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Expression of human beta-defensin 3 in COS-7 cell
Xiao-ye Tuo1, Ming-da Xu, Bi Chen
1Department of Burns and Plastic Surgery, Hospital 304th, Beijing 100037.
Objective:
To establish a cell line for stable expression of human beta-defensin 3 (hBD3).
Methods:
Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofectamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated.
Results:
COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity.
Conclusion:
We successfully established a hBD3-expressing cell line.
