EP0964924A1 - Iron regulated promoter and uses thereof - Google Patents
Iron regulated promoter and uses thereofInfo
- Publication number
- EP0964924A1 EP0964924A1 EP97933590A EP97933590A EP0964924A1 EP 0964924 A1 EP0964924 A1 EP 0964924A1 EP 97933590 A EP97933590 A EP 97933590A EP 97933590 A EP97933590 A EP 97933590A EP 0964924 A1 EP0964924 A1 EP 0964924A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- recombinant
- polypeptide
- sequence
- host cell
- promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 27
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 21
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 39
- 239000013598 vector Substances 0.000 claims abstract description 27
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 18
- 239000012634 fragment Substances 0.000 claims abstract description 15
- 241001465754 Metazoa Species 0.000 claims abstract description 7
- 230000028993 immune response Effects 0.000 claims abstract description 6
- 230000001939 inductive effect Effects 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 45
- 229920001184 polypeptide Polymers 0.000 claims description 44
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 44
- 210000004027 cell Anatomy 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 21
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 20
- 210000000349 chromosome Anatomy 0.000 claims description 16
- 102000053602 DNA Human genes 0.000 claims description 14
- 241000607142 Salmonella Species 0.000 claims description 12
- 241000243796 Trichostrongylus colubriformis Species 0.000 claims description 12
- 108020004511 Recombinant DNA Proteins 0.000 claims description 10
- 101000607560 Homo sapiens Ubiquitin-conjugating enzyme E2 variant 3 Proteins 0.000 claims description 9
- 102100039936 Ubiquitin-conjugating enzyme E2 variant 3 Human genes 0.000 claims description 9
- 108050001049 Extracellular proteins Proteins 0.000 claims description 8
- 102000040739 Secretory proteins Human genes 0.000 claims description 8
- 108091058545 Secretory proteins Proteins 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 6
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 5
- 230000037431 insertion Effects 0.000 claims description 5
- 102100034343 Integrase Human genes 0.000 claims description 2
- 108010061833 Integrases Proteins 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 description 44
- 108090000623 proteins and genes Proteins 0.000 description 42
- 241001494479 Pecora Species 0.000 description 15
- 230000010354 integration Effects 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 11
- 210000003578 bacterial chromosome Anatomy 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 108010005774 beta-Galactosidase Proteins 0.000 description 8
- KDHHWXGBNUCREU-HOTGVXAUSA-N Ferric-aerobactin Chemical compound CC(=O)N(O)CCCC[C@@H](C(O)=O)NC(=O)CC(O)(C(O)=O)CC(=O)N[C@H](C(O)=O)CCCCN(O)C(C)=O KDHHWXGBNUCREU-HOTGVXAUSA-N 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 102000005936 beta-Galactosidase Human genes 0.000 description 7
- 230000002759 chromosomal effect Effects 0.000 description 7
- 235000013601 eggs Nutrition 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 6
- 108010015268 Integration Host Factors Proteins 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 230000002238 attenuated effect Effects 0.000 description 5
- 238000007918 intramuscular administration Methods 0.000 description 5
- 244000045947 parasite Species 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000701835 Salmonella virus P22 Species 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 229960001212 bacterial vaccine Drugs 0.000 description 4
- 235000003642 hunger Nutrition 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000037351 starvation Effects 0.000 description 4
- 230000005875 antibody response Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 101150062334 int gene Proteins 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical group N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 2
- 241001131785 Escherichia coli HB101 Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- 241000244206 Nematoda Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 108010052418 (N-(2-((4-((2-((4-(9-acridinylamino)phenyl)amino)-2-oxoethyl)amino)-4-oxobutyl)amino)-1-(1H-imidazol-4-ylmethyl)-1-oxoethyl)-6-(((-2-aminoethyl)amino)methyl)-2-pyridinecarboxamidato) iron(1+) Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101150007653 Arsa gene Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010054278 Lac Repressors Proteins 0.000 description 1
- 241001180649 Myrcia group Species 0.000 description 1
- 102000009661 Repressor Proteins Human genes 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 101150111036 arsB gene Proteins 0.000 description 1
- AQLMHYSWFMLWBS-UHFFFAOYSA-N arsenite(1-) Chemical compound O[As](O)[O-] AQLMHYSWFMLWBS-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000007940 bacterial gene expression Effects 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- OEUUFNIKLCFNLN-LLVKDONJSA-N chembl432481 Chemical compound OC(=O)[C@@]1(C)CSC(C=2C(=CC(O)=CC=2)O)=N1 OEUUFNIKLCFNLN-LLVKDONJSA-N 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 101150046339 fur gene Proteins 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001415 gene therapy 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
- 238000009396 hybridization Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 230000028744 lysogeny Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000009021 pre-vaccination Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000001262 western blot Methods 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
-
- 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
Definitions
- This invention relates to promoters and their use for the expression of polypeptides and in particular their use in live vaccines.
- the present invention also provides a method of integrating foreign genes into the chromosome of bacteria and to bacterial vaccines expressing foreign genes.
- the Esche ⁇ chia coli bacterium displays a characteristic response to iron starvation which is shared by many other aerobic and facultative anaerobic microorganisms, such as Salmonella typhimu ⁇ um. Under conditions where the iron concentration is less than about 5 ⁇ M the transcription of a collection of genes or groups of genes scattered through the chromosome is coordinately activated in a manner resembling an iron- controlled regulon. Iron control is in all instances mediated by negative regulation via the product of the fur gene, the absence of which triggers the expression of the genes to constitutive levels. The Fur protein also regulates expression of the aerobac tin-mediated iron transport regulon.
- the present inventors have developed a system involving the use of a novel hybrid iron-regulated promoter which can be induced to hyper-express polypeptide encoding genes.
- the promoter can provide an optimal polypeptide dose to the host immune system.
- the inventors mean a DNA molecule having a nucleic acid sequence which (as a portion of a larger DNA molecule) is effective in inducing the expression of a polypeptide- encoding gene or genes localised downstream of the promoter DNA sequence.
- the polypeptide may comprise one or more antigenic determinants of a pathogenic organism, and may be derived from a virus, bacterium, fungus, yeast or parasite.
- Live attenuated Salmonella strains are currently being developed world-wide as carriers of foreign antigens from viral, bacterial and parasite origin. These recombinant salmonellae are being used to immunise the animal or human host to elicit a protective immune response against the respective infection. As mentioned above these recombinant Salmonella- based vaccines suffer from a number of drawbacks.
- One major problem is that they typically carry the foreign antigen gene on self-replicating plasmids. These plasmids are often unstable and are lost from the bacterial cell in vivo resulting in a loss of the foreign antigen gene and consequently the foreign protein. This plasmid loss results in a reduced antigen dose being presented to the host immune system with a consequent sub-optimal protective immune response.
- the present invention consists in an iron-regulated promoter comprising the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence.
- the iron-regulated promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1.
- the present invention consists in a recombinant
- DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding a polypeptide.
- the promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1.
- polypeptide includes at least one at least one epitope. It is preferred that the polypeptide includes B-cell and/or T-cell epitopes. In another embodiment the polypeptide includes at least one CTL epitope.
- a preferred polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
- the present invention consists in a recombinant vector, the vector comprising an iron-regulated promoter and a site for insertion of a sequence encoding at least one polypeptide such that the inserted sequence is in frame with the iron-regulated promoter, wherein the iron-regulated promoter comprises the DNA sequence shown in Fig. 1. or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence.
- the vector further includes an attP sequence and preferably the vector further includes a sequence encoding integrase protein.
- the vector further includes a sequence encoding at least one polypeptide inserted at the insertion site. It is preferred that the encoded polypeptide includes at least one epitope. Typically the encoded peptide will B and/or T-cell epitopes.
- the encoded polypeptide includes at least one CTL epitope.
- the sequence encoding the polypeptide may encode a plurality of CTL epitopes. Such an arrangement is disclosed in PCT/AU95/00461 the disclosure of which is incorporated herein by reference.
- the encoded polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
- the present invention consists in a recombinant host cell, the host cell including a recombinant DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig, 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding at least one polypeptide.
- the recombinant DNA molecule is inserted into the host cell chromosome.
- the host cell is a bacterium, preferably Gram negative, and more preferably the bacterium is Escherichia coli or
- Salmonella species and preferably the Salmonella species is Salmonella typhimu ⁇ um.
- the encoded polypeptide includes at least one at least one epitope. It is preferred that the polypeptide includes B-cell and/or T-cell epitopes. In another embodiment the polypeptide includes at least one CTL epitope.
- a preferred polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
- the present invention consists in a composition for use in inducing an immune response in an animal, the composition comprising a recombinant host cell of the present invention and an acceptable carrier.
- the term "functionally equivalent nucleic acid sequence" is intended to cover minor variations in the promoter sequence which do not result in a promoter having substantially lower activity from the promoter defined from residues 284 to 409 in Fig. 1.
- the vector of the present invention includes the bacteriophage P22 int gene and an attP region. When this vector is introduced into a suitable bacterium, the bacteriophage P22 int gene is expressed to produce the int protein.
- a short DNA sequence called attP (attachment) in the vector is homologous to the attB sequence found in the bacterial chromosome.
- the bacterial chromosome expresses a protein called IHF (integration host factor) and that protein interacts with the expressed int protein from the vector which causes the homologous recombination of the vector DNA and the chromosomal DNA between attP and attB sequences.
- IHF integration host factor
- attB and attP recombine and stably integrate the vector into the bacterial chromosome.
- the vector includes a DNA molecule encoding a foreign gene, upon integration, the bacterium is capable of expressing that gene from its chromosome to produce the foreign protein.
- the bacterium is an attenuated vaccine bacterium and the heterologous protein is derived from a virus, bacterium, fungus, yeast or parasite. More preferably, the bacterium is a S. typhimurium strain and the polypeptide is the 37 kD extra cellular/secretory protein from the nematode parasite of sheep Trichostrongylus colubriformis.
- the present inventors have developed a site-specific chromosomal integration system to integrate foreign antigen genes into the chromosome of attenuated S. typhimurium strains. The advantages of this system include:
- the system does not use mobile genetic elements (e.g. transposons) and therefore once the vaccine strain is constructed the genotype and phenotype will not be altered by genes hopping around to other sites in the bacterial chromosome. 3.
- the efficiency of chromosomal integration approaches 100% i.e. all bacterial cells that are transformed by this DNA are found to have the heterologous polypeptide genes integrated in the bacterial chromosome.
- the system is "user-friendly” and requires a single step cloning event to clone the gene of interest into the "chromosomal integration vector" followed by transformation of the attenuated S. typhimurium strain of choice.
- This system is highly versatile and can be adapted for integration of genes into the chromosomes of a wide range of Gram-negative bacteria as well as eucaryotic cells.
- the applications of this technology range widely and can be used as (a) basic-research tool to study chromosomally integrated genes in Gram- negative bacteria and Eucaryotic cells, (b) preparation of live attenuated vaccines, (c) tissue specific expression in gene therapy, and (d) development of transgenic plants and animals.
- Figure 1 shows the complete DNA sequence encoding a hybrid iron- regulated promoter according to the present invention
- Figure 2 shows the construction of plasmids including promoters according to the present invention
- Figure 3 shows details of the plasmid used for chromosomal integration in Salmonella
- Figure 4 shows anti-37KD serum titers
- Figure 5 shows anti ⁇ -galactosidase serum antibody titers
- Figure 6 shows egg counts
- Figure 7 shows worm count in sheep intestinal lumen.
- the promoter was initially cloned as an EcoRI/BamHI fragment is plasmid pSU207 (Fig. 2).
- Plasmid pHBl ⁇ (Fig. 2) is a pBR322 based plasmid carrying a polylinker.
- PCR primers HB38 and HB39 were designed to amplify part of the aerobactin gene promoter between positions 10 and 394 on the aerobactin promoter sequence. This region carriers minor and major promoters P2 and P2 respectively, along with the primary and secondary Fur binding sites.
- the Shine Dalgarno and downstream DNA sequence of the aerobactin promoter were not amplified.
- the PCR primers carried the Notl and Pad restriction enzyme cleavage sites and the shortened aerobactin promoter was subcloned into the Notl/Pacl sites of pHBl58 to give plasmid designated pHBl64 (Fig. 2).
- the 37 kD extracellular/secretory protein encoding gene from nematode parasite of sheep Trichostrongylus colubriformis was cloned as a lacZ gene fusion in plasmid pHbl67(Fig. 2). Upstream of the 5' region of the 37 kD gene sequence was engineered a DNA sequence carrying the Pad restriction enzyme site followed by Shine Dalgarno sequence (AGGA), a 7 bp spacer sequence (AACAGCT) and a translation start codon (ATG).
- the Pacl/Xbal fragment form plasmid pHBl67 (Fig. 2) carrying the upstream region, along with the 37 kD/7 ⁇ cZ genes was subcloned into the Pacl/Xbal sites of plasmid pHBl64 to give plasmid designated pHBl70(Fig. 2).
- This fusion of the aerobactin promoter (paer) with the upstream region provided a novel iron-regulated promoter according to the present invention.
- This promoter is unique in carrying the aerobactin promoter gene sequences for Pi and P2, as well as designed primary and secondary Fur binding sites, and designed Shine Dalgarno, spacer and ATG sequences.
- the important sequence of the iron-regulated promotor lies from DNA sequence residues 284 to 409 inclusive. This region includes the -35 (residues 284 to 289) and -10 (residues 307 to 312) regions of minor promotor P2, the -35 (residues 340 to 345) -10 (residues 363 to 368) regions of major promotor Pi, the primary (residues 333 to 363) and secondary
- Plasmid pHBl70 was transformed into E. coli strain HB101 and S. typhimurium ⁇ ro ⁇ -strain.
- the strains were grown in vitro under iron-rich conditions in Luria Broth with 200 ⁇ M FeCl 3 .
- Cultures for iron starvation were grown under identical conditions but with the inclusion of 100 ⁇ M 2,2'- dipyridyl (iron chelator).
- the FeCl 3 and 2,2'-dipyridyl concentrations were optimised to obtain maximal repression and induction respectively without affecting growth rate or viability of the organisms.
- Bacteriophage P22 integrates its genome into the chromosome of S. typhimurium by the following mechanism:
- P22 attaches to the bacterial cell surface and injects its DNA into the bacterial cell.
- the injected DNA circularises and expresses the INT (integration) protein encoded by the int gene.
- the P22 genome has a short DNA sequence called the attP (attachment) which is homologous to the attB sequence found in the Salmonella chromosome.
- the bacterial chromosome expresses a protein called the IHF (integration host factor).
- the INT and IHF proteins interact to homologously recombine the attP and attB sequences thereby integrating the P22 genome into the bacterial chromosome (lysogeny).
- Plasmid pNEB193 purchased from New England Biolabs
- plac/lac gene fragment which encodes the lac repressor protein, was PCR amplified from plasmid pLOF/Ars and cloned into the Pacl/Xbal restriction sites of plasmid pNEBl93 to result in plasmid pHBl78.
- the ptac promoter gene fragment from plasmid pKK223-3 was PCR amplified and cloned into the Sacl/Kpnl sites of plasmid pHBl78 to result in plasmid pHBl79.
- the p ⁇ er/ArsA/ArsB gene fragment which encodes Arsenite resistance was PCR amplified from plasmid pLOF/Ars and cloned into the Hindi II site of plasmid pHBl79 to result in plasmid pHBl ⁇ O.
- the bacteriophage P22 int attP region including its ribosome binding site-spacer-translation start (ATG) was PCR amplified from the P22 phage genome. This gene fragment was cloned into the Kpnl/Pacl site of plasmid pHBl ⁇ O to result in plasmid pHBl ⁇ l. Transformation of plasmid pHB181 into Salmonella typhimurium strain and confirmation of chromosomal integration.
- Plasmid pHBl ⁇ l was transformed into S. typhimurium strain H4004 and transformants were selected on Brain Heart Infusion agar containing Ampicillin. The transformation efficiency was 10° colony forming units (c.f.u.) per microgram of DNA and was the same as control plasmid DNA. Plasmid DNA from individual transformants were analysed by agarose gel electrophoresis and the results showed that the plasmid had integrated into the chromosome. DNA from control E. coli cells harbouring the same plasmid revealed the presence of the plasmid. Southern hybridisation with the plasmid as a probe was used to confirm that the plasmid had integrated into the chromosome.
- the groups include: 1. S. typhimurium aroA- [strain 4335] - oral (10 1 1 organisms).
- the plasmid used for this integration is shown schematically in Fig 3. 6. Same as (5) - intramuscular (10 9 organisms).
- the serum was analysed in ELISA assay to detect antibodies to the T. colubriformis 37kD polypeptide and to the ⁇ -galactosidase reporter polypeptide.
- the serum antibody titers are shown in Figs. 4 and 5.
- T. colubriformis eggs were counted in fecal samples at various time points. Results of egg counts are shown in Fig. 4. The sheep were euthanased about 2 months after commencement of challenge and the intestinal linings were scraped to collect T. colubriformis worms. The worm count data is shown in Fig. 5.
- S. typhimurium aroA- carrying the plasmid pHBl70 (iron regulated promoter-37kD-lacZ) showed no serum antibody response to either 37kD or the ⁇ -galactosidase polypeptides.
- worm count data (Fig. 5) parallels that of egg counts and the i.m. vaccinated sheep (chromosomally integrated) had a significant (p ⁇ 0.05) reduction of 61% in worm burden.
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPO2164A AUPO216496A0 (en) | 1996-09-06 | 1996-09-06 | Inducible promoters |
AUPO216496 | 1996-09-06 | ||
AUPO245496 | 1996-09-19 | ||
AUPO2454A AUPO245496A0 (en) | 1996-09-19 | 1996-09-19 | Site-specific chromosomal integration system |
AUPO7801A AUPO780197A0 (en) | 1997-07-09 | 1997-07-09 | Site-specific chromosomal integration system incorporating inducible promoter |
AUPO780196 | 1997-07-09 | ||
PCT/AU1997/000503 WO1998010064A1 (en) | 1996-09-06 | 1997-08-08 | Iron regulated promoter and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0964924A1 true EP0964924A1 (en) | 1999-12-22 |
EP0964924A4 EP0964924A4 (en) | 2001-04-18 |
Family
ID=27157946
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97933590A Withdrawn EP0964924A4 (en) | 1996-09-06 | 1997-08-08 | Iron regulated promoter and uses thereof |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0964924A4 (en) |
AR (1) | AR008171A1 (en) |
BR (1) | BR9711692A (en) |
CA (1) | CA2265883A1 (en) |
NZ (1) | NZ334649A (en) |
UY (1) | UY24698A1 (en) |
WO (1) | WO1998010064A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994019482A1 (en) * | 1993-02-22 | 1994-09-01 | The General Hospital Corporation | Heterologous antigens in live cell vaccine strains |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE190660T1 (en) * | 1990-12-18 | 2000-04-15 | Gen Hospital Corp | IMPROVED VACCINES |
-
1997
- 1997-08-08 BR BR9711692-0A patent/BR9711692A/en unknown
- 1997-08-08 NZ NZ334649A patent/NZ334649A/en unknown
- 1997-08-08 CA CA002265883A patent/CA2265883A1/en not_active Abandoned
- 1997-08-08 EP EP97933590A patent/EP0964924A4/en not_active Withdrawn
- 1997-08-08 WO PCT/AU1997/000503 patent/WO1998010064A1/en not_active Application Discontinuation
- 1997-09-05 UY UY24698A patent/UY24698A1/en not_active Application Discontinuation
- 1997-09-08 AR ARP970104095A patent/AR008171A1/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994019482A1 (en) * | 1993-02-22 | 1994-09-01 | The General Hospital Corporation | Heterologous antigens in live cell vaccine strains |
US5747028A (en) * | 1993-02-22 | 1998-05-05 | The President And Fellows Of Harvard College | Immunizing compositions comprising Vibrio cholerae expressing heterologous antigens |
Non-Patent Citations (5)
Also Published As
Publication number | Publication date |
---|---|
UY24698A1 (en) | 1998-02-12 |
CA2265883A1 (en) | 1998-03-12 |
WO1998010064A1 (en) | 1998-03-12 |
EP0964924A4 (en) | 2001-04-18 |
NZ334649A (en) | 2000-08-25 |
AR008171A1 (en) | 1999-12-09 |
BR9711692A (en) | 2000-10-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Galyov et al. | Nucleotide sequence of the Yersinia pestis gene encoding F1 antigen and the primary structure of the protein: Putative T and B cell epitopes | |
JPH07502646A (en) | Bacterial expression vector containing DNA encoding a lipoprotein secretion signal | |
CA2323634A1 (en) | Lactobacilli harboring aggregation and mucin binding genes as vaccine delivery vehicles | |
WO1990011687A1 (en) | VACCINES CONTAINING AVIRULENT phoP-TYPE MICROORGANISMS | |
EP0544685A1 (en) | Mycobacterial expression vector | |
WO1991013157A1 (en) | Shuttle plasmid for escherichia coli and mycobacteria | |
Cárdenas et al. | Stability, immunogenicity and expression of foreign antigens in bacterial vaccine vectors | |
JP2007332149A (en) | Vaccine against mycobacterial infection | |
US5238823A (en) | Interleukin-2-leukotoxin gene fusions and uses thereof | |
Rezaei et al. | Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis | |
Kramer et al. | Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains | |
JPH05317039A (en) | Manipuration of number of copied gene in bordetella | |
EP0931093B1 (en) | Helicobacter pylori live vaccine | |
JPH09508012A (en) | Surface layer protein expression | |
US7118758B1 (en) | Transformed bacteria producing CS6 antigens as vaccines | |
JPH07504082A (en) | M. Paratuberculosis promoter and its use for the expression of immunogenic sequences | |
AU737981B2 (en) | Iron regulated promoter and uses thereof | |
WO1998010064A1 (en) | Iron regulated promoter and uses thereof | |
WO1988005817A1 (en) | Expression of the p. falciparum circumsporozoite protein by yeast | |
CA2730741C (en) | Self-replicating vector lacking an antibiotic-resistance gene | |
Moore et al. | Foreign gene expression in Corynebacterium pseudotuberculosis: development of a live vaccine vector | |
WO2000005252A1 (en) | Vaccine comprising a non-toxic immunogenic derivative of clostridium botulinum type d neurotoxin | |
JPH06121688A (en) | Protective plasmodium falciparum hybrid protein containing partial sequence of malaria antigen hrpii and serp and its preparation and use | |
CN116983397B (en) | Streptococcus iniae DNA vaccine, preparation method and application | |
US5895756A (en) | Non-antibiotic system for selection of recombinant mycobacteria |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19990311 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20010307 |
|
AK | Designated contracting states |
Kind code of ref document: A4 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
17Q | First examination report despatched |
Effective date: 20030808 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20031219 |