Haemophilus parasuis (H. parasuis) is associated with meningitis, polyserositis, polyarthritis and bacterial pneumonia. At present, its prevention and control is difficult because of the lack of suitable subunit vaccines. Nowadays, high-throughput methods, immunoproteomics, are available to screen for more vaccine candidates. A protein extraction method for H. parasuis and two-dimensional electrophoresis (2-DE) were optimized to provide high-resolution profiles covering pH 3 to 10. Twenty immunoreactive spots were excised from gels after strict comparison between 2-DE Western blot membranes and the relevant gels. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-TOF-MS successfully identified 16 different proteins. Fifteen of them were reported as immunoreactive proteins in H. parasuis for the first time. In addition, recombinant HP5-7 (ABC transporter, periplasmic-binding protein) showed immunoreactivity both with hyperimmune rabbit serum and convalescent swine serum. Four recombinants of the 14 successfully expressed genes showed immunoreactivity with hyperimmune rabbit serum.
- MeSH
- 2D gelová elektroforéza MeSH
- bakteriální proteiny chemie genetika imunologie MeSH
- Haemophilus parasuis chemie klasifikace genetika imunologie MeSH
- hemofilové infekce imunologie mikrobiologie veterinární MeSH
- hmotnostní spektrometrie MeSH
- králíci MeSH
- molekulární sekvence - údaje MeSH
- nemoci prasat imunologie mikrobiologie MeSH
- prasata MeSH
- proteomika MeSH
- protilátky bakteriální analýza imunologie MeSH
- séroskupina MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Čína MeSH
The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer's guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5 × 10(3) CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture.
- MeSH
- bakteriologické techniky ekonomika metody MeSH
- barvení a značení metody MeSH
- časové faktory MeSH
- digoxigenin metabolismus MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- faktory virulence genetika MeSH
- hybridizace nukleových kyselin metody MeSH
- infekce vyvolané Escherichia coli mikrobiologie veterinární MeSH
- kur domácí MeSH
- lidé MeSH
- membránové proteiny genetika MeSH
- oligonukleotidové sondy genetika MeSH
- přenašečství mikrobiologie veterinární MeSH
- proteiny z Escherichia coli genetika MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
