野生型CENP-E基因siRNA表达载体的构建与有效干扰质粒的筛选研究  

Construction and Identification of CENP-E Targeted Recombinant Plasmids for RNA Interference

作　　者：陈珊[1] 林超 

机构地区：[1]内江医科学校临床检验教研室,四川内江641003 [2]内江市中医院检验科,四川内江641000

出　　处：《保健医学研究与实践》2010年第3期7-11,共5页Health Medicine Research and Practice

摘　　要：目的为探索有丝分裂中的重要基因CENP-E在非整倍体的形成及肿瘤发生中的作用,设计并构建了以野生型CENP-E中Q2-Q3片段为靶点的RNA干扰表达载体pCENP-Es,筛选其中对野生型CENP-E有显著干扰作用的表达质粒。方法根据已知的野生型CENP-E中Q2-Q3片段的mRNA序列,选择设计4条带发卡结构的核苷酸序列,克隆到载体pgenesil-1中并测序分析。用脂质体转染的方法将干扰质粒与表达质粒pDSRED-Q2-Q3共转染293T细胞,72h后,荧光倒置显微镜观察转染效率,并收集细胞,通过RT-PCR方法检测不同干扰质粒对野生型CENP-EmRNA的干扰效果。结果 4个野生型CENP-E的siRNA片断被成功克隆到pgenesil-1质粒载体中,插入片段测序结果与设计的序列完全一致。RT-PCR结果表明,针对1号和4号干扰质粒共转染时干扰效果最好,干扰效率可达84.47%。结论成功构建了能表达野生型CENP-EshRNA(smallhairpinRNA)重组质粒,同时通过共转染技术,筛选到干扰效果达80%以上的干扰片断,为体内研究CENP-E基因及蛋白在肿瘤发生过程中功能打好基础。Objective To identify the role of important mitosis gene CENP-E in aneuploidy formation and tumorigenesis,the expression vectors of CENP-E for RNA interference as pCENP-Es were designed and contructed.Method The interfering sequences of CENP-E were designed according to the sequence of CENP-E in GenBank.Four pairs of oligonucleotides were synthesized and inserted into plasmid pgenesil-1 to generate siRNA expression vector,and were identified by sequence analysis.The recombinant plasmid pCENP-Es and CENP-E expression plasmid pDSRED-Q2-Q3 was cotransfected into the cultured 293T cell line.The interfering eficiency was detected by RT-PCR.Results Four CENP-E siRNA frames were successfully inserted into the plasmid vector pgenesil-1,and the recombinant plasmids were sent to the sequence analysis.The result of RT-PCR indicates that the siRNA expression vector 1 and 4 got the best interfering eficiency as 84.47%.Conclusion The siRNA recombinant can be constructed successfully by RNAi technique for inhibiting CENP-E expression.

关 键 词：CENP-E RNA干扰 短发夹RNA RT-PCR 

分 类 号：R346[医药卫生—基础医学]