To construct and express the fusion protein Stx2B-IntiminC300 of EHEC 0157 :H7, and to further investigate its immunoprophyiactic potential, the gene of Stx2B (stx2b) from EHEC 0157:H7 chromosome was cloned into pMD18...To construct and express the fusion protein Stx2B-IntiminC300 of EHEC 0157 :H7, and to further investigate its immunoprophyiactic potential, the gene of Stx2B (stx2b) from EHEC 0157:H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokaryotic expression plasmid pET-28a (+)-eaeC300, which was constructed previously. The recombinant pasmid pET-28a(+)-stx2b-eaeC300 was transformed into E. coli BL21(DE3). After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophyiactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with Al(OH)3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC 0157: H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-IntiminC300 was successfully cloned into pET-28a (+) vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25% of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-IntiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophyiactic potential.展开更多
文摘To construct and express the fusion protein Stx2B-IntiminC300 of EHEC 0157 :H7, and to further investigate its immunoprophyiactic potential, the gene of Stx2B (stx2b) from EHEC 0157:H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokaryotic expression plasmid pET-28a (+)-eaeC300, which was constructed previously. The recombinant pasmid pET-28a(+)-stx2b-eaeC300 was transformed into E. coli BL21(DE3). After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophyiactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with Al(OH)3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC 0157: H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-IntiminC300 was successfully cloned into pET-28a (+) vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25% of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-IntiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophyiactic potential.
