Construction and prokaryotic expression of the fusion protein Stx2B-IntiminC300 of EHEC O157: H7 and its immunoprophylactic potential

Objective To construct and prokaryotically express the fusion protein of Stx2B and IntiminC300 of EHEC O157∶H7, and to investigate the immunoprophylactic potential of the fusion protein Stx2B IntiminC300. Methods The coding gene of Stx2B ( stx2b ) was amplified from EHEC O157∶H7 chromosome by PCR and then cloned into pMD18 T vector. Thereafter, the gene was cut from pMD18 T vector and cloned into prokaryotic expression plasmid pET 28a(+) eaeC300 which was previously constructed, and the recombinant plasmid pET 28a(+) stx2b eaeC300 was transformed into E.coli BL21(DE3). After the induced expression, the protein of Stx2B IntiminC300 was isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS PAGE), Western blot and N terminal amino acid residual sequencing. Then the fusion protein was primary purified by ion exchange chromatography for immunization of BALB/c mice. Results The gene of Stx2B was successfully cloned into pET 28a(+) eaeC300 . The results of SDS PAGE and Western blot assay showed that the molecular weight of the expressed product was 43kD, and the expression rate was about 25%. The result of the N terminal amino acid residual sequencing is identical to that of the molecular design. The fusion protein Stx2B IntiminC300 elicited high titer antiserum and has certain protective efficacy. Conclusion The fusion protein Stx2B IntiminC300 of EHEC O157∶H7 was successfully constructed and well expressed in prokaryotic expression system. After preliminarily purified, it has shown certain immunoprophylactic potential.