抑制聚ADP核糖聚合酶1活性的短发夹RNA载体构建  

Construction of short hairpin RNA vector of inhibiting poly ADP-ribor polymerase activity

作　　者：沙焱[1] 庄志雄[1] 何云[1] 胡大林[1] 胡恭华[1] 杨建平[1] 涂晓志[1] 

机构地区：[1]中山大学公共卫生学院

出　　处：《卫生研究》2006年第1期10-12,共3页Journal of Hygiene Research

基　　金："973"国家重点基础研究发展计划基金资助项目(No.2002CB512904;2002CB512903);国家自然科学基金资助项目(No.30571592)

摘　　要：目的构建抑制人聚ADP核糖聚合酶1(hPARP1)活性的短发夹RNA(shorthairpinRNA,shRNA)表达载体。方法化学合成2对编码短发夹RNA序列的、靶向hPARP1基因的寡核苷酸,各69对碱基,退火,然后利用BamHⅡ及EcoRⅠ与pSIRENRetroQ载体连接。用EcoRⅠ及BglⅡ切取其中U6启动子及下游的shRNA部分,与pEGFPC1载体重组构建pEGFPC1shRNA载体。结果重组构建的pEGFPC1P1、pEGFPC1P2、pEGFPC1N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330个碱基成功插入到预计位点。结论载体的成功构建,为进一步研究聚ADP核糖聚合酶在DNA修复过程中的功能打下基础。Objective To construct expressing vector of short hairpin RNA（shRNA） in order to inhibit human PARP1 activity. Methods 2 pairs of 64 base oligos for hairpin RNA expression which targeted PARP1 gene were chemically synthesized and annealed then ligased with pSIREN-RetroQ vector with BamH Ⅱ and EcoR T . Cut by EcoR Ⅰ and Bgl Ⅱ ,shRNA and its upstream U6, which have 330 bp, were inserted into the same treated pEGFP-C1 vecter to construct GFP expression plasmids that inhibited hPARP1 protein shRNA plasmid （pEGFP-CIP）. Oligos with a scrambled sequence were used as a negative control. Results Recombinant pEGFP-CIPI, pEGFP-C1P2 and pEGFP-C1N vectors was identified by digestion with EcoR Ⅰ and Bgl Ⅱ and confirmed by sequencing analysis with U6 primer. The results demonstrated that the 330 bp had been inserted into the expected site. Furthermore, the insertion sequence was exactly correct. Conclusion pEGFP-C1-shRNA system has been constructed successfully. This w ill facilitate the study of PARPI＇s DNA repairing function.

关 键 词：RNAI SHRNA PEGFP-C1 

分 类 号：R994.6[医药卫生—毒理学] Q783.2[医药卫生—药学]