Immunoproteomic Approach for Screening Vaccine Candidates from Bacterial Outer Membrane Proteins
Outer membrane proteins (OMPs) are unique to Gram-negative bacteria and have been revealed as potential vaccine candidates for conferring protection against infections in recent years. Immunoproteomics is a powerful technique that is ideally suited to scr
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Introduction Gram-negative bacteria have two types of outer membrane proteins (OMPs), lipoproteins, which anchor to the inner leaflet of the outer membrane, and integral OMPs. Integral OMPs are unique to Gram-negative bacteria and adopt a β-barrel architecture with their external sequences exposed to the extracellular environment [1]. These features instill OMPs with essential physiological and virulence functions [2], and OMPs have also been suggested as potential vaccine candidates for conferring protection against infection [3]. The external short sequences of OMPs might act as epitopes to induce specific antibody responses and are thus promising candidates for the development of vaccines. For this reason, in recent years, research has focused on the determination of the immunogenic characteristics of diverse OMPs of diverse bacterial species. For example, in V. parahaemolyticus, a model marine bacterium, a few OMPs, such as OmpW and LamB, have been found to be immunogenic and to provide immune protection [4, 5]. These potential vaccine candidates were mainly identified one at a time. For rapid and comprehensive characterization of new vaccine candidates, immunoproteomics, the combination of proteomic technologies and immunological methods, has been proposed [6, 7], and it is now considered to be a powerful technique suitable for the screening and identification of potential vaccine antigens. This chapter outlines the procedure of screening and determination of potential vaccine candidates from OMPs in V. parahaemolyticus using an immunoproteomic strategy.
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_34, © Springer Science+Business Media New York 2016
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Materials
2.1 Bacterial Strains and Plasmids
1. V. parahaemolyticus RIMD 2210633, Escherichia coli DH5α, and E. coli BL21 (DE3), pET-28a and pET-32a.
2.2
1. ICR mice with the average weight of 20 g (see Note 1).
Animals
2.3 Bacterial Culture Medium
1. Luria-Bertani (LB) liquid medium: (1 L) 1 % (w/v) tryptone, 0.5 % (w/v) yeast extract, and 1 % (w/v) NaCl. Weigh 10 g tryptone, 5 g of yeast extract, and 10 g NaCl, and transfer to a 1-L glass baker. Add 950 mL of deionized water to dissolve these materials. Mix the solution and adjust its pH to 7.0 with 1 N NaOH (~1 mL). Adjust the final volume of the solution to 1 L with deionized water. Sterilize the medium by autoclaving it for 20 min at 15 psi (1.05 kg/cm2) on the liquid cycle. If antibiotic is needed, allow the solution to cool to approximately 55 °C, and add ampicillin or kanamycin at a final concentration of 50 μg/mL. Store at room temperature or 4 °C. 2. LB solid medium: Prepare LB medium as above, but add 15 g/L agar before autoclaving. After autoclaving, cool to approximately 55 °C and add antibiotic (if needed). To prepare plates, pour medium into petri dishes. After hardening, invert and store at 4 °C. 3. High salt medium: LB medium c
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