HSP2 recombinant in BL21 (DE3) pLysS strain was expressed at high level, but had varied in variable conditions from 22 - 370C. After simple steps of processing, HSP2 in soluble form could be purified by ion-exchange chromatography through CM-sepharose on FPLC system
Escherichia coli ; proteins ; isolation & purification

Animals ; Cattle ; Chickens ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; isolation & purification ; pathogenicity ; Sheep ; Swine

Child ; China ; Diarrhea ; diagnosis ; microbiology ; Escherichia coli Infections ; diagnosis ; Humans ; Shiga-Toxigenic Escherichia coli ; isolation & purification

OBJECTIVETo understand the distribution of virulence gene and the epidemiological characteristics of diarrheagenic Escherichia(E.) coli (DEC) from diarrheal patients in Beijing.

METHODSStool specimens from diarrheal patients were cultured which were collected from the hospitals under sentinel surveillance program, during 2012-2013. DNA was examined by real-time PCR.

RESULTS253 out of 6 370 specimens were positive for DEC detection with the rate as 4.0%. A total number of 262 DEC strains were isolated. Two different pathotypes of DEC strains with mixed infection, were isolated from 9 specimens. Different pathotypes would show the following profiles: 42.8% for enteropathogenic E. coli (EPEC) including 42.0% atypical and 0.8% typical; 38.9% for enterotoxigenic E. coli (ETEC) including 24.8% st positive, 9.9% lt positive and 4.2% st and lt both positive;15.3% for enteroaggregative E. coli(EAEC);2.7% for enteroinvasive E. coli (EIEC); one strain STEC with serotype O26:K60. ETEC had obvious characteristics on age. All kinds of DEC were isolated throughout the year with seasonal fluctuation.

CONCLUSIONDEC isolates from diarrheal patients in Beijing were dominated by EPEC and ETEC, with atypical ones accounted for the majority of EPEC. One specimen was found under mixed infection. Pathotypes DEC were found to have different age and seasonal distributions.

China ; epidemiology ; Diarrhea ; microbiology ; Enteropathogenic Escherichia coli ; genetics ; isolation & purification ; Enterotoxigenic Escherichia coli ; genetics ; isolation & purification ; Epidemics ; Escherichia coli ; genetics ; isolation & purification ; Escherichia coli Infections ; epidemiology ; microbiology ; Genotype ; Humans ; Virulence

Escherichia coli ; classification ; genetics ; isolation & purification ; Escherichia coli Infections ; urine ; Humans ; Urinary Tract Infections ; microbiology ; urine ; Urine ; microbiology

China ; epidemiology ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; classification ; isolation & purification ; metabolism ; Humans ; Shiga Toxin 1 ; biosynthesis ; isolation & purification ; Shiga Toxin 2 ; biosynthesis ; isolation & purification ; Sorbitol

Annexin A5 ; biosynthesis ; isolation & purification ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo evaluate the detective efficacy of Chromogenic Coliform and Escherichia Coli Agar (CCEA).

METHODSA new chromogenic medium CCEA prepared by Huankai laboratory was used to compare with a classical medium of violet red bile agar (VRBA), and other two Chromogenic media Agar I and Agar II by detecting separately 11 reference strains, thirteen sterile samples with Coliform or E.coli and other four samples, and the accordant rates of detection were observed.

RESULTSCCEA had the good selectivity. To seven kinds of quality strains in the resultant analysis, CCEA with VRBA and Agar I had not shown salience difference (P > 0.05), and CCEA with Agar II had significant difference (P < 0.05). CCEA showed more advantages than the Agar II. To thirteen sterile samples with Coliform or E.coli in resultant analysis, CCEA with Agar I and Agar II had shown no significant difference (P > 0.05), while CCEA with VRBA had significant difference (P < 0.05). CCEA might be more advantageous than the VRBA. In analysis of the four actual samples of Coliform, CCEA with VRBA, Agar I and Agar II showed no significant difference (P > 0.05). The accordant rates were 90%, 71.88%, 86.25% and 81.25% respectively, showing CCEA > Agar I > Agar II > VRBA. To two actual samples of E.coli in the resultant analysis, the CCEA with Agar I and Agar II had not shown significant difference (P > 0.05). The accordant rates were 100% respectively.

CONCLUSIONSThe CCEA might be more advantageous than the VRBA, having the same efficacy as with Agar I and Agar II.

Bacteriological Techniques ; Culture Media ; Enterobacteriaceae ; isolation & purification ; Escherichia coli ; isolation & purification

OBJECTIVETo obtain highly purified intimin encoded by the eae gene and study its adhesion activity.

METHODSThe eae gene was amplified from enterohemorrhagic Escherichia coli O157:H7 (EHEC) chromosome by PCR and cloned into pMD19-T vector. The eae gene was cut from pMD19-T vector and subcloned into the prokaryotic expression plasmid pET28a(+), and expressed in E.coli BL21(DE3). The recombinant protein was purified with Ni(2+)-chelating affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purified intimin was detected by immunofluorescence staining to test its adhesion.

RESULTSThe 2805-bp eae gene fragment was obtained, and the recombinant expression plasmid pET28a(+)-eae was successfully expressed in E.coli BL21 (DE3). The molecular weight of the recombinant protein was 97 000. Purified recombinant intimin was recognized by rabbit anti-O157 antiserum, and bound to the surface of HEp-2 cells as revealed by immunofluorescence staining.

CONCLUSIONHighly purified and immunoreactive intimin has been successfully obtained, which can adhere to the surface of HEp-2 cells. The acquisition of recombinant intimin provides the basis for studying its interaction with the host receptors during EHEC O157:H7 infection.

Adhesins, Bacterial ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Animals ; Blotting, Western ; Cell Adhesion ; Cell Line ; Cloning, Molecular ; Escherichia coli ; genetics ; Escherichia coli O157 ; Escherichia coli Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Plasmids ; genetics

Tumor necrosis factor(TNF-alpha) plays an improtant role in the process of anti-infection and anti-cancer. It can both protect and make damage to the host. In order to find new way of inhibiting its host-damaging activity, An E. coli flagella displayed random peptide library was constructed and TNF-alpha antagonist peptides were screened using the peptide library. After 5 rounds of panning and DNA sequencing, six peptide sequences were obtained. Two of them(TBP2, TBP3) have the same sequence frame of V--N-WG. After primary comparation of TNF-alpha binding ability, four peptides were synthesised and purified with RP-HPLC. The activity of inhibiting TNF-alpha was detected with L929 cell and MTT method. The data show that TBP2 and TBP3 can inhibit 90% of TNF-alpha activity when TNF-alpha gives about 30% cell toxicity on L929. The two sequences have not been reported.
Escherichia coli ; genetics ; Peptide Library ; Peptides ; isolation & purification ; pharmacology ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors