Purified nontypable Haemophilus influenzae P5 protein as a vaccine for nontypable Haemophilus influenzae infection
a technology of nontypable haemophilus influenzae and p5 protein, which is applied in the direction of peptides, sense disorders, drug compositions, etc., can solve the problem that the antibodies of capsules cannot be effective at preventing nthi infections
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example 1
Purification of P5
Protein extracts of P5 are obtained from both NTHi and Hib strains by differential detergent extraction of outer membranes. Following Zwittergent octylglucoside 3-14 and Zwittergent 3-14 and 0.5M NaCl extractions the P5 protein is solubilized with 1% sarcosyl in 50 mM Tris-HCl, pH8, 5 mM Na.sub.2 EDTA. The extracts are adjusted to 1% Zwittergent.TM. 3-14 in the same buffer and dialyzed against 10 fold excess of 1% Zwittergent.TM. 3-14 in 50 mM Tris-HCl, pH8, 5 mM Na.sub.2 EDTA (3.times.) over 24 hours. The dialyzed extract is then passed through an anion exchange (DEAE) column and a cation exchange (S) column connected in tandem. The columns are separated and the S column is eluted with an increasing salt gradient in the same Zwittergent containing buffer. The purified P5 protein is eluted as a single peak as analyzed by SDS-PAGE (FIG. 1).
example 2
Protease Digestion, Peptide Isolation, and Protein Sequencing
Purified NTHi P5 and Hib P5 are used directly to determine the amino acid sequence .[.(FIG. 2).]. .Iadd.(FIGS. 2A and 2B).Iaddend. (SEQ ID NO:1). Total sequence of the NTHi Protein (SEQ ID NO:1) is determined by obtaining overlapping peptides by protease digestion with several proteases including endopeptidases Lys-C. Arg-C, Glu-C, papain, trypsin, and chymotrypsin. Cyanogen bromide also is used to cleave the protein, at methionine residues to create large fragments. The peptides are isolated using a microbore High Performance Liquid Chromatograph (HPLC) and the sequences determined with a protein seqencor. Alignment of the peptides utilizes overlapping peptides and homology to the E. coli OmpA protein .[.(FIG. 2).]. .Iadd.(FIGS. 2A and 2B).Iaddend..
example 3
Whole formalin fixed Haemophilus Influenzae cells from several different strains are prepared from mid-log phase cells grown to OD.sub.490 =1.0 in BHI-XV media. Cells are washed twice in Phosphate Buffered Saline (PBS) (7 mM NaHPO4-7H20, 2 mM KH2PO4, 2 mM CK1, 137 mM NaCl pH7.4) then resuspended in PBS with 0.3% formalin and incubated at room temperature for 1-2 hours. Formalin is removed by washing cells in PBS and resuspending cells in PBS to a final concentration OD.sub.620 =0.2. Seventy-five microliters of cells are then added to the wells of microtiter plates and dried overnight at 37.degree. C. Plates are blocked with PBS-0.1% Tween-20 for one hour and washed in a microplate washer. One Hundred microliters of antiserum diluted in phosphate buffered saline containing 0.15 mM CaCl.sub.2, 0.5 mM MgCl.sub.2, 1% gelatin and 0.3% Tween-20 (PCM-GT) are added to the wells and plates are incubated at 37.degree. C. for two hours. After washing, bound antibodies wer...
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