Neisseria meningitidis antigens and compositions
A composition and antibody technology, applied in the direction of drug combinations, antibodies, microorganisms, etc., can solve the problems of incompleteness and inability to overcome antigenic variation.
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Embodiment 1
[0390] Using the procedure described above, the following oligonucleotide primers were employed in a polymerase chain reaction (PCR) assay to clone the indicated ORFs:
[0391] Table 1 is used for the oligonucleotide in the PCR of embodiment 2-10
[0392]
[0393] Positioning of ORFs
[0394]The following DNA and amino acid sequences are qualified with headings of the form: [g, m, or a][#].[seq or pep], where "g" indicates that the sequence is from Neisseria gonorrhoeae and "m" indicates that the sequence is from meninges Neisseria B, and "a" indicates that the sequence is from N. meningitidis A; "#" indicates the sequence number; "seq" indicates the DNA sequence, and "pep" indicates the amino acid sequence. For example, "g001.seq" refers to the N. gonorrhoeae DNA sequence numbered 1. When the suffix "-1" is present for these sequences, it indicates additional sequences found for the same ORF, therefore, the ORF data for sequence nomenclature without and with suffix apply...
Embodiment 2
[0637] Expression of ORF 919
[0638] The primers described in Table 1 for ORF 919 were used to map and clone ORF 919. The predicted gene 919 was cloned in pET vector and expressed in E. coli. Protein expression products and purified products were analyzed by SDS-PAGE. In panel A) the analysis of the purification of the 919-His fusion protein is shown. Mice were immunized with purified 919-His, and serum was used for Western blot analysis (Panel B), FACS (Panel C), bactericidal test (Panel D) and ELISA analysis (Panel E). Symbols: M1, molecular weight marker; PP, purified protein; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the location of the major recombinant protein product (A) and N. meningitidis immunogen-reactive bands (B). These experiments confirmed that 919 is a surface-exposed protein and a useful immunogen. Hydrophobicity map, antigenic index, and amphipathic region of ORF 919 in Figure...
Embodiment 3
[0640] Expression of ORF 279
[0641]The primers for ORF 279 described in Table 1 were used to map and clone ORF 279. The predicted gene 279 was cloned in pGex vector and expressed in E. coli. Protein expression products and purified products were analyzed by SDS-PAGE. In panel A) the analysis of the purification of the 279-GST fusion protein is shown. Mice were immunized with purified 279-GST, and serum was used for Western blot analysis (Panel B), FACS (Panel C), bactericidal test (Panel D) and ELISA analysis (Panel E). Symbols: M1, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle (vesicle) preparation. Arrows indicate the location of the major recombinant protein product (A) and N. meningitidis immunogen-reactive bands (B). These experiments confirmed that 279 is a surface-exposed protein and a useful immunogen. Hydrophobicity map, antigenic index, and amphipathic region of ORF 279 in Figure 11 availabl...
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