hTRXPR39融合基因cDNA克隆的构建及鉴定

山东大学学报(医学版)

hTRXPR39融合基因cDNA克隆的构建及鉴定

阮喜云¹，毕建忠²，刘庆勇³，张士宝³，杨广笑⁴，王全颍⁴

（1. 山东大学附属济南市中心医院神经内科，济南 250013； 2．山东大学第二医院神经内科，济南 250033；
3. 山东大学附属济南市中心医院泌尿外科，济南 250013； 4．西安华广生物工程有限公司，西安 710025 ）

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-03-16 发布日期:2009-03-16
通讯作者: 毕建忠

Construction and identification of recombinant plasmids expressing hTRXPR39

RUAN Xiyun¹,　BI Jianzhong²,　LIU Qingyong¹,　ZHANG Shibao³,　YANG Guangxiao⁴,　WANG Quanying⁴

(1. Department of Neurology ,Jinan Central Hospital Affiliated to Shandong University, Jinan 250013, China;
2. Department of Neurology,Second Hospital Affiliated to Shandong University, Jinan 250033, China;
3. Department of Urology, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013, China;
4. Xi′an Hua Guang Biological Engineering Limited Company, Xi′an 710025, China)

Received:1900-01-01 Revised:1900-01-01 Online:2009-03-16 Published:2009-03-16
Contact: BI Jianzhong

摘要/Abstract

摘要： 目的研究人硫氧还蛋白（hTRX）和抗菌肽PR39基因在脑缺血疾病治疗中的作用，构建hTRXPR39cDNA融合基因。方法设计合成PR39、hTRX正向和反向引物，采用PCR方法，扩增获得两端具有BamHI和Ecor721、Eco721和EcoI酶切位点的PR39 cDNA和hTRX cDNA片段，并分别克隆到pGEMT easy中，转化细菌，筛选阳性克隆，酶切鉴定并测序。BamH I和EcoR721双酶切pGEMThTRX和pGEMTPR39，将获取的PR39/BamHI，EcoR721片段克隆到pGEMThTRX/BamHI，EcoR721载体中，构建pGEMThTRXPR39载体。结果经DNA测序证实，修饰过的hTRX cDNA、PR39 cDNA片段的核酸序列和推导的氨基酸同数据库资料一致。重组质粒pGEMThTRXPR39酶切图谱证实hTRXPR39融合肽cDNA已克隆到pGEMT表达载体中。结论成功克隆出具有EcoR721和EcoRI、BamH I和EcoR721酶切位点的hTRX cDNA和PR39 cDNA片段，并构建pGEMThTRXPR39载体。

关键词: 脑缺血, 硫氧还蛋白, 基因融合, 抗菌肽PR39

Abstract: To explore the effects of hTRX and PR39 as target genes on cerebral ischemic disease, and to construct a fusion gene of hTRXPR39 cDNA. MethodsThe forward and reverse primers of PR39 were designed and synthesized. By means of PCR, the fragment encoding PR39 was gained, including EcoR721 and BamH I restriction enzyme sites, and the new hTRX cDNA including EcoR721 and EcoRI restriction enzyme sites was also gained. Then the synthesized fragments were easily cloned into vector pGEMT. The positive clone was identified by restriction enzymes, and then the cloned amplified fragments were sequenced by the dideoxymediated chaintermination method. The cloned hTRX and PR39 cDNA were compared with the GeneBank sequence by the DNASIS. After pGEMThTRX and pGEMTPR39 were both digested by BamH I and EcoRI, the PR39/BamHI, EcoRI was cloned into the recombinant vector pGEMThTRX/BamHI,EcoRI .The recombinant vector pGEMThTRXPR39 was gained. ResultsSequences of modified hTRX and PR39 were consistent with those of the gene bank. After analyzing the ORF of the cloned hTRXPR39 cDNA by the DNASIS, we found that amino acids encoded by the cloned hTRXPR39 cDNA were identical to published results. ConclusionThe sequence of hTRX including EcoR721 and EcoRI restriction enzyme sites and sequence of PR39 including EcoR721 and BamH I restriction enzyme sites were successfully obtained by PCR. Also, the recombinant vector pGEMThTRXPR39 was successfully constructed.

Key words: Brain ischemia, Thioredoxin, gene fusion, Antibacterial peptide PR39

中图分类号:

R743

阮喜云1 ，毕建忠2 ，刘庆勇3 ，张士宝3 ，杨广笑4 ，王全颍4
. hTRXPR39融合基因cDNA克隆的构建及鉴定[J]. 山东大学学报(医学版), 2009, 47(03): 30-34.

RUAN Xiyun¹,　BI Jianzhong²,　LIU Qingyong¹,　ZHANG Shibao³,　YANG Guangxiao⁴,　WANG Quanying⁴.

Construction and identification of recombinant plasmids expressing hTRXPR39

[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2009, 47(03): 30-34.

参考文献

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