Meningococcal vaccine development: characterization of lipoproteins Ag473 and NMB1946

• Main Authors: Li-Hsiu Lin, 林俐秀
• Other Authors: Chiou-Ying Yang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/fr3zac

碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 97 === Ag473 is a lipoprotein in Neisseria meningitidis which exist in outer membrane of different N. meningitidis species. The antibody generates from E. coli BL21（DE3） which expressed Ag473-3His do not cross reaction with human neuroblast, such finding make this lipoprotein a potential vaccine candidate. After trypsin digestion of lipoprotein Ag473-3His, lipopeptides were purified by reverse phase medium（POROS beads）. Matrix-assisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF）inditify the N-terminal lipopeptide of Ag473-3His have different fatty acid modification. According to molecular weight, lipopeptides have lipid moiety and will link with fatty acid, C16、C17、C18 or C19. LCMSD analysis of the trypsin digests show there were two groups, di-acyl and tri-acyl, lipid modified peptides. Each group consists of different fatty acid（C16 to C19）. Further fragmentation also confirmed that the N-terminal position of Cys（R3） is modified by the fatty acid, palmitic acid, and that on the diacylglyceryl position（R1 and R2）, one is palmitic acid and the other one is an unsaturated fatty acid of different carbon length（C16 to C19）, differing by CH2 group. Based on reverse vaccinology, protein sequence prediction in MC58 screen for other vaccine antigens. After construction, expression in Escherichia coli system and purification recombinant protein, N-terminal sequence and mass spectrometer analysis indicated NMB1946 is also a lipoprotein. Western blotting data show that antiserum NMB1946 generates from purified NMB1946 protein in mouse have effective titer. To confirm this antiserum specificity, 60 different N. meningitidis strains cell lysate will test with antiserum NMB1946 by western blotting technique, antiserum NMB1946 is specific to all of the N. meningitidis strains. Enzyme-linked immunosorbent assay（ELISA）and flow cytometry demonstrated NMB1946 is not an epitope in outer membrane surface. Mass spectrometer analysis show NMB1946 and Ag473-3His have the same lipid modification structure.