喉癌eIF4E基因的体外转录  

In vitro transcription of eIF4E in laryngeal carcinoma

作　　者：滕博[1] 金春顺[1] 文连姬[1] 崔树勋[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：《中国耳鼻咽喉颅底外科杂志》2008年第4期251-254,共4页Chinese Journal of Otorhinolaryngology-skull Base Surgery

基　　金：吉林大学青年创新基金(K203)

摘　　要：目的克隆喉癌eIF4E基因编码区的cDNA序列,并构建其体外转录载体,获得eIF4E基因编码区mRNA,以便进一步的体外实验研究。方法利用逆转录多聚酶链反应(RT-PCR)法,以喉癌Hep-2细胞mRNA为模板,扩增获得全长eIF4E编码区cDNA,与pMD18T连接作全自动测序,并利用基因重组技术构建eIF4E基因mRNA体外转录载体pGEM-7zf+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[a-32P]UTP标记的靶eIF4E基因编码区mRNA。结果经测序证实获得的eIF4E基因cDNA序列与GeneBank一致,并成功构建了体外转录质粒pGEM-7zf+eIF4E。结论成功构建了体外转录质粒pGEM-7zf+eIF4E,为进一步研究eIF4E的功能奠定了基础。Objective To clone the cDNA sequence of eIF4 E coding area in Hep-2 cell, to construct its transcriptive vector, and transcript the eIF4E mRNA in vitro. Methods Using Hep-2 cell eDNA as template, full-length eIF4E was obtained with the technique of RT-PCR followed by automatic sequencing of pMD18T-eIF4E, and its in vitro transcription plasmid pGEM-7zf ＋ eIF4E was constructed with recombinant DNA technique. Then pGEM-7zf ＋ eIF4E plasimid was transcripted in vitro with T7 trascription kit, and eIF4E mRNA was labelled with [ a-32P] UTP. Results Sequencing proved that the cloned eIF4E eDNA was completely identical with that of Gene Bank, and the recombinant in vitro transcriptive plasmid pGEM-7 zf ＋ eIF4E was constructed successfully. Conclusion eIF4E eDNA is successfully cloned and an in vitro transcriptive plasmid pGEM-7 zf ＋ eIF4E is constructed.

关 键 词：真核细胞翻译起始因子-4E 体外转录 逆转录多聚酶链反应 喉肿瘤 

分 类 号：R739[医药卫生—肿瘤] Q343.1[医药卫生—临床医学]