神经营养素4前导序列介导Exendin-4表达质粒的构建及鉴定

神经药理学报 ›› 2012, Vol. 2 ›› Issue (3): 22-28.

神经营养素4前导序列介导Exendin-4表达质粒的构建及鉴定

王俊红^1,4 ，郭永红² ，焦杨¹ ，张静¹ ，徐静¹ ，谢璇¹ ，杨广笑³ ，王全颖³ ，王枫⁴

1. 西安交通大学医学院第二医院内分泌科，西安，710004，中国
2. 西安交通大学医学院第二医院传染科，西安，710004，中国

3. 西安华广生物工程有限公司，西安，710025，中国

4. 第四军医大学基础医学部预防医学系，西安，710032，中国

出版日期:2012-06-26 发布日期:2013-12-25
通讯作者: 王枫，E-mail：wangfeng@fmmu.edu.cn
作者简介:王俊红，主治医师，硕士研究生；研究方向：糖尿病及并发性的基因治疗；+86-029-87679343, E-mail： jhwangdoctor@sohu.com
基金资助:
陕西省社发攻关项目(No.2011K14-06-04)

Identification and Construction of Plasmid Expressing Exendin-4 mediated NT4 Leading Sequence

WANG Jun-hong, GUO Yong-hong, JIAO Yang, ZHANG Jing, XU Jing, XIE Xuan, YANG Guang-xiao, WANG Guan-ying, WANG Feng

1. Department of Endocrine, the Second Hospital, Medical College of Xi'an Jiaotong University, Xi'an, 710004, China
2. Department Of Infectious Disease, the Second Hospital,Medical College of Xi'an Jiaotong University, Xi’an, 710004, China

3. Huaguang Biology Co., Ltd, Xi’an, 710025, China
4. Department of Nutrition and Food Hygiene, the Fourth Military Medical University, Xi’an, 710032, China

Online:2012-06-26 Published:2013-12-25
Contact: 王枫，E-mail：wangfeng@fmmu.edu.cn
About author:王俊红，主治医师，硕士研究生；研究方向：糖尿病及并发性的基因治疗；+86-029-87679343, E-mail： jhwangdoctor@sohu.com
Supported by:
陕西省社发攻关项目(No.2011K14-06-04)

摘要/Abstract

摘要： 目的：构建神经营养素4前导序列介导的可分泌表达Exendin-4的重组腺伴病毒载体，为探究Exendin-4用于2型糖尿病临床治疗提供实验基础。方法：使用互补引物/模板法及Ｔ载体克隆法扩增Exendin-4的cDNA克隆，连接于NT4前导肽序列的3′端，融合基因NT4- Exendin-4亚克隆于穿梭质粒pSSHG，转染HEK-293细胞，包装病毒，斑点杂交方法测定重组病毒滴度。结果：经酶切、测序证实克隆出Exendin-4 cDNA，成功构建同一开放读码框ORF的NT4前导肽-Exendin-4cDNA克隆，得到高滴度（2.5×10⁹pfu·L^-1）的重组腺相关病毒。结论：通过分子克隆技术成功制备了Exendin-4的重组腺伴病毒载体pSSHG /NT4- Exendin-4并成功包装了较高浓度的重组病毒，为进一步研究Exendin-4功能及应用于临床打下基础。

关键词: 质粒构建, Exendin-4, NT4, 腺伴病毒载体

Abstract: Objective: To identify and construct a recombinant adeno-associated virus (AAV) vector that can secrete Exendin-4 mediated by NT4 leading sequence. Methods: ·L^-1). Conclusion: The recombinant virus pSSHG/NT4 - Exendin - 4 was successfully constructed by molecular cloning. The relatively high levels of recombinant virus were obtained. These results provide the basis for further studies that examine the function of Exendin-4.Exendin-4 cDNA was cloned by complementary primer/template PCR and T carrier cloning method. The Exendin-4 gene was linked to terminal of the signal peptide of NT4 leading sequence. The fusion gene of NT4- Exendin-4 was subcloned into the shuttle plasmid of pSSHG. The recombinant virus was transfected and packaged by human embryo kidney 293 cells and the virus titer was measured by quantitative dot-blot hybridization. Results: Exendin-4 cDNA cloning was confirmed by restriction enzyme digestion and DNA sequencing. The NT4 leading sequence and Exendin-4 cDNA clone were successful constructed in the same ORF and high titer of recombinant AAV was obtained (2.5×10⁹pfu

Key words: plasmid construction, Exendin-4, NT4, adeno-associated virus (AAV)

中图分类号:

R587.1

王俊红, 郭永红, 焦杨, 张静, 徐静, 谢璇, 杨广笑, 王全颖, 王枫. 神经营养素4前导序列介导Exendin-4表达质粒的构建及鉴定[J]. 神经药理学报, 2012, 2(3): 22-28.

WANG Jun-hong, GUO Yong-hong, JIAO Yang, ZHANG Jing, XU Jing, XIE Xuan, YANG Guang-xiao, WANG Guan-ying, WANG Feng. Identification and Construction of Plasmid Expressing Exendin-4 mediated NT4 Leading Sequence[J]. ACTA NEUROPHARMACOLOGICA, 2012, 2(3): 22-28.

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