WO1993010246A1 - Expression membranaire de genes heterologues - Google Patents
Expression membranaire de genes heterologues Download PDFInfo
- Publication number
- WO1993010246A1 WO1993010246A1 PCT/US1992/009659 US9209659W WO9310246A1 WO 1993010246 A1 WO1993010246 A1 WO 1993010246A1 US 9209659 W US9209659 W US 9209659W WO 9310246 A1 WO9310246 A1 WO 9310246A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dna
- polypeptide
- dna segment
- localizing
- seq
- Prior art date
Links
- 239000012528 membrane Substances 0.000 title claims abstract description 78
- 108090000623 proteins and genes Proteins 0.000 title claims description 114
- 230000014509 gene expression Effects 0.000 title abstract description 39
- 210000004027 cell Anatomy 0.000 claims abstract description 111
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 105
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 93
- 229920001184 polypeptide Polymers 0.000 claims abstract description 89
- 239000000427 antigen Substances 0.000 claims abstract description 44
- 108091007433 antigens Proteins 0.000 claims abstract description 43
- 102000036639 antigens Human genes 0.000 claims abstract description 43
- 229960005486 vaccine Drugs 0.000 claims abstract description 35
- 210000001322 periplasm Anatomy 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims description 77
- 239000013598 vector Substances 0.000 claims description 58
- 241000607142 Salmonella Species 0.000 claims description 48
- 238000000034 method Methods 0.000 claims description 48
- 102000004169 proteins and genes Human genes 0.000 claims description 44
- 239000012634 fragment Substances 0.000 claims description 31
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 20
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 18
- 238000003780 insertion Methods 0.000 claims description 17
- 230000037431 insertion Effects 0.000 claims description 17
- 230000002238 attenuated effect Effects 0.000 claims description 14
- 108010049048 Cholera Toxin Proteins 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 102000009016 Cholera Toxin Human genes 0.000 claims description 11
- 210000000170 cell membrane Anatomy 0.000 claims description 8
- 230000000890 antigenic effect Effects 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 239000013599 cloning vector Substances 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- 230000001018 virulence Effects 0.000 claims description 5
- 230000001086 cytosolic effect Effects 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 230000037452 priming Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 28
- 108020004707 nucleic acids Proteins 0.000 abstract description 18
- 102000039446 nucleic acids Human genes 0.000 abstract description 18
- 238000010276 construction Methods 0.000 abstract description 17
- 239000013604 expression vector Substances 0.000 abstract description 10
- 230000004044 response Effects 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 230000004927 fusion Effects 0.000 description 64
- 210000004379 membrane Anatomy 0.000 description 63
- 101150009573 phoA gene Proteins 0.000 description 47
- 239000013612 plasmid Substances 0.000 description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 38
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 26
- 238000002360 preparation method Methods 0.000 description 26
- 101150087320 ctxB gene Proteins 0.000 description 24
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 23
- 108020001507 fusion proteins Proteins 0.000 description 22
- 102000037865 fusion proteins Human genes 0.000 description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 19
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 18
- 239000008188 pellet Substances 0.000 description 18
- 239000000203 mixture Substances 0.000 description 16
- 239000000523 sample Substances 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 230000028993 immune response Effects 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 11
- 229930027917 kanamycin Natural products 0.000 description 11
- 229960000318 kanamycin Drugs 0.000 description 11
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 11
- 229930182823 kanamycin A Natural products 0.000 description 11
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 238000012163 sequencing technique Methods 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 9
- 238000002955 isolation Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 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 7
- 229920001817 Agar Polymers 0.000 description 7
- 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 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 230000002163 immunogen Effects 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 229940016590 sarkosyl Drugs 0.000 description 7
- 108700004121 sarkosyl Proteins 0.000 description 7
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 7
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 0.000 description 6
- 206010008631 Cholera Diseases 0.000 description 6
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000004480 active ingredient Substances 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 239000013611 chromosomal DNA Substances 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 210000004837 gut-associated lymphoid tissue Anatomy 0.000 description 6
- 238000003119 immunoblot Methods 0.000 description 6
- 150000003839 salts Chemical group 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000002741 site-directed mutagenesis Methods 0.000 description 6
- 238000002255 vaccination Methods 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000007984 Tris EDTA buffer Substances 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 230000005875 antibody response Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 102000012410 DNA Ligases Human genes 0.000 description 4
- 108010061982 DNA Ligases Proteins 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 4
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000004520 agglutination Effects 0.000 description 4
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000002759 chromosomal effect Effects 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 239000013600 plasmid vector Substances 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 229910001868 water Inorganic materials 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000008940 Alkaline Phosphatase assay kit Methods 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108091027305 Heteroduplex Proteins 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- -1 for example Chemical class 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000009851 immunogenic response Effects 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 2
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 241001167018 Aroa Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108090000204 Dipeptidase 1 Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101100491986 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) aromA gene Proteins 0.000 description 2
- 108010040721 Flagellin Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 2
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 108010059712 Pronase Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 101100323035 Sphingomonas sp phoK gene Proteins 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 101150037081 aroA gene Proteins 0.000 description 2
- 102000006635 beta-lactamase Human genes 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000009795 derivation Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 210000005256 gram-negative cell Anatomy 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 229940126578 oral vaccine Drugs 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000013492 plasmid preparation Methods 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000017105 transposition Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- QZCJOXAIQXPLNS-UHFFFAOYSA-N 1,1,2,2,3,3,4,4,4a,5,5,6,6,7,7,8,8,8a-octadecafluoronaphthalene 4-(2-aminoethyl)benzene-1,2-diol Chemical compound NCCc1ccc(O)c(O)c1.FC1(F)C(F)(F)C(F)(F)C2(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C2(F)C1(F)F QZCJOXAIQXPLNS-UHFFFAOYSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 101710187573 Alcohol dehydrogenase 2 Proteins 0.000 description 1
- 101710133776 Alcohol dehydrogenase class-3 Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 101150076489 B gene Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241000908115 Bolivar Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000037164 Collema parvum Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- HBHMVBGGHDMPBF-GARJFASQSA-N Cys-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N HBHMVBGGHDMPBF-GARJFASQSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241001522878 Escherichia coli B Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- FLLRAEJOLZPSMN-CIUDSAMLSA-N Glu-Asn-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FLLRAEJOLZPSMN-CIUDSAMLSA-N 0.000 description 1
- LPHGXOWFAXFCPX-KKUMJFAQSA-N Glu-Pro-Phe Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O LPHGXOWFAXFCPX-KKUMJFAQSA-N 0.000 description 1
- 102000030595 Glucokinase Human genes 0.000 description 1
- 108010021582 Glucokinase Proteins 0.000 description 1
- GVVKYKCOFMMTKZ-WHFBIAKZSA-N Gly-Cys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)CN GVVKYKCOFMMTKZ-WHFBIAKZSA-N 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 1
- 101100256651 Homo sapiens SENP6 gene Proteins 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- ULNXMMYXQKGNPG-LPEHRKFASA-N Met-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N ULNXMMYXQKGNPG-LPEHRKFASA-N 0.000 description 1
- HAQLBBVZAGMESV-IHRRRGAJSA-N Met-Lys-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O HAQLBBVZAGMESV-IHRRRGAJSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 108010087702 Penicillinase Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- APMXLWHMIVWLLR-BZSNNMDCSA-N Phe-Tyr-Ser Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(O)=O)C1=CC=CC=C1 APMXLWHMIVWLLR-BZSNNMDCSA-N 0.000 description 1
- 102000001105 Phosphofructokinases Human genes 0.000 description 1
- 108010069341 Phosphofructokinases Proteins 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 239000004353 Polyethylene glycol 8000 Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- ZCXQTRXYZOSGJR-FXQIFTODSA-N Pro-Asp-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZCXQTRXYZOSGJR-FXQIFTODSA-N 0.000 description 1
- DYMPSOABVJIFBS-IHRRRGAJSA-N Pro-Phe-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N[C@@H](CS)C(=O)O DYMPSOABVJIFBS-IHRRRGAJSA-N 0.000 description 1
- KHRLUIPIMIQFGT-AVGNSLFASA-N Pro-Val-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHRLUIPIMIQFGT-AVGNSLFASA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 108020005115 Pyruvate Kinase Proteins 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 101150038317 SSP1 gene Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 101100125020 Schizosaccharomyces pombe (strain 972 / ATCC 24843) pss1 gene Proteins 0.000 description 1
- 101100018019 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ssc1 gene Proteins 0.000 description 1
- 102100023713 Sentrin-specific protease 6 Human genes 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 241000589884 Treponema pallidum Species 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- HJSLDXZAZGFPDK-ULQDDVLXSA-N Val-Phe-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N HJSLDXZAZGFPDK-ULQDDVLXSA-N 0.000 description 1
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607447 Yersinia enterocolitica Species 0.000 description 1
- LUXUAZKGQZPOBZ-SAXJAHGMSA-N [(3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (Z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O LUXUAZKGQZPOBZ-SAXJAHGMSA-N 0.000 description 1
- HDRRAMINWIWTNU-PRJDIBJQSA-N [[(5r)-5-(2-amino-6-oxo-3h-purin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1CCC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HDRRAMINWIWTNU-PRJDIBJQSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 101150042732 aroC gene Proteins 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 101150065227 ctx gene Proteins 0.000 description 1
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- URGJWIFLBWJRMF-JGVFFNPUSA-N ddTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)CC1 URGJWIFLBWJRMF-JGVFFNPUSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 210000005205 gut mucosa Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000008944 intestinal immunity Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 229950009506 penicillinase Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 101150028857 phoP gene Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 description 1
- 229940085678 polyethylene glycol 8000 Drugs 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- NLJUOWMZPAVXHU-UHFFFAOYSA-N prop-2-enamide;urea Chemical compound NC(N)=O.NC(=O)C=C NLJUOWMZPAVXHU-UHFFFAOYSA-N 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 101150063569 slgA gene Proteins 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 108010044241 tetanus toxin fragment C Proteins 0.000 description 1
- TXEYQDLBPFQVAA-UHFFFAOYSA-N tetrafluoromethane Chemical compound FC(F)(F)F TXEYQDLBPFQVAA-UHFFFAOYSA-N 0.000 description 1
- 230000007888 toxin activity Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 101150108727 trpl gene Proteins 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 229940098232 yersinia enterocolitica Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- protective vaccines 35 from epitopes of known antigens of eukaryotic, viral or prokaryotic pathogens by taking advantage of the synthetic capacities of transformed host cells. Examples include tumor specific proteins which might be expressed and utilized to stimulate an immune response. Oral vaccines have stimulated research because of the ease of administration and, more importantly, in some instances the unsatisfactory protection a orded from parenteral injection. Vaccination against cholera, for example, gives short-term protection, thus provoking developmental work toward an oral vaccine that would presumably stimulate mucosal intestinal immunity more efficiently (Sanchez et al. , 1990).
- cytoplas ic proteins may not produce a high immunogenic response and heterologous proteins from recombinant DNA molecules expressed cytoplasmically often exhibit a diminished antibody reactivity (Sanchez, et al . , 1990).
- surface expressed epitopes of bacteria are expected generally to elicit the greatest humoral response; however, factors controlling surface expression of heterologous proteins have not been defined and there is no way to assure that any given fusion protein will localize to a host cell membrane surface.
- Vaccines are the most cost effective medical intervention known to prevent disease. However, effective vaccines are available for relatively few diseases. Successful immunization against infectious organisms often requires a multicomponent host immune response against a variety of antigenic determinants. Orally administered vaccines, especially live attenuated vaccines, induce specific cell-mediated effector responses and elicit secretory IgA (slgA) responses.
- slgA secretory IgA
- SlgA is important because of its effectiveness at mucosal surfaces. SlgA production and cell effector responses are mediated through the delivery of antigens to gut- associated lymphoid tissue (GALT) . Stimulation of GALT can lead to effective cell and humoral defense at all mucosal surfaces and provide systemic protection (1,2).
- heterologous epitopes it is generally believed that the export of heterologous epitopes to the Salmonella cell surface enhances their immunogenicity (11) .
- Investigators have used recombinant DNA methods to express heterologous epitopes as inserts in Salmonella flagellin (9) and the lamB encoded polypeptide of E. coli (10) .
- a significant antibody response to the heterologous surface-expressed epitopes was observed.
- a limitation of these systems is the relatively small number of epitopes which can be inserted into the laraB and flagellin genes. This is important as single (or few) epitopes may not result in the broad-based immune response which characterizes today's most successful vaccines.
- nucleic acid segments defined by SEQ ID NO:l encode a polypeptide product which when fused to a heterologous polypeptide will direct that polypeptide to the outer membrane of a bacterial cell.
- nucleic acid sequences While particular nucleic acid sequences have been defined it is nevertheless contemplated that nucleic acid sequences will be found to vary. It is expected that analogous sequences with similar functions may be found in other gram-negative bacteria such as E. coli .
- the invention concerns expression vectors that are constructed to include any of the DNA segments herein disclosed.
- DNA may be fused directly with a gene of interest and used in an expression system to produce heterologous polypeptides as hybridization probes for, e.g., identifying related sequences, as primers or even as building blocks for the construction of mutant or variant sequences.
- a particularly useful application of the DNA segements of this invention is to achieve directed expression of heterologous polypeptides.
- polypeptides will be expressed on the inner membrane periplasmic space, the outer membrane of the host cell, or on the surface of the outer membrane of the host cell.
- the pZIP plasmids of Figure 2 and Figure 3 have been constructed.
- fusion polypeptides are exported to the inner membrane/periplasmic space or to the outer membrane of the host cell.
- pZIP-OUT directs the export of fusion polypeptides to the outer membrane and may also direct a heterologous peptide to the external surface of a gram-negative host cell.
- pZIP-OUT is a vector which expresses bipartite fusion which includes a DNA segment capable of exporting the fusion product to the external membrane of a gram- negative cell.
- the other part of the chimeric gene is a p oA gene segment lacking signal and expression segments.
- a variety DNA segments may be inserted into the phoA segment at suitable restriction sites to create a tripartite fusion.
- pZIP-IN plasmid shown in Figure 3.
- This plasmid directs the export of polypeptides to the inner membrane/periplasmic space.
- the construction of the plasmid is bipartite. Part of the alkaline phosphatase gene lacking signal and expression sequences is fused with a DNA sequence that contains an exportation sequence capable of directing its fusion polypeptide to an inner membrane/periplasmic space.
- reporter genes encoding detectable polypeptides include ⁇ - lactamase and alkaline phosphatase genes. Reporter genes may be conveniently fused in frame downstream of the disclosed nucleic acid sequences with or without other DNA ragments/segments. Moreover, restriction sites in the gene sequence of the reporter gene may be used for insertion of a desired DNA fragment(s) .
- heterologous polypeptides expressed in the inner membrane or periplasmic space isolation of the heterologous polypeptide may be affected by cell lysis and other procedures utilized in the isolation of a desired fusion protein.
- Heterologous fusion proteins exported to the outer membrane of the host cell may be isolated from the outer membrane directly. Typical procedures include separation of inner and outer cell membranes and then isolation of the fusion polypeptide from membranous material.
- antigenic proteins are expressed on the surface of the host cell.
- Selected epitopes of eukaryotic viral or prokaryotic pathogens expressed on the surface of a host cell may be used for vaccine development. Tumor specific genes could be expressed and utilized to stimulate an immune response.
- Whole cells expressing immunogenic epitopes might be used for agglutination-based screening tests.
- Surface expressed polypeptides of other organisms might be identified by screening recombinant libraries for specific surface expressed polypeptides.
- cholera toxin B subunit may be expressed on the surface of a Salmonella harboring the pZIP-OUT plasmid vector hereinabove described.
- cholera toxin B subunits When expressed from Salmonella strain TA2362 harboring plasmid pRSPl ⁇ , cholera toxin B subunits agglutinated in the presence of specific antibody, indicating exposure of epitopic regions on the external membrane surface of formalin-fixed cells.
- Antigens or epitope(s) are selected and a gene encoding these moieties is inserted into one or more of the recombinant vectors disclosed.
- Appropriate host cells are transformed and after screening or trans ormants one is selected which expresses the antigen or epitopes for which a vaccine is desired.
- Vaccines may then be prepared by a variety of methods.
- Antigens on the surface of appropriate host cells may be safely administered orally. For example, attenuated Salmonella orally administered could stimulate an immune response on gut mucosa.
- whole cells or cell fragments containing the membrane-bound antigen may be suitably injected into a mammal to generate an immune response. In any event, it is expected that the immunogenicity of an antigen or epitope may be significantly enhanced when expressed on the surface of a bacterial cell.
- the invention concerns primers capable of priming amplification of selected portions of disclosed DNA segments.
- Primers hybridize to DNA and serve as initiation sites for synthesis of a portion of the gene.
- Nucleotide primers are designed to bind at separate sites on opposing duplex strains thereby defining the intervening sequence as the portion to be amplified.
- Nucleic acid molecules to be employed as primers whether DNA or RNA will generally include at least a 10 nucleotide segment of the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:2.
- the 10 base pair size is selected as a general lower limit in that sizes smaller than 10 bases hybridization stabilization may be become a problem. However, as the size of the primer decreases too much below 7-8 bases, non-specific hybridization may occur with other genes having complimentary sequences over short stretches.
- Primers may be utilized for several purposes. For example, primers may be used to amplify selected portions of the disclosed DNA segments. Certain primer combinations may more efficiently generate DNA encoding polypeptides that more effectively target to inner or outer membranes. Additionally, primers prepared from the disclosed DNA may be used to. amplify regions of DNA from other related organisms in order to identify similar targeting sequences. Once amplified products are obtained probes which referred to nucleic acid molecules employed to detect DNA sequences through hybridization procedures may be employed to detect and isolate selected DNA fragments. Like primers, probes may be DNA or RNA and are generally of similar size usually including at least a 10 nucleotide segment or more, often of 220 or 21 base pairs. Probes may be labeled, for example, by radio labeling, to assist in identification of nucleic acid sequences.
- kits useful for the expression of fusion proteins are also envisioned comprising separate containers, each having suitably aliquoted reagents for performing the foregoing methods.
- the containers may include one or more vectors, examples being the vectors of claim 19, particular embodiments of which are shown schematically in Figures 4 and 5.
- Suitable containers might be vials made of plastic or glass, various tubes such as test tubes, metal cylinders, ceramic cups or the like.
- Containers may be prepared with a wide range of suitable aliquots, depending on applications and on the scale of the preparation. Generally this will be an amount that is conveniently handled so as to minimize handling and subsequent volumetric manipulations.
- Most practitioners will prefer to select suitable endonucleases from common supplies usually on hand; however, such restriction endonucleases could also be optionally included in a kit preparation.
- Vectors supplied in kit form are preferably supplied in lyophilized form, although such DNA fragments may also be taken up in a suitable solvent such as ethanol, glycols or the like and supplied as suspensions. For most applications, it would be desirable to remove the solvent which for ethanol, for example, is a relatively simple matter of evaporation.
- FIG. 1 illustrates the cloning of phoA gene fusion from TnpnoA insertion mutants and construction of tribrid gene fusions.
- Tnp ⁇ oA is a derivative of Tn5 which encodes E. coli alkaline phosphatase, minus the signal sequence and expression signals, inserted into the left IS50L element (21) .
- Random transposition of TnphoA results in an active insertion only when the phoA gene sequence is fused in frame downstream of the promoter and export signals of a target gene (A) .
- the point at which the phoA sequence joins the target gene is referred to as the fusion joint (FJ) .
- the remaining portion of the gene begins at the distal joint (DJ) .
- kanamycin resistance gene e.g., Baj ⁇ HI
- phoA gene sequence e.g., -Efi-ndlll
- Plasmids carrying phoA gene fusions can then be used as exposition vectors (B) .
- the Sspl and Pvull restriction sites in phoA provide blunt ended sites at which in frame insertions (IF) of a gene of interest (GOI) can be inserted.
- GOI must also be consistent with the phoA frame at the insertion site.
- the resulting tribrid gene fusions contain the expression and export signals of the target gene fused in frame with the phoA and GOI sequences.
- FIG. 2B shows the DNA sequence across the
- IS50L/p oA sequence was observed.
- a stop codon in this ORF was observed at position -99. Multiple stop codons in all reading frames were present in sequences -150 to - 200.
- Two putative translation start codons (AUG) were present at positions -84 and -51.
- the predicted amino acid sequence of the coding region is shown above the nucleotide sequence.
- the IS50L and the beginning of the phoA derived sequences are underlined.
- FIG. 2 schematically shows plasmid pZIP-OUT contains a 4.5 Kb -ffindlll chromosomal fragment from invasion-attenuated S. typhimurium TnphoA insertion mutant TAP 43 inserted into pBR322 at the -Hindlll site. It expresses a 46 Kd PHOA fusion protein which localizes to the outer membrane.
- Figure 3 shows plasmid pZIP-IN which contains a -Ba-mHI chromosomal fragment from S . typhimurium Tnp oA insertion mutant TAG 28, inserted into pBR322 at the SaraHI site. It expresses a 55 kd PhoA fusion protein ⁇ 'hich localizes to the inner membrane.
- Figure 4 is an immunoblot analysis of Salmonella membrane preparations using mouse anti-alkaline phosphatase. S. typhimurium TA 2362 harboring pBR322 showed no reaction in the total envelope (TE) .
- TA 2362 harboring pZIP-OUT showed a 46 Kd PHOA fusion in the TE and after separation of the inner and outer membrane by treatment with 0.5% sarkosyl, the majority of the fusion protein was associated with the outer membrane (OM) .
- TA 2362 harboring pZIP-IN showed a 55 Kd PhoA fusion protein in the TE and after separation of the inner and outer membrane by treatment with 0.5% sarkosyl, the majority of the fusion protein was found associated with the inner membrane (IM) . All lanes were loaded with membrane preparations from an equal amount of cells.
- Figure 5 is an immunoblot analysis of urea extracts (SURF) using anti-alkaline phosphatase as the primary antibody.
- S. typhimurium TA 2362 harboring pBR322 showed no reacting polypeptides to the alkaline phosphatase antibodies.
- TA 2362 harboring pZIP-OUT showed a PhoA fusion at 46 Kd.
- TA 2362 harboring pZIP-IN showed no reacting polypeptides with the same antisera. Lanes were loaded with an equivalent amount of extract prepared from an equivalent number of whole cells.
- Figure 6 shows the derivation of plasmid pRSP18 from pZIP-OUT in which the final 294 base pairs of ⁇ txB have been inserted in frame (IF) with the phoA gene sequence at the PvuII site.
- the ctx gene sequence is from pRIT10810 which encodes the entire ctxB gene (22) .
- Figure 7 shows the derivation of plasmid pIMB13 from pZIP-IN in which the final 294 base pairs of ctxB have been inserted in frame (IF) with the phoA gene sequence at the Sspl site.
- the ctxB gene sequence is from PRIT10810 which encodes the entire ctxB gene (22) .
- Figure 8 is a schematic representation of the CtxB fusion from pRSP18 and pIMB13 that results in exportation of the 32 kDa CtxB protein to the outer and inner membranes, respectively.
- Figure 9 is an immunoblot analysis of urea extracts (SURF) using affinity purified anti-CTB as the primary antibody.
- S. typhimurium TA 2362 harboring pRIT10810 which encodes cytoplasmically expressed CTB showed no reaction.
- TA 2362 harboring pRSPl ⁇ showed a CTB tribrid fusion protein at 32 Kd.
- TA 2362 harboring pIMB13 showed no reactivity to anti-CTB antibodies.
- Lanes were loaded with equal amounts of extract from equivalent numbers of whole cells.
- Figure 10 is a proposed protocol for insertion of a fragment of HIV gpl60 gene into pZIP-OUT.
- Figure 11 shows the sequence of export specific signal in pZIP-IN. Promoter and regulatory sequences are underlined. IS50L and phoA sequences from pZIP-IN are shown. The ORF is shown in capital letters.
- the present invention relates to nucleic acid segments encoding particular polypeptides capable of forming fusion proteins that export to particular areas of a host cell. These nucleic acid segments are useful in constructing vectors that allow expression of heterologous proteins in appropriately transformed host cells.
- Polypeptides may be localized within the inner membrane/periplasmic space or on the outer membrane surface.
- Antigens or epitopic regions of antigens localized on host cell membranes have particular potential for vaccine development and antibody production.
- a heterologous gene expression system has been developed which utilizes a virulence-attenuated Salmonella as a carrier for a plasmid expression system (pZIP-OUT) which can direct the products of large segments of heterologous genes to the outer membrane (Fig. 2) .
- Recombinant DNA techniques are utilized to fuse the reading frame of the gene to be expressed with Salmonella export specifying sequences.
- Figure 1. Several cloning sites are possible which allow maintenance of the proper reading frame and produce tribrid fusion polypeptides which contain Salmonella export specifying sequences, the heterologous gene sequences and phoA gene sequences. Recombinants which export the tribrid fusion protein are selected through the loss of phoA activity and appearance of the predicted fusion polypeptide on the surface of the outer membrane.
- a tribrid fusion has been constructed which encodes virtually the entire cholera toxin B subunit (ctxB) gene.
- Figure 6 evaluated its subcellular localization in Salmonella.
- the DNA of the present invention was isolated from Salmonella typhimurium, strain TAP43, an invasion attenuated strain.
- Invasion attenuated refers to species which have lost one or more virulence factors affecting the efficiency by which Salmonella invades epithelial cells. Isolation of an attenuated strain of Salmonella was considered useful in developing the present invention because such strains may be used to deliver heterologous antigens to the gut of an animal. Salmonella given orally tends to establish an infection in the intestinal mucosa, leading to an immune response. The presence of a desired antigen is expected to stimulate a response to that species, as well as to the Salmonella or other host antigens.
- Tnp oA is a transposon derivative of Tn5 in the phoA gene which lacks a promoter, translation initiation site, signal sequence DNA and the first five amino acids of its protein.
- TnphoA inserts into a foreign gene in the correct orientation and reading frame, gene fusions are generated, coding for hybrid proteins which have alkaline phosphatase activity if transported beyond the inner membrane. Detection of such activity is generally accomplished with an alkaline phosphatase indicator dye, allowing visualization of colored colonies for successful gene fusions that lead to export of heterologous gene products.
- Part of the present invention contemplates vaccine preparation and use.
- antigens or epitopes of antigens, will be readily expressed in localized regions of a host cell using the methods disclosed.
- Expression vectors incorporating the DNA segment encoding exportation polypeptides directing products to a host cell outer membrane surface are expected to be particularly useful. Epitopic regions of antigens, well exposed at a membrane surface, may elicit high immunogenic responses, providing a route to vaccines or antibody production.
- vaccines which contain peptide sequences as active ingredients are generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference.
- such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
- the preparation may also be emulsified.
- the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
- the vaccines are conventionally administered parenterally, by injection, for example, either ⁇ ubcutaneously or intramuscularly.
- Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
- suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol t lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
- the proteins may be formulated into the vaccine as neutral or salt forms.
- Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
- the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
- the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient t r vaccination. Suitable regimes for initial administr ion and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations. The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
- Various methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C.
- agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab)
- parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
- physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
- the vaccine will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations.
- the vaccinations will normally be administered from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies.
- the course of the immunization may be followed by assays for antibodies for the supernatant antigens.
- the assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
- the invention also contemplates the use of disclosed nucleic acid segments in the construction of expression vectors or plasmids and use in host cells.
- the following is a general discussion relating to such use and the particular considerations in practicing this aspect of the invention.
- prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention.
- strains such as E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. ' coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative rather than limiting.
- prokaryotes may also be preferred for expression.
- the aforementioned strains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325) , bacilli such as Bacillus subtilus , or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans , and various Pseudomonas species may be used.
- plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
- the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
- E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g. ,
- the pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
- the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.
- promoters most commonly used in recombinant DNA construction include the ⁇ -lactamase (penicillinase) and lactose promoter systems (Chang et al. , 1978; Itakura et al., 1977; Goeddel et al. , 1979) and a tryptophan (trp) promoter system (Goeddel et al . , 1979; EPO Appl. Publ. No. 0036776) .
- eukaryoti ⁇ microbes such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are available.
- Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are available.
- the plasmid YRp7 for example, is commonly used (Stinchcomb et al. , 1919 ; Kingsman et al., 1979; Tschemper et al . , 1980).
- This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977) .
- Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al . , 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al . , 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. .
- the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
- promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
- Any plasmid vector containing a yeast- compatible promoter, origin of replication and termination sequences is suitable.
- cultures of cells derived from multicellular organisms may also be used as hosts.
- any such cell culture is workable, whether from vertebrate or invertebrate culture.
- vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973).
- useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293 and MDCK cell lines.
- Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
- control functions on the expression vectors are often provided by viral material.
- promoters are derived from polyoma, Adenoyirus 2, and most frequently Simian Virus 40 (SV40) .
- the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al . , 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the -Efindlll site toward the -Bgll site located in the viral origin of replication.
- promoter or control sequences normally associated with the desired gene sequence provided such control sequences are compatible with the host cell systems.
- An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g. , Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. Also contemplated within the scope of the present invention is the use of the disclosed DNA as a hybridization probe. While particular examples are provided to illustrate such use, the following provides general background for hybridization applications taking advantage of the disclosed nucleic acid sequences of the invention.
- the DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to S. typhimurium gene sequences.
- nucleic acid probes of an appropriate length are prepared based on a consideration of the sequence, e.g., as shown SEQ ID NO:l and SEQ ID NO:2 or derived from flanking regions of these genes.
- the ability of such nucleic acid probes to specifically hybridize to the s. typhimurium gene sequences lend them particular utility in a variety of embodiments.
- the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other g ⁇ etic constructs.
- the preferred nucleic acid sequence employed for hybridizations or assays includes sequences that are complementary to at least a 10 to 40, or so, nucleotide stretch of the selected sequence, such as that shown in Figure 1 or Figure 2, SEQ ID N0:1 or SEQ ID NO:2.
- a size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective.
- Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
- nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired.
- Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.
- nucleotide sequences of the invention are important for their ability to selectively form duplex molecules with complementary stretches of S. typhimurium gene segments.
- relatively stringent conditions for applications requiring a high degree of selectivity, one will typically desire to employ relatively low salt and/or high temperature conditions, such as provided by 0.02 M-0.15 M NaCl at temperatures of 50°C to 70°C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
- the present invention is envisioned as useful in the cloning of nucleic acids encoding certain exportation polypeptides. Identification of other exportation polypeptides in addition to the 46 kDa and 55 kDa proteins should be possible,using methods analogous to those disclosed herein. One method would be to produce a cDNA library using mRNA obtained from mutant S. typhimurium strains. Although the production of cDNA libraries from bacteria is not commonly done because of the usual absence of poly-A tails on prokaryotic messages, a cDNA library could be constructed from S . typhimurium mRNA.
- a method of preparing variants of the S. typhimurium exportation polypeptides is site-directed mutagenesis.
- This technique is useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, derived from the 46 kDa or 55 kDa protein sequence, through specific mutagenesis of the underlying DNA.
- the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
- Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
- a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
- the technique of site-specific mutagenesis is well known in the art as exemplified by publications (Adelman et al . , 1983). As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
- Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing et al. , 1981). These phage are readily commercially available and their use is generally well known to those skilled in the art.
- site-directed mutagenesis in accordance herewith is performed by first obtaining a single- stranded vector which includes within its sequence a DNA sequence which encodes an export polypeptide.
- An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Crea et al . (1978) .
- This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
- DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
- This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence.
- appropriate cells such as E. coli cells
- clones are selected which include recombinant vectors bearing the mutated sequence.
- sequence variants of the selected exportation polypeptide gene using site-directed mutagenesis is provided as a means of producing potentially useful exportation species and is not meant to be limiting as there are other ways in which sequence variants of the exportation polypeptide gene may be obtained.
- recombinant vectors encoding the desired gene may be treated with mutagenic agents to obtain sequence variants (see, e . g. , a method described by Eichenlaub, (1979) for the mutagenesis of plasmid DNA using hydroxylamine) .
- TnphoA is a derivative of Tn5 which encodes E. coli alkaline phosphatase, minus the signal sequence and expression signals, inserted into the left IS50L element. Random transposition of TnphoA results in an active insertion only when the phoA gene sequence is fused inframe downstream of the promoter and export signals of a target gene A, Figure 1. Plasmids containing phoA gene fusions can then be used as exposition vectors, Figure 1, (B) .
- the SSP1 and the PvuII restriction sites in phoA are blunt ended sites at which inframe insertions (IF) of a gene of interest (GOI) can be inserted.
- IF inframe insertions
- GOI gene of interest
- Figure 1 is a schematic representation of typical phoA fusions and illustrating cloning of successful fusions.
- the point at which the phoA sequence joins the target gene is referred to as the fusion joint (FJ) .
- the remaining portion of the gene begins at the distal joint (DJ) .
- Utilizing restriction enzymes which cut either downstream of the kanamycin resistance gene (e . g . , BamHI ) or the phoA gene sequence (e .g. , HindllX) allows cloning of phoA gene fusions, provided the target gene is not cleaved ("R”) .
- the fusion joint including all the phoA gene fusions and upstream Salmonella sequences, were cloned into the Hind TL or BaroHI site of pBR322, Figures 2 and 3. Plasmids containing phoA gene fusions were then used as exposition vectors. Cells produced fusion polypeptides that had alkaline phosphatase activity, indicated by the formation of blue colonies on agar supplemented with the indicator dye (5-bromo-4-chloro-3- indolylphosphate) .
- nucleic acid segments isolated from a strain of Salmonella similar functions may be obtained from nucleic acid segments from other Salmonella strains and even other microorganisms.
- the nucleic acid sequences identified and the corresponding encoded polypeptides are useful in developing methods of producing a wide variety of heterologous proteins as well as expression vectors for localizing polypeptides in selected areas of a host cell.
- This plasmid contains a chimeric gene including a DNA segment from a strain of Salmonella fused with a segment of the alkaline phosphatase gene lacking signal and expression sequences. When expressed in a suitable host cell, the fusion product is localized to the inner membrane/periplasmic space of the host cell.
- pZIP-IN Figure 3 is a derivative of pBR322 containing a BamHI fragment encoding alkaline phosphatase activity and kanamycin resistance inserted at the BamHI site.
- the BamHI fragment was cloned from a chromosomal DNA preparation of the TnphoA insertion mutant TAG28, which was constructed by TnphoA mutagenesis (see above) of S. typhimurium TA2361 (phoN mutant derived from LT2) .
- Chromosomal DNA was prepared from 50 ml of overnight growth of TAG28 in L-broth with vigorous shaking at 37°C.
- the bacterial culture was precipitated and washed once in phosphate buffered saline (pH 7.0).
- the washed bacterial pellet was resuspended in 10, ml of ice cold ET buffer [10 mM EDTA, 10 mM Tris-HCl (pH 8.0)]. Lysozyme was added to a concentration of 0.1 mg/ml and incubated for 15 minutes at 37 ⁇ C.
- sarkosyl-pronase solution (10% sarkosyl, 5 mg/ml pronase in ET buffer) was added and the solution was incubated for 1 hr at 37°C. The solution was then extracted 3 times with TE [(10 mM Tris HCl, 1 mM EDTA (pH 8.0)] saturated phenol followed by 3 extractions with chloroform:isoamyl alcohol (24:1). The aqueous phase was transferred to a 50 ml beaker on ice and one- half volume of 7.5 M ammonium acetate was added. Three volumes of ice cold absolute ethanol was gently layered on top of the solution.
- the chromosomal DNA was precipitated onto a glass rod by gently stirring the solution to mix the interface.
- the precipitated DNA was rinsed once in 70% ice cold ethanol and dissolved overnight in 2 ml of TE buffer at 4°C.
- the concentration of DNA was quantitated by measuring the O.D. at 260 nm.
- TAG 28 chromosomal DNA 2 ⁇ g was digested with BamHI at 37°C for 2 hrs. The solution was extracted once with TE saturated phenol, followed by 2 extractions with chloroform:isoamyl alcohol (24:1). The aqueous phase was removed and the DNA precipitated by the addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol followed by centrifugation in a microcentrifuge. 0.2 ⁇ g of pBR322 DNA was digested with BaraHI and prepared for ligation as above. Ligation of the vector DNA (pBR322) and TAG 28 chromosomal DNA was performed by overnight incubation at 4°C in 20 ⁇ l of IX commercial (Promega) ligase buffer and 2 U of T4 DNA ligase.
- pZIP-IN was isolated from the ligation reaction by transformation of subcloning efficiency DH5 ⁇ competent cells. 5 ⁇ l of the ligation mixture was added to 50 ⁇ l of DH5 ⁇ competent cells and incubated on ice for 30 minutes. Cells were heat shocked for 30 seconds by incubating in a 37°C water bath. Cells were cooled on ice for 2 minutes and 0.950 ml of L-Broth was added to the tube. Cells were incubated for 1 hr at 37°C.
- Transformants with alkaline phosphatase activity and kanamycin resistance were selected by plating 0.1 ml of the bacterial culture on the L-agar plates containing 50 ⁇ g/ml kanamycin and 40 ⁇ g/ml BCIP (5-bromo-4-chloro-3- indolyl phosphate) , followed by overnight incubation at 37°C. The following day, kanamycin resistant colonies were visible and all were blue, indicating the transformants had alkaline phosphatase activity. This was confirmed by alkaline phosphatase assays, Western blotting with monoclonal antibodies to alkaline phosphatase, and DNA sequencing of the fusion joint.
- Figure 3 shows a partial restriction map of pZIP-IN.
- the following example illustrates the construction of pZIP-OUT, Figure 2.
- the plasmid is constructed from a DNA segment of Salmonella and a PhoA DNA segment lacking signal and expression sequences. When expressed from a suitable host cell, the fusion protein is localized to the outer membrane of the host cell.
- Genomic DNA was isolated from strain TAP43. A 25 ml culture in LB broth was grown overnight at 37°C with shaking. The cells were harvested by centrifugation, and the pellet washed once in PBS. The washed pellet was resuspended in 10 mis of cold TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). One ml of a 1 mg/ml lysozyme solution was added, and the mixture was incubated in a 37°C water bath for fifteen minutes. After this incubation, 1.2 ml of 10% sarkosyl, 5 mg/ml pronase in TE buffer was added, and incubation continued at 37°C for 1- 2 hours, until cell lysis occurred.
- TE buffer 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
- the lysate was then extracted twice with an equal volume of phenol, once with phenol/chloroform, and once with chloroform. To the final extraction, a half-volume of 7.5 M ammonium acetate was added. The solution was mixed gently and placed on ice. Two volumes of ice-cold absolute ethanol were layered on top of the lysate, and the chromosomal DNA was collected at the interface by spooling on a glass rod. The spooled DNA was rinsed once in 70% ethanol, and then allowed to dissolve off of the glass rod into TE buffer overnight at 4°. The buffer, containing the dissolved DNA, was then ethanol-precipitated.
- the purified chromosome was collected by centrifugation and resuspended in a small volume of TE buffer. 1-5 ⁇ g of the purified DNA was restricted with -ffindlll, and then phenol/chloroform extracted and ethanol precipitated. The sample was collected by centrifugation, the pellet washed once with 70% ethanol, and dried under vacuum.
- Vector pUC18 was also restricted with Hindi , extracted, and precipitated in the same manner.
- the J ⁇ i-ndlll fragments of the genomic DNA were then ligated into the -ffindlll site of pUC18 with T4 DNA ligase.
- the DNA was transformed into competent DH5 ⁇ cells and plated on L-agar supplemented with ampicillin and BCIP (5-bromo-4-chloro-3-indolyl phosphate) , both at 40 ⁇ g/ml. Blue colonies, indicating the presence of an active alkaline phosphatase fusion in the transformant, were selected and analyzed by restriction mapping.
- Transformant 43-17 contained a 4.5 kp Hindlll insert in the pUCl ⁇ vector. 3.1 kp of this insert consisted of phoK sequences, with the remaining 1.4 kp being derived from Salmonella chromosomal sequences.
- Salmonella-phoK fusion contained within this Hindlll fragment was designated as the pZIP-OUT cassette. This cassette was subsequently cloned into the Findlll sites of the vectors pBR322 and pAT153. The general structure of pZIP-OUT is shown in Figure 2.
- the following example illustrates how DNA may be fused to the gene segments of plasmid pZIP-IN, shown in this example with a portion of the cholera subunit B gene.
- pIMB13 is a derivative of pZIP-IN in which the final 294 base pairs of ctxB have been inserted in frame with the phoA gene sequence at the Sspl site.
- the inserted fragment containing the ctxB gene sequence is from pRIT10810 which encodes the entire ctxB gene.
- the Sspl site in the pBR322 portion of pZIP-IN was eliminated as follows. 2 ⁇ g of a plasmid preparation of pZIP-IN was digested with Seal and EcoRV. Both enzymes cut at a single site within the pBR322 portion of the vector and generate compatible blunt ends.
- the digested DNA was precipitated and ligation was performed in 20 ⁇ l of IX ligase buffer containing 1 U of T4 DNA ligase overnight at 4°C.
- DH5 ⁇ frozen competent cells were transformed with 5 ⁇ l of the ligation reaction mixture.
- Transformants were selected on L-agar plates containing 50 ⁇ g/ml kanamycin. Colonies were then replicated to L-agar plates containing 40 ⁇ g/ml ampicillin. Loss of ampicillin resistance encoded by pZIP-IN indicated that the segment from Seal (3844) to -B ⁇ oRV (185) which contained the Sspl site (4168) had been eliminated. The resulting plasmid p ⁇ S28-l contained a single Sspl site in the phoA sequence which generates an in-frame blunt end cut.
- pIMB13 was constructed from p ⁇ S2 ⁇ -l as follows.
- the ctxB sequence encoded by pRITlO ⁇ lO contains an Sspl site which generates an in-frame blunt end cut near the 5' end of the structural gene.
- pRITlO ⁇ O also contains an Sspl site in the pBR322 portion of the vector. Digestion of pRITlOl ⁇ lO with Sspl generates 2 fragments, one of which contains the 3' final 294 base pairs of ctxB. 2 ⁇ g of p ⁇ S2 ⁇ -l and 2 ⁇ g of pRITlO ⁇ lO were digested with Sspl .
- White kanamycin resistant colonies were picked for isolation and screened for expression of a ctxB fusion protein by Western blotting of total envelope fractions with affinity purified anti- ctxB.
- a DH5 ⁇ strain harboring a derivative of pZIP-IN encoding a ctxB gene fusion was identified and the plasmid was designated pIMB13.
- the following example is an example of a tripartite fusion prepared from plasmid pZIP-OUT.
- This plasmid may be used to express a fusion polypeptide from suitable host cells.
- the DNA inserted in this example is a segment from cholera B toxin subunit.
- pRSPl ⁇ The construction of the trihybrid fusion, pRSPl ⁇ , was accomplished as follows. Plasmid pRITlO ⁇ lO, containing the cholera toxin B gene, was first restricted with Ec ⁇ R and Pstl. The ends generated by these restrictions were repaired with Klenow, and the vector was ligated back together. This created a .8 kp deletion in pRITlO ⁇ lO, eliminating an undesirable Sspl site in the vector. This deleted pRITlO ⁇ lO was then restricted with Hindlll and Sspl. pZIP-OUT (in vector pUCl ⁇ ) was doubly restricted with Hindlll and PvuII.
- This example illustrates the procedure for extracting and separating bacterial membranes. After isolation of the membrane fragments, they were analyzed for localization of fusion peptides.
- alkaline phosphatase assays were performed to test for enzyme activity in membrane fractions of host cells in which alkaline fusion proteins were expressed.
- Alkaline phosphatase activity encoded by pZIP-IN and pZIP-OUT was confirmed by spectrophotometric assay using the chromogenic alkaline phosphatase substrate para- nitrophenol phosphate (PNPP) .
- PNPP para- nitrophenol phosphate
- One ml of overnight bacterial cultures was pelleted for 15 seconds in a microcentrifuge. The pellet was washed once in 1 M Tris- HC1 (pH 8.0) and resuspended in 1 ml of 1 M Tris-HCl (pH 8.0). The O.D. 600 of the bacterial suspension was recorded. 50 ⁇ l of chloroform and 50 ⁇ l of 0.1% SDS were added to permeabilize the cells. Samples were vortexed briefly.
- the following example illustrates the expression of a ctxB polypeptide from an attenuated Salmonella strain with localization of the ctxB to the surface of the outer cell membrane.
- the following example illustrates the procedures contemplated as useful for creating an immune response in a mammal elicited with virulence attenuated Salmonella strains expressing antigens on the surface of the intact cell.
- CTB is used as an illustration.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP92925134A EP0613500A1 (fr) | 1991-11-15 | 1992-11-12 | Expression membranaire de genes heterologues |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US79225291A | 1991-11-15 | 1991-11-15 | |
US07/792,252 | 1991-11-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993010246A1 true WO1993010246A1 (fr) | 1993-05-27 |
Family
ID=25156267
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1992/009659 WO1993010246A1 (fr) | 1991-11-15 | 1992-11-12 | Expression membranaire de genes heterologues |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0613500A1 (fr) |
AU (1) | AU3130893A (fr) |
CA (1) | CA2123675A1 (fr) |
WO (1) | WO1993010246A1 (fr) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2273708A (en) * | 1992-12-14 | 1994-06-29 | Zeneca Ltd | Protein/cell membrane association assay |
WO1996011708A1 (fr) * | 1994-10-18 | 1996-04-25 | Board Of Regents, The University Of Texas System | Expression membranaire de genes heterologues |
WO2000044878A1 (fr) * | 1999-01-28 | 2000-08-03 | Werner Lubitz | Compartimentage de polypeptides recombinants dans des cellules hotes |
WO2001055169A1 (fr) * | 2000-01-27 | 2001-08-02 | Loma Linda University | Vaccins a base de plantes transgeniques |
US7422747B2 (en) | 1997-10-07 | 2008-09-09 | Loma Linda University | Transgenic plant-based vaccines |
US7700104B2 (en) | 1998-09-04 | 2010-04-20 | Emergent Product Development Uk Limited | Attenuated Salmonella SP12 mutants as antigen carriers |
US7842290B2 (en) | 1994-12-09 | 2010-11-30 | Emergent Product Development Uk Limited | Identification of genes |
US7887816B2 (en) | 1999-05-10 | 2011-02-15 | Emergent Product Development Uk Limited | Attenuated microorganisms for the treatment of infection |
US8703153B2 (en) | 2008-06-16 | 2014-04-22 | Prokarium Ltd. | Salmonella vectored vaccines against Chlamydia and methods of use |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0355737A2 (fr) * | 1988-08-24 | 1990-02-28 | BEHRINGWERKE Aktiengesellschaft | Expression de protéines fonctionnelles homologues et hétérologues sur la membrane extérieure de E.coli et autres bactéries gramnégatives |
EP0368819A1 (fr) * | 1988-09-16 | 1990-05-16 | Aktiebolag Vitec | Expression de la sous-unité liante de la toxine chloérique à l'aide de promoteurs étrangers et/ou de séquences guides |
DE3901681A1 (de) * | 1989-01-21 | 1990-07-26 | Behringwerke Ag | Signalpeptid fuer die sekretion von peptiden in escherichia coli, verfahren zu seiner gewinnung und seine verwendung |
EP0407259A1 (fr) * | 1989-06-26 | 1991-01-09 | Commissariat A L'energie Atomique | Protéines hybrides entre une enzyme extracytoplasmique et au moins une autre protéine, leur procédé de préparation, ainsi que leurs applications |
-
1992
- 1992-11-12 WO PCT/US1992/009659 patent/WO1993010246A1/fr not_active Application Discontinuation
- 1992-11-12 CA CA002123675A patent/CA2123675A1/fr not_active Abandoned
- 1992-11-12 EP EP92925134A patent/EP0613500A1/fr not_active Withdrawn
- 1992-11-12 AU AU31308/93A patent/AU3130893A/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0355737A2 (fr) * | 1988-08-24 | 1990-02-28 | BEHRINGWERKE Aktiengesellschaft | Expression de protéines fonctionnelles homologues et hétérologues sur la membrane extérieure de E.coli et autres bactéries gramnégatives |
EP0368819A1 (fr) * | 1988-09-16 | 1990-05-16 | Aktiebolag Vitec | Expression de la sous-unité liante de la toxine chloérique à l'aide de promoteurs étrangers et/ou de séquences guides |
DE3901681A1 (de) * | 1989-01-21 | 1990-07-26 | Behringwerke Ag | Signalpeptid fuer die sekretion von peptiden in escherichia coli, verfahren zu seiner gewinnung und seine verwendung |
EP0407259A1 (fr) * | 1989-06-26 | 1991-01-09 | Commissariat A L'energie Atomique | Protéines hybrides entre une enzyme extracytoplasmique et au moins une autre protéine, leur procédé de préparation, ainsi que leurs applications |
Non-Patent Citations (5)
Title |
---|
ABSTR. GEN. MEET. AM. SOC. MICROBIOL. vol. 91, 1991, page 34 J. MONCRIEF ET AL. 'Surface expression of a PhoA fusion protein from an invasion-attenuated Salmonella-typhimurium' 91st general meeting of the american society for microbiology 1991, Dallas, Texas, USA, may 5-9 1991 abstract B-55 * |
ABSTR. GEN. MEET. AM. SOC. MICROBIOL. vol. 92, 1992, page 225 L. PHILLIPS ET AL. 'Salmonella exposition vectors derived from TnPhoA fusion strains' 92nd general meeting of the american society for microbiology, New Orleans, Louisianna, USA, may 26-30, 1992 abstract H-256 * |
GENE vol. 104, no. 2, 15 August 1991, ELSEVIER, AMSTERDAM, NL; pages 147 - 153 F. BREITLING ET AL. 'A surface expression vector for antibody screening' * |
MOLECULAR MICROBIOLOGY vol. 3, no. 12, December 1989, DEVONSHIRE PRESS, TORQUAY, GB; pages 1669 - 1683 H.W. STOKES AND R.M. HALL 'A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons' * |
PROC. NATL. ACAD SCI. vol. 82, no. 23, December 1985, NATL. ACAD SCI., WASHINGTON, DC,US; pages 8129 - 8133 C. MANOIL AND J. BECKWITH 'TnphoA: A transposon probe for protein export signals' * |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2273708B (en) * | 1992-12-14 | 1996-08-21 | Zeneca Ltd | Method for identifying compounds which modulate protein/cell membrane association |
US5776675A (en) * | 1992-12-14 | 1998-07-07 | Zeneca Limited | Identification of compounds modulating protein/cell membrane association |
GB2273708A (en) * | 1992-12-14 | 1994-06-29 | Zeneca Ltd | Protein/cell membrane association assay |
WO1996011708A1 (fr) * | 1994-10-18 | 1996-04-25 | Board Of Regents, The University Of Texas System | Expression membranaire de genes heterologues |
US7842290B2 (en) | 1994-12-09 | 2010-11-30 | Emergent Product Development Uk Limited | Identification of genes |
US7422747B2 (en) | 1997-10-07 | 2008-09-09 | Loma Linda University | Transgenic plant-based vaccines |
US7700104B2 (en) | 1998-09-04 | 2010-04-20 | Emergent Product Development Uk Limited | Attenuated Salmonella SP12 mutants as antigen carriers |
US7955600B2 (en) | 1998-09-04 | 2011-06-07 | Emergent Product Development Uk Limited | Attenuated salmonella SP12 mutants as antigen carriers |
CN100359001C (zh) * | 1999-01-28 | 2008-01-02 | 沃纳·卢比茨 | 宿主细胞中重组多肽的区室化 |
US6610517B1 (en) | 1999-01-28 | 2003-08-26 | Werner Lubitz | Compartmentalization of recombinant polypeptides in host cells |
WO2000044878A1 (fr) * | 1999-01-28 | 2000-08-03 | Werner Lubitz | Compartimentage de polypeptides recombinants dans des cellules hotes |
US7887816B2 (en) | 1999-05-10 | 2011-02-15 | Emergent Product Development Uk Limited | Attenuated microorganisms for the treatment of infection |
WO2001055169A1 (fr) * | 2000-01-27 | 2001-08-02 | Loma Linda University | Vaccins a base de plantes transgeniques |
US8703153B2 (en) | 2008-06-16 | 2014-04-22 | Prokarium Ltd. | Salmonella vectored vaccines against Chlamydia and methods of use |
Also Published As
Publication number | Publication date |
---|---|
AU3130893A (en) | 1993-06-15 |
EP0613500A1 (fr) | 1994-09-07 |
CA2123675A1 (fr) | 1993-05-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5356797A (en) | Membrane expression of heterologous genes | |
Engleberg et al. | DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity | |
DE69131014T2 (de) | Rekombinante avirulente salmonella antifruchtbarkeitsimpfstoffe | |
AU722088B2 (en) | Compositions and methods for administering BORRELIA DNA | |
Hannavy et al. | TonB protein of Salmonella typhimurium: a model for signal transduction between membranes | |
JP3242106B2 (ja) | 組換えdna由来コレラトキシンサブユニット類似体 | |
US5571718A (en) | Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7 | |
CA2265852A1 (fr) | Introduction ciblee d'antigenes dans la voie de traitement du cmh de classe i au moyen d'une proteine de fusion de la toxine du bacille du charbon | |
US6737521B1 (en) | Delivery and expression of a hybrid surface protein on the surface of gram positive bacteria | |
WO1993010246A1 (fr) | Expression membranaire de genes heterologues | |
US20040023325A1 (en) | Altered OspA of Borrelia burgdorferi | |
EP0646176B1 (fr) | Apport et expression d'une proteine de surface hybride sur la surface de bacteries gram positif | |
JP2887238B2 (ja) | ポリペプチド | |
DE69227898T2 (de) | Rekombinante hybride porinepitope | |
Mallavia et al. | The genetics of Coxiella burnetii: etiologic agent of Q fever and chronic endocarditis | |
WO1993024636A1 (fr) | Emploi de la proteine oprf dans l'expression d'oligopeptides a la surface de cellules bacteriennes | |
WO1996011708A1 (fr) | Expression membranaire de genes heterologues | |
US7601350B2 (en) | Antibodies that bind M. tuberculosis polypeptides | |
EP0556182A1 (fr) | Adn capable d'une integration a specificite de site dans des mycobacteries | |
JPH02502784A (ja) | コクシジウム感染の診断用ポリペプチドとその組換えdna法による製造法 | |
US20040076976A1 (en) | Aopb gene, protein,homologs, fragments and variants thereof, and their use for cell surface display | |
US6210948B1 (en) | Expression and secretion of heterologous polypeptides from caulobacter | |
CA2079027A1 (fr) | Expression accrue de polypeptides recombinants a faible poids moleculaire | |
Pohlner et al. | Serological properties and processing in Escherichia coli K12 of OmpV fusion proteins of Vibrio cholerae | |
WO1997008322A1 (fr) | Gene mpt83 extrait de mycobacterium tuberculosis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AT AU BB BG BR CA CH CS DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO PL RO RU SD SE UA |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE BF BJ CF CG CI CM GA GN ML MR SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2123675 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1992925134 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1992925134 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1992925134 Country of ref document: EP |