Reporter gene assay for detection of shellfish toxins.
OBJECTIVE
To explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry.


METHODS
A reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfectants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software.


RESULTS
Dose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX2,3 from 0 to 16 ng/mL.


CONCLUSIONS
pEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.
Yang
Wei-Dong
WD
School of Life Science and Technology, Jinan University, Guangzhou 510632, Guangdong, China. tywd@jnu.edu.cn
Wu
Min-Yi
MY
Liu
Jie-Sheng
JS
Peng
Xi-Chun
XC
Li
Hong-Ye
HY
D000818:Animals; D001681:Biological Assay; D045744:Cell Line, Tumor; D017930:Genes, Reporter / Q000502:physiology*; D049452:Green Fluorescent Proteins; D057097:Harmful Algal Bloom / Q000502:physiology; D006801:Humans; D010957:Plasmids; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D012758:Shellfish / Q000032:analysis*; D015222:Sodium Channels; D014118:Toxins, Biological / Q000737:chemistry* / Q000633:toxicity*
D016760:Proto-Oncogene Proteins c-fos; D015222:Sodium Channels; D014118:Toxins, Biological; D049452:Green Fluorescent Proteins