Primary determination for activity and expression of Stx 2a’-LHRH chimeric toxin / 中国病理生理杂志

ABSTRACT

AIM: To express chimeric toxin Stx2a’-LHRH a nd to investigate the cytotoxic activity of recombinant toxin Stx2a’-LHRH to huma n carcinoma cells.METHODS: Stx2a’-LHRH sequences that added the res tri ction endonucleases NcoⅠ and EcoRⅠ at the 5' and 3 ends were amplified by PCR a nd digested with appropriate restriction enzymes. The digested fragment was subc loned into the vector obtatined by digestion of plasmid pET-28a(+) with NcoⅠ an d 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-?-D-galactoside (IPTG). T h e cytotoxity of Stx2a’-LHRH to Hep-2 cells was observed under the microscop y. RESULTS: Recombitant plasmid pET-SL was constructed successfu lly and the clones expressing pET-SL stablely were obtained. A special electroph oretic band in SDS-PAGE (a glycoprotein of 28kD) was noted. Stx2a’-LHRH killed He lp-2 cells clearly. CONCLUSION: In this study, construction of c himeric toxin Stx2a’-LHRH and its expression were described. Moreover, it has o bvious cytotoxity to Hep-2 cell. These finding could open up new vistas in the s tudy of targeted durgs.