特异性脱氧核酶对喉癌真核细胞起始因子4E基因mRNA的体外切割作用  

Target cleavage of eIF4E mRNA with 10-23DNAzyme in laryngeal carcinoma in vitro

作　　者：滕博[1] 辛丁[1] 文连姬[1] 王君影[1] 崔树勋[1] 金春顺[1] 

机构地区：[1]吉林大学第二医院耳鼻咽喉科,吉林长春130041

出　　处：《中国耳鼻咽喉头颈外科》2007年第11期659-661,共3页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：吉林大学青年创新基金项目(K205)

摘　　要：目的研究特异性脱氧核酶(10-23DNAzyme)对喉癌真核细胞起始因子4E(eukaryotici nitiation factor-4E,eIF4E)基因mRNA的体外切割作用。方法设计合成针对原癌基因eIF4EmRNA编码区的1102、1289、1226位点的3个10-23DNAzyme-eIF4E,用内切酶XhoI将含有eIF4E基因mRNA的pGEM-7zf+eIF4E质粒消化成线性。以此为模板体外转录出用[α-32P]UTP标记的靶eIF4E基因编码区mRNA。在37℃,pH7.5,Mg2+离子50mmol/L条件下,分别与3个10-23DNAzyme-eIF4E(1μmol/L)混合进行切割反应,并比较不同Mg2+离子浓度下酶对靶mRNA的切割活性。反应产物在8%变性聚丙烯酰胺凝胶上电泳分离并行放射自显影,应用凝胶成像分析仪评价酶的切割效率。结果在设定条件下,3个10-23DNAzyme-eIF4E对eIF4E基因mRNA均有切割活性,且随着Mg2+浓度越高,酶的切割活性增强。结论3个10-23DNAzyme-eIF4E能有效切割eIF4E基因编码区mRNA,且切割效率与Mg2+浓度有关。OBJECTIVE To study the cleavage activity of 10-23DNAzyme to eIF4E mRNA in vitro, METHODS Three 10-23DNAzyme-eIF4E were designed to cleave at different point of eIF4E mRNA and their catalytic activity were observed in vitro. pGEM-7zf＋eIF4E plasmid was transcript in vitro with T7 transcription kit, and eIF4E mRNA was labeled with [α-^32p]UTP. Under the condition of 37℃, pH7.5, Mg^2＋ 50 mmol/L, the three 10-23DNAzyme-eIF4Es were mixed with substrate RNA individually. The cleaved products were separated with 8 % denatured polyacrylamide gel electrophoresis and displayed by autoradiography. Meanwhile, the activity of DRz3 to the substrate RNA at different Mg^2＋ concentration was compared. RESULTS The agitation was remarkably suppressed in group Ⅰ and group Ⅱ. But the time of extubation and waking time was delayed in group Ⅱ compared with that in group Ⅰ. CONCLUSION The synthesis 10-23DNAzyme-eIF4E can efficiently cleave target eIF4E mRNA in vitro, and its activity have some relation to the concentration of Mg^2＋ ion.

关 键 词：DNA 催化性 真核细胞起始因子4E 基因疗法 喉肿瘤 

分 类 号：R739.63[医药卫生—肿瘤]