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|本期目录/Table of Contents|

NT4-TAT-His-PR39融合基因cDNA的构建及意义

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2009年第6期
页码:
805-809
栏目:
基础研究
出版日期:
2009-11-05

文章信息/Info

Title:
Construction and significance of fusion gene NT4-TAT-His-PR39 cDNA
作者:
郝跃文; 孙立军; 刘莹
第四军医大学西京医院放射科，陕西 西安 710032
Author(s):
HAO Yue-wen;  SUN Li-jun;  LIU Ying
Department of Radiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi, China
关键词:
PR39; 神经生长因子4; TAT; 6×His; 基因克隆
Keywords:
PR39;  neurotrophin 4;  TAT;  6×His;  gene clone
分类号:
Q781
DOI:
-
文献标识码:
A
摘要:
目的: 探讨PR39对急性缺血心肌的保护作用，构建可分泌表达PR39的神经生长因子4（NT4）-TAT-His-PR39融合基因cDNA。方法: 采用互为模板、引物的PCR技术，制备两端含有酶切位点的PR39 cDNA片段。通过常规分子生物学方法，将其与编码NT4信号肽及引导肽、TAT及6×His基因序列用T4 DNA连接酶相连，将NT4-TAT-His-PR39装入质粒pBV220中。结果: 融合基因NT4-TAT-His-PR39的长度为421 bp，限制性内切酶切图谱显示，目的带的大小略超过marker的400 bp的位置，证实已经成功地将PR39 cDNA重组到NT4-TAT-His序列的下游。基因测序结果及DNAsis软件分析结果表明，NT4-TAT-His-PR39的全序列与实验设计的序列完全一致。结论: 成功地构建能分泌表达PR39、并具穿膜功能和标签的融合基因NT4-TAT-His-PR39。
Abstract:
AIM: To explore the protection of PR39 for acute myocardial ischemia and to construct the NT4-TAT-His-PR39 cDNA secreting and expressing PR39. METHODS: PR39 cDNA was obtained by PCR twice, which contained restriction enzyme sites on the two extremities. PR39 cDNA was connected with the signal peptide and leader peptide of neurotrophin 4 (NT4) cDNA, TAT cDNA, 6×His cDNA by molecular biological method. The NT4-TAT-His-PR39 was inserted into the plasmid of pBV220. RESULTS: The length of fusion gene was 421 bp, and restriction enzyme digestion confirmed that the gene expression box of NT4-TAT-His-PR39 was successfully constructed. DNA sequencing and DNAsis software analysis results indicated that NT4-TAT-His-PR39 gene sequence was completely consistent with that of the experimental design. CONCLUSION: The NT4-TAT-His-PR39 fusion gene is successfully constructed in this experiment, which secretes PR39, crosses cellular membrane, and contains a 6×His tag.

参考文献/References

［1］ Muinck ED, Nagy N, Tirziu D, et al.Protection against myocardial ischemia-reperfusion injury by the angiogenic Masterswitch protein PR 39 gene therapy: the roles of HIF1alpha stabilization and FGFR1 signaling［J］. Antioxid Redox Signal, 2007, 9(4):437-445.

［2］ Li J, Post M, Volk R, et al. PR39, a peptide regulator of angiogenesis［J］. Nat Med, 2000, 6(1):49-55.

［3］ Lee PH, Ohtake T, Zaiou M, et al. Expression of an additional cathelicidin antimicrobial peptide protects against bacterial skin infection［J］. Proc Natl Acad Sci USA, 2005, 102(8):3750-3755.

［4］ Wu J, Parungo C, Wu G, et al. PR39 inhibits apoptosis in hypoxic endothelial cells: role of inhibitor apoptosis protein-2［J］. Circulation, 2004, 109(13):1660-1667.

［5］ 佘华宁,孙乃学,郑玉萍,等. 神经营养素-4-神经短肽NAPVSIPQ融合基因对兔眼虹膜色素上皮细胞的转染和保护作用［J］.中华眼科杂志, 2006, 42(11):1008-1012.

［6］ 杨宇，吴江，杨欣,等. NT4-NAP融合基因原核表达载体的构建及在大肠杆菌中的表达［J］. 中华神经科杂志, 2004, 37(3):260-261.

［7］ Zhao M, Weissleder R. Intracellular cargo delivery using Tat peptide and derivatives［J］. Med Res Rev, 2004, 24(1):1-12.

备注/Memo

备注/Memo:
收稿日期：2008-8-15.通讯作者:孙立军,主任医师,主要从事心血管系统疾病影像诊断的研究Email:sunlijun_fmmu@sina.com 作者简介:郝跃文,住院医师,硕士Email:1982_edifier@163.com
更新日期/Last Update: 2009-09-30