Construction Of A Recombinant Plasmid PEGFP-c-fos And Application In Detection Of HAB Toxins

Objective: To establish a rapid cell-based assay for detection the harmful algal bloom (HAB) toxins. First, to construct a recombinant plasmid pEGFP-c-fos with c-fos promoter and EGFP, and then transfect it into Human bladder transitional cell carcinoma BIU-87 cell; Second, based on the changes of the expression of GFP in the BIU-87 cell which induced by the aconitine and HAB toxins, the concentration of the HAB toxins could be detected.Methods: Plasmid pEGFP and c-fos promoter which amplified by PCR were digested by Vspl and EcoRI respectively, and then ligated with T4-DNALigase. The recombinant plasmid pEGFP-c-fos which were identified by sequencing were transfected into BIU-87 cell through LipofectAMINE2000, and the positive transfectants were obtained by G418. The changes of the expression of GFP in BIU-87 cell that induced by the aconitine and HAB toxins, GTX were detected with fluorescence microscope, and quantitatively measured with Image-pro Plus software.Conclusions: A recombinant plasmid pEGFP-c-fos was constructed and a new pEGFP-c-fos-BIU-87 cell strain was established successfully. The dose-effect relationship between the intensity of green fluorescence and the level of GTX was detected, and elementarily, a rapid cell-based assay for detection HAB toxins was established.