Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (41): 7719-7722.doi: 10.3969/j.issn.1673-8225.2011.41.029

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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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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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