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

Objective: A recombinant plasmid pEGFP-c-fos with c-fos promoter and EGFP was constructed, and then transfected into T24 cell. The positive transfectants were obtained by screening. Based on the changes of the expression of EGFP in the T24 cell which induced by the HABs toxins, a new method to detect HABs toxins was founded in receptor level.Methods: The c-fos with cohesive terminus was obtained after amplified by PCR and then digested by VspI and EcoRI, and then ligated with the plasmid pEGFP digested by the same restriction enzymes. The recombinant plasmid pEGFP-c-fos, which was identified by PCR, digesting, and sequencing, was transfected into T24 cell through LipofectAMINE2000. The positive transfectants were obtained by G418. The changes of the expression of EGFP in T24 cell with addition of increasing NSP or PSP were detected by Laser scanning confocal microscope, and quantitative analysis was carried out by Image-pro Plus software.Conclusions: A recombinant plasmid pEGFP-c-fos was constructed and a new pEGFP-c-fos-T24 cell strain was established successfully. The dose-effect relationship between the intensity of green fluorescence and the level of NSP or PSP was detected. A rapid cell-based assay for detection HABs toxin was establishedprimarily.