PTEN基因真核荧光表达载体的构建及其在喉癌细胞株

J4 ›› 2009, Vol. 35 ›› Issue (5): 817-820.

PTEN基因真核荧光表达载体的构建及其在喉癌细胞株

陈鸥¹|李野¹| 赵明¹|金春顺¹|金玲²|姜艳芳³

1．吉林大学第二医院耳鼻咽喉科|吉林长春130041；2.吉林大学中日联谊医院检验科| 吉林长春 130033；3. 吉林大学第一医院感染症科|吉林长春 130021

收稿日期:2008-12-30 出版日期:2009-09-28 发布日期:2009-09-28
通讯作者: 金春顺 E-mail:jinchunshun@163.com
作者简介:陈鸥（1958-）|女|吉林省长春市人|教授|医学博士|主要从事喉癌的临床研究。
基金资助:
吉林省科技厅科技发展计划项目资助课题（200505109）

Construction of |PTEN |gene |eukaryotic |expressing |vector and |PTEN |gene |expression |in |laryngeal |Hep-2 cell |line

CHEN Ou¹, LI Ye¹, |ZHao-Meng¹, JIN Chun-Shun¹, JIN Ling², JIANG Yan-Fang³

1.Department of Otorhinolaryngology, Second Hospital, Jilin University, Changchun |130041,China;2.Department of Clinical Laboratory, China-Japan Union Hospital, Jilin University, Changchun 130033,China;3. Department of Infectious Diseases,First Hospital,Jilin University, Changchun 130021,China

Received:2008-12-30 Online:2009-09-28 Published:2009-09-28

摘要/Abstract

摘要：

目的: 构建人PTEN基因的真核荧光表达载体并在Hep-2喉癌细胞中高效表达，阐明PTEN基因在喉癌细胞株中的抑癌效果并为喉癌的基因治疗奠定基础。方法: 从人喉黏膜组织中提取总RNA，经逆转录多聚酶链反应（RT-PCR）得到PTEN全基因。克隆入PGEM-T easy载体上，经过PCR和酶切鉴定为阳性的克隆，进行测序分析。将所获得的基因定向克隆入Pegfp-C1载体中构建PTEN真核荧光表达载体，并将其转入Hep-2细胞中进行表达，Western blotting检测其表达。结果: 反转录PCR扩增后的产物，在约1 400 bp处可观察到特异性条带，序列分析表明与GenBank中的PTEN基因序列相同。PCI-PTEN真核载体转染Hep-2细胞后的Western Blotting表明表达产物与抗人PTEN单克隆抗体有特异性免疫反应。结论：成功构建出PTEN真核荧光表达载体，诱导产生蛋白经检测证实为PTEN蛋白。

关键词: 抑癌基因;PTEN基因;基因克隆;基因表达

Abstract:

Abstract:Objective To construct eukaryotic expressing vector of PTEN gene and to express the gene in the Hep-2 cell line and to clarify theraputic effect of PTEN gene in the Hep-2 cell line,and may laid foundation for further study of PTEN gene treatment of laryngeal carcinoma.Methods PTEN gene was amplified from human normal laryngeal mucosa by RT-PCR and the fragment of the cDNA was cloned into the PTEN-Teasy vector.After the selection of the positive clone by DNA sequencing and restriction enzyme analysis,the fragment of the cDNA was cloned into the eukaryotic expressing vector of pEGFG-C1.The recombinant plasmid transfected into Hep-2 cell line and the gene transient expression was observed.with Western blotting.Results A 1 400 bp DNA fragment was amplified with RT-PCR.The sequence analysis showed it was consistant with the sequence of PTEN gene in GenBank.Western blotting analysis provided strong evidence that PTEN gene was expressed successfully in transfected Hep-2 cell line.Conclusion The eukaryotic expressing vector of PTEN gene is constructed and PTEN protein is induced to express successfully.

Key words: anti-oncogene;PTEN gene;gene cloning; , gene expression

中图分类号:

R734

陈鸥, 李野, 赵明, 金春顺, 金玲, 姜艳芳. PTEN基因真核荧光表达载体的构建及其在喉癌细胞株[J]. J4, 2009, 35(5): 817-820.