人硫氧还蛋白-1基因克隆及其重组载体的构建与鉴定

山东大学学报(医学版) ›› 2010, Vol. 48 ›› Issue (11): 41-45.

人硫氧还蛋白-1基因克隆及其重组载体的构建与鉴定

石岩岩1，丁士刚1，蒋素贞2，鲁凤民2，张静1，刘琳娜1，王晔1

北京大学 1.第三医院消化科； 2.医学部病原生物学系，北京 100191

收稿日期:2010-06-09 出版日期:2010-11-16 发布日期:2010-11-16
通讯作者: 丁士刚（1963- ），男，博士，教授，博士生导师，主要从事上消化道疾病的诊治研究。 E-mail: dingshigang222@163.com
作者简介:石岩岩（1988- ），女，硕士研究生，主要从事硫氧还蛋白与胃癌关系研究。
基金资助:
国家自然科学基金资助项目（30770980）

Cloning of the human thioredoxin-1 gene and construction and identification of its recombinant vector

SHI Yan-yan1, DING Shi-gang1, JIANG Su-zhen2, LU Feng-min2, ZHANG Jing1， LIU Lin-na1， WANG Ye1

1. Department of Gastroenterology, Peking University Third Hospital, Beijing 100191, China；
2. Department of Microbiology, Peking University Health Science Center, Beijing 100191, China

Received:2010-06-09 Online:2010-11-16 Published:2010-11-16

摘要/Abstract

摘要：

目的克隆人硫氧还蛋白-1（human thioredoxin-1, hTrx1）基因，并构建含有该目的基因的重组载体。方法以人胃癌细胞系BCG823、MNK45的总RNA为模板，设计含有酶切位点的特异性引物，用逆转录PCR的方法，扩增hTrx1编码蛋白的cDNA片段，连接至EZ-T载体，形成EZ-T-hTrx1。对EZ-T-hTrx1进行双酶切后将目的片段连接至pcDNA3.1myc-His，形成pcDNA3.1myc-His-hTrx1，并进行双酶切、测序鉴定。结果双酶切鉴定显示hTrx1 cDNA成功连入pcDNA3.1myc-His质粒；测序结果显示目的片段连入质粒，且与GenBank（NM003329）完全一致。该实验成功构建出含hTrx1的重组载体pcDNA3.1myc-His-hTrx1。结论 hTrx1基因的克隆以及重组载体pcDNA3.1myc-His-hTrx1的成功构建，为进一步探讨hTrx1的生物学活性及其对肿瘤发生的作用机制奠定了基础。

关键词: 人硫氧还蛋白-1；重组载体；逆转录PCR；双酶切

Abstract:

Objective To clone the human thioredoxin-1 gene (hTrx1) and construct the recombinant vector containing the target gene. Methods The cDNA gene of human thioredoxin was amplified by RT-PCR from gastric cancer cells BCG823 and MNK45, using specific primers containing double endonuclease digesting sites. The hTrx1 cDNA was then inserted into the EZ-T vector to construct the EZ-T-hTrx1 recombinant vector. The next step was to double digest the EZ-T-hTrx1 recombinant vector and insert the target gene into the pcDNA3.1myc-His to construct the pcDNA3.1myc-His-hTrx1 recombinant vector, which was identified by double digestion and DNA sequencing. Results By double digestion, the hTrx1 cDNA was successfully inserted into the pcDNA3.1myc-His vector; and through DNA sequencing, the sequence of the target gene was inserted and was in accordance with GenBank(NM003329). The pcDNA3.1myc-His-hTrx1 recombinant vector was successfully gained. Conclusion The successful cloning of the hTrx1 gene and construction of its recombinant vector pcDNA3.1myc-His-hTrx1 lays the foundation for the study of the biological activity of hTrx1 and mechanism of its function in tumor genesis.

Key words: Human thioredoxin-1; Recombinant vector; Reverse transcription-PCR; Double endonuclease digestion

中图分类号:

R318.14

石岩岩1，丁士刚1，蒋素贞2，鲁凤民2，张静1，刘琳娜1，王晔1. 人硫氧还蛋白-1基因克隆及其重组载体的构建与鉴定[J]. 山东大学学报(医学版), 2010, 48(11): 41-45.

SHI Yan-yan1, DING Shi-gang1, JIANG Su-zhen2, LU Feng-min2, ZHANG Jing1， LIU Lin-na1， WANG Ye1 . Cloning of the human thioredoxin-1 gene and construction and identification of its recombinant vector[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(11): 41-45.

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