Twenty-four monoclonal antibodies were produced by immunizing BALB/c mice with Rickettsia tsutsugamushi Boryong strain and used for the analysis of antigenic characteristics of R.tsutsugamushi Boryong strain and antigenic heterogeneity of R.tsutsugamushi by indirect immunofluorescent(IF) test. R. tsutsugamushi Kato, Karp, Gilliam, TA686, TA716, TA763, TC586, TH1817, and Boryong were used for the analysis of antigenic heterogeneity of R.tsutsugamushi. Five monoclonal antibodies were reactive with 27-kDa protein, four monoclonal antibodies were reactive with 47-kDa protein, and eight monoclonal antibodies were reactive with 56-kDa protein of R.tsutsugamushi Boryong strain. The reactive protein of seven monoclonal antibodies could not be identified by immunoblotting method. All monoclonal antibodies to 27-kDa protein and three monoclonal antibodies to 47-kDa protein, and five monoclonal antibodies to 56-kDa protein were reactive with three to eight strains among nine strains of R. tsutsugamushi tested. One monoclonal antibody reactive to 47-kDa protein(KI18) and two monoclonal antibodies reactive to 56-kDa protein(KI36, and KI37) reacted with all the strains of R. tsutsugamushi tested. Strain-specific monoclonal antibody(KI58) could be found among antibodies which were reactive with 56-kDa protein. There was no strain which showed same reactivity pattern to these 24 monoclonal antibodies among nine strains. From this results, it could be concluded that Boryong strain is antigenically different from other strains of R.tsutsugamushi and antigenic heterogeneity of R.tsutsugamushi is due to the antigenic diversity of several proteins of R. tsutsugamushi including 56-kDa protein.
Animals ; Antibodies, Monoclonal/*immunology ; Antigens, Bacterial/*analysis ; Bacterial Proteins/analysis ; Mice ; Mice, Inbred BALB C ; Orientia tsutsugamushi/*immunology ; Species Specificity

Streptomyces coelicolor is the model species among streptomycetes. Until now, proteomic analyses of S. coelicolor have been conducted using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, few integral membrane proteins were identified due to the hydrophobic and low-abundance nature of these proteins. In this work, 154 possible inner membrane proteins from S. coelicolor were identified using high pH-proteinase K sample preparation method and multidimensional protein identification technology, among them 44 are integral membrane proteins containing at least one transmembrane domain, most peptides and their corresponding proteins were identified experimentally for the first time.
Bacterial Proteins ; analysis ; Cell Membrane ; chemistry ; Genome, Bacterial ; genetics ; Mass Spectrometry ; methods ; Membrane Proteins ; analysis ; Proteome ; analysis ; genetics ; Streptomyces coelicolor ; chemistry ; genetics

OBJECTIVETo explore the application of biosensor technology in the determination of endotoxin-neutralizing materials.

METHODSAfter mixing polymyxin B (PMB) with endotoxin in certain concentration, the neutralizing ratio of PMB to endotoxin was assessed by biosensor technique and limulus amebocyte lysate test respectively. The results from the two methods were compared.

RESULTSThe neutralizing ratio of PMB to endotoxin as assessed by biosensor technology was 0.35 microg to 1 ng, while that by dynamic turbidimetric and chromogenic limulus amebocyte lysate (LAL) technique was 0.5 mg to 1 ng and 1 mg to 1 ng, respectively. The results obtained by biotechnology were similar to that by biosensor technique.

CONCLUSIONBiosensor technology was an accurate, convenient and rapid method for the determination of potency of endotoxin-neutralizing materials.

Bacterial Proteins ; analysis ; Biosensing Techniques ; methods ; Endotoxins ; analysis ; Lipid A ; analysis ; Polymyxin B ; analysis ; Reproducibility of Results ; Sensitivity and Specificity

Bacterial Proteins ; analysis ; China ; Proteome ; analysis ; Proteomics ; Yersinia pestis ; chemistry ; genetics

OBJECTIVETo analyze the genetic differences of Orientia tsutsugamushi (Ot) Sta56 gene between Shandong isolates and other strains deposited in GenBank.

METHODSPCR and restriction fragment length polymorphism (RFLP) were used to amplify the complete sequence of Ot-Sta56 gene. RFLP profiles of Ot were predicted by a computer program according to their complete sequences of Ot-Sta56 gene. PCR amplicon from XDM2 strain was sequenced and analyzed by Clustal X (1.8) and PHYLIP software.

RESULTSThe complete sequences (about 1.6 kbp) of Ot-Sta56 gene were amplified from B16 strain (isolated from patients), FXS2 strain (isolated from A. agrarius) and XDM2 strain. Four species of restriction endonucleases (Hha I, Hinf I, Hae III, Pst I) were used to digest the PCR amplicons from the 3 isolates. When comparing with the RFLP profiles of prototype Ot, the RFLP profiles of PCR amplicons from the 3 isolates were similar to those of Japan Kawasaki strain, but were quite different from the international reference strains Gilliam, Karp, Kato. Results from DNA sequence analysis showed that the complete sequence of Ot-Sta56 gene homology to Japan Kawasaki strain of XDM2 strain was 97%, and deduced amino acid sequence was 92%.

CONCLUSIONData from the complete sequence of Sta56 gene indicated that the genotypes of Ot isolates in Shandong province were similar, but with distinction from the Kawasaki strain.

Amplified Fragment Length Polymorphism Analysis ; Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; Bacterial Typing Techniques ; classification ; DNA, Bacterial ; genetics ; Genes, Bacterial ; Membrane Proteins ; genetics ; Orientia tsutsugamushi ; genetics ; isolation & purification ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

OBJECTIVETo construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry.

METHODSMatrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques were used to identify 12 recombinant proteins (10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori). A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-ClinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FlexAnalysis softwares.

RESULTSModels of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities: RC=100%, mean CVA=98.7% (the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively) and PPV=95%. This model can be used to classify the bacteria and their recombinant, which only requires 3.7×103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks (9, 16, 9, and 14 for the four stages, respectively) was found in the various phase of recombinant bacteria.

CONCLUSIONMALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.

Bacterial Proteins ; analysis ; Cloning, Molecular ; Escherichia coli ; Mass Spectrometry ; Organisms, Genetically Modified ; Peptide Mapping ; Recombinant Proteins ; analysis

OBJECTIVEIn this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis (LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA (enzyme-linked immunosorbent assay) kit for LB.

METHODSSix recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic (ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models.

RESULTSTwo IgG (VlsE and OspC B.g) and two IgM (OspC B.g and OspC B.a) antigens were left by the logistic regression model screened. VlsE had the highest specificity for syphilis samples in the IgG test (87.7%, P<0.05). OspC B.g had the highest diagnostic value in the IgM test (AUC=0.871). Interactive effects between OspC B.a and Fla B.g could reduce the specificity of the ELISA.

CONCLUSIONThree recombinant antigens, OspC B.g, OspC B.a, and VlsE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research.

Antigens, Bacterial ; blood ; Bacterial Proteins ; analysis ; China ; Enzyme-Linked Immunosorbent Assay ; veterinary ; Lyme Disease ; diagnosis ; Recombinant Proteins ; analysis ; Sensitivity and Specificity ; Serologic Tests ; veterinary

OBJECTIVETo investigate the drug resistance of flavobacterium and its ability to produce BLA (beta-lactamases) and ESBLs (Extended-spectrum beta-lactamases).

METHODSThe production of BLA and ESBLs from 6 clinical isolated flavobacterium strains was determined by nitrocefin disc test and double-disc synergy method, respectively. The antibiotic susceptibilities of the strains were determined by Kirby-Bauer disc diffusion test and the agar dilution method and the MIC was assessed.

RESULTSAll the six flavobacteria were BLA-producing strains and more than 80% of them were ESBLs-producing, and they were highly resistant to beta-lactamase antibiotics (MIC 32 - 256 mg/L), but susceptible to fluoroquinolones and cephalosporin with beta-lactamase inhibitors (MIC 0.125 - 8 mg/L).

CONCLUSIONMost of the flavobacteria in nosocomial infections were beta-lactamase-producing and were highly resistant to beta-lactamase antibiotics. Fluoroquinolones and beta-lactamase antibiotics with lactamase inhibitors should be the first choice for the management of infection caused by flavobacterium.

Anti-Bacterial Agents ; pharmacology ; Drug Resistance, Bacterial ; drug effects ; Flavobacterium ; drug effects ; enzymology ; Humans ; Membrane Proteins ; analysis ; metabolism ; Microbial Sensitivity Tests ; Ribosomal Proteins ; analysis ; metabolism

OBJECTIVETo evaluate the biological characteristics of Campylobacter jejuni (CJ) cultured on different culture media and their expression abundance of outer membrane proteins (OMPs).

METHODSCJ was cultured on the improved Bull's medium yolk agar, improved Bull's blood agar or improved Bull's agar for 48 h. The biological characteristics of the bacteria, including the colony feature, morphology, motility, biochemistry, and results of indirect fluorescence test were observed and compared. OMP of the cultured CJ was extracted using 0.2 mol/L and glycine-hydrochloride buffered solution (pH 2.2) and identified by SDS-PAGE to compare the expression abundance of the OMPs with molecular weight of 28-31 kD.

RESULTSCJ exhibited typical biological characteristics with larger cell body and more rapid growth on improved Bull's medium yolk agar than those on improved Bull's blood agar and improved Bull's agar. The bacteria grown on improved Bull's medium yolk agar showed also greater expression abundance of the OMPs with molecule mass between 28 kD and 31 kD.

CONCLUSIONImproved Bull's medium yolk agar allows rapid growth of CJ with typical biological characteristics and enhanced expression of the OMPs with molecular weight of 28 -31 kD, and can be widely used in CJ subunit vaccine development, CJ epidemiological survey, CJ food safety examination, and CJ quarantine.

Bacterial Outer Membrane Proteins ; analysis ; metabolism ; Campylobacter jejuni ; growth & development ; metabolism ; Culture Media

OBJECTIVETo identify antigenic proteins secreted by Streptococcus suis (S. suis) type 2 strain SC84.

METHODSTwo-dimensional electrophoresis (2-DE), western-blot assay and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis were performed to search and identify antigenic proteins secreted by S. suis strain SC84, which triggered an outbreak of the disease in Sichuan province,China, in 2005.

RESULTSA total number of 14 western blot spots were found on PVDF membrane. 11 spots which could be found the existence of matching protein on coomassie G-250-stained 2-DE gel were identified by MALDI-TOF MS. The 11 proteins, all located at extra-cellular or cell wall, were classified into 8 kinds of proteins. Among of them, muramidase-released protein (MRP), suilysin (Sly) and extra-cellular factor (EF) were the known antigenic proteins, but several proteins such as putative 5'-nucleotidase, ribo-nucleases G and E, and predicted metal-loendo-peptidase were newly found antigenic proteins. All the identified protein were found to have had the coding gene in genomic of S. suis strain 05ZYH33, isolated from patients in Sichuan province, China in 2005.

CONCLUSIONThe newly found proteins could be used as voluntary antigens for detection and vaccination of S. suis.

Bacterial Proteins ; analysis ; immunology ; Humans ; Proteomics ; Streptococcal Infections ; Streptococcus suis ; immunology ; isolation & purification ; metabolism