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http://ir.nhri.org.tw/handle/3990099045/5209
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| Title: | Biochemical characterizations of Escherichia coli-expressed protective antigen Ag473 of Neisseria meningitides group B |
| Authors: | Sung, JWC;Hsieh, SY;Lin, CL;Leng, CH;Liu, SJ;Chou, AH;Lai, LW;Lin, LH;Kwok, Y;Yang, CY;Chong, P |
| Contributors: | Vaccine Research and Development Center |
| Abstract: | 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 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 (5 mg/mL) was not affected after several freeze-thaw cycles. These biophysical and immunological properties make rAg473 a good vaccine candidate against NmB. |
| Date: | 2010-11 |
| Relation: | Vaccine. 2010 Nov;28(51):8175-8182. |
| Link to: | http://dx.doi.org/10.1016/j.vaccine.2010.09.091 |
| JIF/Ranking 2023: | http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0264-410X&DestApp=IC2JCR |
| Cited Times(WOS): | https://www.webofscience.com/wos/woscc/full-record/WOS:000286288700019 |
| Cited Times(Scopus): | http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=78649655552 |
| Appears in Collections: | [冷治湘] 期刊論文
[劉士任] 期刊論文
[莊再成] 期刊論文
[宋旺洲] 期刊論文
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| SCP77958025236.pdf | | 883Kb | Adobe PDF | 932 | View/Open |
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