國家衛生研究院 NHRI:Item 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:	[Chih-Hsiang Leng] Periodical Articles [Shih-Jen Liu] Periodical Articles [Pele Choi-Sing Chong] Periodical Articles [Wang-Chou Sung] Periodical Articles

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