摘要
目的构建并筛选大鼠聚腺苷二磷酸核糖聚合酶-1(PARP-1)基因的RNA干扰(RNAi)表达质粒,为职业性疾患的基因治疗探索新途径。方法根据基因序列数据库中报道的PARP-1基因序列及短发夹RNA(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-1 RNAi质粒,酶切及测序鉴定正确后,以脂质体Li-pofectamineTM2000介导转染大鼠骨髓间充质干细胞。48 h后,采用逆转录聚合酶链反应(RT-PCR)技术检测转染细胞中PARP-1 mRNA的转录水平,筛选有效的PARP-1 RNAi质粒。结果 4种重组PARP-1 RNAi质粒经酶切及测序证明构建正确。转染后48 h,转染质粒shRNA-PARP-1-532的细胞PARP-1基因抑制效果最明显,mRNA的转录水平下降了78%,可作为后续实验的有效质粒。结论成功构建并筛选出PARP-1基因的RNAi表达质粒,为探索PARP-1基因在疾病中的作用奠定了基础。
Objective To construct and screen the RNAi expression vector for rat poly（ADP-ribose） polymerase-1（PARP-1）gene and provide a novel route for the gene therapy of occupational diseases. 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 transfected to rat bone mesenchymal stem cells in mediation of LipofectamineTM2000.The transcription levels of PARP-1 mRNA were determined by real-time reverse transcription polymerase chain reaction（RT-PCR） 48 hours after transfection to screen the valid RNAi expression vectors targeting PARP-1 gene. Results Restriction analysis and sequencing proved that 4 recombinant RNAi expression vectors targeting PARP-1 gene were constructed correctly.The transcription level of PARP-1 mRNA in bone mesenchymal stem cells transfected with recombinant plasmids shRNA-PARP-1-532 decreased more remarkable.It was used as the valid RNAi expression vectors targeting PARP-1 gene for further study. Conclusion The RNAi expression vector for rat PARP-1 gene was successfully constructed and screened,which laid a foundation of study on the effect of PARP-1 gene in the diseases.
出处
《中国职业医学》
CAS
北大核心
2012年第1期1-4,共4页
China Occupational Medicine
基金
国家自然科学基金资助项目(30972500)
广东省自然科学基金项目(7301507)
湛江市科技攻关计划项目(2011C3104004)
