CN101294162B - 用于犬和人埃利希病的免疫诊断的p156抗原及其用途 - Google Patents
用于犬和人埃利希病的免疫诊断的p156抗原及其用途 Download PDFInfo
- Publication number
- CN101294162B CN101294162B CN2008100879600A CN200810087960A CN101294162B CN 101294162 B CN101294162 B CN 101294162B CN 2008100879600 A CN2008100879600 A CN 2008100879600A CN 200810087960 A CN200810087960 A CN 200810087960A CN 101294162 B CN101294162 B CN 101294162B
- Authority
- CN
- China
- Prior art keywords
- albumen
- ehrlichieae
- fragrance
- rich
- dog
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 208000000292 ehrlichiosis Diseases 0.000 title claims abstract description 22
- 239000000427 antigen Substances 0.000 title abstract description 18
- 108091007433 antigens Proteins 0.000 title abstract description 18
- 102000036639 antigens Human genes 0.000 title abstract description 18
- 241000282465 Canis Species 0.000 title abstract description 5
- 241000605312 Ehrlichia canis Species 0.000 claims abstract description 64
- 229940051998 ehrlichia canis Drugs 0.000 claims abstract description 63
- 241000605317 Anaplasmataceae Species 0.000 claims description 57
- 239000003205 fragrance Substances 0.000 claims description 50
- 210000002966 serum Anatomy 0.000 claims description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 14
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 13
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 13
- 210000004027 cell Anatomy 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 229960005486 vaccine Drugs 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 108010052285 Membrane Proteins Proteins 0.000 claims description 3
- 102000018697 Membrane Proteins Human genes 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 238000009396 hybridization Methods 0.000 claims description 3
- 241000238631 Hexapoda Species 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 108060001084 Luciferase Proteins 0.000 claims description 2
- 239000005089 Luciferase Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 2
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 2
- 210000004962 mammalian cell Anatomy 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims 1
- 239000012530 fluid Substances 0.000 claims 1
- 238000001890 transfection Methods 0.000 claims 1
- 235000013311 vegetables Nutrition 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 42
- 108090000288 Glycoproteins Proteins 0.000 abstract description 13
- 102000003886 Glycoproteins Human genes 0.000 abstract description 12
- 241000894007 species Species 0.000 abstract description 6
- 241000605310 Ehrlichia chaffeensis Species 0.000 abstract description 3
- 206010020429 Human ehrlichiosis Diseases 0.000 abstract description 2
- 229940031626 subunit vaccine Drugs 0.000 abstract description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 34
- 239000012634 fragment Substances 0.000 description 20
- 238000000034 method Methods 0.000 description 19
- 230000009182 swimming Effects 0.000 description 19
- 235000018102 proteins Nutrition 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 238000003745 diagnosis Methods 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 9
- 241000605314 Ehrlichia Species 0.000 description 8
- 108700026244 Open Reading Frames Proteins 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 7
- 150000001720 carbohydrates Chemical class 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 210000004898 n-terminal fragment Anatomy 0.000 description 6
- 230000001154 acute effect Effects 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 150000004676 glycans Chemical class 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 210000004900 c-terminal fragment Anatomy 0.000 description 4
- 230000037029 cross reaction Effects 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 230000000405 serological effect Effects 0.000 description 4
- 229940048102 triphosphoric acid Drugs 0.000 description 4
- 108090000604 Hydrolases Proteins 0.000 description 3
- 230000014107 chromosome localization Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000605282 Ehrlichia ewingii Species 0.000 description 2
- 108700005084 Multigene Family Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000009616 inductively coupled plasma Methods 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000008521 reorganization Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 235000010384 tocopherol Nutrition 0.000 description 2
- 235000019731 tricalcium phosphate Nutrition 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 208000030090 Acute Disease Diseases 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 241000605281 Anaplasma phagocytophilum Species 0.000 description 1
- 241000605280 Anaplasma platys Species 0.000 description 1
- 208000031504 Asymptomatic Infections Diseases 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 208000032420 Latent Infection Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 206010033661 Pancytopenia Diseases 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 241001123227 Saccharomyces pastorianus Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- SRBFZHDQGSBBOR-QMKXCQHVSA-N alpha-L-arabinopyranose Chemical compound O[C@H]1CO[C@@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-QMKXCQHVSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000003229 cytophilic effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- XQTWDDCIUJNLTR-CVHRZJFOSA-N doxycycline monohydrate Chemical compound O.O=C1C2=C(O)C=CC=C2[C@H](C)[C@@H]2C1=C(O)[C@]1(O)C(=O)C(C(N)=O)=C(O)[C@@H](N(C)C)[C@@H]1[C@H]2O XQTWDDCIUJNLTR-CVHRZJFOSA-N 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 208000001780 epistaxis Diseases 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 230000037230 mobility Effects 0.000 description 1
- 208000011309 nasal bleeding Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 101150053109 p28 gene Proteins 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
- Y10S530/825—Bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及用于犬和人埃利希病的免疫诊断的P153和P156抗原及其用途。具体为从犬埃利希氏体(p153基因)和恰菲埃利希氏体(p156基因)克隆了编码两种免疫反应性糖蛋白的序列。这两种糖蛋白是种特异性的免疫反应性直向同源物，可以用作犬埃利希氏体和恰菲埃利希氏体的亚单位疫苗和用于血清学和分子诊断。
Description
本发明专利申请是国际申请号为PCT/US2003/034916，国际申请日为2003年11月4日，进入中国国家阶段的申请号为“200380108238.6”，发明名称为“用于犬和人埃利希病的免疫诊断的P153和P156抗原及其用途”的发明专利申请的分案申请。
相关申请的交叉引用关系
本申请要求美国临时专利申请60/423573的权益，该申请提交于2001年11月4日，现已放弃。
联邦资助说明
本发明部分依靠联邦政府提供的资金，即国立过敏和传染病研究所(National Institute of Allergy and Infectious Diseases)基金编号：AI31431而完成。因此，联邦政府对于本发明享有一定权利。
发明背景
技术领域
本发明主要涉及分子和免疫诊断领域。更具体地，本发明涉及来源于犬埃利希氏体(Ehrlichia canis)和恰菲埃利希氏体(Ehrlichia chaffeensis)的种特异性免疫反应蛋白的直向同源物(～200kDA)，用于种特异性诊断犬埃利希病和人嗜单核细胞埃利希病。
相关技术的描述
犬单核细胞埃利希病是一种潜在的致死性蜱传播狗类疾病，该疾病主要是由立克次氏体(犬埃利希氏体(Huxsoll等人，1970))引起，在世界范围内广泛分布。犬埃利希氏体是一种专性细胞内细菌，表现出对单核细胞和巨嗜细胞的嗜性(Nyindo等人，1971)，能在脊椎动物宿主体内引起持续感染 (Harrus等人，1998)。该病的特征分三个阶段：持续2-4周的急性感染阶段；亚临床感染阶段，在此阶段狗可能维持感染好几年，但不显现出任何临床症状；此后是慢性阶段，在此阶段由于骨髓再生不良，很多狗的病况进行性恶化预后更差(Troy等人，1990)。
犬埃利希氏体感染犬科动物引起埃利希病。犬埃利希病包括急性阶段和慢性阶段。急性阶段的特征为发热、浆状的眼鼻排泄物、厌食、抑郁、和体重减轻。慢性阶段的特征除了更严重的急性病临床症状外，还有严重的全血细胞减少症、鼻出血、血尿、粪便带血。如果在疾病进程早期进行治疗，狗对于强力霉素的治疗反应良好。然而，慢性感染的狗对于该抗生素反应不佳。因此，早期诊断对于犬埃利希病的治疗尤为重要。
急性阶段治疗此疾病对其最佳预后很重要。血液学异常(诸如：白血球减少症和血小板减少症)常常可为犬埃利希病提供有用的证据，也是最初诊断的重要因素(Troy等人，1990)。然而，因为犬埃利希病的临床表现是非种特异性的，这给诊断带来了困难。
用血清学方法，如：间接荧光抗体(IFA)检测，来诊断犬埃利希病由于其简便、可靠和成本效应(cost effectiveness)低已经成为标准的方法(Troy等人，1990)。然而，间接荧光抗体检测的缺点包括：该方法不能做出种特异性诊断，因为它与也感染狗的其它密切相关的埃利希体属类(恰菲埃利希氏体，E.ewingii，Anaplasma phagocytophilum，以及A.platys)会发生抗原交叉反应。主观的解释也可能由于交叉反应性抗原而得到假阴性或假阳性结果。据报道，已开发的种特异性检测犬埃利希氏体的其它诊断方法，如聚合酶链式反应(PCR)，比细胞培养分离法更灵敏，但是这种方法需要专门训练和昂贵的仪器(McBride等人，1996)。病原体的分离很费时，仅有少数几家实验室一直成功地使用这一方法。而且，采用这一方法来确定具体的病因，还需要其它检测方法来鉴定该分离物。
已报道犬埃利希氏体和恰菲埃利希氏体之间存在血清学交叉反应性抗原。一些主要的血清学交叉反应性蛋白的分子量为28-30kDa(Chen等人，1997；Rikihisa等人，1994)，现在已知这些蛋白是由同源性多基因家族所编码(Ohashi等人，1998a，b)。在犬埃利希氏体和恰菲埃利希氏体中分别有22个和25个基因同源但不完全相同，而p28基因相同并进行了测序。在犬 埃利希氏体和恰菲埃利希氏体的P28蛋白之间观察到类似的种内和种间菌株的同源性，从而解释了这些蛋白所产生的血清学交叉反应性(McBride等人，1999)。
最近一项报道表明恰菲埃利希氏体的rP28蛋白在诊断人类嗜单核细胞埃利希病(HME)中不敏感(Yu等人，1999a)。其根本原因似乎是不同株的恰菲埃利希氏体中P28蛋白有变异性(Yu等人，1999b)。与之相反，分布于不同地域的犬埃利希氏体中鉴定到的P28基因却是保守的，而且证明犬埃利希氏体的rP28可用于犬埃利希病的诊断(McBride等人，1999；Ohashi 1998a)。已克隆了其它同源免疫反应性蛋白(包括犬埃利希氏体(gp140)和恰菲埃利希氏体的糖蛋白(gp120))(Yu等人，1997，2000)。恰菲埃利希氏体的rgp120的反应性与用于嗜人单核细胞埃利希病血清诊断的间接荧光抗体具有很好的相关性，而且采用犬埃利希氏体的rgp140的初步研究提示它可能是一种敏感可靠的免疫诊断抗原(Yu等人，1999a，2000)。
以往技术缺乏用于犬埃利希氏体和恰菲埃利希氏体的血清学和分子诊断的特异性抗原，以及缺少采用这些诊断抗原的方法。本发明填补了该领域长久已来的需求。
发明概述
已鉴定了犬埃利希氏体的一种强免疫反应性的43kD蛋白(p43)(美国专利号：6355777)。作为一种免疫诊断抗原，p43蛋白与间接荧光抗体检测相比具有96％的准确性，为犬埃利希氏体感染提供了种特异性的诊断方法。进一步的研究显示犬埃利希氏体的p43代表了一种推测分子量为153kD蛋白质的N末端部分，是报道的埃利希氏体属类中的最大的免疫反应性蛋白质。用蛋白凝胶电泳分析重组表达的p153蛋白片段，结果显示其分子量大于预测的分子量(～10至30％)，在N-和C-末端片段中存在聚糖(carbohydrateglycan)，表明p153是一种糖蛋白。
对可获得的恰菲埃利希氏体基因组序列(95％)进行了BLASTn搜索，在恰菲埃利希氏体中鉴定到编码p153蛋白直向同源物(orthologh)的基因。犬埃利希氏体的p153基因(4263bp)和恰菲埃利希氏体的p156基因(4389bp)具有相似的染色体定位，在同源性(～87％)脱氧鸟苷三磷酸三磷酸水解酶基因以及 开放阅读框之前同源性(～90％)的基因间序列的下游。在该糖蛋白基因之间观察到了核酸序列的同源性(50％)，这支持了先前有关p43基因片段遗传多样性的发现，而p153和p156蛋白的氨基酸序列相似性为32％。分子量为200kD的天然犬埃利希氏体蛋白能与用p153N-末端区域(p43)所产生的抗血清反应，提示该天然蛋白已经过翻译后修饰。类似地，包含恰菲埃利希氏体p156蛋白N-末端区的重组蛋白比预计的(～200kD)迁移更多，而且在该重组蛋白上还测到了碳水化合物。在这段N-末端片段上鉴定到有一个主要的免疫反应表位。染色体定位、氨基酸的同源性、以及生物物理特征都支持以下结论：即p153和p156糖蛋白(命名为gp200s)是种同源性免疫反应性直向同源物。
在犬埃利希氏体p153蛋白的N-(P43)和C-末端区域以及恰菲埃利希氏体p156直向同源物的N-末端区域中所鉴定出的主要免疫反应性表位，可用于血清学诊断和疫苗。而且，编码这些蛋白的基因是种特异性的，可用于分子诊断学的发展。
本发明的其它和更深入的方面、特征、和优点，将通过以下本发明提供的优选实施例描述得以显现。给出这些实施例的目的在于说明。
附图简要描述
可以获得并详细了解上述的本发明特征、优点和目的、以及其它一些显而易见的特征、优点和目的。参考附图中加以解释的某些实施例，可以对上面简要概括的本发明进行更具体的阐述。这些附图也是本说明书的一部分。然而，须注意，附图用于说明本发明的优选实施例，因而不认为是对于发明范围的限制。
图1A和1B显示恰菲埃利希氏体p156(最上面一行)和犬埃利希氏体p153(最下面一行)蛋白质直向同源物的Lipman-Pearson氨基酸序列排列对比。氨基酸的相同性，保守替换(：)和半保守替换(.)表示在中间。
图2A和2B显示犬埃利希氏体p153(A)和恰菲埃利希氏体(B)重组蛋白片段用抗V5抗体检测得到的表达情况。犬埃利希氏体p153，泳道1；N-末端片段(1107bp，nt-1-1107)，泳道2，内部片段(910bp，nt1080-1990)；泳道3，内部片段(1000bp，nt-1950-2950)；和泳道4，C-末端片段(1280bp，nt-2940-4220)。恰菲埃利希氏体p156，泳道1，N-末端片段(1545bp， nt-125-1675)；泳道2，内部片段(1365bp，nt-1685-3050)；和泳道3，C-末端(1365bp，nt-2950-4315)。
图3A显示犬埃利希氏体10p153重组片段的Western免疫印迹。泳道1，N-末端片段(1107bp，nt-1-1107)；泳道2，内部片段(910bp，nt1080-1990)；泳道3，内部片段(1000bp，nt-1950-2950)，和泳道4，C-末端片段(1280bp，nt-2940-4220)。
图3B显示在对应于犬埃利希氏体p153的纯化重组片段上检测到碳水化合物，该重组片段用pRSET表达载体在大肠杆菌内表达。将结合于该重组蛋白的聚糖进行氧化并用生物素标记后，用链霉亲和素-碱性磷酸酶检测。
图4A显示恰菲埃利希氏体的p156重组片段(泳道1-3)与人血清(左图)和狗血清(右图)的Western印迹图。泳道1，恰菲埃利希氏体p156的N-末端片段(1545bp，nt-125-1675)；泳道2，内部片段(1365bp，nt-1685-3050)；以及泳道3，C-末端(1.365bph nt-2950-4315)。所表达的重组蛋白代表约95％恰菲埃利希氏体p156蛋白。
图4B显示三种对应的重组恰菲埃利希氏体p156蛋白(泳道1-3)的碳水化合物的检测。
图5显示犬埃利希氏体全细胞裂解物中蛋白质与来自犬埃利希氏体感染狗的多克隆抗血清的Western印迹图(泳道1)，和与抗重组p43(gp200)多克隆兔血清的Western印迹图(泳道2)，及与抗重组gp140多克隆兔血清的Western印迹图(泳道3)。
本发明的具体描述
据先前报道，犬埃利希氏体p43基因序列是1173bp(美国专利：6355777)，但是进一步分析揭示DNA测序错误导致产生一个人造中止密码子，和截短的基因序列。采用引物-接头基因步移方法，在原有p43克隆中2.4kbp片段的下游又确定了另外一个4.5kbp序列。不完整的p43基因序列补全后，显示了一个4263bp的开放阅读框，其编码预计分子量为153kD的蛋白质(命名为p153)。p153基因的上游有一个编码脱氧鸟苷三磷酸三磷酸水解酶的开放阅读框，p153基因前还有一段基因间非编码区，它们在犬埃利希氏体和恰菲埃利希氏体之间具有很高的同源性(分别为87％和90％)。
对恰菲埃利希氏体基因组和2.4kbp的p43克隆进行BLASTn搜索鉴定到高度同源性的核苷酸序列。关于该上游同源性编码序列和基因间核苷酸序列，在相同的染色体位置上发现了与犬埃利希氏体p153大小几乎相等的一个大开放阅读框(4389bp)，该阅读框编码的蛋白质具有预计的分子量156kD(p156)。发现犬埃利希氏体p153和恰菲埃利希氏体p156基因之间具有核酸序列同源性(约50％)；然而，该蛋白质的氨基酸全序列的相似性仅为32％(图1)。
在大肠杆菌中表达的基因构建物，代表了恰菲埃利希氏体p156蛋白(nt-125-1670；nt-1685-3050；nt 2950-4315)和四个犬埃利希氏体p153蛋白的片段(nt-1-1107(p43)；nt-1080-1990；nt-1950-2950；nt-2940-4220)，这些构建物在大肠杆菌中进行表达。犬埃利希氏体N-末端(nt 1-1107)和C-末端(nt-2940-4220)重组表达蛋白表现出很强的免疫反应性(图3A)。然而，恰菲埃利希氏体p156只有N-末端片段(nt-125-1670)具有免疫反应性(图4A)。
犬埃利希氏体(nt-1-1107和nt-2940-4420)和恰菲埃利希氏体p156重组蛋白片段(nt-125-1607)在SDS-PAGE上的迁移率大于预计值，表明该片段发生过翻译后修饰。随后，在犬埃利希氏体p153和恰菲埃利希氏体p156肽片段上检测到碳水化合物(图3B和4B)。
抗-p43抗体能与犬埃利希氏体全细胞裂解物中的一个约200kD的天然蛋白反应。而且，该200kD的蛋白也可被犬埃利希氏体感染的狗血清所识别(图5)。先前鉴定为p43(p153的N-末端部分)的一部分基因序列在GenBank中被指定为登录号AF252298。修正后编码p153的序列在GenBank中被指定为登录号AY156950。
染色体定位、氨基酸同源性、和生物物理性质支持以下结论，即p153和p156糖蛋白(命名为gp200s)是种特异性的免疫反应性直向同源物。这些蛋白对疫苗的开发具有潜在的用途，也可用作灵敏可靠的血清诊断用抗原来诊断埃利希氏体属感染。先前的发现也支持这一点，该发现表明犬埃利希氏体p43具有免疫反应性，用作血清诊断性抗原(美国专利：6355777)。与抗p43抗体之间的反应和间接荧光抗体滴度(IFA)＞40的样品之间，具有100％的相关性，并能和间接荧光抗体滴度＜40的数个样品发生反应。几份间接荧光抗体阴性的样品能与p43抗体产生弱反应，这提示p43蛋白可能是一种更 为敏感的血清诊断性抗原。本发明所提供的结果表明p43是更大的犬埃利希氏体p153蛋白的一部分。
本发明涉及分离的编码犬埃利希氏体免疫反应性表面蛋白p153和恰菲埃利希氏体p156蛋白的多聚核苷酸。优选该分离的多聚核苷酸编码具有SEQID NO：1和2中所示氨基酸序列的蛋白质。此外，由于遗传密码的简并性，该DNA可以在核苷酸序列上有所不同。
本发明还包括含有这些分离的多聚核苷酸和在细胞中表达该DNA所必须的调节序列的载体；分离和纯化的p153和p156蛋白质；以及抗这些蛋白质的抗体。
本发明还涉及p153和p156蛋白在制备犬和人埃利希病疫苗中的用途。此外，还提供通过检测狗血清是否与该p153或p156蛋白反应来确定狗或人是否感染了埃利希氏体属类的方法。所用的蛋白可以是重组的，可用Western印迹分析来检测血清对这些蛋白质的反应。由于和先前分离的犬埃利希氏体p28蛋白的反应也是犬埃利希氏体感染的可靠标志，诊断可以包括检测对犬埃利希氏体p153蛋白、gp140蛋白、和p28抗原的免疫反应性。
本发明还涉及测定狗或人是否被埃利希氏体属类感染的血清学诊断试剂盒。该试剂盒包括本文所述的固定化蛋白(p153或p156)、合适的狗血清稀释缓冲液、连接有报告分子的抗狗血清第二抗体、和检测该报告分子的合适的试剂。可行的固定抗原的方法包括将抗原连到膜或者微量滴定板上。报告分子可以是荧光素酶、辣根过氧化酶、β-半乳糖苷酶、或荧光标记。
本发明还涉及测定狗是否被埃利希氏体属类感染的PCR扩增方法。提取潜在感染的狗或人的血液DNA，用犬埃利希氏体p153基因或恰菲埃利希氏体p156基因的特异性寡核苷酸引物进行PCR扩增。所得的PCR扩增产物用凝胶电泳等方法按大小分离，检测到适当大小的产物表明已感染了埃利希氏体属类。
本发明还涉及一套PCR检测p153和p156基因的试剂盒。该试剂盒包括提取血液DNA的试剂、p153或p156的特异性寡核苷酸、和用于PCR扩增的试剂。
依照本发明，可以采用本领域技术人员所用的常规分子生物学、微生物学、和重组DNA技术。文献中对这些技术的详细说明。参见，例如， Maniatis，Fritsch&Sambrook，“分子克隆：实验手册(1982)”；“DNA克隆：实用方法，”卷I和II(D.N.Glover编著1985)；“寡核苷酸合成”(M.J.Gait编著1984)；“核酸杂交”(B.D.Hames & S.J.Higgins等编著(1985))；“转录和翻译”(B.D.Hames & S.J.Higgins等编著(1984))；“动物细胞培养”(R.I.Freshney，编著(1986))；“固定化细胞和酶”(IRL PRESS，(1986))；“分子克隆实用指南”(1984)。
本文所用的术语“宿主”不仅包括原核细胞，还包括酵母、植物和动物细胞等真核细胞。可用编码本发明蛋白的重组DNA分子或基因以任何本领域普通技术人员熟知的任何技术转化宿主。原核宿主可以包括大肠杆菌、鼠沙门氏菌、粘质沙雷氏菌、和枯草芽孢杆菌。真核宿主包括酵母菌(如：毕赤巴斯德酵母菌)、哺乳动物细胞和昆虫细胞。
通常，把含有能促进插入的DNA片段的有效转录的启动子序列的表达载体和宿主结合使用。该表达载体通常含有复制启始区、启动子、中止子、以及能够提供转化细胞表型筛选的特定基因。可用本领域已知的方法发酵培养转化的宿主以达到最佳细胞生长。可以用本领域技术人员熟知的方法来构建含有合适的转录和翻译调控信号的表达载体。例如，参见Sambrook等人，1989，分子克隆：实验手册(第二版)(冷泉港出版社，纽约)中所述的技术。
本文所用的术语“引物”指一种寡核苷酸，可以是天然存在的(如存在于纯化的限制性消化片段中)或是人工合成的，该寡核苷酸能在合适的条件下(即可诱导引物延伸而合成与核酸链互补的片段)作为合成起始点。所述条件包括：存在核苷酸和诱导试剂(如：DNA聚合酶)以及适当的温度和pH。引物可以是单链或双链的，必须足够长而能在诱导试剂存在下引发所需的延伸产物的合成。引物的确切长度取决于很多因素，包括温度、引物来源和使用的方法。例如，应用于诊断时，根据靶序列的复杂性，寡核苷酸引物一般包含15-25个或更多个核苷酸。也可使用核苷酸数目较少的引物。
本文选用的引物与特定靶向DNA序列的不同链“基本”互补。这就意味着该引物必须足以与其各自的链互补杂交。因此，引物序列不需要反映模板的确切序列。例如，可将非互补性核苷酸片段结合于该引物的5’末端，而引物序列的其它部分可与该链互补。或者，可将非互补碱基或更长的序列插入该引物中，只要该引物序列与所述序列足够互补或能与其杂交，形成合成延 伸产物的模板。
给出以下实施例目的是说明本发明的各种实施方案，而不意味着以任何方式限制本发明。
实施例1
犬埃利希氏体p153和恰菲埃利希氏体p156蛋白质的特征鉴定
如前所述(McBride等人，2001；美国专利号：6355777)，从Lambda Zap II表达库中鉴定到的犬埃利希氏体p43蛋白基因。最初的2.4-kb克隆包含一个编码脱氧鸟苷酸-三磷酸三磷酸水解酶基因的开放阅读框(ORF)和下游截短的、位于p43基因片段之前的229bp的基因间序列。采用引物-接头PCR法以犬埃利希氏体的基因组DNA(Jake，北卡罗莱那株)作为模板，测定该p43开放阅读框的全长序列。扩增子直接以用于扩增的引物测序或克隆到TOPO/TA中进行序列分析。用犬埃利希氏体(E.canis)p43的全长克隆(2.4-kb)进行恰菲埃利希氏体基因组序列的BLASTn检索鉴定，得到恰菲埃利希氏体的直向同源物(p156基因)。
将犬埃利希氏体p153和恰菲埃利希氏体p156基因分成长片段(1至1.5kbp)，克隆到pUni/V5-His-TOPO Echo供者载体中，并与pBAD Thio-E或pRSET Echo受者表达载体重组。用阿拉伯糖或IPTG诱导后，重组蛋白表达4小时。膜标记后，用检测糖蛋白的免疫印迹试剂盒(Bio-Rad)对所表达的重组蛋白进行聚糖检测。在大肠杆菌中表达前面所述的恰菲埃利希氏体重组Dsb蛋白(McBride等人，2002)，作为对埃利希氏体进行糖蛋白检测研究时的阴性对照蛋白。将犬埃利希氏体全细胞裂解物用梯度胶(4-12％Bis-Tris，Novagen)进行凝胶电泳分离，用半干转移单元(Bio-Rad)(semidry transfer unit)转移至干净的硝酸纤维膜上。如前所述(McBride等人，2001)进行免疫印迹分析。
讨论
含有犬埃利希氏体p153N-末端(p43)部分的克隆具有很强的免疫反应性，使得该克隆得到最初鉴定并分析(McBride等人，2001)。与间接荧光抗体试验检测狗犬埃利希氏体抗体的结果相比，p43表现出优良的敏感性和特异 性。而且，由于抗重组p43多克隆抗体并不与恰菲埃利希氏体感染的DHS2细胞反应，因此p43似乎能够提供种特异性检测。在恰菲埃利希氏体中鉴定到p153的直向同源物(p156)，此直向同源物(也称为定向进化同源物)是基因多样性的表现，氨基酸同源性程度低，支持了先前的发现(即p43蛋白是种特异性抗原)，p43也因此可能是极好的种特异性免疫诊断性抗原。主要的线性B细胞抗原表位存在于p153蛋白的N-(p43)区域和C-末端区域。
P43重组蛋白所显示的分子量大于预期值(约30％或约10kD)，这一点是最初并未认识到的。先前报道的埃利希氏体的糖蛋白gp120和gp140比预期值大60％至100％。尽管(电泳时)分子量的漂移程度要小得多，但p43蛋白是一种糖蛋白，这在碳水化合物检测中得到了附着聚糖的证实。与p43蛋白的发现相一致的是，表达的恰菲埃利希氏体p156重组基因片段表现的分子量大于预期值，而且在这些片段上检测到了碳水化合物。此外，犬埃利希氏体p153的C-末端片段也表现出分子量大于预期值(约10％或6kD)。
当鉴定出p43基因时，从全细胞裂解物中对应的犬埃利希氏体天然蛋白质却不与抗p43抗血清反应。根据本发明提供的发现，这一分歧可能归因于以下事实：即p43基因代表一个不完全的开放阅读框，而该阅读框并不编码43kD蛋白。此外，由于此蛋白分子量很大(＞150kD)，故需对凝胶电泳的条件给予特别注意才能获得免疫印迹法对该蛋白的鉴定结果相一致。犬埃利希氏体全细胞裂解物中的200kD蛋白与抗p43多克隆抗体起很强的免疫反应。该蛋白的分子量与预测的偶联了某些聚糖使其分子量有所增加的p153的分子量相一致。这一发现同样与恰菲埃利希氏体p156重组片段几乎代表整个开放阅读框的分子量相一致。
埃利希氏体属类的糖蛋白是在致病细菌中最早被特征鉴定的此类蛋白质。至今已发现的埃利希氏体的糖蛋白，可被受感染的病人和动物的抗体一致和强烈地识别。这些独特的暴露于表面的免疫反应性蛋白质在疫苗开发中具有潜在价值，而且这些蛋白可以是亚单位疫苗的重要组分。
本文中引用了以下参考资料：
Chen，等人，1997.采用Western免疫印迹法分析罹患嗜人单核细胞埃利希病的病人的抗体应答，该病可以由不同株的恰菲埃利希氏体和犬埃利希氏体 (Ehrlichia canis)所引起CIin.Diagn.Lab.Immunol.4：731-735.
Harrus，等人，1998.感染犬埃利希氏体后20-34个月的狗的埃利希氏体DNA的扩增.J.Clin.Microbiol.36：73-76.
Huxsoll，D.L.，P.K.Hildebrandt，和R.M.Nims.1970.热带犬全血细胞减少症.J.Am.Vet.Med.Assoc.157：1627-1632.
McBride，等人，1996.狗急性犬埃利希氏体感染的PCR检测.J.Vet.Diagn.Invest.8：441-447.
McBride，等人，1999.Clin.Diag.Lab.Immunol.6：392-399.
McBride，等人，2001.用重组蛋白对犬埃利希氏体感染的免疫诊断.J.Clin.Microbiol.39：3 15-322.
McBride，等人，2002.埃利希氏体属类的一种免疫反应性DsbA样硫-二氧化硫氧化还原酶的鉴定和功能分析。Infect.Immun.70：2700-2703.
Nyindo，等人，1971.热带犬全血细胞减少症：病原体--犬埃利希氏体的体外培养.Am.J.Vet.Res.32：1651-1658.
Ohashi，等人，1998a.编码犬埃利希氏体的免疫显性的30kD的主要外膜蛋白的多基因的克隆和特征分析以及该重组蛋白在血清学诊断中的应用。J.Clin.Microbiol.36：2671-2680.
Ohashi，等人，1998b.多态性多基因家族编码的恰菲埃利希氏体的免疫显性主要外膜蛋白.Infect.Immun.66：132-139.
Rikihisa等人，1994.恰菲埃利希氏体，犬埃利希氏体，或E.ewingii感染狗和人的Western免疫印迹分析.J.Clin.Microbiot.32：2107-2112.
Troy，G.C.and S.D.Forrester.1990.犬埃利希病，p.404-4 18.C.E.Green编著，狗和猫的传染性疾病.W.B.Sauders Co.，Philadelphia.
Yu，等人，1997.编码恰菲埃利希氏体的120kDa免疫显性蛋白的基因的克隆和测序.Gene 184：149-154.
Yu，等人，1999a.恰菲埃利希氏体重组蛋白用于嗜人单核细胞埃利希病血清学诊断的比较。J.Clin.Microbiot.37：2568-2575.
Yu，等人，1999b.从人体中分离的恰菲埃利希氏体28-kD外膜蛋白基因的遗传多样性.J.Clin.Microbiol.37：1137-1143.
Yu，等人，2000.编码犬埃利希氏体120-kD蛋白的基因的分子克隆和特征 鉴定以及将该重组120-kD蛋白应用于犬埃利希病的血清学诊断.J.Clin.Microbiol.38：369-374.
在本说明书中提及的任何专利或出版物代表本发明涉及领域技术人员的水平。这些专利和出版物纳入本文作为参考，就好像各篇出版物被单独而具体地纳入作为参考一样。
本领域的技术人员不难理解：本发明十分适于实施其目标，并获得所述的和其本身所有的结果和优点。这些实施例连同本文所述的方法、步骤、处理、分子，和特殊的化合物以优选实施例为代表，都是示范性的，并不用于限制本发明范围。在不违背所附权利要求书范围所界定的本发明的精神下，本领域技术人员可以进行改动和作为其它用途。
序列表
Claims (15)
1.一种分离的DNA，其编码氨基酸序列如SEQ ID NO：1所示的156kDa恰菲埃利希氏体免疫反应性表面蛋白。
2.一种载体，其特征在于，其含有权利要求1所述的DNA和在细胞中表达该DNA所必需的调控元件。
3.一种用权利要求2所述的载体转染的宿主细胞。
4.如权利要求3所述的宿主细胞，其特征在于，所述细胞选自下组：细菌细胞、哺乳动物细胞、植物细胞或昆虫细胞。
5.一种分离的恰菲埃利希氏体156kDa免疫反应性表面蛋白，所述蛋白的氨基酸序列如SEQ ID NO：1所示。
6.一种抗体，其特征在于，该抗体针对权利要求5所述的156kDa蛋白。
7.一种抗犬埃利希病疫苗，其特征在于，该疫苗含有权利要求5所述的156kDa蛋白。
8.如权利要求5所述的蛋白在制备检测试剂中的用途，所述检测试剂通过测定获自人或狗的测试血清与所述蛋白的反应来测定所述人或狗是否感染了恰菲埃利希氏体。
9.如权利要求8所述的用途，其特征在于，所述的蛋白是一种重组蛋白和/或所述诊断测定采用Western印迹分析方法进行。
10.如权利要求8所述的用途，其特征在于，所述的检测试剂与另一含有犬埃利希氏体p28蛋白和/或p153蛋白的检测试剂联合使用。
11.如权利要求5所述的蛋白在制备试剂盒中的用途，所述试剂盒包含：
a)固定于载体上的如权利要求5所述的蛋白；
b)用于稀释狗血清的缓冲液；
c)连接有报告分子的抗狗血清第二抗体；和，
d)检测所述报告分子的试剂。
12.如权利要求11所述的试剂盒，其特征在于，所述的载体是膜或微量滴定板和/或所述的报告分子选自荧光素酶、辣根过氧化酶、β-半乳糖苷酶或荧光标记物。
13.如权利要求1所述的DNA在制备用于测定人或狗是否感染了恰菲埃利希氏体的试剂或试剂盒中的用途。
14.如权利要求13所述的用途，其特征在于，所述试剂是与权利要求1所述的DNA杂交的特异性寡核苷酸。
15.如权利要求14所述的用途，其特征在于，所述试剂盒包含：
a)提取血液中DNA所用的试剂；
b)与权利要求1所述的DNA杂交的特异性寡核苷酸；和
c)PCR扩增所用的试剂。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US42357302P | 2002-11-04 | 2002-11-04 | |
| US60/423,573 | 2002-11-04 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003801082386A Division CN100386430C (zh) | 2002-11-04 | 2003-11-04 | 用于犬和人埃利希病的免疫诊断的p153和p156抗原及其用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101294162A CN101294162A (zh) | 2008-10-29 |
| CN101294162B true CN101294162B (zh) | 2011-03-30 |
Family
ID=32312680
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008100879600A Expired - Fee Related CN101294162B (zh) | 2002-11-04 | 2003-11-04 | 用于犬和人埃利希病的免疫诊断的p156抗原及其用途 |
| CNB2003801082386A Expired - Fee Related CN100386430C (zh) | 2002-11-04 | 2003-11-04 | 用于犬和人埃利希病的免疫诊断的p153和p156抗原及其用途 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003801082386A Expired - Fee Related CN100386430C (zh) | 2002-11-04 | 2003-11-04 | 用于犬和人埃利希病的免疫诊断的p153和p156抗原及其用途 |
Country Status (13)
| Country | Link |
|---|---|
| US (2) | US7204992B2 (zh) |
| EP (1) | EP1581638B1 (zh) |
| JP (1) | JP4494974B2 (zh) |
| KR (1) | KR101063177B1 (zh) |
| CN (2) | CN101294162B (zh) |
| AT (1) | ATE372378T1 (zh) |
| AU (1) | AU2003290575B2 (zh) |
| BR (1) | BRPI0315980B1 (zh) |
| CA (1) | CA2504762A1 (zh) |
| DE (1) | DE60316189T2 (zh) |
| ES (1) | ES2293057T3 (zh) |
| MX (1) | MXPA05004804A (zh) |
| WO (1) | WO2004042037A1 (zh) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7842473B2 (en) * | 2005-04-04 | 2010-11-30 | Idexx Laboratories, Inc. | Ehrlichia canis DIVA (differentiate infected from vaccinated animals) |
| US7842474B2 (en) | 2005-04-04 | 2010-11-30 | Idexx Laboratories, Inc. | Ehrlichia canis DIVA (differentiate infected from vaccinated animals) |
| BRPI0611820B8 (pt) | 2005-06-16 | 2021-05-25 | Res Found Dev | método de identificar uma infecção por e. canis ou e. chaffeensis |
| PT2187958T (pt) | 2007-09-11 | 2017-03-17 | Yissum Res Dev Company Of The Hebrew Univ Of Jerusalem | Vacina atenuada de erliquiose |
| WO2009039414A2 (en) | 2007-09-21 | 2009-03-26 | Idexx Laboratories, Inc. | Methods and compositions for detection of ehrlichia chaffeensis (vlpt) |
| EP2045816A1 (en) * | 2007-10-01 | 2009-04-08 | Paul Scherrer Institut | Fast readout method and swiched capacitor array circuitry for waveform digitizing |
| EP3527221B1 (en) | 2007-10-31 | 2022-02-23 | Idexx Laboratories, Inc. | Ehrlichia canis diva (differentiate infected from vaccinated animals) |
| KR101588736B1 (ko) | 2008-01-10 | 2016-01-26 | 리서치 디벨롭먼트 파운데이션 | 얼리키아 샤피엔시스에 대한 백신 및 진단제 |
| ES2596952T3 (es) | 2009-11-20 | 2017-01-13 | Abaxis, Inc. | Péptidos, dispositivos y procedimientos para la detección de anticuerpos de Ehrlichia |
| ES2661088T3 (es) * | 2010-07-02 | 2018-03-27 | Intervet International B.V. | Vacuna contra Ehrlichia canis y métodos asociados |
| BR112015008036B1 (pt) | 2012-10-11 | 2022-09-13 | Zoetis Services Llc | População de peptídeos isolados, método para detectar em uma amostra um anticorpo, método para diagnosticar erliquiose monocítica e/ou granulocítica, método para detectar a presença de anticorpos e kit |
| US9194870B2 (en) | 2014-01-21 | 2015-11-24 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Anaplasma antibodies |
| US9442112B2 (en) | 2014-04-04 | 2016-09-13 | Abaxis, Inc. | Compositions and methods for identifying Ehrlichia species |
| EP3996741A1 (en) * | 2019-07-12 | 2022-05-18 | Research Development Foundation | Ehrlichia vaccines and immunogenic compositions |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6355777B1 (en) * | 2000-04-28 | 2002-03-12 | Research Development Foundation | P43 antigen for the immunodiagnosis of canine ehrlichiosis and uses thereof |
| US6436399B1 (en) * | 1998-04-09 | 2002-08-20 | The Ohio State University Research Foundation | Nucleic acid encoding the major outer membrane protein of the causative agent of human granulocytic ehrlichiosis and peptides encoded thereby |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6653128B2 (en) * | 1996-10-17 | 2003-11-25 | University Of Florida | Nucleic acid vaccines against rickettsial diseases and methods of use |
| US6207169B1 (en) * | 1997-03-21 | 2001-03-27 | Corixa Corporation | Compounds and methods for the diagnosis and treatment of Ehrlichia infection |
| US6403093B1 (en) * | 1997-03-21 | 2002-06-11 | Mayo Foundation For Medical Education And Research | Methods to detect granulocytic ehrlichiosis |
| WO1999052370A1 (en) * | 1998-04-09 | 1999-10-21 | The Ohio State Research Foundation | Nucleic acids encoding outer membrane protein of human granulocytic ehrlichiosis agent |
| US6043085A (en) * | 1998-08-27 | 2000-03-28 | Research Development Foundation | Ehrlichia canis 120-kDa immunodominant antigenic protein and gene |
| US6544517B1 (en) * | 1998-09-18 | 2003-04-08 | The Ohio State University Research Foundation | Outer membrane protein of Ehrlichia canis and Ehrlichia chaffeensis |
| US6512005B2 (en) | 2001-02-28 | 2003-01-28 | Taro Pharmaceutical Industries, Ltd. | Process for synthesis of pure warfarin acid, warfarin alkali metal salts and corresponding clathrates |
-
2003
- 2003-11-04 EP EP03783110A patent/EP1581638B1/en not_active Expired - Lifetime
- 2003-11-04 CN CN2008100879600A patent/CN101294162B/zh not_active Expired - Fee Related
- 2003-11-04 BR BRPI0315980A patent/BRPI0315980B1/pt not_active IP Right Cessation
- 2003-11-04 US US10/701,038 patent/US7204992B2/en not_active Expired - Fee Related
- 2003-11-04 MX MXPA05004804A patent/MXPA05004804A/es active IP Right Grant
- 2003-11-04 ES ES03783110T patent/ES2293057T3/es not_active Expired - Lifetime
- 2003-11-04 CA CA002504762A patent/CA2504762A1/en not_active Abandoned
- 2003-11-04 AU AU2003290575A patent/AU2003290575B2/en not_active Expired - Fee Related
- 2003-11-04 CN CNB2003801082386A patent/CN100386430C/zh not_active Expired - Fee Related
- 2003-11-04 DE DE60316189T patent/DE60316189T2/de not_active Expired - Fee Related
- 2003-11-04 AT AT03783110T patent/ATE372378T1/de not_active IP Right Cessation
- 2003-11-04 WO PCT/US2003/034916 patent/WO2004042037A1/en active IP Right Grant
- 2003-11-04 KR KR1020057007983A patent/KR101063177B1/ko not_active IP Right Cessation
- 2003-11-04 JP JP2004550409A patent/JP4494974B2/ja not_active Expired - Fee Related
-
2007
- 2007-03-12 US US11/684,832 patent/US7754224B2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6436399B1 (en) * | 1998-04-09 | 2002-08-20 | The Ohio State University Research Foundation | Nucleic acid encoding the major outer membrane protein of the causative agent of human granulocytic ehrlichiosis and peptides encoded thereby |
| US6355777B1 (en) * | 2000-04-28 | 2002-03-12 | Research Development Foundation | P43 antigen for the immunodiagnosis of canine ehrlichiosis and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| DE60316189T2 (de) | 2007-12-20 |
| AU2003290575A1 (en) | 2004-06-07 |
| CA2504762A1 (en) | 2004-05-21 |
| EP1581638B1 (en) | 2007-09-05 |
| US7204992B2 (en) | 2007-04-17 |
| EP1581638A4 (en) | 2005-12-28 |
| ES2293057T3 (es) | 2008-03-16 |
| JP4494974B2 (ja) | 2010-06-30 |
| KR101063177B1 (ko) | 2011-09-08 |
| JP2006505270A (ja) | 2006-02-16 |
| US20070218082A1 (en) | 2007-09-20 |
| WO2004042037A1 (en) | 2004-05-21 |
| KR20050084679A (ko) | 2005-08-26 |
| US20040121433A1 (en) | 2004-06-24 |
| CN101294162A (zh) | 2008-10-29 |
| US7754224B2 (en) | 2010-07-13 |
| DE60316189D1 (de) | 2007-10-18 |
| BRPI0315980B1 (pt) | 2016-03-29 |
| MXPA05004804A (es) | 2005-12-05 |
| CN100386430C (zh) | 2008-05-07 |
| CN1735684A (zh) | 2006-02-15 |
| AU2003290575B2 (en) | 2009-10-29 |
| EP1581638A1 (en) | 2005-10-05 |
| ATE372378T1 (de) | 2007-09-15 |
| BR0315980A (pt) | 2005-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7754224B2 (en) | P153 and p156 antigens for the immunodiagnosis of canine and human ehrlichioses and uses thereof | |
| Farencena et al. | Characterization of a new isolate of Chlamydia trachomatis which lacks the common plasmid and has properties of biovar trachoma | |
| McBride et al. | Immunodiagnosis of Ehrlichia canis infection with recombinant proteins | |
| WO2001079274B1 (en) | Tuberculosis antigens and methods of use thereof | |
| CN104650195A (zh) | Ev71病毒 vp1重组抗原及其单克隆抗体与应用 | |
| Noordhoek et al. | A new attempt to distinguish serologically the subspecies of Treponema pallidum causing syphilis and yaws | |
| Brank et al. | Development of a recombinant antigen for antibody-based diagnosis of Mycoplasma bovis infection in cattle | |
| Tanaka et al. | Molecular characterization of three rabbit rotavirus strains | |
| CN101970463A (zh) | 用于检测恰菲埃里希氏体(vlpt)的方法和组合物 | |
| Dettori et al. | Evaluation of Western immunoblotting technique in the serological diagnosis of human syphilitic infections | |
| CN102533663B (zh) | 口蹄疫杂交瘤细胞株、单克隆抗体、检测试剂和试剂盒 | |
| US6699674B2 (en) | Expression and refolding of truncated recombinant major outer membrane protein antigen (R56) of orientia tsutsugamushi and its use in antibody based detection assays and vaccines | |
| CN104710537A (zh) | 柯萨奇病毒ca16 vp1重组抗原及其单克隆抗体与应用 | |
| Bowie et al. | Potential value of major antigenic protein 2 for serological diagnosis of heartwater and related ehrlichial infections | |
| NZ520600A (en) | Novel therapeutic compositions for treating infection by Lawsonia spp | |
| EP0811067B1 (en) | Equine arteritis virus peptides, antibodies and their use in a diagnostic test | |
| CN101228276A (zh) | 多毛短螺旋体的基因和蛋白质及其用于诊断和治疗的用途 | |
| US6045804A (en) | Method for detecting B. burgdorferi infection | |
| EP0458918A1 (en) | Method for obtaining a recombinant protein and its utilization in the assay for the african swine pest virus (aspv) | |
| Aradaib et al. | Current status and future prospects of epizootic haemorrhagic disease of deer-a review. | |
| CN1318646A (zh) | 编码伤寒沙门氏菌特异性和抗原性外膜蛋白的dna序列 | |
| US20090068218A1 (en) | Antigens for Vaccination Against and Detection of Mycoplasma Suis | |
| CN108410785B (zh) | 一种制备细菌菌影的方法 | |
| CN102933597B (zh) | 毛线虫属的抗原性多肽及其用途 | |
| CN118388664A (zh) | 副猪嗜血杆菌融合抗原及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110330 Termination date: 20111104 |