CN1120315A - 35/31 KDA subunit of the entamoeba histolytica adherence lectin - Google Patents
35/31 KDA subunit of the entamoeba histolytica adherence lectin Download PDFInfo
- Publication number
- CN1120315A CN1120315A CN94191690A CN94191690A CN1120315A CN 1120315 A CN1120315 A CN 1120315A CN 94191690 A CN94191690 A CN 94191690A CN 94191690 A CN94191690 A CN 94191690A CN 1120315 A CN1120315 A CN 1120315A
- Authority
- CN
- China
- Prior art keywords
- kilodalton
- lectin
- entamoeba histolytica
- subunit
- antibody
- 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.)
- Pending
Links
- 239000002523 lectin Substances 0.000 title claims abstract description 120
- 108090001090 Lectins Proteins 0.000 title claims abstract description 113
- 102000004856 Lectins Human genes 0.000 title claims abstract description 112
- 241000224432 Entamoeba histolytica Species 0.000 title claims abstract description 84
- 229940007078 entamoeba histolytica Drugs 0.000 title claims description 83
- 238000000034 method Methods 0.000 claims abstract description 47
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 claims abstract description 32
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 claims abstract description 32
- 229960005486 vaccine Drugs 0.000 claims abstract description 32
- 230000000692 anti-sense effect Effects 0.000 claims abstract description 6
- 230000001717 pathogenic effect Effects 0.000 claims description 77
- 150000001413 amino acids Chemical class 0.000 claims description 44
- 108020004414 DNA Proteins 0.000 claims description 37
- 235000001014 amino acid Nutrition 0.000 claims description 33
- 239000012634 fragment Substances 0.000 claims description 31
- 238000000746 purification Methods 0.000 claims description 31
- 239000000523 sample Substances 0.000 claims description 30
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 27
- 210000004027 cell Anatomy 0.000 claims description 22
- 230000036039 immunity Effects 0.000 claims description 20
- 238000003752 polymerase chain reaction Methods 0.000 claims description 19
- 230000004927 fusion Effects 0.000 claims description 16
- 239000002773 nucleotide Substances 0.000 claims description 15
- 210000003812 trophozoite Anatomy 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 230000004044 response Effects 0.000 claims description 9
- 108091034117 Oligonucleotide Proteins 0.000 claims description 8
- 230000000295 complement effect Effects 0.000 claims description 8
- 239000012472 biological sample Substances 0.000 claims description 4
- 208000004881 Amebiasis Diseases 0.000 claims description 3
- 206010001980 Amoebiasis Diseases 0.000 claims description 3
- 230000001464 adherent effect Effects 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 230000008521 reorganization Effects 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 230000002238 attenuated effect Effects 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 108091033319 polynucleotide Proteins 0.000 claims 3
- 239000002157 polynucleotide Substances 0.000 claims 3
- 102000040430 polynucleotide Human genes 0.000 claims 3
- 241000700605 Viruses Species 0.000 claims 2
- 230000000735 allogeneic effect Effects 0.000 claims 2
- 108020004511 Recombinant DNA Proteins 0.000 claims 1
- 241000700618 Vaccinia virus Species 0.000 claims 1
- 230000009918 complex formation Effects 0.000 claims 1
- 239000002398 materia medica Substances 0.000 claims 1
- 230000003612 virological effect Effects 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 66
- 102000004169 proteins and genes Human genes 0.000 abstract description 52
- 239000000463 material Substances 0.000 abstract description 7
- 238000003556 assay Methods 0.000 abstract description 5
- 235000018102 proteins Nutrition 0.000 description 51
- 239000000059 antiamebic agent Substances 0.000 description 41
- JROGBPMEKVAPEH-GXGBFOEMSA-N emetine dihydrochloride Chemical compound Cl.Cl.N1CCC2=CC(OC)=C(OC)C=C2[C@H]1C[C@H]1C[C@H]2C3=CC(OC)=C(OC)C=C3CCN2C[C@@H]1CC JROGBPMEKVAPEH-GXGBFOEMSA-N 0.000 description 41
- 229930004094 glycosylphosphatidylinositol Natural products 0.000 description 35
- 238000001962 electrophoresis Methods 0.000 description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 23
- 238000002372 labelling Methods 0.000 description 23
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 22
- 239000002299 complementary DNA Substances 0.000 description 19
- 201000010099 disease Diseases 0.000 description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 18
- 239000003550 marker Substances 0.000 description 18
- 210000002966 serum Anatomy 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 229930182830 galactose Natural products 0.000 description 15
- 239000012528 membrane Substances 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 14
- 230000029087 digestion Effects 0.000 description 14
- 238000009396 hybridization Methods 0.000 description 14
- 238000001114 immunoprecipitation Methods 0.000 description 13
- 102000004196 processed proteins & peptides Human genes 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 230000002503 metabolic effect Effects 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 238000004809 thin layer chromatography Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 11
- 208000015181 infectious disease Diseases 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 244000052769 pathogen Species 0.000 description 11
- 241000193830 Bacillus <bacterium> Species 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 10
- 239000000499 gel Substances 0.000 description 10
- 229940067626 phosphatidylinositols Drugs 0.000 description 10
- 150000003905 phosphatidylinositols Chemical class 0.000 description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 9
- 108700022034 Opsonin Proteins Proteins 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 230000001900 immune effect Effects 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 150000002337 glycosamines Chemical class 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- IPCSVZSSVZVIGE-UHFFFAOYSA-M hexadecanoate Chemical compound CCCCCCCCCCCCCCCC([O-])=O IPCSVZSSVZVIGE-UHFFFAOYSA-M 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 238000005063 solubilization Methods 0.000 description 8
- 230000007928 solubilization Effects 0.000 description 8
- 206010024652 Liver abscess Diseases 0.000 description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 230000015556 catabolic process Effects 0.000 description 7
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 7
- 238000006731 degradation reaction Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 229910052938 sodium sulfate Inorganic materials 0.000 description 7
- 235000011152 sodium sulphate Nutrition 0.000 description 7
- 241000699694 Gerbillinae Species 0.000 description 6
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 150000001720 carbohydrates Chemical class 0.000 description 6
- 210000000172 cytosol Anatomy 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 229940105132 myristate Drugs 0.000 description 6
- 229910017604 nitric acid Inorganic materials 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 241000096130 Toxopus brucei Species 0.000 description 5
- 206010000269 abscess Diseases 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000000890 antigenic effect Effects 0.000 description 5
- 238000000376 autoradiography Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 230000009615 deamination Effects 0.000 description 5
- 238000006481 deamination reaction Methods 0.000 description 5
- 230000002550 fecal effect Effects 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000000833 heterodimer Substances 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 239000012925 reference material Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 235000021360 Myristic acid Nutrition 0.000 description 4
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 4
- 239000007997 Tricine buffer Substances 0.000 description 4
- 238000001261 affinity purification Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 4
- 238000003119 immunoblot Methods 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- 230000005012 migration Effects 0.000 description 4
- 238000013508 migration Methods 0.000 description 4
- 229920000682 polycarbomethylsilane Polymers 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- -1 streptomycin sulfates Chemical class 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 241000252203 Clupea harengus Species 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 108700005084 Multigene Family Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 150000002314 glycerols Chemical class 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 235000019514 herring Nutrition 0.000 description 3
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 125000001419 myristoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 244000045947 parasite Species 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 238000004451 qualitative analysis Methods 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000011537 solubilization buffer Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 241000224431 Entamoeba Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 208000035126 Facies Diseases 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 2
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 108050008598 Phosphoesterases Proteins 0.000 description 2
- 102000002067 Protein Subunits Human genes 0.000 description 2
- 108010001267 Protein Subunits Proteins 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000000776 antibody secreting cell Anatomy 0.000 description 2
- 210000000628 antibody-producing cell Anatomy 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000008614 cellular interaction Effects 0.000 description 2
- 206010009887 colitis Diseases 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- 238000009795 derivation Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 150000002338 glycosides Chemical group 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000003345 scintillation counting Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- BHNQPLPANNDEGL-UHFFFAOYSA-N 2-(4-octylphenoxy)ethanol Chemical compound CCCCCCCCC1=CC=C(OCCO)C=C1 BHNQPLPANNDEGL-UHFFFAOYSA-N 0.000 description 1
- LBCZOTMMGHGTPH-UHFFFAOYSA-N 2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCO)C=C1 LBCZOTMMGHGTPH-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000008102 Ankyrins Human genes 0.000 description 1
- 108010049777 Ankyrins Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000003930 C-Type Lectins Human genes 0.000 description 1
- 108090000342 C-Type Lectins Proteins 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 240000006497 Dianthus caryophyllus Species 0.000 description 1
- 235000009355 Dianthus caryophyllus Nutrition 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000007563 Galectins Human genes 0.000 description 1
- 108010046569 Galectins Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 125000003047 N-acetyl group Chemical group 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 102000009569 Phosphoglucomutase Human genes 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000711897 Rinderpest morbillivirus Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 101150052863 THY1 gene Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 102100029469 WD repeat and HMG-box DNA-binding protein 1 Human genes 0.000 description 1
- 101710097421 WD repeat and HMG-box DNA-binding protein 1 Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 230000003816 axenic effect Effects 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001816 cooling 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
- 238000012258 culturing Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000003104 cytoplasmic structure Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000000799 fusogenic effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000000503 lectinlike effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000000088 lip Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 1
- 229960000282 metronidazole Drugs 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 108091000115 phosphomannomutase Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 102000009076 src-Family Kinases Human genes 0.000 description 1
- 108010087686 src-Family Kinases Proteins 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Natural products CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The 35/31 kDa subunit of the Gal/GalNAc lectin and DNA encoding this protein in various strains of E. histolytica are provided. These materials are useful in vaccines and diagnostic assays, and in antisense methods to interrupt the production of the Gal/GalNAc lectin. Antibodies specific for the 35/31 kDa subunit also are disclosed.
Description
This research is partly subsidized by U.S. government; U.S. government enjoys certain right to this invention.
The application is the U.S. Patent application No.08/045 of application on April 9th, 1993, and 679 part is extended.Technical field
The present invention relates at Entamoeba histolytica (or claiming entamoeba historlytica) (Enta-moeba histolytica) diagnosis of infection and treatment.More specifically, the present invention relates to utilize this amebic Gal/GalNAc lectin gene of coding especially the encode cDNA of 35/31 kilodalton (kDa) light chain subunit and the treatment of carrying out at the antibody of its protein.Background technology
Infection due to Entamoeba histolytica is extremely extensive, and annual estimation influences 4.8 hundred million people.But these philtrums only 10% can develop into similar colitis or liver abscess symptom.According to the hypothesis disease low incidence rate phenomenon owing to having pathogenic form of amebicide and non-pathogenic form.By 1988, found that the patient who symptom finally occurs carries pathogenic " enzyme deme " (zy-modemes).Classify according to the special hexokinase of these enzyme demes and the isozyme of phosphoglucomutase.But, use form generation standard easily form of causing a disease and corresponding nonpathogenic enzyme deme not to be distinguished.
In diagnosis, cause a disease and non-pathogenic strain between difference most important because have only those can be needed treatment by the talent that infection due to Entamoeba histolytica falls ill.In developed country, though can all use known active drug (metronidazole) to treat to each carrier at least economically, situation be still no good.Certainly, it is disadvantageous using these medicines without distinction.In lesser developed countries, the expense of drug administration is too high without distinction, thereby can produce utmost point adverse effect to whole health care resources.
Infected by morbific enzyme deme with fallen ill between, and almost have fabulous dependency by non-pathogenic enzyme deme infection and between not falling ill.It has been generally acknowledged that to have only the pathogenic strain can be, but not the growth of pathogenic strain is earlier with attenuated bacteria, cultivation in a series of culture medium changes again growth under the no xenogenesis biology (promptly not having growth in the presence of the relevant microorganism).What be accompanied by this adaptation is the enzyme general layout feature (Mirelman D.etal., Infect Immun (1986) 54:827-832) that shows pathogenic strain.This is operated in other laboratorys and did not repeat, and recently the research (seeing below) of genome difference is shown the different kind of morbific and nonpathogenic genomorph.
Although above-mentioned general interested and useful result of study is arranged, diagnoses whether the existence of Entamoeba histolytica pathogenic strain is difficult.Since similar in pathogenic strain and the non-pathogenic strain morphology, so microscopic examination is ineffective especially.In fecal specimens and serum, whether detect the antigenic existence of Entamoeba histolytica, but as if these means of testing can not distinguish pathogenic and non-pathogenic strain with elisa technique.People such as Root (Arch Invest Med (Mex) (1987) 9:Supplement 1:203) take the lead in elisa technique, utilize the rabbit polyclonal antiserum to detect amebicide antigen in fecal specimens.After this, the existing various ways that makes in this way, wherein a part is relevant with microscopic study, and all uses polyclonal antiserum.Clearly, can accurately point out by the example of the form of causing a disease rather than non-pathogenic form infection without any a kind of.For example referring to Palacios et al., Arch Invest Med (Mex) (1987) 9:Supplement 1:203; Randall et al., Trans Roy Soc TropMed Hyg (1984) 78:593; Grundy, Trans Roy Soc Trop Med Hyg (1982) 76:396; Ungar, Am J Trop Med Hyg (1985) 34:465.
About to the research summary of fecal specimens in Amebiasis:Human Infection byEntamoeba Histolytica, J.Ravdin, ed. (1988) Wiley MedicalPublishing, pp.646-648.The antigenic similar approach of Entamoeba histolytica of detected characteristics can not distinguish pathogen and non-pathogen (ibid, pp661-663) equally in serum regulating liver-QI abscess fluid.Described in this piece article, by 1988, the unique method that this amebic pathogenic form and non-pathogenic form are distinguished was a feature of determining the isozyme general layout by electrophoresis.
Recently, studies show that of two different research groups, the comparison by the DNA separator can show cause a disease and non-pathogenic strain between difference.Garfinkel, people such as L.I. (InfectImmun (1989) 57:926-931) have developed with dna probe hybridizes with the DNA that separates from the histolytica entamoeba, and has obtained four types restriction fragment length general layout.These general layouts are relevant with morbific/nonpathogenic characteristic.Similarly, Tannich, people such as E. (Proc Matl Acad Sci (1989) 86:5118-5122) have surveyed with probe and have used the not cDNA storehouse of homophyletic structure, and the result shows that the separator that causes a disease is different from non-pathogenic separator in heredity.But these Technology Needs are cultivated from the isolating organism of patient so that obtain sufficient amount and are used for test, therefore waste time and energy.
Strachan, people such as W.D. (Lancet (1988) 561-562) report has been produced two kinds of monoclonal antibodies, respectively called after 22.3 and 22.5.These two kinds of antibody are big two of organizing in the antibody that get from the mice that crosses with the no xenogenesis biological culture thing immunity of pathogenic Entamoeba histolytica strain NIH200/ATCC30458 by standardization program.These monoclonal antibodies are tested with the culture of aggressive that derives from supposition and Non-Invasive strain in immunofluorescence assay.But in this publication document, do not mention how screening antibody, do not know these antibodies yet, and if do not have these specific antibody whether can repeat this experimental result in what kind of target with this specific character.Described should the test needs complete Entamoeba histolytica, therefore can not be used for serum, urine or liver abscess liquid, and can only be used for the firm fecal specimens of collecting.
The protozoon parasite Entamoeba histolytica mediates (Mann to adhesion of host target cell and destruction subsequently by galactose and the specific cell surface lectin of N-acetyl group-D-galactosamine (Gal/GalNAc lectin), et al., Proc NatlAcad Sci (1991) 3248-3252; Petri et al., J Biol Chem (1989) 264:3007-3012; Petri, et al.Infect Immun (1991) 59:97-101; Ravdin, et al., J Exp Med (1980) 152:377-390).This lectin has a light chain and heavy chain, and molecular weight (MW) is 260 kilodaltons; Light molecular weight subunit as described below is 31/35 kilodalton; And the heavy chain molecule amount is about 170 kilodaltons.
Can distinguish causing a disease and existing describe (Petri W.A.et al., Infect Immunol (1990) 58:1802-1806) of the MONOCLONAL ANTIBODIES SPECIFIC FOR method of non-pathogenic form of Entamoeba histolytica recently.These antibodies are on the different epi-positions of lectin 170 kilodalton heavy chains.
Can prevent that to inhibition Entamoeba histolytica from killing (Ravdin et al., J Clin Invest (1981) 68:1305-1313 to the contact of mammalian cell is dependent by the lectin regular adherence; Saffer et al., Exp Parasit (1991) 72:106-108).The heavy subunit of this lectin almost can be discerned by infected widely immune serum and the T cell of going up the patient of aggressive amoebiasis.(Petri?et?al.，InfectImmun(1987)55：2327—2331；Schain?et?al.，Infect?Immun(1992)60：2143—2146)。Show, with the lectin of purification gerbil jird is carried out immunity and can make 67% in liver, to be subjected to Entamoeba histolytica to excite the animal that produces immunne response that protection fully so as not to the amebicide liver abscess is provided.(Petri?et?al.，Infect?Immun(1991)59：97—101)。170 kilodalton subunits are considered to the active constituent (the U.S. patent was authorized on April 2nd, 5,004,608,1991) of vaccine.
Generally speaking, with bonded protein of carbohydrate or lectin be ubiquitous at nature, and relate to a large amount of identifyings, comprising mammal fertilization, cell adhesion in extracellular matrix and immune cell interaction.Interaction at lectin and carbohydrate aspect the specificity of cell attachment process plays pivotal role.Only depend on the peptide and the oligonucleotide of monomeric number and order opposite with quantity of information, carbohydrate has " coding " extremely potential of abundant information, and these information are the position of glycoside units and the appearance position of end group isomery configuration and branch point.After the effect that homing receptor is selected when having found in the lymphocyte process recycling leukocyte and endothelial bound, the importance of carbohydrate in cell and cell adhesion is people's concern day by day.Except mammalian cell, antibacterial and other microorganisms utilize protein-carbohydrate to interact and settle down and attack host tissue.
The Entamoeba histolytica Gal/GalNAc adherence lectin of above-mentioned discussion be from one cause a disease separate the diseased plant obtain and by people's purification such as Petri W.A. 500 times (J Bi-ol Chem (1989) 264:3007-3012).This successful separation and purification can suppress the external adherent mouse monoclonal antibody of amebicide trophozoite by generation to be realized.From the patient who infects, separate and obtain pathogenic strain, in the culture medium of no xenogenesis biology, grow, with the trophozoite immune mouse that supersound process is crossed, from the immortalized cell of mouse spleen, make antibody (Ravdin J.I.et al., Infect Immun (1986) 53:1-5) again.According to supernatant the inhibition ability that trophozoite adheres to target tissue is screened cell.Therefore, the monoclonal antibody of all these reports is general all has immunoreation with the Gal/GalNAc surface adhesion zone of pathogen.The Gal/GalNAc lectin can be by the preparation of galactose affinity chromatography (report in 1987) (Petri W.J.et al., J.Clin Invest (1987) 80:1238-1244).Patients serum with the pathogenic infection symptoms feature (comprising liver abscess and colitis) of Entamoeba histolytica is learned studies show that of cross reaction, adherence lectin is discerned (Petri Jr.W.A.et al., Am J Med Sci (1989 296:163-165) by all test patients serums.
260 kilodaltons of purification and the bonded lectin of galactose are stable in dodecyl sodium sulfate (SDS), but in the presence of beta-mercaptoethanol, be dissociated into heavily (170 kilodalton) and light (35/31 kilodalton) glycoprotein subunit, have disulfide bond between the hint subunit.So used in the literary composition, " light subunit " means any one or both in the 35 or 31 kilodalton isotypes of light glycoprotein subunit.Heavy subunit and light subunit are by different mRNA (the Mann et al. that encodes, Proc Natl Acad Sci (1991) 88:3248-3252), and having different aminoacid forms and amino terminal sequence (Petri et al., J Biol Chem (1989) 264:3007-3012).Only the heavy subunit of identification and the anti-lectin antiserum of mice polyclone can suppress amebic fully and adhere in the Western blotting, the heavy subunit of this hint contains galactose calmodulin binding domain CaM (Petri et al., J.Biol Chem (1989) 264:3007-3012).The cDNA sequence analysis of 170 kilodalton subunits discloses, and it is an inherent memebrane protein, has the extracellular region territory of being rich in cysteine and Cytoplasm afterbody (Mann et al., Proc Natl Acad Sci (1991) supra of a weak point; Tannich et al., Proc Natl Acad Sci (1991) 88:1849-1853).Nearest report shows that this heavy subunit is member (Mann et al., Parasit Today (a 1991) 1:173-176 in the multigene family; Tannich et al., supra).The amino terminal sequence of 35/31 kilodalton subunit shows microheterogeneity, shows to have multiple light subunit isotype.
(Molec Biochem Parasitol (1992) 55:225-228) reported that coding is called the cDNA clone process of the light subunit of 35 kilodaltons of Gal/GalNAc lectin in one piece of nearest article of people such as Tannich, and discloses deduced amino acid (this article is defined as 220 kilodaltons with the total molecular weight of lectin).The 35 kilodalton subunits of the DNA of hint report can not be used as vaccine, in fact, quote and the communicating by letter of the applicant, and this article claims, can not produce antibody at heterodimer or its small subunit with the rabbit of isolating 35 kilodalton molecular immunes.
Previous technology has only shown the immunne response at Entamoeba histolytica 170 kilodalton subunits; In infected animals or use the antibody that does not also detect in the animal of Gal/GalNAc lectin at light subunit.Therefore, according to previous technology, people can not anticipate that light subunit can be used at the vaccine of Entamoeba histolytica or can be used for infection is diagnosed.In addition, also less than immunologic opsonin antibody at this subunit.Invention discloses
Find that 35/31 kilodalton subunit of Entamoeba histolytica Gal/GalNAc lectin is the effective active composition of Entamoeba histolytica vaccine.The structure of 35/31 kilodalton subunit is clear and prepared the recombined material that produces in view of the above.Therefore the invention provides a kind of improvement vaccine of never predicting at Entamoeba histolytica with protective effect.
Before the present invention owing to there is not the special antibody of light subunit, therefore be difficult to 35/31 kilodalton subunit is carried out analysis on the 26S Proteasome Structure and Function, thereby do not know its adhere to the cytolysis that contact dependence in effect.The effort of exploitation vaccine all concentrates entirely on 170 kilodalton subunits.In the present invention, the light subunit (35/31 kilodalton) of the bonded lectin of galactose has been carried out qualitative analysis, to determine the mechanism of its structure and film grappling.Glycosyl-phosphatidyl inositol (GPI) anchor that the aminoacid sequence of deriving from 35/31 kilodalton subunit cDNA has a hypothesis adds signal, and biochemical analysis and immunoassay are supported this discovery.
One aspect of the invention is the 35/31 kilodalton subunit that contains Entamoeba histolytica Gal/GalNAc adherence lectin or the vaccine of its funtion part, this vaccine can cause immunne response and work to be protected from infection due to Entamoeba histolytica.Also can contain the heavy subunit of 170 kilodaltons or its funtion part, this protein or its fragment can be used as active component, and perhaps especially those utilize the expression vector of viral infection and expression system can be used as vaccine to recombinant expression carrier.
Therefore, in other respects, the present invention relates to be used at cell culture condition or produce the reorganization material of 35/31 subunit in immune host's original position.The invention still further relates to the chimeric fusogenic peptide that contains 35/31 kilodalton subunit.Also relate to the DNA complementary strand of generation coding light chain subunit and the reorganization material of complementary oligonucleotide itself on the other hand, they are used for disturbing Entamoeba histolytica in cultivation or growth in position.
Another aspect of the present invention relates to the method and the method and these antibody itself that produce at the antibody of 35/31 kilodalton subunit immunologic opsonin of carrying out immunity with vaccine of the present invention at Entamoeba histolytica.
Another aspect of the present invention relates in biological sample and to detect non-causing a disease or method that whether pathogenic Entamoeba histolytica exists, this method comprises makes polymerase chain reaction (PCR) to sample, wherein use certain the regional primer of 35/31 kilodalton light chain confine the Gal/GalNAc adherence lectin, and under stringent condition with the DNA of amplification with hybridize corresponding to this regional oligomer.By selecting pathogenic or non-pathogenic Entamoeba histolytica dna characteristics zone separately or that both have, can distinguish or definite jointly same realm of these enzymes.Additive method adopts monoclonal antibody of the present invention.The accompanying drawing summary
Fig. 1 a and 1b are the nucleotide sequence and the aminoacid sequence thereof of 35/31 kilodalton subunit of two gene codes in the gene family.
Fig. 2 is the photographic copying figure of two-dimentional SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of the lectin of affinity purification.
Fig. 3 is the peptide figure with CNBr (Bromine cyanide .) digestion back 31 and 35 kilodalton subunits.
Fig. 4 is the autoradiograph with the galactose lectin SDS-PAGE of Palmic acid and myristic acid metabolic marker.
Fig. 5 be [
3H] autoradiograph of lectin SDS-PAGE of glycosamine labelling.
Fig. 6 a shows that the lipoid product that 31 kilodalton bands from the amebicide gross protein are carried out obtaining after the nitric acid deamination carries out the figure as a result of thin layer chromatography.
Fig. 6 b shows that the lipoid product that 31 kilodalton bands from the lectin of immunoprecipitation are carried out obtaining after the nitric acid deamination carries out the figure as a result of thin layer chromatography.
Fig. 7 is the photographic copying figure that 31 and 35 kilodalton subunits of electroelution is carried out the Western blotting with anti-CRD (CRD) antibody.
Fig. 8 a measures the active figure as a result of Entamoeba histolytica protein G PI-PLC (glycosyl-phosphatidyl inositol-phospholipase C).
Fig. 8 b is the figure as a result to the lipoid product thin layer chromatography of measuring from GPI-PLC (TLC).
Fig. 9 a is the photographic copying figure that as probe Entamoeba histolytica DNA is carried out the Southern blotting with the pcr amplified fragment of 35/31 kilodalton subunit.
Fig. 9 b is the photographic copying figure of the Northern blotting of Entamoeba histolytica RNA and 35/31 kilodalton subunit probe.
Figure 10 is the photographic copying figure of the Western blotting of fusion rotein and subunit or isolating subunit and immunity and pre-immune serum.Embodiment of the present invention
The invention provides and to be used to develop at causing a disease and the vaccine of non-pathogenic form Entamoeba histolytica and the material of diagnostic assay.The vaccine of the present invention's exploitation makes to produce becomes possibility at two kinds of subunits of Gal/GalNAc adherence lectin, the especially vaccine of the light subunit of 35/31 kilodalton.Can derive from cell culture or from the experimenter's who is subjected to risk of infection biological sample on carry out diagnostic assay.This is measured and adopts hybridization probe or antibody, and by the design assay method, amebic pathogenic form and non-pathogenic form can be distinguished.In addition, because cDNA is provided, has an opportunity and " antisense " method to produce adherence lectin.
Light subunit DNA sequence can produce the synthetic peptide based on this sequence.These peptides can be made up specifically to contain for example t cell epitope or the B cell epitope of light subunit.For example, a kind of synthetic peptide vaccine that contains the B cell epitope of an expectation of the light subunit of representative has been described wherein referring to embodiment 16.Differentiate and to be known in this area by the method for the synthetic peptide of the antibody recognition of T cell and immune body according to 31/35 kilodalton subunit sequence.
The invention provides the primary amino acid sequence of Entamoeba histolytica galactose, and show that it contains a GPI anchor in conjunction with lectin 35/31 kilodalton subunit.Sequence analysis to light subunit cDNA shows that subunit contains a potential GPI anchor and adds signal; In addition, after handling with PI-PLC, light subunit can with anti-CRD antibody response.(" CRD " refers to CRD, and it is by the specific phospholipase C of phosphatidylinositols (PI-PLC) DG to be downcut and the determinant of exposure from anchor).The lipoid product that the nitric acid deamination of 31 kilodalton subunit isotypes of lipoid labelling discharges moves with phosphatidylinositols, and also the existence with the GPI anchor conforms to.The galactose lectin is a unique example in the GPI-anchor formula albumen, and it is a heterodimer, and other GPI anchor formula albumen of having described at present are monomer or homodimer.The cDNA sequence of 170 kilodalton subunits has shown and has contained 26 aminoacid hydrophobic domains, is 41 amino acid pro waters of c-terminus subsequently, and this conforms to for inherent memebrane protein with it.Therefore these two kinds of lectin subunits use different film anchors to live mechanism.
The isotype of light subunit forms two kinds of lectin heterodimers, and promptly 170/35 and 170/31.Although the Southern blotting shows light subunit and is encoded by a certain gene family, and the direct order-checking of 35 kilodalton protein subunits has also disclosed the microheterogeneity that conforms to the gene that has more than one, but 35 are the cross reaction (embodiment 15) of antigenicity ground and have very similar aminoacid with 31 kilodalton kind types and form and the postdigestive peptide general layout of CNBr.This shows that they contain identical primary amino acid sequence.31 kilodaltons and 35 kilodalton subunits demonstrate the difference labelling with glycosamine, and have markd fatty acid.Their aminoterminal is also different for the acceptance of Edman Degradation.Therefore, the biochemical difference between two kinds of isotypes is complicated.
31 kilodaltons and 35 kilodalton subunits are encoded by homologous genes.Fig. 1 a has shown the nucleotide sequence and the deduced amino acid of the isotype of a member (being called " lgl 1 ") in the corresponding 35/31 kilodalton subunit multigene family.With three residues and the hydrophobic c-terminus zone of drawing down in the aminoterminal homing sequence that residue that short-term marks represents to suppose.With drawing aminoterminal and the CNBr order of residue that solid line marks down for determining by the Edman edman degradation Edman.The glycosylation site that potential N-connects marks with an asterisk.The GPI anchor excision/interpolation site of inferring marks with black circle.The 79-90 aminoacid of sophisticated protein subunit goes out with collimation mark, to having activity (seeing embodiment 16) by synthetic peptide partly in vaccine.Residue 1 (K) is the aminoterminal of mature protein.Determine the aminoacid sequence of aminoterminal (KTN/QDN/GR/KDQF/LSPNYPYG/DKM DN) and CNBr peptide (MSTSYAIPKSV/DI SARAP) with the Edman edman degradation Edman.The residue of leukorrhagia line is represented different with derived sequence, mainly is positioned at the microheterogeneity zone.
Fig. 1 b has shown a clone's of the 35/31 kilodalton subunit of encoding nucleotide sequence and deduced amino acid.This clone is another member (" lgl 2 ") in the multigene family, use with embodiment 12 in the similar manner that proposes obtain.Certainly, the aminoacid sequence that obtains basically with the amino acid sequence homologous of this gene shown in Fig. 1 a.Other cloned sequences of this two kind of 35/31 kilodalton subunit gene that obtains from different Entamoeba histolytica strain are almost identical with sequence Fig. 1 a and the 1b, and a few amino acids difference is at most only arranged between strain.
Shown that Entamoeba histolytica has the GPI-PLC activity.Although there be (Petri et al., J Clin Invest (1987) 20:1238-1244) in lectin with soluble form and form membrane, in cultivating amebicide and culture medium, also do not identify CRD.Covalently bound (inherent memebrane protein seemingly) of light subunit and 170 kilodalton subunits makes the hydrolysis of GPI anchor can not become the effective mechanism that lectin discharges in the Entamoeba histolytica.
Because the heterodimer albumen of this character was not described in the past, so that propose such problem: why Entamoeba histolytica links to each other a GPI anchor formula polypeptide with an inherent membrane polypeptides.The possible answer of this problem will be according to proteinic biochemistry of GPI anchor formula and biological function.Find that GPI anchor formula protein has the lateral diffusion coefficient similar to membrane phospholipid, this makes the enough fast transferrings of GPI anchor formula receptor-like protein mass-energy to the zone that comes in contact with other cells in cell interaction.If the heterodimer structure is dynamic, promptly 35/31 kilodalton subunit dissociates from the inherent film subunit of 170 kilodaltons hypothesis sometimes, is free state and spreads in film, and then performance may be relevant with lectin like this.Secondly, it is believed that GPI anchor formula protein plays certain effect in cell signal.Evidence suggests the GPI anchor formula protein mediation cell-stimulating signal on a large amount of T lymphocytes (being Thy-1, TAP and Ly-6).Immunoprecipitation studies show that, GPI-anchor formula cell surface molecule and p56
LckThere is fixation phenomenon between (a member of Src family tyrosine kinase in a large amount of mammalian cells), GPI-anchor formula protein is linked to each other with known signal transduction molecule.Amebicide contacts dependence and killing target cell when suppressing lectin and can prevent with centrifuging amebicide contact with target cell membrane with galactose.The signal that this hint lectin (also being its GPI anchor most probably) relates to the lysis process is initial.Definition
So literary composition is used, " corresponding to " DNA of certain referent or protein (as lectin 31/35 kilodalton subunit) or its segmental DNA or RNA or its complementary strand that RNA is meant the coding identical function." corresponding to " protein of certain referent is meant the albumen of identical function in the identical or different Entamoeba histolytica strain.
" substantially " homology is meant that homology is fully so that provide necessity hybridization with target when adopting DNA.Similarly, herein " effectively fragment " thus be meant that a fragment with enough sizes provides necessity hybridization with target when adopting DNA.
Nucleotide sequence or epi-position with pathogenic or non-pathogenic strain feature are meant nucleotide sequence or the epi-position that has difference between these kinds.Therefore, nucleotide sequence or epi-position with pathogenic Entamoeba histolytica feature are meant certain nucleotide or epi-position, its corresponding sequence or epi-position with non-pathogenic strain is different, and the corresponding sequence of non-pathogenic strain or epi-position are different with the sequence or the epi-position of pathogenic strain on feature.
The compound nucleotide sequence refers to certain nucleotide sequence, and its part is corresponding to the sequence of Entamoeba histolytica pathogenic strain, and part is corresponding to the sequence of the non-pathogenic strain of Entamoeba histolytica.
" replicon " is meant when being transformed into suitable host Shi Neng and has the dna vector that duplicates of one's own.When being transformed into escherichia coli, use the most continually in the context (replicon) herein.
Diagnostic test relates to the hybridization under the tight degree of different hybridization, and these degree are defined as follows:
Low stringency is corresponding at 0.2 * SSC, among the 0.1%SDS in 37 ℃ wash film (1 * SSC is 0.15M NaCl, and 0.015M sodium citrate, SDS are dodecyl sodium sulfate).
High stringency is corresponding at 0.2 * SSC, among the 0.1%SDS in 65 ℃ the film of washing.
Medium stringency is corresponding at 0.2 * SSC, among the 0.1%SDS in 45 ℃ the film of washing.
So literary composition is used, is meant that about " immunologic opsonin " of certain particular target target described antibody capable is being that the much bigger affinity of the bonded affinity of antigenic determinant combines with target than this antibody and other epi-positions.Required degrees of specificity becomes with environment, but typically, the immunologic opsonin antibody of certain specified target target is to combine with this target is affine, the affinity when this this antibody of affine binding ratio combines with other epi-positions or antigen decision family big at least 1 or 2 or best several magnitude.
Term " antibody " not only refers to immunoglobulin itself, but also refers to the specific immunoglobulin fragment of those molecular immunes that are kept perfectly.These segmental examples are well-known in the art, for example comprise Fab, Fab ' and F (ab ')
2Fragment.Term " antibody " not only comprises the native form of immunoglobulin, but also comprises the immunoglobulin form (the existing technology that is used to modify in this area) of giving desired properties after modified and not changing immunologic opsonin.For example form the chimeric antibody that derives from two kinds and just becoming more feasible.In brief, " antibody " refers to keep any component or the derivative form of immunoglobulin of the immunologic opsonin of immunoglobulin self.
" cause a disease form " of term Entamoeba histolytica is meant that those are invasive and causes the form of the infected's disease." non-pathogenic form " is meant that the person of being carried carries and the form of reveal any symptoms not.
" Gal/GalNAc lectin " refers to the glycoprotein of above-mentioned discovery on the Entamoeba histolytica surface, and it mediates, and amebicide adheres to target cell and this mediation is suppressed by galactose or N-acyl group galactosamine.The Gal/GalNAc lectin refers in particular to the lectin that people such as Petri (ibid) separate and report from pathogenic strain HMI-IMSS, also refer to the corresponding lectin of finding from other Entamoeba histolytica kinds." 35/31 kilodalton subunit " or " light subunit " is meant the small subunit that obtains by reduction Gal/GalNAc lectin and the corresponding small subunit in other kinds.The Gal/GalNAc lectin and the 35/31 kilodalton subunit of preparation purification
With the mode similar to embodiment 2, can from non-pathogenic strain, isolate 35/31 kilodalton subunit, wherein use and immunoreactive antibody to take place as affinity ligand with distinctive lectin epi-position of non-pathogenic form or lectin epi-position pathogenic and that non-pathogenic form is total.
As described below, lectin 35 kilodaltons and 31 kilodalton forms also check order with the CNBr hydrolysis.According to the sequence that obtains, preparation primer be used to increase cD-NA as probe.Use this probe, obtained the cD-NA of the pathogenic form 35/31 kilodalton subunit of coding; Two kinds of isotypes are by same gene code, and have same primary structure.The derivation aminoacid sequence of these isotypes is shown in Fig. 1 a and 1b, above describes.The preparation monoclonal antibody
The immunization protocol of the light subunit of Gal/GalNAc lectin of monoclonal antibody the application of the invention separation described below and purification and preparing.As the use of immunogenic purification and unpack format lectin, and they have greatly promoted suitable MONOCLONAL ANTIBODIES SPECIFIC FOR and discriminating in screening with the availability that obtains in the monoclonal preparation.
For immunity, can use the scheme of standard, and any suitable vertebrates especially mammal such as rat, mice, rabbit etc. can both be used as experiment material.When obtaining enough titres, just can gather in the crops serum.As above Zhi Bei serum can be used as the polyclonal antibody compositions, has found that these compositionss are essential for reclaiming the recombinant clone of expressing the light subunit gene of coding 35/31 kilodalton.
If need monoclonal antibody, then laboratory animal produces the cell of antibody, it is desirable to splenocyte, handles through the immortalization scheme.Most convenient be by Kohler and Millstein initial propose form hybridoma method.But other immortalization technology such as viral infection also can use.
At this moment screen the infinite multiplication cell to produce required monoclonal antibody.Prevailingly, with the standard immunoassay algoscopy supernatant of ELISA or the immortalized cell of RIA method test through cultivating for example, as antigen, and the lectin of purification or subunit are as immunogen with them.The supernatant of positive reaction is further test again.Isolating lectin by using the reduction form as analyze or the Western blotting in antigen, can prove easily and immunoreactivity that 35/31 kilodalton comes base.
Then test the cross reactivity of supernatant and other forms of lectin or subunit again, for example, with the supernatant of the antibody secreting cell that from the experimental subject of the Entamoeba histolytica immunity of causing a disease, makes, test the lectin of its anti-non-pathogenic amebicide purification or the situation of other antigen compositions that contain lectin by immunoassay.Conversely, with the supernatant of the antibody secreting cell of the experimental subject of non-pathogenic form lectin immunity, test itself and pathogenic form lectin or other and contain the cross reactivity of antigenic compositions.
Therefore, the monoclonal antibody formulation that obtains or play immunoreation with the total epi-position of pathogen and non-pathogen, perhaps be that unique epi-position plays immunoreation derived from certain form and to it.Therefore with regard to 35/31 kilodalton subunit, can make three class monoclonal antibodies.One antibody-like is immunologic opsonin to the epi-position of finding on the distinctive 35/31 kilodalton subunit of form that causes a disease, therefore these antibody only can play immunoreation with amebic pathogenic form or with 35/31 kilodalton subunit of its lectin to a considerable extent.The second class monoclonal antibody is immunologic opsonin to the distinctive epi-position of 35/31 kilodalton subunit of non-pathogenic form; Therefore in fact only play immunoreation, and do not play immunoreation with pathogenic form with nonpathogenic amebicide or with its lectin.The 3rd class monoclonal antibody is immunologic opsonin to epi-position pathogenic and the 35/31 kilodalton subunit that non-pathogenic form is common.This antibody-like can play immunoreation with amebicide or with subunit, no matter and its pathogenicity how.
The monoclonal antibody that this area of mentioning in superincumbent background technology one book was reported is to make by utilizing amebicide that the adherent inhibitory action of target cell is carried out method for screening.Therefore, the antibody that prior art produces is different with antibody of the present invention, because up to the present existing technology can't prepare the antibody at 35/31 kilodalton subunit.
Monoclonal antibody of the present invention is to make by the immortalized cell system of cultivating the energy secretory antibody.As known in the art, the cultivation of these cell lines has two kinds of methods usually: the extracorporeal culturing method that carries out with nutrient in road as is generally known, perhaps it is injected into suitable host such as mice, it is bred in vivo, from ascites, reclaim monoclonal antibody subsequently.As used herein, " cultivation " immortalized cell system and " reclaiming monoclonal antibody from culture " all comprise the process of using these two kinds of methods.Vaccine
Similarly, can prepare have with the total epi-position of pathogen and non-pathogen rise immunne response or be the vaccine that unique epi-position plays immunne response derived from certain form and to it.In addition, can prepare at the vaccine of the light subunit of Gal/GalNAc lectin with heavy subunit.The method that is used to formulate vaccine formulation is as known in the art, and vaccine can contain carrier and/or adjuvant.Can determine optimal application scheme (the i.e. minimum antigen that needs in order to produce protective response with technology known in the art.)
The invention provides the DNA of the light subunit of coding 35/31 kilodalton, produce this albumen, comprise and contain this proteic fusion rotein thereby can recombinate.Exist these protein to promote the immunne response of inoculation animal to Entamoeba histolytica in vaccine, suitable fusion rotein includes (but are not limited to) colibacillary maltose-binding protein or glutathione transferase.In addition, with people's such as Wolff method (Science (1990) 247:1465) can be directly with the DNA of the light subunit of coding 35/31 kilodalton as vaccine.Can in protokaryon known in the art or eukaryotic system, produce protein (promptly not being) individually as fusion rotein.These systems include, but is not limited to mammalian cell, yeast cells, insect cell etc.Can use in addition DNA live, express in attenuated vaccine carrier such as rinderpest virus, bacill calmette-guerin, the attenuation salmonella.Mensuration based on DNA
In order to carry out mensuration of the present invention, the standard step of sampling type prepares sample and the wherein contained amebicide of solubilization according to being applicable to.For example handle fecal specimens with reference to people's such as Ungar method (Am J Trop Med Hyg (1985) 34:465).Serum or plasma sample carry out a series of dilutions in phosphate buffer.Although also can use other biological body fluid or biopsy material, feces, serum or plasma sample are preferred.
According to Kawasaki, E.S. " PRC scheme " book (people such as Innis M.A. edits, (1990) Academic Press, the 18th chapter, p.153 and following etc.) in method, extracting DNA from sample.In brief, in this scheme, sample is suspended in after lyophilization among 100 μ l 50mM Tris pH8.3,150mM NaCl, the 0.5%NP40.Add E.C. 3.4.21.64 to 100 μ g/ml.Sample was cultivated 1 hour at 55 ℃, boiled 3 minutes, placed on ice then.After the cooling, sample stayed supernatant after on little centrifuge centrifugal 5 minutes, and it is stored in 4 ℃ or use immediately.Per 50 μ l reactant mixtures use about 5 μ l supernatant to increase as dna profiling in this step.
The preparation of standard is to use the trophozoite of having identified in initial preparation process.About 2 * 10
6Individual trophozoite washs once with PBS, centrifugation, and lyophilization is also preserved in 20 ℃ for further use.In extracting, use the freeze dried amebicide of about 10mg.
Whether can be determined with the existence pathogenic or DNA that non-pathogenic strain is relevant by the technical scheme of utilizing the nucleotide sequence difference between these type strains.All technical schemes all relate to uses pcr amplified dna.
With standard step, carry out the PCR reaction with the primer of coupling target specific regions of design.In a kind of scheme, primer is designed to and desired causing a disease or the distinctive DNA area hybridization of nonpathogenic form.Therefore, have only characteristic area to be amplified out, be used for detection with probe.Select again specifically probe that should the zone.Because there is this zone in the pathogenic strain, so pathogenic strain and non-pathogenic strain can be distinguished according to itself and hybridization ability corresponding to this regional probe.Equally, the hybridization ability according to the corresponding probe of non-pathogenic strain sequence distinctive with it can detect non-pathogenic strain.
In another kind of scheme, such primer is used to distinguish pathogen and non-pathogen.In this scheme, the primer that uses at least a PCR reaction is corresponding to one type or the peculiar sequence of another kind of type strain.Therefore have only desired sequence to be amplified and to be gone out by probe in detecting.In this example, the probe of use can be from causing a disease or non-pathogenic strain, or their complex, and the stringency that can regulate hybridization conditions is to adapt to selected probe.High and low or the medium homology degree that depends on probe of stringency.
When the probe that uses corresponding to zone to be detected, generally adopt high stringency.In above-mentioned first method, need high stringency so as will to cause a disease and non-pathogenic strain distinguish.Rely in the method for characteristic with selection PCR primer at second kind, can use low stringency, because the differentiation of pathogen and non-pathogen was carried out.Detect hybridization with probe with routine techniques and under above-mentioned stringent condition.
The present invention also provides the test kit that is applicable to above-mentioned diagnostic method.These test kits comprise suitable primer, probe, other reagent (if desired) and the explanation of measuring at least.The antisense method
The utilization antisense technology can use DNA of the present invention to control to produce 35/31 kilodalton Gal/GalNAc in conjunction with the lectin subunit.Application process with standard, Remingto ' s Pharmaceutical Sciences one book (Mack Publishing Com-pany for example, Easton, PA publishes, latest edition) method described in offers the amebicide culture with the complementary strand of 35/31 kilodalton or its main sheet segment DNA or offers the host organisms of band infection due to Entamoeba histolytica.Ideally, for handling study subject in the body, complementary strand is to provide and with conventional excipient such as RingerShi solution, HankShi solution etc. is prepared by injection.Carry out Orally administered also effective with proper formula.Although it is general that great majority are used, and as for compartmentalization situation such as entity growth of tumor, also can be surface or local application.The release mechanism that also can use medicine to transmit.
In addition, complementary nucleotide sequence can produce in position, an expression system promptly is provided, and this system contains the DNA of 35/31 kilodalton subunit or from its similar DNA or its active fragment of other Entamoeba histolytica strains in the expression system of " anti-orientation ".Expression system can be designed to for example operate in the mammal experimental subject at host's experimental subject, thereby the control that wherein reverse Entamoeba histolytica coded sequence is positioned at as SU-40 promoter, adenovirus promoter etc. makes complementary strand to be transcribed in position down.When expression system is used for the cell culture of Entamoeba histolytica, can provide expression system by the replicon that holds altogether with the Entamoeba histolytica strain.
The following example is used to set forth the present invention, rather than limits the present invention.Embodiment 1
Cultivation of Entamoeba histolytica and results
In 37 ℃, in the 15ml glass culture tube, in the culture medium TYI-S-33 that is added with 100 units per ml penicillins and 100 μ g/ml streptomycin sulfates (Pfizer) (casein Trypsin enzymolysis thing (trypticase) and yeast extract, iron ion and serum), cultivate axenic Entamoeba histolytica strain HMI1-IMSS.After growth 72 hours, by gather in the crops the amebicide trophozoite in centrifugal 5 minutes, and, wash among the 65mM NaCl pH7.2 (Ravdin et al., 1981) at 75mM Tris (Sigma) in 4 ℃ of 150xg.By adding 50 μ Ci/ml[to TYI-S-33 culture medium
3H] Palmic acid or myristic acid (NewEngland Nuclear) and 37 ℃ cultivate amebicide 24 hours and realize [
3H] palmitate and [
3H] metabolic marker of myristate.Embodiment 2
By monoclonal antibody affinity chromatography purification galactose lectin
The amebicide trophozoite of cultivating the back results in 72 hours by described wash and add be dissolved in 150mM NaCl, 50mM Tris pH 8.3,0.5% Nonidet P40 (Non-idet P-40, NP-40) (Sigma), 5mM ethylenediaminetetraacetic acid (EDTA) (Sigma) and 2mM phenyl methyl sulfuryl fluoride (PMSF) (Sigma) in.The amebicide of solubilization is 15, centrifugal 10 minutes of 000xg, supernatant is splined on a monoclonal antibody affinity column in 4 ℃, (Affi-Gel10) (contains various anti-lectin monoclonal antibody H8-5 with a-protein-purification on the Bio-Rad) to 1-2 milliliters the affinity gel 10 of this affinity column, 7F-4,5B-8, respectively 2 milligrams of 3F-4 and 6D-2.Supernatant spends the night by affinity column with the peristaltic pump recirculation, uses solubilization buffer thorough washing post then.Bonded lectin 0.2N acetic acid pH2.5 eluting, freezing immediately then and lyophilizing.In some preparation, contain in the solubilization buffer 5mM right-chloromercuri phenyl-sulfonic acid (PCMS) is (Sigma) to suppress amebic glycosyl-phosphatidyl inositol-phospholipase C (GPI-PLC).Embodiment 3
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography
Press the standardization program of describing before the Laemmli (1970), carry out SDS-PAGE with 6-15% acrylamide gel.Molecular weight is measured according to the molecular weight marker thing (Rainbow Markers (14,300-200,000)) that derives from Amersham company.Unless indicate in addition, the electrophoretic protein that is useful on all boils in 4%SDS and 10% β-mercaptoethanol.For autoradiography, before drying, SDS-PAGE glue is soaked among Entensi-fy A and the B (DuPont), then at-70 ℃ to the XAR of Kodak-5 exposure one week or longer time.
One dimension SDS-PAGE through the lectin of immunoprecipitation and affinity purification is identified the band of 170,35 and 31 kilodaltons (not demonstrating).Because in the lectin preparation of purification, do not differentiate before the 31 kilodalton bands (Petri et al., J BiolChem (1989) 264:3007-3012), so we wish and can carry out qualitative analysis to itself and 35 kilodalton subunits.All with heterodimer form migration, this shows that they all are coupled to 170 kilodalton subunits (1989, ibid for Petri, et al.) by disulfide bond on non-reduced glue for 31 kilodaltons or 35 kilodalton protein.
In order to determine that whether 31 kilodalton bands are former proteinic subunits of U/I heterotrimer, analyze lectin by two-dimentional gel analysis method.The lectin of purification in 6% tubular type gel under non-reduced condition along continuous straight runs carry out electrophoresis, in 10% slab gel, under reducing condition, carry out the electrophoresis of vertical direction then, dye with Coomassie brilliant blue.The results are shown in Fig. 2, arrow has been pointed out 170,35 and 31 kilodalton subunits.The molecular weight marker thing of representing with kilodalton is listed in the left side of second direction.170 kilodalton subunits are the wide main band on the horizontal direction, with the inferior band of 160-170 kilodaltons.As shown in the figure, 35 and 31 kilodalton subunits are positioned at the below of 170 kilodalton bands in vertical direction, but their separately migrations in the horizontal direction.This hints that they are not heterotrimeric members, but the light subunit of two kinds of different heterodimers.A kind of 170 and 35 kilodalton subunits that contain in these two kinds of heterodimers, another kind contains 170 and 31 kilodalton subunits.Embodiment 4
The aminoacid of the light subunit of lectin is formed
The aminoacid of two kinds of subunits is formed much at one, and forms with the aminoacid that derives from 35/31 kilodalton cD-NA and the 35/31 kilodalton aminoacid delivered is in the past formed and carefully compared.
The aminoacid of table 1 galactose lectin subunit is formed residue 1234
Residue/100 aminoacid Cys ND
aND 1.8 NDAla 4.1 4.5 5.8 6.8Ile 3.6 3.7 4.4 4.4Leu 5.6 5.4 4.4 5.8Asp 9.6 10.8 6.2 7.0Asn ND ND 7.3 NDGly 12.8 10.0 5.1 8.5Glu 12.1 12.1 4.0 9.2Gln ND ND 5.5 NDSer 8.0 6.9 5.1 7.3Val 5.0 4.9 4.7 5.1Tyr 5.0 4.6 8.0 6.6Phe 3.9 4.1 5.1 5.1His 1.4 1.3 1.1 1.9Lys 11.6 12.0 8.4 9.4Met 0.4 0.4 1.4 1.5Pro 4.3 4.4 5.8 5.9Arg 4.5 5.4 6.5 6.9Thr 7.9 9.4 8.0 7.7Trp ND ND 1.5 ND
aND can not survey
Derive according to cDNA in hurdle 3
Observed nuance may be owing to having used different Entamoeba histolytica strain (HM-1:IMSS is used for amino acid analysis, and H-302:NIH is used for cDNA order-checking), perhaps causes owing to the member of each concrete light subunit gene family.Embodiment 5
The Bromine cyanide. digestion of the light subunit of lectin
For further qualitative analysis 31 and 35 kilodalton subunits, with two kinds of subunits of CNBr (Bromine cyanide .) digestion, the peptide that obtains is with N-[three (methylol) methyl] glycine (tricine)-SDS-PAGE analyzes (Fig. 3).Spend the night with CNBr digestion after the 31 and 35 kilodalton subunit dryings of electroelution.The protein of digestion N-[three (methylol) methyl] glycine (tricine)-SDS-PAGE analysis, be transferred to behind the electrophoresis on polyvinylidene fluoride (PVDF) filter membrane, and dye with Coomassie brilliant blue.Electrophoresis road, a left side is 35 kilodalton subunits; Right electrophoresis road is 31 kilodalton subunits.The molecular weight marker thing of representing with kilodalton is listed in the right side.
For two kinds of subunits, the postdigestive fragments of peptides general layout of CNBr is identical basically.This shows, if they are not identical, is exactly closely-related protein so.The CNBr digestion fragment of 31 kilodalton subunits is carried out the not achieving success of trial of determined amino acid sequence with the Edman edman degradation Edman.In addition, the aminoterminal of 31 kilodalton subunits seals for the Edman edman degradation Edman, but the aminoterminal of 35 kilodalton subunits does not seal.Modification after difference between two kinds of protein of this hint is to translate, it has caused the N-terminal sealing of 31 kilodalton subunits.Embodiment 6
Metabolic marker
A. fatty acid: whether have glycosyl-phosphatidyl inositol (GPI) anchor in order to check light subunit, from [
3H] palmitate and [
3H] purification lectin in the amebicide of myristate metabolic marker.The amebicide trophozoite in TYI-SS-33 culture medium with 50 μ Ci/ml[
3H] Palmic acid or [
3H] myristic acid metabolic marker 24 hours.Behind the metabolic marker, amebicide is precipitated, and with 75mM Tris, 65mM NaCl, pH7.2 washs 2 times, adds to be dissolved in 150mM NaCl, 50mM Tris, among pH8.3,0.5%NP-40 (Nonidet P-40), 5mM EDTA, 2mM PMSF and the 5mM PCMS again.The results are shown in Fig. 4. Electrophoresis road 1 and 4 is total amebicide protein; Electrophoresis road 2 and 5 is the protein through the pre-immune serum immunoprecipitation of mice; Electrophoresis road 3 and 6 is the protein through the anti-lectin antiserum immunoprecipitation of mice.Shown in electrophoresis road 1 and 4, a large amount of amebicide protein is also by two kinds of fatty acid labellings.
B.[
3H] the glycosamine labelling with phosphatidylinositols-phospholipase C (cultivation of PI-PLC): compare with 31 kilodalton subunits, 35 kilodalton subunits by [
3H] the glycosamine labelling gets stronger.This shows the glycosylation difference of this two peptide species.The difference of mark intensity can not only be attributable simply to the difference of quantity, because as using shown in the lectin gel of coomassie brilliant blue staining (Fig. 2), two kinds of subunits are abundant equally basically.
SDS-PAGE shows that Bruce trypanosomicide (T.brucei) the surface glycoprotein variant (VSG) in the detergent lysate has low 800-2000 daltonian apparent molecular weights than solubility VSG.For whether the definite migration of lectin on SDS-PAGE is subjected to PI-PLC influence, will [
3H] lectin of glycosamine labelling cultivates with the PI-PLC of wax shape bacillus or PCMS (a kind of PI-PLC inhibitor), carries out SDS-PAGE and radioautographic analysis again.As shown in Figure 5, the Entamoeba histolytica trophozoite is with 50 μ Ci/ml[
3H] glycosamine, there is being 5mM PCMS to have (electrophoresis road A), or having 1 units per ml wax shape bacillus PI-PLC to exist under (electrophoresis road B), 37 ℃ of metabolic markers are 1 hour in the TYI-S that does not contain galactose-33 culture medium.Carry out immunoprecipitation with anti-lectin antiserum then, analyze by SDS-PAGE and autoradiography again.List in the left side with the molecular weight marker thing that kilodalton is represented.[
3H] PI-PLC digestion of lectin of glycosamine labelling all not have significantly to influence to the animal migration of 35 and 31 kilodalton subunits.This hints that the GPI anchor on the 31 kilodalton subunits is opposing wax shape bacillus PI-PLC's.For other GPI anchor formula protein, listed resistance difference from the PI-PLC of separate sources.Embodiment 7
Immunoprecipitation from the amebic galactose lectin of solubilization
As implied above, with [
3H] palmitate carries out metabolic marker and solubilization to amebicide, then 4 ℃ centrifugal 10 minutes to remove insoluble matter.In some test, before adding antibody, the wax shape bacillus PI-PLC (Sigma) with 0.5 unit cultivated 1 hour in 37 ℃ with the amebicide of solubilization.The galactose lectin of solubilization cultivated with the anti-heavy subunit monoclonal antibody of mice or anti-lectin antiserum spend the night, use mice IgG
1Or the pre-immune serum of mice in contrast.Immune complex spends the night in 4 ℃ of precipitations with 50% protein-A sepharose 4B (Sigma) of 50 μ l, and washs 5 times with the solubilization buffer before SDS-PAGE.
The sero-fast immunoprecipitation of anti-lectin with mice shows, by [
3H] the palmitate metabolic marker be 170 and 31 kilodalton subunits rather than 35 kilodalton subunits (Fig. 4, electrophoresis road 3 and 6).Lipoid on the heavy subunit is modified and is not observed, but the cDNA sequence analysis shows that it is an inherent memebrane protein.[
3H] as if the band at 21 kilodalton places of myristate labelling not relevant with lectin, because it is with contrasting the non-immunoprecipitation specifically of antiserum, Fig. 4, electrophoresis road 5 and 6.Embodiment 8
The nitric acid deamination of the lectin of immunoprecipitation
The proteinic nitric acid deamination of GPI anchor formula disconnects glycosamine-(1-6)-inositol glycosidic bond to discharge phosphatidylinositols or acyl phospholipids acyl inositol, and the existence that is used to diagnose the GPI anchor whether.For the character of differentiating that lipoid is modified, from the quilt of amebicide gross protein [
3H] the palmitate labelling, 31 kilodalton bands carry out deaminizating with nitric acid, the lipoid product n-butanol extraction that obtains.When analyzing with thin layer chromatography (TLC), the lipoid product of release accurately moves with phosphatidylinositols.
The amebicide trophozoite in TYI-S-33 culture medium with 50 μ Ci/ml[
3H] palmitate metabolic marker 24 hours.[
3H] the amebicide protein of palmitate labelling carries out immunoprecipitation with anti-lectin antiserum, reuse SDS-PAGE separates, drying, and through autoradiography to differentiate by 31 kilodalton subunits of lipoid labelling.Cut 31 kilodalton bands from gel, rehydrated in deionized water, and electroelution.The protein of electroelution carries out water is dialysed and boiled off moisture.Exsiccant protein resuspending is in the freshly prepd deaminizating buffer of 400 μ l (0.1M sodium acetate (CH
3COONa) pH3.7,0.1%NP-40,0.25M NaNO
2) in, cultivating 12 hours in room temperature, reuse 6 μ l 5M HCl carry out acidify.Product is at the water saturated n-butyl alcohol of 400 μ l: water distributes in (1: 1), and the water saturated n-butyl alcohol of water reuse 400 μ l extracts to remove remaining salt.Butanols is merged, and boils off moisture content, and carries out TLC and analyze.
Briefly, TLC is performed as follows.With the chloroform of lipoid resuspending: in the methanol in 9: 1.Be heated to 80 ℃ of Kieselgel 60 of 30 minutes then before use and be lined with the enterprising circumstances in which people get things ready for a trip analysis of spectrum of silica gel plate (EM Science) of plastics.The solvent system that uses is 4: 4: 1 chloroforms: methanol: water.Each electrophoresis road is cut into 1 centimetre herring bone and analyzes with scintillation counter.The lipoid reference material is that the lipoid with labelling carries out stratographic simultaneously and develops the color in iodine vapor.Reference material is (A) phospholipid acyl chlorides; (B) phosphatidylinositols; (C) PHOSPHATIDYL ETHANOLAMINE; (D) two myristoyl base glycerols.The results are shown among Fig. 6 a.
In order to confirm to represent the light subunit of 31 kilodalton lectins from the 31 kilodalton bands of SDS-PAGE, lectin carries out immunoprecipitation and repeated trials with mouse anti lectin antiserum.Amebicide protein usefulness [
3H] palmitate carries out metabolic marker and uses the detergent solubilization.After the solubilization, carry out immunoprecipitation with the anti-lectin antiserum of mice.Reuse SDS-PAGE separates the lectin subunit.31 kilodalton bands differentiate with autoradiography, cut from gel and by as above step the lipoid component are analyzed.In current chromatograph, allow the solvent front move in an enterprising step of TLC plate, this makes the lipoid of phosphatidylinositols reference material and labelling all become wide band, but radioactive label still moves altogether with phosphatidylinositols, thereby proves the homogeneity (Fig. 6 b) of lipoid on the 31 kilodalton subunits.The release of phosphatidylinositols (a kind of GPI anchor component) has confirmed to have the GPI anchor on 31 kilodalton subunits.Embodiment 9
Carry out the Western blotting with anti-CRD (CRD) antibody
From the GPI anchor of several kinds show have jointly one known be the carbohydrate epi-position of CRD (CRD), this epi-position is by (PI-PLC) downcuts DG and exposes from anchor with the specific phospho-esterase c of phosphatidylinositols.In order to determine whether lectin contains CRD, 31 and 35 kilodalton subunits of preparation among the embodiment 3 are carried out electroelution, cultivate with the PI-PLC of wax shape bacillus, and carry out immunoblotting with anti-CRD antibody.The wax shape bacillus PI-PLC (the specific phospho-esterase c of phosphatidylinositols) of each subunit of 10 micrograms and 0.1 unit cultivated 30 minutes in 37 ℃, carry out electrophoresis by SDS-PAGE, transfer on the PVDF filter membrane, so as to carry out coomassie brilliant blue staining or with 1: 500 the dilution anti-CRD antibody (the Dr.Tamara Doering by Johns Hopkins Uni-versity grants) carry out immunoblotting.Before the immunoblotting, filter membrane spends the night with the protein binding ability of sealing filter membrane blank space in 4 ℃ of cultivations in 5% defatted milk powder (Carnation).With the cultivation time of antibody be at room temperature 1 hour, then in 50mM Tris-HCl, 200mM NaCl, 0.1%Tween 20 pH 7.35/31 washing 3 times, each 10 minutes.The washing first time after an anti antibody is cultivated is at 50 ℃.Two anti antibodys are anti-rabbit igg alkaline phosphatase conjugates (Promega).Trace develops the color in Western Blue (Promega).
The results are shown in Fig. 7.Electrophoresis road 1, the bonded lectin of the galactose of affinity purification; Electrophoresis road 2 and 4,31,35 kilodalton subunits of electrophoresis elution; Electrophoresis road 3 and 5,31 kilodalton subunits of electroelution.Electrophoresis road 1,2 and 3, the protein of coomassie brilliant blue staining.Electrophoresis road 4 and 5; The immunoblotting that carries out with anti-CRD antibody.Two kinds of subunits are all by anti-CRD antibodies ( electrophoresis road 4 and 5).When no PI-PLC, according to trace intensity (not shown), antibody can both carry out combination equally well.
The existing report of the weak reactivity of the VSG of form membrane and anti-CRD antibody, and be likely owing to be exposed to that a CRD epi-position on the GPI anchor of form membrane causes.Anti-CRD antibody does not react with lectin 170 kilodalton subunits, and two independent anti antibodys do not combine (not shown) with 31 or 35 kilodalton subunits yet.Embodiment 10
The fractionated of Entamoeba histolytica cell and to the active mensuration of amebic GPI-PLC
The proteinic discovery hint of GPI anchor formula Entamoeba histolytica has endogenic GPI-PLC activity.For amebicide GPI-PLC is measured, purification [
3H] the myristate labelling VSG and as substrate.Discharge DG from VSG and be used to diagnose GPI-PLC activity.
Amebic cytosol component and film component are pressed the method for describing in the past (Fox et al.J.Biol Chem (1986) 261:15767-15771) and are changed a little and be prepared.Briefly, centrifugal 5 * 10
7The amebicide trophozoite also washs in 5ml 75mM Tris, 65mM NaCl pH7.2, then in same buffer with 1: 1000 DFP (Sigma) cultivated on ice 30 minutes.Trophozoite contains in the 10mM sodium phosphate buffer of 2mMPMSF pH8.0 by the infiltration cell lysis at 5ml then.After 5 minutes, prepared product is by freeze thawing and centrifugal 1 hour in 4 ℃ at 50000xg 37 ℃ of cultivations.Supernatant is the cytosol component after measured, and the precipitate resuspending that obtains is in the fresh lysate buffer and 100, and 000xg is centrifugal 1 hour in 4 ℃.This precipitate is put on ice in the vibration pressure-even pulp crusher, carries out equal pulp in 5ml 1% n-octyl glucoside, 25mM Tris HCl, pH8.0 and 2mM PMSF.The homogenize thing is again 100,000xg in 4 ℃ centrifugal 1 hour, supernatant is the film component after measured.
The active mensuration of amebicide GPI-PLC be with 2 micrograms/15 milliliter [
3H] VSG (surface glycoprotein variant) of myristate labelling is as substrate, in 0.1%NP-40,50mM Tris-HCl, 5mM EDTA pH8.0, undertaken half an hour by described method (Hereld et al., J Biol Chem (1986) 13813-13819) in 37 ℃.Each component contains 1 * 10
7The amebicide equivalent.The wax shape bacillus PI-PLC of 1 unit purification as positive control, will be measured buffer (0.1%NP-40,50mM Tris-HCl pH8.0,5mM EDTA) as negative control.Measure with dithiothreitol, DTT (DTT) and EDTA, 25mM DTT and 5mM EDTA are added the cytosol component.Repeat to be DTT and EDTA and measure, the result represents with the meansigma methods of twice mensuration.After PI-PLC analyzes, n-butyl alcohol evaporates in SpeedVac (Speed Vac), and then be suspended in the 10 microlitre chloroform/methanol (9: 1), and on the thin layer of the silica gel 60 that is lined with plastics (be heated to earlier before use 80 ℃ 30 minutes), carry out chromatograph as solvent with petroleum ether/diethyl ether/acetic acid (80: 20: 1).The electrophoresis road is cut into 0.5 centimetre herring bone, analyzes with scintillation counting technique.
The result represents with the total cpm in the count per minute (cpm) of butanols in mutually/biphase.The wax shape bacillus PI-PLC of 1: the 1 unit purification in hurdle; Hurdle 2: independent buffer; Hurdle 3: Entamoeba histolytica film component; Hurdle 4: Entamoeba histolytica cytosol component.
Shown in Fig. 8 a, (T.Brucei) is different with castellanella brucei, and Entamoeba histolytica contains GPI-PLC in cytosol, and castellanella brucei (T.Brucei) identifies endogenic GPI-PLC activity in the film component.Add 5mM EDTA or 25mM DTT make respectively [
3H] fracture of form membrane VGS increases by 23% and 41% (not shown), and this shows that GPI-PLC is similar to castellanella brucei (T.Brucei), and amebic enzyme also part is subjected to Ca
2+Suppress, and may contain an avtive spot sulfydryl.
In order to confirm that the lipoid that discharges from VSG is amebicide GPI-PLC, with amebicide cytosol component to from [
3H] the organic facies product that go up to downcut of the VSG of myristate labelling carries out TLC.Derive from butanols that GPI-PLC measures and carry out the TLC analysis with the PI-PLC digestion product of the wax shape bacillus of purification.Butanols is evaporated mutually, and the lipoid resuspending was in 9: 1 chloroforms: in the methanol, to the silica gel 60TLC plate that is lined with plastics (be heated to earlier before use 80 ℃ 30 minutes) analyzed as solvent with petroleum ether/diethyl ether/acetic acid (80: 20: 1) again.Each electrophoresis road is cut into 0.5 centimetre herring bone, analyzes with scintillation counting technique.The lipoid reference material develops the color in iodine vapor.Reference material is (A) two myristoyl base glycerols; (B) myristic acid; (C) two myristoyl phosphoric acid.On TLC, demonstrate a single radioactive bands, two myristoyl base glycerols of it and purification and with purification with wax shape bacillus PI-PLC digestion [
3H] the lipoid product of VSG moves (Fig. 8 b) altogether.The identical test of carrying out with amebic film component shows, can not discharge radioactive label and enter the organic facies (not shown).Embodiment 11
Amino acid analysis and Bromine cyanide. digestion
As above the lectin to affinity purification carries out electroelution.The band of 31 and 35 kilodaltons is by at room temperature, with 0.5% Coomassie brilliant blue in acetic acid: isopropyl alcohol: dye in the water (1: 3: 6) and determine that then at acetic acid: methanol: water takes off dyeing in (50: 165: 785).Useful band is by excision and electroelution from gel.The protein of electroelution is analyzed its aminoacid composition at protein and the nucleic acid sequencing center of University ofVirginia.For the mapping of peptide, with 50 microgram dried proteins with the CNBr of 50 microlitres, 70 mg/ml in the solution of 70% formic acid, in dark, digest under the room temperature and spend the night.Postdigestive protein is dried and boils in 2X reduction sample buffer and is used for N-[three (methylol) methyl] glycine (tricine)-SDS-PAGE analyzes (Schagger ﹠amp; Von Jagow, 1987).At N-[three (methylol) methyl] after glycosides propylhomoserin (tricine)-SDS-PAGE analyzed, protein was dyeed to pvdf membrane and with Coomassie brilliant blue by electrophoretic transfer.The aminoterminal of the light subunit of lectin and a CNBr fragment check order with the Edman edman degradation Edman, as described in people such as Mann (Proc Natl Acad Sci (1991) 85:2444-2448).Embodiment 12
Polymerase chain reaction (PCR) and sequencing reaction
Utilize the deviation of Entamoeba histolytica codon, two kinds of merger property of the sequential design that obtains in the order-checking according to embodiment 11 oligonucleotide, and in polymerase chain reaction (PCR), be used as primer so that from the Entamoeba histolytica genomic DNA, amplify the fragment of 400bp.The primer that uses is: 5 ' GCGCAAGCTTCCAAA (C, T) TA (C, T) CCATA (C, T) GG3 ' (justice is arranged) and 5 ' GCGCCTCGAG (T, A) AC (T, A) GA (CT) TTTGGAAT (T, A) G3 ' (antisense).The PCR reaction is carried out with Pfu polymerase (Stratagent).Response time is 93 ℃ 1 minute, 37 ℃ 1 minute and 72 ℃ 3 minutes, totally 30 circulations.The PCR product of 400bp with α [
32P] (Boehringer-Mannheim) carries out labelling to dCTP (Amersham) with random primer dna marker test kit.[
32P] the PCR fragment of labelling is used to the Entamoeba histolytica λ gt 11 cDNA storehouses (the Bruce Torian doctor present (Torian et al.Proc Natl Acad Sci (1990) 87:6358-6362) of Louisiana State University) of screening from H-302:NIH strain.Identify a plaque of hybridizing with the PCR fragment of labelling.Separate this plaque and purification, the plaque of this purification is originated as template DNA, the primer that designs with the flanking sequence according to 400bp fragment and λ gt11 produces extra PCR product.By described method (Mann et al., Proc Natl Acad Sci (1991) 88:3248-3252), prepare the PCR product that is used to check order by polyethylene glycol precipitation.400 bp fragments and subsequent purificn be the single plaque hybridization of order-checking also, and checks order by the dideoxy sequencing method of Sanger with TAQuenase system (USB).
Gels data reads with Sequence Assembly Manager carries out.With FAS-TA software program (Pearson et al, Proc Natl Acad Sci (1988) 85:2444-2448) protein sequence of derivation and the data base of state-run biomedical research foundation (NationalBiomedical Research Foundation), Swiss-prot and Genbank are compared.Insert son from the cDNA of the plaque (being called lgl1) of purification and contain an open reading frame (ORF), (Fig. 1 a) to predict 278 the amino acid whose polypeptide that have that molecular weight of its coding is 32.1 kilodaltons.Analysis to deduced amino acid has identified amino terminal sequence and CNBr sequence and N-terminal 3 aminoacid of determining with the Edman edman degradation Edman of mature polypeptide, and they are the part of memebrane protein and the common homing sequence of secretory protein most probably.(sophisticated proteinic N-terminal-3 and-1 have alanine and glycine respectively, this conforms in these locational p1 amino acids requirements with signal peptide).The remainder of homing sequence comprises not having the aminoterminal translation initiation codon ATG of expection in this clone.
Amino terminal sequence and total amino acids form with the light subunit of describing in the past (Petri et al., 1989) much at one, thereby confirmed the homogeneity (Fig. 1 and table 1) of this cDNA.Aminoacid sequence contains two potential glycosylation sites that connect by N-, this result of study of having supported metabolic marker promptly 35/31 kilodalton subunit [
3H] glycosamine mixes.Between the carbohydrate structure territory of 35/31 kilodalton subunit and classical C type and S type lectin, there is not the similarity on the sequence.FASTA software to complete light subunit aminoacid sequence is searched discovery, there is not significant similarity with other protein among state-run biomedical research foundation (the National BiomedicalResearch Foundation) data base (Person et al., 1988).
Hydropathic profile shows, this polypeptide is hydrophilic relatively and lack membrane spaning domain.The substitute is at c-terminus and identify 7 amino acid whose hydrophobic sequences.Hydrophobic c-terminus zone is that GPI anchor formula protein is peculiar.Although compare with other GPI anchor formula protein of great majority, the c-terminus zone of 7 hydrophobic residues is less relatively, but, the VSG that castellanella brucei (T.Bru-cei) is lived by the serine anchor have short precursor tail and one section only 8 hydrophobic amino acid places stop.The interpolation that it is believed that the GPI anchor is an early stage translation back incident, and wherein the 15-30 c-terminus residue is cut from the nascent protein, subsequently the GPI anchor is connected the tripeptides C-S-A from the aminoterminal of 15 residues to the cDNA coding of hydrophobic c-terminus.These three locational these tripeptides with before the GPI anchor excision/interpolation site of order-checking be consistent, but compare with the shortest signal peptide that identifies at present, be three aminoacid of more close c-terminus.
In about 100 kinds of known GPI ankyrin matter, only have an appointment 20 kinds the GPI attachment site carried out qualitative investigation, and add in the signal at the GPI of other biological and to have found sizable heterogeneity.Therefore, only can't determine that according to sequence information anchor adds the site accurately.To the signal peptide of aminoterminal and c-terminus and the discriminating in potential excision/interpolation site, add the membrane spaning domain and the cytoplasmic structure territory that lack supposition, these all hint GPI anchor formula protein of this cDNA coding.
Another clone who obtains with above-mentioned technical scheme has represented another member (lgl 2) of gene family, is shown in Fig. 1 b.The sequence of lgl 2 comprises the single open reading frame of 864 nucleotide, its 288 amino acid whose polypeptide of encoding, and the molecular weight of the calculating of its mature protein is 32.4 kilodaltons.Embodiment 13
Southern and Northern analyze
A.Southern engram analysis: whether have the light subunit gene of a plurality of lectins in order to analyze Entamoeba histolytica, isolation of genomic DNA from strain HM-1:IMSS, complete with restriction endonuclease BglII and EcoRI digestion, according to the program of delivering (Maniatis et al., Molecular Cloning:A Laboratory Manual (1982)) it is transferred on the Zetabind filter membrane (CUNO) then.The PCR fragment of 400bp α [
32P] dCTP (Boehringer Mannheim) is with the random priming labelling, the degeneration by boiling 10 minutes before use then.Hybridization is carried out at 37 ℃.Washing is carried out in 50 ℃ in 0.1 * SSC, 0.1%SDS.Fig. 9 a has shown that BglII (electrophoresis road A) or EcoRI (electrophoresis road B) digestion back separates with agarose gel electrophoresis and be transferred to result on the nylon leaching film.With kilobase the molecular weight marker thing of expression is listed in the left side.The PCR product of labelling with the BglII dna digestion 8.3 and 4.8kb fragment and with the EcoRI dna digestion must 10.5 and 6.8kb fragment combine.Probe with have a light subunit gene family with postdigestive two the segmental hybridization hint of restriction endonuclease because on 35/31 kilodalton subunit cDNA, do not find restriction enzyme site.Research work in the past identifies microheterogeneity in the aminoacid sequence of 35/31 kilodalton subunit, this is consistent (Petri et al., 1989) with it by more than one coded by said gene.
B.Northern engram analysis:,, analyze then by former described method (Mann et al., Proc Natl Acad Sci (1991) 88:3248-3252) the total RNA of purification Entamoeba histolytica for the Northern engram analysis.By formaldehyde gel electrophoresis purification with separate the total RNA of Entamoeba histolytica, be transferred on the nylon leaching film then.As described in the A paragraph of this embodiment, filter membrane and usefulness [
32P] the 400bp fragment hybridization of 35/31 kilodalton subunit of labelling.Fig. 9 b has shown the result, with kilobase the molecular weight marker thing of expression is listed in the left side.Identify the single mR-NA of about 1.0kb of coding 35/31 kilodalton subunit, this 1.2kb (Mann et al., Proc Natl Acad Sci (1991) 88:3248-3252) with the cDNA size of 837bp and estimation more early is identical well.The mRNA that has single the multiple light subunit of carrying means in the North-ern trace must be that size is close.Embodiment 14
The identification of the light subunit of adherence lectin
35 kilodaltons and 31 kilodalton isotypes can both be discerned at the antiserum of recombinant expressed glutathion-S-transferring enzyme (GST)-light subunit fusion rotein.The cDNA sequence of complete amino acid sequence of the light subunit of 35/31 kilodalton lectin of encoding is expressed with the pGEX expression plasmid in escherichia coli with the form of gst fusion protein.Therefore fusion rotein contains as N-terminal GST, contains the light subunit as c-terminus.Fusion rotein breaks with French press (French press) and is able to purification through electrophoresis and electroelution, is used for immune mouse then.Derive from the fusion rotein and the reaction of the adherence lectin on the Western trace of immune serum and the purification of mice.The results are shown in Figure 10.Electrophoresis road 1 is the fusion rotein that reacts with pre-immune serum; Electrophoresis road 2 is and fusion rotein with the mouse immune seroreaction of GST-35 kilodalton subunit fusion protein immunization; Electrophoresis road 3 is the lectins with the purification of pre-immune serum reaction; Electrophoresis road 4 is and 31/35 kilodalton isotype at the immune serum reaction of GST-35 kilodalton.Confirm that from the result of electrophoresis road 2 and 4 35 kilodaltons and 31 kilodalton subunit isotypes are discerned by antiserum.Embodiment 15
With adherence lectin 35/31 kilodalton subunit immunity gerbil jird
35/31 kilodalton subunit-gst fusion protein with the purification of 50 micrograms in complete FreundShi adjuvant passes through peritoneal injection immunity gerbil jird.After two weeks, experimental animal passes through peritoneal injection with the fusion rotein of 50 micrograms in complete FreundShi adjuvant for the second time.After for the second time around the injection, by being injected directly into liver, the amebicide trophozoite excite animal immune to reply again.Kill animals after two weeks checks whether there is the amebicide liver abscess.In contrast, with the independent immune treated animal of GST protein.With only compare with the animal of GST protein immunity, carry out immunity with 35/31 kilodalton fusion rotein and cause reducing of liver abscess size.
Immunity abscess weight (gram) animal # 1 GSA 6.14
#2?GSA 0.68
#3?GSA 13.87
#4?GSA 1.66
#5?GSA 4.39
#6?GSA 0.33
#7?GST 6.27
#8 GAT-35/31 kilodalton 1.16
#9 GAT-35/31 kilodalton 1.33
#10 GAT-35/31 kilodalton 5.81
#11 GAT-35/31 kilodalton 1.44
#12 GAT-35/31 kilodalton 0.12
#13 GAT-35/31 kilodalton 1.34
#14 GAT-35/31 kilodalton 0.75
#15 GAT-35/31 kilodalton 1.85
Average abscess weight
Average 4.76 1.72
SD (+/- 4.71 1.73S
SE 1.78 0.61 embodiment 16
With the 79-90 amino acid whose synthetic peptide immunity gerbil jird that contains adherence lectin 35/31 kilodalton subunit
The light subunit peptides of the 79-80 aminoacid (aminoacid that goes out with circle in Fig. 1 a) of the 35/31 kilodalton subunit of the sophisticated 1gl 1 of adherence lectin analyzes to be differentiated to be potential B-cell epitope through the hydropathic profile of 35/31 kilodalton aminoacid sequence.For example referring to T.P.Hopp et al., " Prediction of Protein antigenic determi-nants from amino acid sequences, " Proc.Natl.Acad.Sci., USA (1981) 78:3824-3828.Synthetic this peptide also is connected in it on keyhole hemocyanin (KLH) carrier by conventional chemistry of peptides method known in the art.For example referring to U.S. Patent No. 4,708,871, its disclosed content is incorporated herein by reference.With the light subunit of 50 micrograms, perhaps for relatively, with light subunit peptides or the independent immune gerbil jird of GST (" contrast ") with glutathion-S-transferring enzyme (GST) fusion rotein (" light subunit ") formal representation.Pass through peritoneal injection immunity gerbil jird with complete FreundShi adjuvant.After two weeks, pass through the peritoneal injection booster immunization once more with the immunogen of 50 extra micrograms in complete FreundShi adjuvant.After six weeks, by being injected directly into liver, the amebicide trophozoite excite animal immune to reply again.Kill animals after two weeks checks whether there is the amebicide liver abscess.Result in the following table shows that light subunit peptides vaccine and 35 kilodalton subunit fusion product vaccines are almost effectively same.
Immunity abscess weight (gram) has the animal of abscess
Contrast 1.19 ± 0.53 (n=6) 83%
35 kilodalton subunits 0.31 ± 0.09 (n=7)
*100%
35 kilodalton peptides (aminoacid 79-90), 0.40 ± 0.34 (n=6)
#50%
*(p＜0.06, compared with the control)
#(p＜0.20, compared with the control)
Claims (26)
1. a vaccine is characterized in that, it contain with at least a materia medica on 35/31 kilodalton subunit or its function fragment of Gal/GalNAc adherence lectin of the Entamoeba histolytica that mixes mutually of acceptable excipient.
2. vaccine as claimed in claim 1 is characterized in that this lectin is derived from pathogenic strain.
3. vaccine as claimed in claim 1 is characterized in that, also contains 170 kilodalton subunits or its function fragment of the Gal/GalNAc lectin of Entamoeba histolytica.
4. vaccine as claimed in claim 1 is characterized in that, 35/31 kilodalton subunit is the form that is in the fusion rotein that merges mutually with extra aminoacid sequence.
5. vaccine as claimed in claim 4 is characterized in that, this extra aminoacid sequence is glutathion-S-transferring enzyme or its basic fragment.
6. vaccine as claimed in claim 1 is characterized in that, contains a kind of peptide that is made of the 79-90 aminoacid of this 35/31 kilodalton subunit basically as active component.
7. one kind makes the host to the immunifacient method of Entamoeba histolytica, it is characterized in that, comprise: use a kind of 35/31 kilodalton subunit of the Gal/GalNAc adherence lectin that contains Entamoeba histolytica or its function fragment vaccine as active component
And this vaccine can cause the immunne response at the aggressive amebiasis that is caused by the Entamoeba histolytica trophozoite in the host.
8. method as claimed in claim 7 is characterized in that, this active component is this function fragment, and this function fragment is induced the adherent antibody of blocking-up lectin.
9. purification and separated DNA is characterized in that, it is made up of the 35/31 kilodalton subunit of the Gal/GalNAc adherence lectin of coding Entamoeba histolytica or the DNA of its function fragment basically.
10. a recombinant DNA is characterized in that, it contains the 35/31 kilodalton subunit of the Gal/GalNAc adherence lectin connection allogeneic dna sequence DNA, the coding Entamoeba histolytica or the DNA of its function fragment.
11. the 35/31 kilodalton subunit of a Gal/GalNAc adherence lectin that is used to express Entamoeba histolytica or the expression system of its function fragment, it is characterized in that it contains this subunit of coding or its segmental DNA that can connect the control sequence that influences its expression in operation.
12. expression system as claimed in claim 11 is characterized in that, this control sequence is viral control sequence.
13. expression system as claimed in claim 12 is characterized in that, it is arranged in attenuated virus alive.
14. expression system as claimed in claim 13 is characterized in that, this virus is vaccinia virus.
15. a fusion rotein that produces by reorganization is characterized in that, contains the 35/31 kilodalton subunit of Gal/GalNAc adherence lectin of the Entamoeba histolytica that is blended in extra allogeneic amino acid sequence or the aminoacid sequence of its function fragment.
16. method that in the culture of Entamoeba histolytica, suppresses 35/31 kilodalton subunit of generation Gal/GalNAc adherence lectin, it is characterized in that, this culture and the complementary oligonucleotide of the nucleotide sequence of Fig. 1 a or 1b or corresponding oligonucleotide or its effective fragment are contacted;
Wherein, this oligonucleotide is a pathogenic strain, and
Wherein this contact can realize providing expression system of this oligonucleotide to realize under the condition of expressing.
17. one kind is connected in the allos polynucleotide or is in purification and the polynucleotide of unpack format with recombinant forms, it is characterized in that, it contains and the complementary sequence of the nucleotide sequence of Fig. 1 a or 1b or corresponding oligonucleotide or its effective fragment.
18. an expression system is characterized in that, it contains the nucleotide sequence of Fig. 1 a or 1b or corresponding nucleotide sequence or its active fragment of Entamoeba histolytica, and they are in the antisense orientation under the control of compatible control sequence.
Thereby 19. one kind detecting the method that whether has Entamoeba histolytica in the biological sample contain the strand polynucleotide through handling, it is characterized in that,
The sample of this processing is carried out polymerase chain reaction (PCR) amplification, wherein use the Gal/GalNAc adherence lectin of confining Entamoeba histolytica 35/31 kilodalton subunit zone primer and
Under stringent condition, use corresponding to the DNA of this regional oligomer probe and survey amplification.
20. special antibody of 35/31 kilodalton subunit immunity to the Gal/GalNAc adherence lectin of Entamoeba histolytica.
21. antibody as claimed in claim 20 is characterized in that, is monoclonal antibody.
22. antibody as claimed in claim 21 is characterized in that, the epitopic features of pathogenic strain is that immunity is special.
23. antibody as claimed in claim 21 is characterized in that, the epitopic features of non-pathogenic strain is that immunity is special.
24. antibody as claimed in claim 21 is characterized in that, epitopic features pathogenic and non-pathogenic strain is that immunity is special.
25. the immortalized cell that can secrete as antibody as described in the claim 21.
26. one kind is detected the method that whether has Entamoeba histolytica in biological sample, it is characterized in that,
With this sample and antibody described in claim 22-24 any Entamoeba histolytica exist all can with the condition of arbitrary this antibody complex formation under contact; With
The existence that detects this complex whether.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US4567993A | 1993-04-09 | 1993-04-09 | |
| US08/045,679 | 1993-04-09 | ||
| US13411193A | 1993-10-08 | 1993-10-08 | |
| US08/134,111 | 1993-10-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1120315A true CN1120315A (en) | 1996-04-10 |
Family
ID=26723072
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94191690A Pending CN1120315A (en) | 1993-04-09 | 1994-04-06 | 35/31 KDA subunit of the entamoeba histolytica adherence lectin |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0695191A1 (en) |
| CN (1) | CN1120315A (en) |
| AU (1) | AU6530394A (en) |
| CA (1) | CA2157599A1 (en) |
| WO (1) | WO1994023745A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102167747A (en) * | 2011-01-05 | 2011-08-31 | 复旦大学 | Entamoeba histolytica galactose/acetylgalactosamine (Gal/GalNAc) polypeptide fragment, and preparing method and application of polypeptide fragment |
| CN117050155A (en) * | 2023-10-11 | 2023-11-14 | 佛山科学技术学院 | Polymerization site of amoeba perforin, screening method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4769330A (en) * | 1981-12-24 | 1988-09-06 | Health Research, Incorporated | Modified vaccinia virus and methods for making and using the same |
| US4965188A (en) * | 1986-08-22 | 1990-10-23 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme |
| US5135855A (en) * | 1986-09-03 | 1992-08-04 | The United States Of America As Represented By The Department Of Health And Human Services | Rapid, versatile and simple system for expressing genes in eukaryotic cells |
-
1994
- 1994-04-06 EP EP94912959A patent/EP0695191A1/en not_active Withdrawn
- 1994-04-06 CN CN94191690A patent/CN1120315A/en active Pending
- 1994-04-06 AU AU65303/94A patent/AU6530394A/en not_active Abandoned
- 1994-04-06 WO PCT/US1994/003744 patent/WO1994023745A1/en not_active Application Discontinuation
- 1994-04-06 CA CA002157599A patent/CA2157599A1/en not_active Abandoned
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102167747A (en) * | 2011-01-05 | 2011-08-31 | 复旦大学 | Entamoeba histolytica galactose/acetylgalactosamine (Gal/GalNAc) polypeptide fragment, and preparing method and application of polypeptide fragment |
| CN102167747B (en) * | 2011-01-05 | 2013-08-21 | 复旦大学 | Entamoeba histolytica galactose/acetylgalactosamine (Gal/GalNAc) polypeptide fragment, and preparing method and application of polypeptide fragment |
| CN117050155A (en) * | 2023-10-11 | 2023-11-14 | 佛山科学技术学院 | Polymerization site of amoeba perforin, screening method and application thereof |
| CN117050155B (en) * | 2023-10-11 | 2024-01-19 | 佛山科学技术学院 | Polymerization site of amoeba perforin, screening method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2157599A1 (en) | 1994-10-27 |
| WO1994023745A1 (en) | 1994-10-27 |
| EP0695191A1 (en) | 1996-02-07 |
| AU6530394A (en) | 1994-11-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2593643C (en) | A plasmodium vivax hybrid circumsporozoite protein and vaccine | |
| CN110218250A (en) | A kind of preparation method of grass carp component 5 polyclonal antibody | |
| CN1120315A (en) | 35/31 KDA subunit of the entamoeba histolytica adherence lectin | |
| US6193971B1 (en) | Dictyocaulus viviparus antigen for diagnosing lungworm infestation and for vaccination | |
| CN1320165A (en) | Antithrombin nucleotides and proteins from horn fly | |
| CN1289852A (en) | Human nif gene homogenous protein and its coding sequence | |
| CN103459414A (en) | Protein having antigen activity against liver flukes | |
| CN1289850A (en) | Human heat shock protein and its coding sequence | |
| CN1673371A (en) | A new marnifi blue mold cell wall surface antigen I, its coding sequence and application thereof | |
| CN1673372A (en) | A novel marnifi blue mold cell wall surface antigen II its coding sequence and application thereof | |
| CN101052412A (en) | Amebiasis subunit vaccine | |
| EP0434751A1 (en) | A malaria antigen | |
| CN1281898A (en) | Human zinc finger protein isomer and its code sequence | |
| CN1281897A (en) | Human thioredoxin related protein and its code sequence | |
| CN1281043A (en) | Human early endosome antigen protein isomer and its coding sequence | |
| CN1281896A (en) | Human nuclear receptor factor protein an its code sequence | |
| CN1316515A (en) | Human liver cancer related cancer suppressing protein and its coding sequence | |
| CN1289851A (en) | Human micronuclear ribomucleoprotein specific 40KD protein isomer and its coding sequence | |
| CN1269407A (en) | New human ribosomal subunit protein and its code sequence | |
| CN1289848A (en) | Human interleukin enhancer binding factor protein isomer and its coding sequency | |
| CN1264741A (en) | Human adenosine-diphosphate ribosyglucose hydrolase protein and its coding sequence | |
| CN1269365A (en) | New human cell withering-related protein and its code sequence | |
| CN1268571A (en) | New human ribosomal subunit protein and its coding sequence | |
| CN1266898A (en) | Human carboxypeptidase precursor protein and its coding sequence | |
| CN1267733A (en) | Human glucuronic acid transferase protein and its code sequence |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |