A Study On The Mechanism Of Type Ⅰ Allergy At The Level Of Cell And Molecule And The Protein Repelling Inflammatory

Objective: To research the mechanism of immune pathology of type I allergy at the level of cell and molecule. To obtain the cDNA encoding human recombinant phospholipase D2 (rhPLD2) from human daudi cell , which was an NH2 -terminal PLD2 deletion mutant expressed in E.coli; and to investigate the activities of the protein in vive or vitro.Methods: 1) Used transmission electron microscope observing the process of mast cell degranulation induced by anti-IgE-HRP, and the mast cells have stained by ELISA and immunofluorescence. 2) Daudi cells was cultured in Hepes-buffered DMEM that was supplemented with 10% (v/v) fetal bovine serum, 100 units/mL of penicillin and 100 mg/mL of streptomycin, at 37℃ in a humidified, CO2 controlled (5%) incubator. At culture dishes at l*1012 cells/dish., the Daudi cells were deprived of serum by centrifuged at 500 x g for 5 min. 3) we performed reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers that were designed according to the Genbank database. 4) A cDNA encoding hPLD2 was engineered by PCRand introduced into a pET30a(+) vector for expression with a 5' 6X His tag. The recombinant protein was purified and electrophoresed in SDS-polyacrylamide gelelectrophoresis gels in order to identify its molecule weight and purity. The guantity of rhPLD2 was detected with BCA Protein Assay Reagent Kit. The supernatant was subjected to Western blot with 6X His monoclonal antibody albumin free and PLD ( carboxy terminus ) goat polyclonal IgG; respectively. 5) On the other hand, the supernatant was used for assay of PLD activity adopted Amplex Red Phospholipase D Assay Kit. 6) We researched the hypersusceptibility and toxicity of rhPLD2 with guinea pigs, and studied the behave of the model of immediate hypersensitivity animals and the change of the level of eosinophil comparing between the before and after used drugs. 7)To investigate the effect of rhPLD2 meddled in the expression of the genes encoding IL-1B in the murine microglial cell line N9 which was stimulated with LPS.Results: 1) Observed and photographed the kinetic process of mast cell specific degranulation . 2) Set up the new criterior to supervise and diagnosis and treat the asthma. 3) Obtained a recombinant protein which without membrane band site and signal peptide from human Daudi cells .The cDNA sequence can encode a protein with 631 aa of rhPLD2 (accession numbers: AY 178289) . 4) constructed the recombinant expression plasmid pET30a-rhPLD2,and obtained successfully the production of membrane protein from the precipitate of inclusion bodies in E.coli. Expression of this cDNA clone in E. coli shows a functional PLD2 activity similar to that of the natural PLD2. As we predicted that the activity of expressed rhPLD2 was estimated to be 50.9745 raU/mL ( 0.9212mg/mL ) . 5) Founded successfully a animal model of asthma. The principium results showed injected 0.4537 mg/mL or 1.815mg/mL rhPLD2 to animals abdomen which have no hypersusceptibility and toxicity. 6) The protein of rhPLD2 can resist the asthma outbreak. 7) To approve primarily rhPLD2 can inhibit the expression of inflammatory cytokines such as IL-1B in the murine microglial cell line N9 which was stimulated with LPS.Conclusion: 1) The track of mast cell specific degranulation is the membranous aisle in which granule was released . After that, the membranous hole closed and cytoplasma became cellular clearly . 2) We found that only specific sensitized mast cells was stained orange-brown colour. ConclusionThis method can be used to forecast the development of type I allergy, such as: asthma etc. and to conduct some tests related to the degranulated mast cells. 3) The mutation protein of rhPLD2 is a functional protein which possess some biologic and immune activities. 4) A preferable activity of the mutation protein of rhPLD2 can be expressed in E.coli. 5) Themutation protein of rhPLD2 plays an important role which is against to inflammation.