Prokaryotic expression of Tir gene of enterohemorrhagic escherichia coli / 实用医学杂志

Objective To identify the immune activity of the recombinant protein Preli minarily after expressing Tir C-ter minal of enterohemorrhagic Escherichia coli(EHEC) in E.coli BL21/DE3 efficiently. Methods Tir C-ter minal (marked as Tir-C)was amplified by PCR considering the result of Bioinformatics analysis of Tir. The recombinant plasmid(designed as PET-30a(+)-Tir-C)was identified by PCR,sequencing and digested by restriction endonucleases. The positive recombinants were transformed into E.coli BL21/DE3 and induced by IPTG to express the Tir-C. The Tir-C protein was detected by SDS-PAGE and identify the antigenic of the recombinant protein Preli minarily by Western blot. Results The 675bp DNA was gained. The plasmid PET-30a (+)-Tir-C was built. Tir-C was expressed mainly in supernatant of lysis and was purified by Ni+affinity chromatograghy. Concentration of the purified protein is about 500 μg/mL and a unique band was detected with the relative molecular mass of approximately 24KDa by Western blot. Conclusion The recombinant Tir-C was expressed successfully and had immunoreactivity to some extent, which deserves the investigations for vaccine against EHEC.

The combination characteristics of EHEC O157 ∶H7 intimin and it's mutant intiminN916Y with translocated intimin receptor / 中华微生物学和免疫学杂志

Objective To analyze the combination characteristics between Tir-IBD( intimin binding domain) and its ligand intimin or mutant intiminN916Y of EHEC O157 ∶H7.Methods The gene of TirIBD (tir-ibd) from EHEC O157 ∶H7 chromosome was cloned into pMD18-T vector.Thereafter,the amplified gene was cloned into prokaryotic expression plasmid pET-21 a (+).The recombinant pasmid pET-21 a( +)-tir-ibd was transformed into E.coli BL21 (DE3).After inducement,the protein Tir-IBD was successfully expressed and analyzed with SDS-PAGE and Western blot.It was purified by affinity chromatography and ionexchange chromatography and was coupled on the Ni-NTA chip of BIACore 3000.Then the ligand intimin and mutant intiminN916Y were flow through the chip and their combination characteristics were detected.Results In the present study,the gene of Tir-IBD(tir-ibd) was successfully cloned into pET-21a(+).The results of SDS-PAGE and Western blot assay showed that the protein was successfully expressed,which accounts for 15％ of total expression products,and its molecular weight was about 10×103.The purity was above 95％ after purification.After coupled on the Ni-NTA chip of BIACore 3000,their combination characteristics with ligand intimin and mutant intiminN916Y were successfully detected.The equilibrium binding constants Ka was obtained by fitting the data with the BIACore evaluation program ( Version 4.1 ).The result showed that the combination characteristics between Tir-IBD and intimin have some difference compared with that of mutant intiminN916Y and the difference is temperature dependent.Conclusion Tir-IBD of EHEC O157 ∶H7 was successfully constructed and purified.The method to analyze the combination characteristics between Tir-IBD and its ligand intimin or mutant was established.The combination characteristics between Tir-IBD and intimin or mutant intiminN916Y have some temperature dependent difference and the mutated amino acids residue is crucial for their receptor-ligand binding.

Avaliação da interação de Escherichia coli enterohemorrágica (EHEC) pertencente ao sorotipo O157: H7 isoladas de bovinos assintomáticos e de doença humana com células enterocíticas humanas (linhagem Caco-2) / Evaluation of interaction of Enterohaemorrhagic (EHEC) 0157: H7 isolated from asymptomatic cattle and human disease with human enterocitic cells

Reconhecida como agente de doença humana em 1982, E.coli enterohemorrágica (EHEC) pode causar diarréia sanguinolenta, colite hemorrágica e síndrome hemolítica urêmica (SHU). EHEC constitui um subgrupo especialmente virulento das E.coli produtoras de toxina de Shiga (Stx). O fator crítico da sua virulência é a toxina Shiga, capaz de interromper a síntese proteica da célula eucariótica. São conhecidos dois subgrupos de Stx, Stx1 e Stx2. Stx1 possui duas variantes Stx1c e Stx1d. Stx2 possui muitas variantes. Estudos epidemiológicos sugerem que cepas com os perfis toxigênicos Stx2 ou Stx2/Stx2c seriam mais frequentemente associadas a pacientes com SHU. Além da expressão de Stx, EHEC do sorotipo O157:H7 colonizam a mucosa intestinal induzindo a formação de lesões denominadas ‘attaching/effacing’ (A/E). Para a produção da lesão A/E, é necessária a presença de uma ilha de patogenicidade cromossômica denominada LEE, composta por cinco operons, LEE 1 a LEE5. Em LEE 5 são codificadas a adesina intimina e o seu receptor Tir, o qual é translocado por um sistema de secreção tipo III (SSTT) e em LEE 4 são codificadas as proteínas secretadas EspA,B e D. Em EHEC O157:H7 são descritos muitos fatores de virulência, codificados em ilhas de patogenicidade, no cromossomo e no megaplasmídio pO157. Bovinos são o principal reservatório deste patógeno e alimentos de origem bovina e produtos contaminados com fezes de bovinos são causadores de surtos epidêmicos. Em nosso país EHEC O157:H7 é isolada do reservatório animal mas é muito rara a sua ocorrência em doença humana. Notamos que nas cepas bovinas predomina Stx2c, enquanto nas cepas humanas predomina o perfil toxigenico Stx2/Stx2c...

Recognized in 1982 as a human pathogen, enterohemorrhagic Escherichia coli (EHEC) causes bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). EHEC belonging to serotype O157:H7 are mostly important in North America, United Kingdom and Japan. Shiga toxin (Stx) is the critical factor of STEC. Stx is capable to interrupt the protein synthesis of the eukaryotic cell. Two subgroups of Stx are known, Stx1 and Stx2. Two variants of Stx1 are known (Stx1c and Stx1d), but several Stx2 variants have been described. Epidemiological studies suggest that STEC/EHEC strains carrying the toxigenic profiles Stx2 or Stx2/Stx2c are more frequently associated to HUS. Besides the expression of Stx, EHEC O157:H7 colonize the intestinal mucosa inducing the formation of characteristic histopathological lesions denominated “attaching/effacing” (A/E). To the production of A/E lesions, it is necessary the presence of a pathogenicity island called LEE (locus of enterocyte effacement), composed by five operons, LEE 1 to LEE5. An outer membrane adhesin (intimin) and its receptor Tir, which is translocated by a type three secretion sytem (TTSS), are both codified in LEE5 while the secreted proteins EspA, B and D, that constitute part of the SSTT, are codified in LEE4. Cattle are the main reservoir of this pathogen and foods of bovine origin and products contamined with bovine feces are common causes of epidemic outbreaks. In Brazil, EHEC O157:H7 can be isolated from the animal reservoir . Stx2c prevails among the bovine strains, while the toxigenic profiles Stx2 or Stx2/Stx2c are found among the human strains...

Expression, purification and effect of different immunization approaches of Tir from EHEC O157:H7 / 中华微生物学和免疫学杂志

Objective To clone and express translocation intimin receptor(Tir)of enterohemorrhagic Escherichia coli(EHEC)O157:H7,and to analyze the effect of different routes on the induction of immunity to the recombinant protein.Methods The tir eucoding genes were amplified from EHEC O157:H7 strain guangzhou 246 genome,and genes were cloned into the vector pET-30a(+).The pET-30a(+)tir recombinant was transformed into E.coli BL21.and expression was induced bv IPTG.The expressed product was analyzed by SDS-PAGE and purified by Ni-IDA affinity chromatography.The immunized mice sera and fecal against the recombinant protein was detected.Resuits The length of the tir is 1677 bp,with the initiation codon ATG and the termination codon TAA.Double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid pET-30a(+)-tir was constructed.The recombinant protein was expressed in Escherichia coli expression system,and was purified by Ni-IDA affinity chromatography.The mice were able to produce a high serum IgG antibody titer after both subeutaneous and intranasal immunizations.Meanwhile,the intranasal immunization induced serum and fecal IgA antibody titer was significantly higher than that of the subcutaneous immunization group.Conclusion Tir molecule is potential vaccine candidate for preventing EHEC disease.

Recombinant expression and biological activities of fusion protein EspA-Stx2B from enterohemorrhagic E. coil O157:H7 / 中华微生物学和免疫学杂志

Objective To clone the gene encoding protein of EspA and Stx2B from EHEC OI57:H7 by DNA recombinant technology, construct prokaryotic expression vector pET-28a ( + )-espAstx2B, express fusion protein of EspA-Stx2B and to analyze the biological and immunological characteristics of the fusion protein. Methods the sequence encoding the protein of EspA and Stx2B was amplified by PCR from the enterohemorrhagic Escherichia coli strain. The amplified products were connected with linker by recombinant technology and cloned into pET-28a( + ) vector. The vector was then transferred to the host cells E. Coli BL21 strain (DE3). Following, the protein expression was induced by IPTG. The expression quantities and style of fusion protein was then determined by SDS-PAGE. Its immunoreactivity was analyzed by Western blot. Finally, BALB/c mice were injected with the preliminarily purified recombination protein EspA-Stx2B, then oral challenged these mice with EHEC O157-SMR2 and counteracted toxic substances with O157 ultrasonic supernatant. Results The determination of the sequence encoding of the espA-stx2B fusion gene has 100% of consistency with the sequence from GenBank Sakai strain and contrivable linker. This fusion protein EspA-Stx2B was expressed as inclusion body formation and the percentage is approximately 40%. Western blot suggested the fusion protein has excellent immunoreactivity. Titer of antiserum of the mice to EspA-Stx2B increased evidently. EspA-Stx2B could not decrease bacterial number attached to the intestinal tract of mice based on fecal shedding of Oi57 in mice. In the test of death of BAI,B/c causing by conteracting toxic substances with O157 ultrasonic supernatant, immunoprotection of EspA-Stx2B rate was 66.7%. Conclusion A recombinant plasmid that has high performance on expression of EspA-Stx2B prorein was successfully constructed in present study, and the fusion protein has excellent immunoreactivity and immunogenicity. EspA-Stx2B could not decrease bacteria] number attached to the intestinal tract of mice based on fecal shedding of O157 in mice, but evidently decrease the mortality rate of the mice. The antiEspA and anti-Stx2B had immunoprotection effect by different means. These results may provide the foundation for the further development on EHEC O157:H7 double subunit vaccine.

Cloning and expression of Escherichia coli O157∶H7 Stx2B subunit / 医学研究生学报

Objective: To clone,identify and express the Shigalike toxin 2B(Stx2B) subunit gene of EHEC O157∶H7.Methods: A pair of primers were designed based on the Stx2B subunit gene sequence of EHEC O157∶H7.The Stx2B gene was amplified from the EHEC O157∶H7 chromosome by PCR and cloned into the pMD18-T vector.Thereafter,the gene was cut from the pMD18-T vector and cloned into the prokaryotic expression plasmid pET-28a vector.Then the recombinant plasmids were transformed into E. coli BL21(DE3) and the transformed host strain induced by IPTG.The expression protein was detected by SDS-PAGE and Western-blot analyses.Results: The Stx2B gene was successfully cloned into pMD18-T and pET-28a vectors,and the expressed protein identified by SDS-PAGE and Western blot.The molecular mass(Mr) of the expressed product was about 7 500,and the expression rate about 40%. Conclusion: The Stx2B gene was successfully cloned and effectively expressed in the prokaryotic expression system.