| Biochemical characterizations of Escherichia coli-expressed protective antigen Ag473 of Neisseria meningitides group B. | |
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MedLine Citation:
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PMID: 20937316 Owner: NLM Status: In-Process |
Abstract/OtherAbstract:
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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 11,544amu, 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 α-helices (48%), and the overall protein structure was stable up to 60°C and could refold after having been exposed to a temperature cycle from 20 to 90°C. In addition, the solubility of rAg473 (5mg/mL) was not affected after several freeze-thaw cycles. These biophysical and immunological properties make rAg473 a good vaccine candidate against NmB. |
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Authors:
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Jerry Wang-Chou Sung; Shih-Yang Hsieh; Chang-Ling Lin; Chih-Hsiang Leng; Shih-Jen Liu; Ai-Hsiang Chou; Li-Wei Lai; Li-Hsiu Lin; Yan Kwok; Chiou-Ying Yang; Pele Chong |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-10-23 |
Journal Detail:
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Title: Vaccine Volume: 28 ISSN: 1873-2518 ISO Abbreviation: Vaccine Publication Date: 2010 Nov |
Date Detail:
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Created Date: 2010-12-03 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8406899 Medline TA: Vaccine Country: Netherlands |
Other Details:
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Languages: eng Pagination: 8175-82 Citation Subset: IM |
Copyright Information:
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Copyright © 2010 Elsevier Ltd. All rights reserved. |
Affiliation:
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Vaccine Research and Development Center, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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