Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (1): 179-186.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0289

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Eukaryotic Expression Vector Construction of Danio rerio notch3 Gene and Its Expression Analysis

WU Kun-kun1,2(), XU Xing1,2, JI Ce1,2, REN Jian-feng1,2, LI Wei-ming3, ZHANG Qing-hua1,2()   

  1. 1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306
    2. National Pathogen Collection Center for Aquatic Animals,Shanghai Ocean University,Shanghai 201306
    3. Department of Fisheries and Wildlife,Michigan State University,East Lansing 48824,USA
  • Received:2021-03-10 Online:2022-01-26 Published:2022-02-22
  • Contact: ZHANG Qing-hua E-mail:1017676610@qq.com;qhzhang@shou.edu.cn

Abstract:

In order to further study the function of notch3 in Danio rerio,the eukaryotic expression vector of notch3 was constructed and it successfully expressed in eukaryotic cells. The coding sequence(CDS)of notch3 gene in D. rerio was obtained from NCBI online database. According to the sequence,Notch intracellular domain(NICD)was cloned,and then the expression vector pCMV-N3ICD was constructed by homologous recombination. The positive recombinant vector was screened by double enzyme digestion and sequencing,and then transfected into HEK293T cells by liposome method. Finally,the expression of N3ICD was verified by immunofluorescence and Western blot. The results showed that the recombinant plasmids were in accordance with the expected results via enzymatic digestion electrophoresis fragment and sequencing comparison. In Western blot,N3ICD protein expressed normally,and the size was consistent with the predicted one. Cell immunofluorescence showed that the recombinant plasmid expressed in the nucleus of the HEK293T cells. Concurrently,the constructed eukaryotic expression vector N3ICD significantly enhanced the activities of rela and nfkb1 promoters in NF-κB family(nuclear factor-κB).

Key words: Danio rerio, notch3, N3ICD, immunofluorescence, eukaryotic expression vector, NF-κB