To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into ...To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic 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 immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：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 immunoprophylactic potential.展开更多
Objective:This paper provided preliminary description of food contamination derived from Enterohemorrhagic Escherichia coli(EHEC)O104:H4 and EHEC O157:H7 in Wuhan in June 2011.Methods:47 food samples,including vegetab...Objective:This paper provided preliminary description of food contamination derived from Enterohemorrhagic Escherichia coli(EHEC)O104:H4 and EHEC O157:H7 in Wuhan in June 2011.Methods:47 food samples,including vegetables and meat,were subjectively sampled from some restaurants.PCR assays were used to detect EHEC O104:H4 and EHEC O157:H7.Results: The PCR results showed that none of the samples were positive for either EHEC O104:H4 or EHEC O157:H7.Conclusion:Although large outbreaks of gastroenteritis and the hemolytic uremic syndrome caused by EHEC O104:H4 had occurred in some European countries,China has had few outbreaks associated with EHEC O104:H4.This shows that food supply is relatively safe in China.Nevertheless,many ongoing problems of food safety in China are still not solved showing the necessity of further studies on food safety.展开更多
文摘To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic 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 immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：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 immunoprophylactic potential.
文摘Objective:This paper provided preliminary description of food contamination derived from Enterohemorrhagic Escherichia coli(EHEC)O104:H4 and EHEC O157:H7 in Wuhan in June 2011.Methods:47 food samples,including vegetables and meat,were subjectively sampled from some restaurants.PCR assays were used to detect EHEC O104:H4 and EHEC O157:H7.Results: The PCR results showed that none of the samples were positive for either EHEC O104:H4 or EHEC O157:H7.Conclusion:Although large outbreaks of gastroenteritis and the hemolytic uremic syndrome caused by EHEC O104:H4 had occurred in some European countries,China has had few outbreaks associated with EHEC O104:H4.This shows that food supply is relatively safe in China.Nevertheless,many ongoing problems of food safety in China are still not solved showing the necessity of further studies on food safety.
