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Construction of eukaryotic expression vector of Klotho gene and its stable expression in TCMK-1 cell line

SHEN Yue1, LU Li-ming2, QIAN Ying-ying1, GUAN Xue-jing1, QI Yuan-yuan2, NI Zhao-hui1, QIAN Jia-qi1, YAN Yu-cheng1   

  1. 1.Department of Renal Division, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China; 2.Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Online:2014-05-28 Published:2014-05-30
  • Supported by:

    National Natural Science Foundation of China, 81170687; New Otstanding Young Scientists Training Project of Shanghai Municipal Health Bureau, 2011XYQ015； Major Basic Research Project of Science and Technology Commission of Shanghai Municipality, 12DJ1400200; Key Basic Research Project of Science and Technology Commission of Shanghai Municipality, 11JC1410802

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Abstract:

Objective To construct the eukaryotic expression vector of the mouse Klotho gene and to acquire mouse kidney epithelial cell line TCMK-1 with stable expresses Klotho. Methods The Klotho-FLAG gene was amplified by PCR and cloned to the corresponding site of eukaryotic expression vector pCD-CXIN (CMV-MCS-IRES-Neomycin). Once confirmed by enzyme digestion and sequencing, the recombinant pCD-CXIN-Klotho plasmids were transfected to TCMK-1 cells by lipidosome and a cell line that stably expressed Klotho was obtained by G418 screening. The expression of Klotho mRNA was detected by Real-Time PCR, and the expression of Klotho protein was detected by Western blotting. Results The recombinant Klotho gene expression vector with FLAG tag was constructed, and the cell line obtained by transfection and G418 screening presents higher expression of Klotho mRNA and protein compared with that of control groups. Conclusion The recombinant Klotho expression vector has been successfully constructed and the TCMK-1 cell line with stable expression of Klotho is obtained, which might be constructive for the research on the functions of Klotho protein in mouse kidney epithelial cells.

Key words: Klotho, internal ribosome entry sites sequence, FLAG tag, TCMK-1 cells