Primary determination for activity and expression of Stx2a'-LHRH chimeric toxin

AIM:To express chimeric toxin Stx2a'-LHRH and to investigate the cytotoxic activity of recombinant toxin Stx2a'-LHRH to human carcinoma cells.METHODS:Stx2a'-LHRH sequences that added the restriction endonucleases NcoⅠ and EcoRⅠ at the 5' and 3 ends were amplified by PCR and digested with appropriate restriction enzymes. The digested fragment was subcloned into the vector obtatined by digestion of plasmid pET-28a(+) with NcoⅠ and EcoRⅠ. E. coli BL21 (DE3) cells were transformed with plasmids of interst and cultured in LB medium containing ampicillin. Expression of the recombinant protein was induced by the addition of isopropylthio-β-D-galactoside (IPTG). The cytotoxity of Stx2a'-LHRH to Hep-2 cells was observed under the microscopy. RESULTS:Recombitant plasmid pET-SL was constructed successfully and the clones expressing pET-SL stablely were obtained. A special electrophoretic band in SDS-PAGE (a glycoprotein of 28kD) was noted. Stx2a'-LHRH killed Help-2 cells clearly. CONCLUSION:In this study, construction of chimeric toxin Stx2a'-LHRH and its expression were described. Moreover, it has obvious cytotoxity to Hep-2 cell. These finding could open up new vistas in the study of targeted durgs.