Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1995-06-07
2001-08-07
Saidha, Tekchend (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S198000, C435S252300, C435S252330, C435S320100, C536S023100, C536S023200, C536S023500, C530S350000
Reexamination Certificate
active
06270763
ABSTRACT:
1. FIELD OF THE INVENTION
2. BACKGROUND OF THE INVENTION
2.1. Biochemical Aspects of Insect Venom Phospholipase
2.2. T and B Cell Epitopes of Phospholipase
2.3. Modulation of T and B Cell Responses
3. SUMMARY OF THE INVENTION
3.1. Abbreviations
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Isolation of a Vespid Venom Phospholipase Gene
5.2. Expression of a Polypeptide Comprising a Vespid Venom Phospholipase or Fragment Thereof
5.3. Identification and Purification of the Expressed Polypeptide
5.4. Derivatives and Analogs of Vespid Venom Phospholipase
5.5. Assays with Recombinant Vespid Venom Phospholipase or Fragments Derivatives or Analogs Thereof
5.6. Therapeutic and Diagnostic uses of the Vespid Venom Phospholipase or Fragments Derivatives or Analogs Thereof
6. EXAMPLE: VESPID VENOM PHOSPHOLIPASE A
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6.1. Materials and Methods
6.1.1. Isolation and Characterization of Dol m I and its CNBr Peptides
6.1.2. Dol m I-specific cDNA
6.1.3. Phospholipase and Lipase Assays
6.2. Results
6.2.1. Partial Amino Acid Sequence of Dol m I
6.2.2. cDNA Sequence of Dol m I
6.2.3. Lipase Activity of Hornet Phospholipase
6.3. Discussion
7. YELLOW JACKET PHOSPHOLIPASE A
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8. DEPOSIT OF MICROORGANISMS
1. FIELD OF THE INVENTION
The present invention is directed to nucleic acids encoding a vespid venom allergen phospholipase, or fragments thereof, recombinant vectors comprising such nucleic acids, and host cells containing the recombinant vectors. The invention is further directed to expression of such nucleic acids to produce recombinant vespid venom phospholipase or recombinant fragments thereof. Such a phospholipase allergen and fragments thereof are useful for diagnosis of allergy and for therapeutic treatment of allergy.
2. BACKGROUND OF THE INVENTION
2.1. Biochemical Aspects of Insect Venom Allergens
Insect sting allergy to bees and vespids is of common occurrence. The vespids include hornets, yellowjackets and wasps (Golden, et al., 1989, Am. Med. Assoc. 262:240). Susceptible people can be sensitized on exposure to minute amounts of venom proteins as less than 10 &mgr;g of proteins is injected into the skin on a single sting by a vespid (Hoffman and Jackson, 1984, Ann. Allergy. 52:276).
There are many species of hornets (genus Dolichovespula), yellowjackets (genus Vespula) and wasp (genus Polistes) in North America (Akre, et al., 1980, “Yellowjackets of America North of Mexico,” Agriculture Handbook No. 552, US Department of Agriculture). The vespids have similar venom compositions (King, et al., 1978, Biochemistry 17:5165; King, et al., 1983, Mol. Immunol. 20:297; King, et al., 1984, Arch. Biochem. Biophys. 230:1; King, et al., 1985, J. Allergy and Clin. Immunol. 75:621; King, 1987, J. Allergy Clin. Immunol. 79:113; Hoffman, 1985, J. Allergy and Clin. Immunol. 75:611). Their venom each contains three major venom allergens, allergens A
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(37 kd), hyaluronidase (43 kd) and antigen 5 (23 kd) of as yet unknown biologic function.
In addition to the insect venom allergens described above, the complete amino acid sequence of several major allergens from different grass (Perez, et al., 1990, J. Biol. Chem. 265:16210; Ansari, et al., 1989, Biochemistry 26:8665; Silvanovich, et al., 1991, J. Biol. Chem. 266:1204), tree pollen (Breiteneder, 1989, EMBO J. 8:1935; Valenta, et al., 1991, Science, 253:557), weed pollen (Rafnar, et al., 1991, J. Biol. Chem. 266:1229; Griffith, et al., 1991, Int. Arch. Allergy Appl. Immunol. 96:296), mites (Chua, et al., 1988, J. Exp. Med. 167:175), cat dander (Griffith, et al., 1992, Gene. 113:263), and mold (Aruda, et al., 1990, J. Exp. Med. 172:1529; Han, et al., 1991, J. Allergy Clin. Immunol. 87:327) have been reported in the past few years. These major allergens are proteins of 10-40 kd and they have widely different biological functions. Nearly all allergens of known sequences have a varying extent of sequence similarity with other proteins in our environment.
2.2. T and B Cell Epitopes of Allergens
Antibody responses to proteins require the collaboration of T helper and B lymphocytes and antigen presenting cells (APC). The antigen receptors of B cells are the membrane-bound antibody (Ab) molecules, which recognize and bind immunogens directly. The antigen receptors of T cells (TCR) only recognize and bind complexes of antigenic peptide-MHC class II molecule. Immunogens are first processed by APC into peptides that are presented on the surface of APC in association with the MHC class II molecules (Unanue, 1992, Current opinion in Immunol 4:63). As MHC molecules are highly polymorphic in individuals, they have different specificity of binding antigenic peptides (Rothbard and Gefter, 1991, Ann. Rev. Immunol. 9:527). This is one mechanism for genetic control of immune response.
T helper cells are activated when the antigen receptor binds the peptide-MHC complex on the surface of APC. Activated T cells secrete lymphokines. In mice (Street and Mosmann, 1991, FASEB J. 5:171) and apparently in humans (Wierenga, et al., 1990, J. Immunol. 144:4651; Parronchi, et al., 1991, Proc. Natl. Acad. Sci. USA. 88:4538) the T helper cells can be divided into different types on the basis of their patterns of lymphokine production. Primarily, T helper cells divide into two groups: TH1 cells producing IL-2 and IFN-&ggr;, and TH2 cells producing IL-4 and IL-5. These lymphokines in turn influence the antigen-activated B cells to differentiate and proliferate into plasma cells secreting Abs of different isotypes. IL-4 is one lymphokine known to influence IgE synthesis (Finkelman, et al., 1990, Ann. Rev. Immunol. 8:303).
It is believed that the entire accessible surface of a protein molecule can be recognized as epitopes by the antigen receptors of B cells, although all epitopes are not necessarily recognized with equal likelihood (Benjamin, et al., 1984, Ann. Rev. Immunol. 2:67). B cell epitopes of a protein are of two types: topographic and linear. The topographic type consists of amino acid residues which are spatially adjacent but may or may not be sequential adjacent. The linear type consists of only sequentially adjacent residues. X-ray crystallographic data of Ag-Ab complex indicate the size of their complementary binding region to have 16-17 amino acid residues (Amit, et al., 1986, Science 233:747) but peptide mapping suggests that less than about 8 residues contribute significantly to the binding process of a linear epitope (Appel, et al., 1990, J. Immunol. 144:976). Allergens, like other protein antigens, can have both types of B cell epitopes or only one. For example, vespid antigen 5s have both types and bee venom melittin appears to have only one B cell epitope of linear type (King, et al., 1984, J. Immunol. 133:2668).
T cell epitopes of proteins consist of only the linear type since they are peptides that have been processed in the lysosomes of APC by proteases of unknown specificity (Unanue, 1992, Curr. Op. Immunol. 4:63). Analysis of naturally processed antigenic peptides bound to MHC class II molecules indicates that their size range from about 13 to 17 amino acid residues, but analysis of synthetic peptide-MHC class II molecule complex for their T cell proliferate response suggests a minimal size of about 8 amino acid residues (Cf. Rudensky et al., 1991, Nature 353:622). Studies suggest that T cell epitopes are distributed throughout the entire protein molecule, and they may function as major or minor determinants depending on the MHC haplotype of the immunized host (Roy, et al., Science 244:572; Gammon, et al., 1987, Immunol. Rev. 98:53; O'Hehir et al., Ann. Rev. Immunol. 9:67).
Hypersensitivity of the immediate type is known to be caused by the presence of allergen-specific IgE. IgE is found in the circulation and bound to specific IgE-Fc receptors on mast cells and basophils. Cross-linking of cell-bound IgE by allergens leads to relese of histamine, leukotrienes and other chemical mediators that cause the allergic symptoms. IgE is one isotype of immunoglobulin. As pointed out above, lymphokines secreted by T cells influence isotyp
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