Expression of human beta-defensin 3 in COS-7 cell.
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.


CONCLUSIONS
We successfully established a hBD3-expressing cell line.
Tuo
Xiao-ye
XY
Department of Burns and Plastic Surgery, Hospital 304th, Beijing 100037.
Xu
Ming-da
MD
Chen
Bi
B
Chai
Jia-ke
JK
Sheng
Zhi-yong
ZY
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000187:drug effects; D005822:Genetic Vectors; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects; D014162:Transfection; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
C422950:DEFB103A protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins; D023083:beta-Defensins