WO1992001703A1 - Production de l'antigene majeur de piline cs1 et compositions pour ce dernier comprenant des vaccins et des sondes diagnostiques - Google Patents

Production de l'antigene majeur de piline cs1 et compositions pour ce dernier comprenant des vaccins et des sondes diagnostiques Download PDF

Info

Publication number
WO1992001703A1
WO1992001703A1 PCT/US1991/005217 US9105217W WO9201703A1 WO 1992001703 A1 WO1992001703 A1 WO 1992001703A1 US 9105217 W US9105217 W US 9105217W WO 9201703 A1 WO9201703 A1 WO 9201703A1
Authority
WO
WIPO (PCT)
Prior art keywords
csi
major
antigen
pilin
pilin antigen
Prior art date
Application number
PCT/US1991/005217
Other languages
English (en)
Inventor
June R. Scott
Jose Perez-Casal
Original Assignee
Emory University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Emory University filed Critical Emory University
Publication of WO1992001703A1 publication Critical patent/WO1992001703A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the cloning of the structural gene for the major CSI pilin antigen of enterotoxigenic Escherichia coli. More particularly, this invention relates to methods and compositions for the cloning and expression of this gene, molecular probes for the accurate identification of enterotoxigenic E. coli in infected body tissues and fluids, and vaccines utilizing at least part of the major CSI pilin antigen as an immunogen.
  • ETEC E. coli
  • Enterotoxigenic strains of E. coli which colonize the intestinal epithelium, are an important cause of diarrheal disease in infants, in travelers, and in animals.
  • virulent ETEC strains must possess an adhesin, usually associated with pili on the bacterial surface, which mediates species-specific colonization of host cells.
  • CFA/I colonization factor antigens I, II and IV
  • CFA/II colonization factor antigens I, II and IV
  • CFA/II was defined as one serological entity, but later studies indicated that it is composed of three antigenically different structures: CSI, CS2, and CS3 (coli surface antigens) .
  • CFA/IV is also composed of three antigenically distinct members, CS4, CS5, and CS6. Electron microscopy shows CFA/I, CSI, CS2, CS4, and CS5 to be pili and CS3 to be fine, flexible "fibrils". CS6 has not yet been resolved in the electron microscope.
  • the genes required for expression of different pili show three types of structural organization.
  • the genes required for production of the pilus structure are closely linked in a single region, either on the chromosome or on a plasmid. This type of organization occurs for the pap- related pili of uropathogenic E. coli. for the afimbrial adhesion AFA-1 also found on uropathogenic E. coli. for K88 and K99 found on ETEC strains that infect animals, for the F pili of E. coli K12, and for the fibrillar CS3 adherence structure of human ETEC strains.
  • proteins encoded in two regions of a single plasmid are required for expression of the pilus. This is exemplified by the CFA/I pilus in clinical isolates of ETEC strains. Region 1 contains the structural gene for the major pilin antigen, while region 2 possesses a regulatory gene, cfaR.
  • CSI and CS2 The third type of organization occurs for CSI and CS2. These coli surface antigens are never produced in the same strain and expression of their major antigens requires the presence of a plasmid, implying a genetic contribution from the non-plasmid DNA of the host as well.
  • the only region of the plasmid required for expression CSI or CS2 encodes a positive regulatory protein named Rns, which has strong homology with cfaR and can substitute for it functionally.
  • the present invention relates to the cloning and sequencing of the structural gene, named cooA (coli surface antigen one) , for the major CSI pilin antigen of enterotoxigenic Escherichia coli.
  • Subclones that produce the CSI antigen were isolated; these clones were generated from a Sau3AI cosmid library composed of the total DNA from an E. coli strain containing the structural gene but not rns.
  • To test for production of CSI by the clones western blots were performed on total cell extracts whereas heat extracts were used to test for the presence of the CSI antigen on the surface of the cells.
  • cooA The location of cooA was determined by performing western blots, with and without the plasmid that expresses Rns, on total cell extracts of strains containing these subclone plasmids or plasmids constructed from restriction endonuclease-cleaved products of these plasmids.
  • the area in which cooA was located was sequenced and a vector containing the DNA sequence that codes for the CSI antigen was constructed. This vector was used to transform a microbial host that also contained the operon encoding Rns, the positive regulator of CSI expression. Production of the major CSI pilin antigen can then be obtained by culturing this microbial host and recovering the CSI antigen from the culture.
  • Hybridization indicated that in a CSl-producing ETEC strain, cooA is located on a plasmid different from the one encoding Rns, the positive regulator of CSI antigen expression.
  • the amino acid sequence shows that the CSI antigen and CfaB, the major subunit of CFA/I pilin are similar, although there is no antigenic cross- reactivity between the two proteins.
  • the present invention provides for a vaccine comprised of an immunologically effective amount of the CSI antigen, or a portion of this antigen that has at least one immunoreactive and antigenic determinant.
  • a DNA probe capable of binding to the DNA sequence that codes for the CSI antigen, or a portion of this antigen can be constructed and used for the diagnostic identification of the CSI antigen in body tissues or fluids of animals.
  • one of the objectives of this invention is to provide the DNA sequence coding for the major CSI pilin antigen of enterotoxigenic E. coli or any portion of the sequence that has at least one immunoreactive and antigenic determinant of this CSI antigen.
  • the present invention provides for a vector containing the DNA sequence that codes for this CSI antigen and a microbial host transformed by this vector.
  • this invention provides a process that produces the major CSI pilin antigen by culturing a microbial host that contains both the DNA sequence that codes for the CSI pilin antigen and rns. the operon encoding the CSI positive regulatory protein, followed by recovery of the CSI antigen from the culture.
  • the DNA probe of this invention may be complementary to the complete major CSI pilin antigen gene or only a portion of the gene so long as the probe permits specific hybridization to at least a portion of the DNA sequence for the CSI pilin antigen.
  • the instant invention provides a method for diagnosing the presence of the major CSI pilin antigen in an animal by detecting hybridization of this DNA probe with DNA obtained from body tissues or fluids of the animal.
  • a vaccine comprised of an immunologically effective amount of the major CSI pilin antigen, or any portion of the antigen having at least one immunoreactive and antigenic determinant, and a physiologically acceptable carrier.
  • a vaccine comprised of a synthetic immunorecessive epitope of the major CSI pilin antigen containing an antigenic determinant common to another type of pili.
  • Figure 1 shows a restriction map of CSI clones and their expression.
  • Figure 2 shows the nucleotide sequence of cooA.
  • Figure 3 shows a comparison of the amino acid sequences obtained by translation of cooA and cfaB.
  • E. coli K12 strains do not express the colonization factors CSI and CS2.
  • E. coli LMC10 a lac-deletion restriction-negative derivative of the strain C921b-2 was used as the genetic source for the CSI operon.
  • E. coli strain C921b-2 was derived from the wild type ETEC strain C921b-1 (which expresses CSI and CS3) by selection of spontaneous mannose-resistant hae agglutination (MRHA)- negative variants using the method disclosed in Smyth, J. Gen. Microbiol.. 128:2081-2096 (1982).
  • C921b-2 has the genes for CSI, but, because it has lost the plasmid encoding rns and CS3, none of the CFA/II antigens are expressed.
  • E. coli K12 strains that were transduced or transformed in the cloning process were DH5alpha (recAl qyrA96) and JM83 (Str) (Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982)). Additional strains used to express different colonization factors were: H10407 (CFA/I) (Evans et al., Infect. Immun.. 12:656-667 (1975)), 271-6 (CSI) (obtained from J. Kaper) , C921b-1 (CSI and CS3) (Smyth, J. Gen. Microbiol..
  • E9034a (CS3) (Levine et al.. Infect. Immun.. 44:409-420 (1984)), E11881E (CS4 and CS5) (Wolf et al.. Infect. Immun.. 57:164-173 (1989)), E8775 (CS4 and CS6) (Thomas et al., J. Gen. Microbiol.. 131:2319-2326 (1985)), E17018A (CS5 and CS6) (Wolf et al.), E4133A (CS6) (Wolf et al.), and 2230 (AFA-1) (Labigne-Roussel et al.. Infect.
  • Strain C91f-6 is derived from C91f, which expresses CS2 and CS3, by loss of the rns-containin ⁇ . plasmid (Smyth) . This strain has the CS2 but not the CS3 genes.
  • the cloning vectors used were pUCl ⁇ (Ap) , pUC19 (Ap) (Messing et al.. Gene. 19:269-276 (1982)), pJFlO (Cm, Km) (obtained from Joachim Frey) , and pHC79 (Ap, Tc) (Collins et al., Gene. 11:291-298 (1980)).
  • the plasmid pEU2040 contains rns cloned into pHSG576 (possessing the pSClOl replication origin and resistance to Cm) (Takeshita, Gene. 61:63-74 (1987)).
  • Plasmid pEU506 is a pHC79-derived cosmid into which the CS2 operon has been cloned.
  • Media for the bacterial cultures included luria broth (LB) (Scott, Virolo ⁇ v. 62:344-349 (1974)), CFA agar, tryptone.
  • Antibiotics used included ampicillin (50 ⁇ g/ml) , chloramphenicol (30 ⁇ g/ml) , and kanamycin (30 ⁇ g/ml) .
  • E. coli IMC10 Total DNA from E. coli IMC10 was digested with Sau3AI and ligated to BamHI-cleaved pHC79. Ligated DNA packaged into lambda phage was used to transduce E. coli DH5alpha harboring pEU2040. Transductants were selected on tryptone plates containing ampicillin and chloramphenicol. Of the 635 colonies tested, 48 were positive. One, which showed the presence of CSI in a heat extract as determined by the below-discussed western blot analysis, was chosen for further study. The plasmid it contained was designated pEU405. Plasmid pEU405 was digested with Hindlll. self- ligated, and transformed into E. coli JM83 harboring pEU2040.
  • Plasmids from CSl-positive colonies contained an approximately 36 kb insert. DNA from one of these plasmids was partially digested with Sail, self-ligated and used to transform JM83 carrying pEU2040.
  • One CSl- positive clone containing an approximately 14 kb insert was named pEU452.
  • PEU452 and their expression is shown in Figure 1.
  • Figure 1 bold lines represent cloned DNA.
  • the vectors employed were pHC79 for pEU452, pEU600, pEU601, and pEU602; pJFlO for pEU603; pUCl ⁇ for pEU606 and pEU608; pUC19 for pEU605 and pEU607.
  • the letters above the bold line in pEU452 represent restriction endonuclease sites: S:SalI; P:PstI; E:EcoRI; M:SmaI; C:ClaI; X:XbaI.
  • deletions of pEU452 were constructed to determine the smallest DNA fragment containing the CSI structural gene. Deletion of the approximately 2.3 kb Sail fragment of pEU452 generated pEU600, deletion of the approximately 4.5 kb S al segment of pEU600 created pEU601, and deletion of the approximately 4.8 kb Clal fragment gave pEU602.
  • pEU603 was constructed by cloning the approximately 2.4 kb EcoRI fragment of pEU600 into the EcoRI site of pJFlO.
  • pEU606 and pEU605 were made by cloning the approximately 2.1 Pstl-Xbal fragment of pEU600 into pUC18 and pUC19, respectively.
  • the plasmids pEU608 and pEU607 resulted from inserting the approximately 700 bp Clal-Xbal fragment of pEU605 into pUCl ⁇ and pUC19, respectively.
  • Antiserum to CSI was prepared by immunizing rabbits with a formalin-treated suspension of the CS1- expressing strain C921b-1 grown on CFA agar mixed with Freunds' complete adjuvant (Sigma, St. Louis, MO) . Subsequent repeated absorptions with the E. coli strains C91f and E9034a, which express CS2 and CS3, respectively, rendered the antiserum specific.
  • Colony immunoblots were performed on colonies grown at 37°C and transferred to nitrocellulose filters.
  • the filters were washed three times with 2% bovine serum albumin (BSA) , and followed by 30 minutes incubation in IM-2 (per liter: 6.06 g Tris base, 8.77 g NaCl, 2.5 g gelatin, 1.89 g EDTA, 10 ml Tween 20, 0.02% NaN 3 , pH 7.4) containing 2% BSA.
  • IM-2 per g Tris base, 8.77 g NaCl, 2.5 g gelatin, 1.89 g EDTA, 10 ml Tween 20, 0.02% NaN 3 , pH 7.4
  • Polyclonal anti-CSl (1:250 dilution) was added to the filters and incubated at room temperature for 1 hour.
  • the filters were then washed three times in IM-2 + 2% BSA and incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase (Sigma, St. Louis, MO) (1/500 dilution) in IM-2 + 2% BSA for 1.5 hours at room temperature.
  • the filters were washed three times in IM-2 and incubated for 15 minutes in 50 mM Tris (pH 10) .
  • the alkaline phosphatase was assayed with BCI substrate (5- bromo-4-chloro-3-indolyl-phosphate-p-toluidine salt) (Bachem, Torrence, CA) and visualized with nitro blue tretrazolium (Sigma, St. Louis, MO).
  • Nucleotide sequencing on both strands of a 703 bp fragment containing the cooA gene was carried out from plasmid DNA using the Sanger protocol (Sanger et al., Proc. Natl. Acad. Sci. USA. 74:5463-5467 (1977), as modified for supercoiled templates by Zagursky et al. (Zagursky et al.. Gene Anal. Tech.. 2:89-94 (1985) and using the T7 DNA polymerase (Sequenase; Stratagene, LaJolla, CA) . Secondary structure of the protein was predicted using the algorithm of Chou and Fasman (Chou et al., Adv. Enzvmol.. 47:45-141 (1978). Homology searches were performed on the GenBank DNA database by using the FASTP algorithm (Lipman et al., Science. 227:1435-1441 (1985) .
  • Filters were baked in a vacuum oven at 80°C for 2 hours and placed in polyethylene bags.
  • a prehybridization mixture (50% formamide, 0.75 M NaCl, 75 M sodium citrate (5X SSC), 5X Denhardt's solution (Maniatis et al., Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1982)), 100 ⁇ g of denatured calf thymus DNA per ml, 200 ⁇ g of tRNA per ml, and 0.1% SDS) was added and the filters were incubated at 2°C for a minimum of 3 hours.
  • Denatured radioactive probe (8 - 24 ng at a specific activity of about 7 X 10 5 cpm/ng) was added and the bags were resealed and incubated at 42°C for 12 to 16 hours. The filters were washed five times in 0.2X SSC-0.1% SDS at 42°C and exposed to Kodak XAR-5 film with a Lightning-Plus intensifying screen for 12 hours at -80°C.
  • the calculated percent mismatch is 21.88% and for the 285 bp Clal-Mspl probe, the permitted mismatch is calculated to be 21.78%.
  • CSI pilus To begin the analysis of the operon encoding the CSI pilus, a Sau3AI cosmid library made from total DNA of strain LMC10 was screened by colony immunoblots with antiserum specific for CSI. One clone expressing CSI contained the plasmid pEU405. After a restriction map for 12 pEU405 was generated, subclones were isolated as discussed above and as indicated in Figure 1.
  • CSI antigen was present on the cell surface of E. coli K12 strain JM83 carrying pEU2040 (which expresses rns) and pEU452 or pEU600, but no antigen was detectable in the same strain carrying pEU601, pEU602, pEU603, pEU606 or pEU608.
  • CSI was detected in total cell extracts of JM83 containing pEU605 or pEU607, but not in heat extracts. This indicates that the structural gene for CSI, which we call cooA. is located between the Xbal and Clal sites that delimit the cloned DNA in pEU607 (see Figure 1) .
  • sequence analysis of plasmids pEU605 and pEU608, as shown in the nucleotide sequence of Figure 2, reveals an open reading frame of 513 nucleotides, from position 114-626, encoding a predicted product of 171 amino acids with a molecular weight of 17.5 kd.
  • the putative Shine-Dalgarno sequence TGGAGTT is underlined.
  • the arrow between Ala and Val residues represents the probable cleavage site for signal peptidase.
  • the location of an inverted repeated sequence that may function as a signal for termination of transcription is delineated by arrows at positions 664-672 and 681-688.
  • the ATG codon at nucleotide 114 is probably the start of translation because this codon is preceded by a potential ribosome-binding site, TGGAGTT, centered at position 107, which matches in 5 of 7 positions with the consensus.
  • TGGAGTT potential ribosome-binding site
  • FIG. 3 shows a comparison of the amino acid sequences obtained by translation of cooA and cfaB.
  • the FASTP program was used to align the 171 amino acid cooA product with the 170 amino acid cfaB polypeptide. Identical residues are shown by colons (:) , and conservative changes by periods (.) . The second digit of the number is above the designated amino acid and the proposed signal sequence of both proteins is underlined.
  • the 170 amino acid CfaB product has 92% similarity and 55% identity with CooA. Both proteins share a similar hydropathy pattern, although their predicted pi values differ considerably (7 for CooA and 9.28 for CfaB).
  • cooA probes were hybridized to a cosmid encoding CS2 as shown in Figure 2.
  • the restriction endonuclease sites for Clal and Mspl delimit the 285 bp fragment used as a probe that encodes the CooA amino terminal region and a 113 bp upstream region.
  • the second probe was a 318 bp fragment internal to cooA flanked by Hhal sites. No homology was found with either cooA probe used.
  • E. coli LMCIO harbors three plasmids whose molecular weights vary from 5 kb to more than 60 kb.
  • the high frequency of CSl-positive clones obtained in this study suggests that there is more than one copy of cooA in LMC10.
  • the two cooA probes described above and in Figure 2 were hybridized to pEU452 ( Figure 1) and to purified plasmid DNA from IMC10 on Southern blots.
  • the expression of the CSI pilus one of the antigens involved in colonization of the human intestinal epithelium by enterotoxigenic strains of E. coli. is dependent upon the presence of Rns, a plasmid- encoded positive regulatory protein.
  • Rns a plasmid- encoded positive regulatory protein.
  • the rns gene contains only 28% G + C. This suggests that rns was recently introduced into EL coli from another organism.
  • Neither CSI nor CS2 the pili regulated by Rns, is encoded on the plasmid carrying rns. If the genes that rns regulates were introduced with it, they might also have a low G + c content, although they are genetically unlinked.
  • the sequence of cooA indicates that it is composed of 45% G + C; thus, it is much closer to the composition of the normal E. coli genome than to rns.
  • the translated DNA sequence of cooA suggests the presence of a signal peptide cleaved between the alanine at position 23 and the valine at position 24. This is in conformity with all other E. coli fimbrial proteins because they all possess signal sequences that end with an alanine residue.
  • the predicted cooA signal peptide has three positively-charged residues (lysine) at positions 2, 4, and 5 followed by a core of 12 hydrophobic residues (residues 12-23) .
  • the first 28 amino acids (VEKTISVTASVDPTVDLLQSDGSALPND) of the antigen match the predicted sequence if cleavage occurs between positions 23 and 24.
  • the molecular weight calculated for the predicted mature protein is 15.2 kd, which is in close agreement with the size observed on SDS-PAGE (16 kd) .
  • the amino acid content predicted for the CooA protein is consistent with that obtained by Hall et al., J. Bacteriol.. 171:6372-6374 (1989) from analysis of the CSI protein, but differs somewhat from that obtained by Smyth et al. (Microbial Surface Components and Toxins in Relation to Pathogenesis. Plenum Press, London (in press)). Smyth et al. find a total amino acid content of 172, of which 21 residues are glycine, whereas the instant invention and Hall et al. find the protein to have 171 amino acids of which only 11 residues are glycine. It is possible that the differences may be caused by the use of different strains.
  • the genetic organization of the CSI and CS2 pili differs from that of other pilins previously described.
  • Rns a positive regulator required for expression of CSI or CS2
  • Rns-independent a positive regulator required for expression of CSI or CS2
  • cooA is located on a plasmid different from the plasmid containing rns and CS3.
  • the structural gene for CS2 has not yet been located, the paucity of CS2 clones in a library from a CS2 ETEC strain suggests a chromosomal location for this gene.
  • CSI and CFA/I extends to the sequence of the major pilin antigens.
  • the only protein identified as similar to CooA was CfaB.
  • the amino acid level there is 92% similarity and 55% identity between the predicted sequences of these two proteins.
  • two different cooA DNA probes show no homology to an ETEC strain expressing CFA/I.
  • the N-terminal amino acid sequences of several other colonization factors are very similar to that of CooA, we find no homology between the cooA DNA probes and DNA from ETEC strains producing any other colonization factor.
  • either of these cooA probes are of potential epidemiological value to assess the frequency with which CSl-expressing strains are responsible for infections in human populations.
  • ETEC strains are responsible for numerous cases of diarrhea in travelers and infants and the spread of such disease in developing countries is frequently epidemic. Prevention of infection with such strains might be achieved by the use of synthetic peptide vaccines containing antigenic determinants common to as many types of pili as possible. The sequence similarity of CooA and CfaB raises the hope of common epitopes. Thus, although rabbit antisera against both proteins are not cross- reactive, it is possible that immunization with a synthetic immunorecessive epitope present in both proteins would be successful.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne le clonage du gène de structure appelé cooA pour le gène majeur de piline CS1 de Escherichia coli entérotoxycogène. On a isolé des sous-clones produisant l'antigène CSI, on a déterminé l'emplacement et la séquence de cooA, et on a élaboré un vecteur contenant cette séquence d'ADN. On a utilisé ce vecteur afin de transformer un hôte de manière à permettre la production de l'antigène de piline CS1. On peut formuler l'antigène de piline CS1, ou une partie de cet antigène, pour l'utiliser comme immunogène dans un vaccin permettant une protection contre la diarrhée des voyageurs et des enfants. On peut construire des sondes d'ADN complémentaires au gène cooA afin de permettre la détection de souches d'expression CS1 de E. coli dans des tissus et des liquides biologiques infectés.
PCT/US1991/005217 1990-07-24 1991-07-23 Production de l'antigene majeur de piline cs1 et compositions pour ce dernier comprenant des vaccins et des sondes diagnostiques WO1992001703A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US55753590A 1990-07-24 1990-07-24
US557,535 1990-07-24

Publications (1)

Publication Number Publication Date
WO1992001703A1 true WO1992001703A1 (fr) 1992-02-06

Family

ID=24225823

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/005217 WO1992001703A1 (fr) 1990-07-24 1991-07-23 Production de l'antigene majeur de piline cs1 et compositions pour ce dernier comprenant des vaccins et des sondes diagnostiques

Country Status (2)

Country Link
AU (1) AU8417191A (fr)
WO (1) WO1992001703A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038171A1 (fr) * 1995-06-02 1996-12-05 Department Of The Army, Us Government Procede visant a dresser des anticorps contre l'e. coli de la famille cs4cfa/1
WO1998005687A1 (fr) * 1996-08-02 1998-02-12 Department Of The Army, Us Government Anticorps monoclonal permettant d'agglutiner e. coli possedant une proteine de la famille cs4-cfa/i
US7404961B2 (en) 1996-08-02 2008-07-29 The United States Of America As Represented By The Secretary Of The Army Peptides responsive to antibodies against consensus peptide of the CS4-CFA/I family proteins
US7566540B2 (en) 1996-08-02 2009-07-28 The United States Of America As Represented By The Secretary Of The Army Monoclonal antibody which agglutinates E. coli having the CS4-CFA/I family protein

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4443549A (en) * 1981-10-19 1984-04-17 Molecular Genetics, Inc. Production of monoclonal antibodies against bacterial adhesins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4443549A (en) * 1981-10-19 1984-04-17 Molecular Genetics, Inc. Production of monoclonal antibodies against bacterial adhesins

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 112, No. 19, issued 07 May 1990, HAMERS et al., "The nucleotide sequence of the first two genes of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli", Abstract No. 173127f; & MICROB. PATHOG. 6(4), 297-390. *
JOURNAL OF GENERAL MICROBIOLOGY, Vol. 128, 1982, SMYTH: "Two Mannose-resistant Haemagglutins on Enterotoxigenic Escherichia coli of Serotype 06:K15:H16 or H-Isolated from Travellers and Infantile Diarrhoea", 2081-2096. *
NATURE, Vol. 313, issued 28 February 1985, JACOBS et al., "Isolation and characterization and cDNA clones of human erythropoietin", pages 806-810. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Vol. 78, No. 6, issued June 1981, HOPP et al., "Prediction of protein antigenic determinants from amino acid sequences", pages 3824-3828. *
SCIENCE, Vol. 222, issued 18 November 1983, YOUNG et al., "Yeast RNA Polymerase II Genes: Isolation with Antibody Probes", pages 778-782. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038171A1 (fr) * 1995-06-02 1996-12-05 Department Of The Army, Us Government Procede visant a dresser des anticorps contre l'e. coli de la famille cs4cfa/1
US5914114A (en) * 1995-06-02 1999-06-22 The United States Of America As Represented By The Secretary Of The Army Method of raising antibodies against E. coli of the family CS4-CFA/I
WO1998005687A1 (fr) * 1996-08-02 1998-02-12 Department Of The Army, Us Government Anticorps monoclonal permettant d'agglutiner e. coli possedant une proteine de la famille cs4-cfa/i
US7404961B2 (en) 1996-08-02 2008-07-29 The United States Of America As Represented By The Secretary Of The Army Peptides responsive to antibodies against consensus peptide of the CS4-CFA/I family proteins
US7566540B2 (en) 1996-08-02 2009-07-28 The United States Of America As Represented By The Secretary Of The Army Monoclonal antibody which agglutinates E. coli having the CS4-CFA/I family protein

Also Published As

Publication number Publication date
AU8417191A (en) 1992-02-18

Similar Documents

Publication Publication Date Title
Perez-Casal et al. Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli
Barlow et al. The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins
Luo et al. Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73
Logan et al. Evidence for posttranslational modification and gene duplication of Campylobacter flagellin
US5330754A (en) Membrane-associated immunogens of mycobacteria
Guerry et al. Role of two flagellin genes in Campylobacter motility
US5427788A (en) Pertussis toxin and use in vaccines
JP4271231B2 (ja) 髄膜炎菌（Ｎｅｉｓｓｅｒｉａｍｅｎｉｎｇｉｔｉｄｉｓ）のプロテイナーゼＫ抵抗性表面蛋白質
US5871943A (en) Immunoassay comprising a truncated pneumococcal surface protein A (PspA)
Garcia et al. The afimbrial adhesive sheath encoded by the afa‐3 gene cluster of pathogenic Escherichia coli is composed of two adhesins
CA2098448A1 (fr) Fragments d'adn codant pour les sous-unites du recepteur de la transferrine de neisseria meningitidis
JP2007289194A (ja) 空胞形成毒素欠損Ｈ．ｐｙｌｏｒｉおよび関連する方法
Oaks et al. Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens
Alm et al. Variation in antigenicity and molecular weight of Campylobacter coli VC167 flagellin in different genetic backgrounds
O'Toole et al. Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus
Puohiniemi et al. A strong antibody response to the periplasmic C-terminal domain of the OmpA protein of Escherichia coli is produced by immunization with purified OmpA or with whole E. coli or Salmonella typhimurium bacteria
Hu et al. Morganella morganii urease: purification, characterization, and isolation of gene sequences
Nuijten et al. Localization of immunogenic regions on the flagellin proteins of Campylobacter jejuni 81116
NO316623B1 (no) Gen som koder for overflateantigen fra en ikke-typebestembar Haemophilusstamme, slikt protein i seg selv samt fremgangsmate ved fremstilling derav
AU644829B2 (en) Recombinant vaccine for porcine pleuropneumonia
Pedroni et al. Cloning of a novel pilin‐like gene from Bordetella pertussis: homology to the fim2 gene
Gerlach et al. Characterization of two genes encoding antigenically distinct type-1 fimbriae of Klebsiella pneumoniae
Tennent et al. Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ
AU618803B2 (en) Pharmaceutical composition containing fibronectin binding protein
WO1992001703A1 (fr) Production de l'antigene majeur de piline cs1 et compositions pour ce dernier comprenant des vaccins et des sondes diagnostiques

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

NENP Non-entry into the national phase

Ref country code: CA