Purification And Characterization Of Outer Membrane Proteins P6 From Nontypeable Haemophilus Influenzae

Nontypeable Haemophilus influenzae (NTHi) is one kind of the paragenesis fungus , which often settles down in the nasopharynx and the latter mouth swallows of the upper respiratory tract and is highly reaching 80% healthy children as well as 40% healthy adults swallows to have NTHi. NTHi is the human some disease's important cause. They first reside temporarily in the nasopharynx department, then arrives at the middle ear through the eustachian tube, and also infects sinusitis and/or the throat, as well as causes the respiratory tract to infect repeatedly, and most often causes tympanitis and the sinusitis inflammation. NTHi has been the babies, infants and the child tympanitis's main disease germ, which causes tympanitis approximately 1/4～1/3 of all tympanitises. The serology research and the antibiotic curative effect investigation prompt that NTHi is the substantial cause of the lower respiratory tract infections together with chronic blocking lungs disease (COPD). Recently some researches prompt that Haemophilus influenzae is also the leading cause of the AIDS patient bacterial pneumonia. In the past ten years, the morbidity of the newborn pyemia NTHi caused went up, and it might cause 50% patient death, especially in the premature infant the mortality reached highly 90%. Therefore, no matter tympanitis happening or the treatment expense, it is a national major healthy problem. Following the antibiotic resistivity of these microphyte enhancement, the treatment is facing a biggish challenge. Therefore, developing one kind of secure and effective vaccine to prevent NTHi infection already appears especially important. Because this microphyte does not have the capsule polysaccharide, therefore the focus is seeking one kind of the NTHi outer membrane proteins as the foundation vaccine. The outer membrane of nontypeable H.influenzae contains five major proteins: P1 (47,000Da),P2 (39,000Da),P4 (30,000Da),P5 (27,000Da),andP6 (16,600Da).P1, P2, P4, and P5 are targets for bactericidal antibodies. All of these outer membrane proteins are targets for bactericidal antibodies.P1 , P2, and P5 are heterogeneous,while P4 and P6 are antigenically stable and highly conserved.Therefore,Regardless of its immunogenicity or the conservation,P6 protein should receive serious consideration for inclusion in vaccine to prevent infection caused by nontyeable H. influenzae.This experiment distilled the P6 according to its characteristic, which namely in buffer B (0.1M Tris-0.5M NaCl-1% SDS-0.1% 2-mercaptoethanol, pH 8.0), the pellet was incubated at 37℃for 30min, and it was the only insoluble protein.To solubilize P6,the pellet were resuspended in buffer A (0.01 M Tris-0.15 M NaCl , pH 7.4 ) and incubated at 65℃for 30min. The first, cell envelopes were obtained after ultrasonic disruption of the cells by differential centrifugation.The membrane preparation was incubated at 37℃for 30min in buffer B, and after high speed centrifugation ,. The insoluble residu was treated more than three times.To solubilize P6,the pellet was suspended in buffer A and incubated at 65℃for 30min,then 100,000×g for 60 min at room temperature .Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein demonstrated a single band.Image analysis software AlphaDigiDocTM showed that its purity was 100%, molecular weight was 16.73 kDa.IEF-PAGE electrophoresis and AlphaEaseFC software analysis showed that the isoelectric point of the P6 protein was 5.49.The production rate was the 86.6ug/g wet microphyte.NIH mice were immunized by the subcutaneous injection of different dosage and injections.P6-specific immunoglobulin G(IgG) antibody titers in serum were determined by an enzyme-linked immunosorbent assay (ELISA).The results showed thatrdifferent doses of immunization could induce IgG antibody and titers could be up to 1:10240,and P6 protein had a good immunogenicity. We selected the best conditions to immune mice,then attacked by NTHi SSI p/225 strain,and observed protective effect of active immunization.The results showed that:mice attacked by 20LD50,the experimental group survival rate was 80 percent,while the control group survival rate was negative .It showed that P6 protein on the adult mouse had a certain extent of the protection.