Screening method
View Patent ↗The invention relates to methods for the identification of novel receptors and/or novel ligands. The invention is based on the concept that parasites have developed a number of biologically active compounds in their saliva to cope with the defense mechanisms of the host organisms on which they feed and to manipulate the biological functions of host molecules to their advantage. These saliva compounds can be used to identify novel molecules within the host systems. Furthermore, these saliva compounds can be used to elucidate the functions of molecules whose existence is already known.
1. A method for identifying a receptor or ligand with a biological function in a mammalian host organism, said method comprising the steps of:
(a) providing a sample of biological material of a mammalian host organism that is suspected of containing the receptor or ligand, wherein said biological material is a heterogenous protein preparation of a biological fluid, wherein said biological fluid is blood, lymph or cerebrospinal fluid, or a protein expression library, wherein said protein expression library is a wound site library, a lambda wound site expression library or a phage display library;
(b) bringing said sample of biological material from a mammalian host organism that is suspected of containing the receptor or ligand into contact with a preparation of saliva or salivary glands of a tick, wherein said preparation comprises a plurality of proteins and peptides and said plurality of proteins and peptides is immobilized to a support or tagged to aid in isolation of complexes formed by tick salivary gland proteins or peptides and mammalian receptors or ligands; and
(c) isolating from the heterogenous protein preparation, one or more mammalian receptors or ligands that bind to a parasite salivary protein or peptide of the preparation.
2. A method according to claim 1 , wherein said sample of mammalian host biological material is isolated from blood.
3. A method according to claim 1 , wherein said sample of mammalian host biological material comprises one or more proteins isolated from the host organism.
4. A method according to claim 3 , wherein said one or more proteins isolated from the host organism are produced by recombinant means.
5. A method according to claim 4 , wherein said one or more recombinant proteins are generated by expression from a host gene cloned under the control of a heterologous promoter.
6. A method according to claim 5 , wherein said one or more recombinant proteins are the products of a gene expression library.
7. A method according to claim 1 , wherein said preparation of saliva is a preparation of salivary extract.
8. A method according to claim 7 , wherein said preparation of salivary extract is a fraction of a salivary extract.
9. A method according to claim 1 , wherein said sample of mammalian host biological material is brought into contact with an expression library derived from the salivary gland of at least one tick species.
10. A method according to claim 1 , wherein said method includes a step of affinity purification, antigen panning, plasmon resonance detection, or comprises a competitive binding assay or yeast two hybrid system.
11. A method according to claim 10 wherein either said preparation of the saliva or salivary glands of a tick or the biological material from the mammalian host organism is tagged to aid the isolation of the host receptor or ligand bound to said parasite salivary compound.
12. A method according to claim 11 wherein said preparation of the saliva or salivary glands of a tick is bound to a support.
13. The method according to claim 12 wherein said support is a resin.
14. A method for identifying a receptor or ligand with a biological function in a mammalian host organism, said method comprising the steps of:
(a) providing a sample of biological material of a mammalian host organism that is suspected of containing the receptor or ligand, wherein said biological material is a heterogenous protein preparation of a biological fluid, wherein said biological fluid is blood, lymph or cerebrospinal fluid, or a protein expression library, wherein said protein expression library is a wound site library, a lambda wound site expression library or a phage display library;
(b) bringing said sample of biological material from a mammalian host organism that is suspected of containing the receptor or ligand into contact with a preparation of saliva or salivary glands of at least one tick species, wherein said preparation comprises a plurality of proteins and peptides and said plurality of proteins and peptides is immobilized to a support or tagged to aid in isolation of complexes formed by tick salivary gland proteins or peptides and mammalian receptors or ligands; and
(c) isolating from the heterogenous protein preparation, one or more mammalian receptors or ligands that bind to a parasite salivary protein or peptide of the preparation.