초록 ▼

Disclosed herein are methods for the chemiluminescent assay of a variety of analytes of interest. The methods are adaptable to the determination of microbial species in both liquid samples and on solid surfaces. The disclosed methods can be used with rapid, self-contained chemiluminescence assay dev

Disclosed herein are methods for the chemiluminescent assay of a variety of analytes of interest. The methods are adaptable to the determination of microbial species in both liquid samples and on solid surfaces. The disclosed methods can be used with rapid, self-contained chemiluminescence assay devices, or can be used with novel sampling devices and conventional microbial analysis techniques involving growth of microbial samples on appropriate culturing media. The specificity of the methods can be enhanced with the use of immunospecific reagents. The sensitivity of the technique can be increased by 3 to 6 orders of magnitude by first converting all DNA in the sample to inorganic phosphates before generating the emission signal. The breadth of applicability of the disclosed methods can be enhanced through the selection of appropriate enzyme-catalyzed reactions where one of the products of enzymatic oxidation is hydrogen peroxide.

대표청구항 ▼

Disclosed herein are methods for the chemiluminescent assay of a variety of analytes of interest. The methods are adaptable to the determination of microbial species in both liquid samples and on solid surfaces. The disclosed methods can be used with rapid, self-contained chemiluminescence assay dev

Disclosed herein are methods for the chemiluminescent assay of a variety of analytes of interest. The methods are adaptable to the determination of microbial species in both liquid samples and on solid surfaces. The disclosed methods can be used with rapid, self-contained chemiluminescence assay devices, or can be used with novel sampling devices and conventional microbial analysis techniques involving growth of microbial samples on appropriate culturing media. The specificity of the methods can be enhanced with the use of immunospecific reagents. The sensitivity of the technique can be increased by 3 to 6 orders of magnitude by first converting all DNA in the sample to inorganic phosphates before generating the emission signal. The breadth of applicability of the disclosed methods can be enhanced through the selection of appropriate enzyme-catalyzed reactions where one of the products of enzymatic oxidation is hydrogen peroxide. like polynucleotide of claim 6; and a non-native promoter linked to said polynucleotide in a way to control the expression of said polynucleotide. 12. A vector comprising the M protein gene-like polynucleotide of claim 6. 13. A cell comprising the M protein gene-like polynucleotide of claim 6. 14. A method for inhibiting transport of RNA, proteins or RNA-protein complexes between nucleus and cytoplasm of a cell comprising the step of exposing the cell to sufficient quantity of a VSV M protein-like polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1 and the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, such that the transport of RNA, proteins or RNA-protein complexes between nucleus and cytoplasm of the cell is inhibited. 15. The method of claim 14, wherein the amino acid sequence is at least 45% similar to amino acids 47-229 of SEQ ID NO:1. 16. The method of claim 14, wherein the amino acid sequence is at least 50% similar to amino acids 47-229 of SEQ ID NO:1. 17. The method of claim 14, wherein the amino acid sequence is at least 60% similar to amino acids 47-229 of SEQ ID NO:1. 18. The method of claim 14, wherein the amino acid sequence is at least 80% similar to amino acids 47-229 of SEQ ID NO:1. 19. The method of claim 14, wherein the amino acid sequence further contains tryptophan and an aromatic residue at positions corresponding to positions 91 and 105 of SEQ ID NO:1. 20. The method of claim 19, wherein the aromatic residue is selected from tyrosine and phenylalanine. 21. The method of claim 20, wherein the VSV M protein-like polypeptide is a vesiculovirus M protein. 22. The method of claim 21, wherein the vesiculovirus is selected from chandipura virus (CV), spring viremia of carp virus (SVCV), and piry virus (PV). 23. The method of claim 22, wherein the vesiculovirus is CV. 24. The method of claim 14, wherein exposing a cell to a sufficient quantity of a VSV M protein-like polypeptide is achieved by synthesizing the VSV M protein-like polypeptide inside the cell. 25. The method of claim 14, wherein the VSV M protein-like polypeptide is obtained by extraction from the polypeptide's native virus or a host cell expressing the polypeptide. 26. A method for inhibiting transport of RNA, proteins or RNA-protein complexes between nucleus and cytoplasm of a cancer cell comprising the step of exposing the cancer cell to sufficient quantity of a VSV M protein-like polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1 and the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, such that the transport of RNA, proteins or RNA-protein complexes between nucleus and cytoplasm of the cancer cell is inhibited. 27. A method for inhibiting import of proteins or RNA-protein complexes from cytoplasm into nucleus of a cell comprising the step of exposing the cell to sufficient quantity of a VSV M protein-like polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1 and the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, such that the import of proteins or RNA-protein complexes from cytoplasm into nucleus of the cell is inhibited. 28. A method for inhibiting export of nucleic acids from nucleus to cytoplasm of a cell comprising the step of exposing the cell to sufficient quantity of a VSV M protein-like polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1 and the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, such that the export of nucleic acids from nucleus to cytoplasm of the cell is inhibited. 29. A method for inhi biting transport of RNA, proteins or RNA-protein complexes between nucleus and cytoplasm of a cell comprising the step of: analyzing an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1 to determine a smaller fragment that retains the ability to inhibit nucleocytoplasmic transport, wherein fragments of the amino acid sequence are compared to determine which segments of the amino acid sequence can be deleted without loss of transport inhibition function; and exposing a cell to sufficient quantity of a VSV M protein-like polypeptide which comprises the smaller fragment such that transport of RNA, proteins or RNA-protein complexes across the nuclear envelope is inhibited. 30. A method for screening for an agent that can alter the activity of an M protein comprising the steps of: introducing into the nucleus of a cell a VSV M protein-like polypeptide comprising an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, and wherein the polypeptide inhibits the transport of a molecule between the nucleus and cytoplasm of the cell and the molecule is selected from a RNA, a protein and a RNA-protein complex; exposing the cell to a test agent; and determining the nucleocytoplasmic transport rate of the molecule before and after exposing the cell to the agent. 31. A method for identifying a nuclear export element comprising the steps of: exposing a cell to sufficient quantity of an M protein-like polypeptide which comprises an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, so that export of RNA between the nucleus and cytoplasm is inhibited; and selecting an RNA molecule that is exported in the presence of the M protein-like polypeptide and examining the molecule for the presence of a nuclear export element. 32. A method for identifying a nuclear import element comprising the steps of: exposing a cell to sufficient quantity of an M protein-like polypeptide which comprises an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, so that import of proteins from cytoplasm into the nucleus is inhibited; and selecting a protein molecule that is imported in the presence of the M protein-like polypeptide and examining the imported molecule for the presence of a polypeptide that can function as a nuclear export element when attached to another protein. 33. An isolated polypeptide consisting of an amino acid sequence selected from amino acids 1-57 of VSV M protein and amino acids 23-57 of VSV M protein. 34. An isolated polynucleotide consisting of a nucleotide sequence that encodes the polypeptide of claim 33. 35. A chimeric protein comprising a non-M protein polypeptide and a polypeptide having an amino acid sequence selected from amino acids 1-229 of VSV M protein, amino acids 47-229 of VSV M protein, amino acids 1-57 of VSV M protein, and amino acids 23-57 of VSV M protein. 36. An isolated nucleic acid comprising a nucleotide sequence that encodes the chimeric protein of claim 35. 37. A method for introducing a non-M protein polypeptide into the nucleus of a cell comprising the step of linking the non-M protein polypeptide to a second polypeptide having an amino acid sequence selected from amino acids 1-229 of VSV M protein, amino acids 47-229 of VSV M protein, amino acids 1-57 of VSV M protein, and amino acids 23-57 of VSV M protein such that the non-M protein polypeptide can enter into the nucleus of a cell along with the second polypeptide.