中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (41): 7719-7722.doi: 10.3969/j.issn.1673-8225.2011.41.029

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

真核表达载体p4CCL20-ZsGreen1-DR的构建与鉴定

王  勇1，王志中1，钟  兵1，王  恒1，邹庆华1，陈  渝2   

  1. 解放军第三军医大学西南医院，1风湿科，2烧伤研究所，创伤、烧伤、复合伤国家重点实验室，重庆市  400038
  • 收稿日期:2011-04-19 修回日期:2011-06-25 出版日期:2011-10-08 发布日期:2011-10-08
  • 作者简介:王勇☆，男，1970年生，湖南省吉首市人，汉族，2009年解放军第三军医大学毕业，博士，副教授，主要从事风湿病的发病机制研究。
  • 基金资助:

    重庆市科委自然基金面上项目(2008BB5133)；国家自然基金面上项目(30973827)。

Construction and identification of p4CCL20-ZsGreen1-DR eukaryotic expression vector

Wang Yong1, Wang Zhi-zhong1, Zhong Bing1, Wang Heng1, Zou Qing-hua1, Chen Yu2   

  1. 1Department of Rheumatology, 2 Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University of Chinese PLA, Chongqing  400038, China
  • Received:2011-04-19 Revised:2011-06-25 Online:2011-10-08 Published:2011-10-08
  • About author:Wang Yong☆, Doctor, Associate professor, Department of Rheumatology, Southwest Hospital, Third Military Medical University of Chinese PLA, Chongqing 400038, China wangyongjhy@tom.com
  • Supported by:

    the Natural Science Foundation of Chongqing Science and Technology Commission (General Program), No. 2008BB5133*; the National Natural Science Foundation of China, No. 30973827*

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摘要：

背景：抗炎药物高通量筛选体系的建立，可为相关药物的研究提供一个理想的技术平台。
目的：构建以核因子κB顺式作用元件4×CCL20基序为增强子，以SV40为启动子，以ZsGreen1-DR为报告基因的真核表达载体p4CCL20-ZsGreen1-DR。
方法：以PGL2-control质粒为模板，PCR扩增目的片段SV40，两侧引入KpnⅠ/BamHⅠ酶切位点，克隆至pZsGreen1-DR质粒的KpnⅠ/BamHⅠ酶切位点中，构建成pSV40-ZsGreen1-DR载体。将4×CCL20基序双链DNA克隆到pSV40-ZsGreen1- DR载体的BglⅡ和EcoRⅠ酶切位点之间，构建p4CCL20-ZsGreen1-DR重组质粒。
结果与结论：经过DNA测序分析证实p4CCL20-ZsGreen1-DR重组质粒构建成功。该重组质粒可作为抗炎药物高通量筛选体系的基础。

关键词: 载体构建, CC亚族趋化因子配体20, 不稳定型绿色荧光蛋白, 测序分析

Abstract:

BACKGROUND: It is necessary to establish a high throughput screening system for anti-inflammatory drugs for rheumatoid arthritis.
OBJECTIVE: To construct an eukaryotic expression vector p4CCL20-ZsGreen1-DR with the NF-B cis-acting element 4×CCL20 motif as an enhancer, SV40 as a promoter, and ZsGreen1-DR as a reporter gene.
METHODS: The target fragment SV40 was PCR amplified using PGL2-control plasmid as a template. KpnⅠ/Bam HⅠ restriction sites were introduced into the flank of the target fragment. Then, pSV40-ZsGreen1-DR vector was constructed by cloning the target fragment into pZsGreen1-DR plasmid. Finally, p4CCL20-ZsGreen1-DR plasmid was constructed by cloning the double strand DNA of 4×CCL20 motif (with BglⅡ and EcoRⅠ sticky ends at the 5’ and 3’ terminus, respectively) into the corresponding restriction sites of pSV40-ZsGreen1-DR vector (upstream of SV40 promoter).
RESULTS AND CONCLUSION: DNA sequencing demonstrated successful construction of p4CCL20-ZsGreen1-DR plasmid. The construction of p4CCL20-ZsGreen1-DR plasmid might be useful to establish a high throughput screening system for anti-inflammatory drugs.

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