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|標題:||Biochemical characterizations of Escherichia coli-expressed protective antigen Ag473 of Neisseria meningitides group B||作者:||Sung, J.W.C.
|關鍵字:||Lipopeptides;Lipoproteins;N-Acyl-S-diacylglycerylcysteine;Mass;spectroscopy;Reverse-phase high pressure liquid chromatography;(RP-HPLC);Matrix-assisted laser desorption ionization time of flight;(MALDI-TOF);Electrospray ionization (ESI);Tandem gas chromatography;and mass spectroscopy (GC-MS);Circular dichroism (CD);nf-kappa-b;fatty-acid;lipid modification;lipoprotein;vaccine;protein;tlr2||Project:||Vaccine||期刊/報告no：:||Vaccine, Volume 28, Issue 51, Page(s) 8175-8182.||摘要:||
Polysaccharide-based vaccines against Neisseria meningitidis (Nm) serogroups A,C,Y and W135 have been available since 1970, but similar vaccine candidates developed for Nm group B (NmB) have not been successful due to both poor immunogenicity and their potential immunological cross-reactivity with human neurological tissue. In previous reports, a protective antigen and vaccine candidate, Ag473, was identified using proteomics and NmB-specific bactericidal monoclonal antibody. To initiate human phase one clinical trials, antigen production and characterization, pre-clinical toxicology and animal studies are required. In the present study, we report the biochemical characterization of Escherichia coli-expressed recombinant Ag473 (rAg473). Using MALDI-TOF mass analysis, chromatographically purified rAg473 was found to have two major isoforms that have molecular masses of 11,306 and 11,544 amu, respectively. The isoforms were separated using RP-HPLC and pooled into two fractions. Based on the chromatogram, the ratio of lipoproteins in fractions #1 and #2 was found to be 1-2. GC-MS analysis of lipoproteins was performed, and the acylated fatty acids were identified. The results indicated that the first lipoproteins in fraction #1 contained the lipids palmitic acid (C16:0), cyclopropaneoctanoic acid (C17:1) and, predominately, stearic acid (C18:0). A different lipid composition of cyclopropaneoctanoic acid (C17:1), oleic acid (C18:1) and, predominately, palmitic acid (C16:0) was found in the second lipoprotein fraction. Both lipoprotein isoforms were tested and found to have Toll-like receptor (TLR) agonist activity in stimulating cytokine secretion from THP-1 cells. Circular dichroism (CD) analysis showed the secondary structure of rAg473 to be dominated by alpha-helices (48%), and the overall protein structure was stable up to 60 degrees C and could refold after having been exposed to a temperature cycle from 20 to 90 degrees C. In addition, the solubility of rAg473 (5 mg/mL) was not affected after several freeze-thaw cycles. These biophysical and immunological properties make rAg473 a good vaccine candidate against NmB. (c) 2010 Elsevier Ltd. All rights reserved.
|Appears in Collections:||分子生物學研究所|
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