小鼠PARP-1基因RNAi表达质粒的构建与筛选  

Construction and Screening of RNAi Expression Vector for Mouse Poly(ADP-Ribose)Polymerase-1 Gene

作　　者：王铁君[1] 韩成敏[2] 李星花[3] 尹金植[1] 陈玉丙[1] 王红勇[1] 李英普[1] 

机构地区：[1]吉林大学第二医院肿瘤放疗科,长春130012 [2]吉林大学口腔医院内镜中心,长春130012 [3]吉林大学第一医院呼吸内科,长春130012

出　　处：《中国生物制品学杂志》2009年第2期143-146,共4页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目;吉林省科委白求恩专项基金(200705224)

摘　　要：目的构建并筛选小鼠聚腺苷酸二磷酸核糖转移酶-1(PARP-1)基因的RNAi表达质粒,为肿瘤基因治疗探索新途径。方法根据GenBank中报道的PARP-1基因序列及shRNA设计原则,设计、合成用于构建RNAi质粒的寡核苷酸,构建4种重组PARP-1RNAi质粒,酶切及测序鉴定正确后,以脂质体LipofectamineTM2000介导转染小鼠Lewis肺癌细胞株。48h后,采用RT-PCR技术检测转染细胞中PARP-1基因mRNA的转录水平,筛选有效的PARP-1RNAi质粒。结果4种重组PARP-1RNAi质粒经酶切及测序证明构建正确。转染后48h,转染质粒pGPU6/GFP/Neo-PARP-1-1308和pGPU6/GFP/Neo-PARP-1-1837的Lewis细胞PARP-1基因mRNA的转录水平降低,转染pGPU6/GFP/Neo-PARP-1-1308组下降更明显,以其作为后续实验的有效质粒。结论已成功构建并筛选出PARP-1基因的RNAi表达质粒,为PARP-1基因RNAi治疗肿瘤的研究奠定了基础。Objective To construct and screen the RNAi expression vector for mouse poly （ADP-ribose） polymerase-1 （PARP-1） gene and provide a novel route for the gene therapy of tumors. Methods The oligonucleotides were designed and synthesized according to the PARP-1 gene reported in GenBank and the general principle of shRNA design, based on which 4 recombinant RNAi expression vectors targeting PARP-1 gene were constructed, identified by restriction analysis and sequencing, then transfeeted to mouse lung cancer Lewis cell strain in mediation of LipofectamineTM 2000. The transcription levels of PARP-1 mRNA were determined by RT-PCR 48 h after transfection to screen the valid RNAi expression vectors targeting PARP gene. Results Restriction analysis and sequencing proved that 4 recombinant RNAi expression vectors targeting PARP-1 gene were constructed correctly. Both the transcription levels of PARP-1 mRNA in Lewis cells transfected with recombinant plasmids pGPU6/GFP/Neo-PARP-1-1308 and pGPU6/GFP/Neo-PARP-1-1837 decreased. However, more remarkable decrease of the transcription levels was observed in Lewis cells transfected with pGPU6/GFP/Neo-PARP-1-1308 which was used as the valid RNAi expression vectors targeting PARP-1 gene for further study. Conclusion The RNAi expression vector for mouse PARP-1 gene was successfully constructed and screened, which laid a foundation of study on treatment of tumors with PARP-1 gene.

关 键 词：聚腺苷酸二磷酸核糖转移酶-1 SIRNA 肿瘤 基因治疗 

分 类 号：Q782[生物学—分子生物学]