基因片段序列分析,结果表明64个碱基成功插入到预计位点,并且序列完全一致。结论载体的成功构建,为进一步研究MAGE-1在肿瘤中的功能打下基础,可用于分析基因功能、肿瘤治疗等。

Objective To construct expressing vector of siRNA in order to inhibit MAGE - 1 activity. Methods Sixty four base - pair oligos for hairpin RNA expression which targeted MAGE - 1 gene were chemically synthesized and annealed, pSUPER vector was linearized with Bgl Ⅱ and Hind Ⅲ. Finally, annealed oligos were inserted into the treated pSUPER to construct RNAi plasmid（pSUPER - MAGE - 1 ）. The reconstructed RNAi plasmid must be identiffed by electrophoresis after digestion with EcoR Ⅰ and Hind Ⅲ and be confirmed by sequencing analysis. Results Recombinant pSUPER - MAGE - 1 vector was identified by digestion with EcoR Ⅰ and Hind Ⅲ, and confirmed by sequencing analysis. The results demonstrated that 64 bp had been inserted the expected site. Furthermore, the insertion sequence was exactly correct. Conclusion pSUPER - MAGE - 1 RNAi system has been constructed successfully. This will facilitate the study of MAGE - 1 activity inhibition and function.