Construction Of A Recombinant Plasmid HF443-GFP And Application In Detection Of HAB Toxins

Objection: A novel rapid cell-based assay was established for detection theharmful algal bloom (HAB) toxin. The first, A recombinant plasmid HF443-GFP which contained a c-fos promoter and GFP gene was constructed, and then transfected into Human bladder transitional cell carcinoma BIU-87 cell; The second, GTX, a HAB toxin and aconitine, a sodium channel activator were added into the cell culture system. The expression of GFP induced by aconitine was detected in the system. Based on the changes in the expression of GFP blocked by GTX, the level of GTX was finally deduced.Methods: The green fluorescent protein(GFP) gene amplified by PCR wasdigested by Hind III and Xba I, and then ligated with the plasmid HF443 which digested by the same restriction endonuclease. The resulting plasmid HF443-GFP was identified with sequencing. The BIU-87 cell was transfected by the recombinant plasmid HF443-GFP through LipofectAMINE2000. The behavior of expression of GFP in BIU-87 cell was observed under the fluorescent microscope.Conclusions: A new recombinant plasmid HF443-GFP was constructedsuccessfully. The sequence of the GFP was identified as same as the reported sequence. When aconitine were added into BIU-87 cell culture system transfected with plasmid HF443-GFP, green fluorescence was observed, which suggested that the aconitine can induced the expression of GFP in the BIU-87 cell. In the toxin detection experiment, with the increase of the level of GTX, the intensity of green fluorescence decreased. A certain dose-effect relationship was observed. These results indicated that GTX may inhibit the expression of GFP. This system can be used in detection of HAB toxin.