EP0377711A1 - Serotyping dna probes - Google Patents

Serotyping dna probes

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Publication number
EP0377711A1
EP0377711A1 EP89907053A EP89907053A EP0377711A1 EP 0377711 A1 EP0377711 A1 EP 0377711A1 EP 89907053 A EP89907053 A EP 89907053A EP 89907053 A EP89907053 A EP 89907053A EP 0377711 A1 EP0377711 A1 EP 0377711A1
Authority
EP
European Patent Office
Prior art keywords
dna
enterobacteriaceae
fragment
antigen
serotype
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Withdrawn
Application number
EP89907053A
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German (de)
English (en)
French (fr)
Inventor
Paul Alexander Manning
Michael William Heuzenroeder
Danny Werner Beger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Luminis Pty Ltd
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Luminis Pty Ltd
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Filing date
Publication date
Application filed by Luminis Pty Ltd filed Critical Luminis Pty Ltd
Publication of EP0377711A1 publication Critical patent/EP0377711A1/en
Withdrawn legal-status Critical Current

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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

  • the present invention relates to the identification and isolation of DNA fragments which may be utilised inter alia as DNA probes and in live vaccine carrier strains.
  • the outer membrane of the cell wall of Gram negative bacteria comprises proteins, phospholipids and lipopolysaccharide (LPS) and of these the LPS is by far the most abundant molecule on the cell surface.
  • the LPS is composed of three regions: the innermost is the lipid-A moiety which forms the outer leaflet of the lipid bilayer of the outer membrane; the central region is the core oligosaccharide, and outermost is the O-antigen side-chain consisting of a series of repeat units usually of specific sugars. The sequence and types of sugars and their linkages is what determines the O-serotype of the bacterium.
  • the diagnosis of disease requires the specific identification of the aetiological agent and this usually involves isolation of the organism on selective media and then subjecting it to a battery of biochemical tests which can take days before a positive identification can be made. It may then be necessary to serotype the organism especially if the epidemiology of the disease is to be examined. Serotyping can usually only proceed after the type of organism has been determined, and again is time consuming. Thus, a more rapid means of both diagnosis and serotyping would be of considerable benefit especially since many of the Enterobacteriaceae can cause life-threatening disease and are in fact the major causes of infant mortality in developing countries such as India, Bangladesh and much of South America. It is accordingly an object of the present invention _ _ .
  • a DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae. which probe detects the presence of the rfb region of an Enterobacteriaceae.
  • the fragment of DNA contains genes encoding for the synthesis of the O-antigen of the 0101, 0157 or 02 serotype of an Escherichia coli.
  • the Escherichia coli is an Enterotoxigenic E.coli (ETEC) .
  • a DNA probe kit including a first DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae, which probe detects the presence of the rfb region of an Enterobacteriaceae; and a second DNA probe including a fragment of DNA which detects the presence of the rfb region of an Enterobacteriaceae in a manner different to said first probe such that the first and second probes are serotype specific.
  • the genes responsible for the production of the O-antigen of the lipopolysaccharide of members of the Enterobacteriaceae are encoded within the gene cluster referred to as the rfb locus.
  • the structure of the O-antigen also varies.
  • the serotype of the bacterium is different.
  • this region of the bacterial chromosome represents a set of genes which are directly related to the serotype of the organism, and thus variation in this region is absolutely related to the change in the serotype.
  • the DNA probes according to the present invention make it possible to directly analyse the variation at rfb and correlate this with the serotype and thus provide a rapid means of serotyping an organism, as well as being able to differentiate one species from another.
  • a DNA probe including a plasmid cloning vector; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae inserted into a suitable site in the plasmid cloning vector.
  • the fragment of DNA may include the entire rfb locus of the Enterobacteriaceae or a fragment thereof.
  • the Enterobacteriaceae may be an E.coli of the 01, 0157 or 02 serotype.
  • Enterobacteriaceae is an E.coli of the 0101 serotype
  • a fragment having a minimum length of approximately 8.9 Kb and a maximum length of approximately 11.8 Kb is required for 0101 0-antigen biosynthesis when it is introduced into E.coli K-12.
  • the plasmid cloning vector may be of any suitable type.
  • the plasmid cloning vector ⁇ HC79 has been found to be suitable.
  • the fragment of DNA may be inserted into the BamHI site on the plasmid pHC79.
  • the plasmid so formed has been designated pPM 1301. By deleting rfb DNA a derivative pPM 1305 has been derived.
  • a DNA probe including the plasmid pPM 1305 or a digestion derivative thereof.
  • Fragments of the clone pPM 1305 may be produced by partial digestion as described below. At present six fragments of pPM 1305 have been envisaged. These are designated plasmids pPM 1321, pPM 1322, pPM 1323, pPM 1324, pPM 1325, pPM 1326, pPM 1330 and pPM 2218. Samples of the plasmids are maintained in the Culture Collection of the Universith of Sydney, North Terrace, Sydney, South Australia. These are illustrated in Figure 6 below.
  • a DNA probe including a plasmid from which rfb DNA has been deleted and selected from plasmids pPM 1305, pPM 1322, pPM 1323, pPM 1324, pPM 1325, pPM 1326, pPM 1330 and pPM 2218 and mixtures thereof.
  • the plasmids pPA1343, pPA1344 and pPA1345 are examples of clones containing the 02 rfb region. Accordingly in a preferred form, there is provided a DNA probe including a plasmid selected from plasmids pPA1343, 1344 or 1345 or a digestion derivative thereof. This has great potential in the development of vaccines against diseases caused by Enteobacteriaceae where the O-antigen is a protective antigen, that is, where the protection is serotype specific. These clones also provide a basis for developing further probes. Similarly this approach has also been used to clone the genes for the 0157 rfb region. Plasmid pPM1354 represents an example of such a clone.
  • a DNA probe including a plasmid pPM1354, or a digestion derivative thereof. This clone has potential for both vaccine development and for specific probes.
  • E.coli of the 0157 serotype are responsible for haemorrhatic uraemic syndrome and are referred to as
  • E.coli Enterohaemorratic E.coli and EHEC. This is a severe condition and can often lead to death, and vaccines against this disease are not presently available.
  • the advantages of such probes include: the presence of rfb and closely linked DNA can be detected even in the absence of gene expression, a problem which may arise if the O-antigen is incompatible with the core region of the recipient organism, or if the recipient is expressing its own O-antigen and effectively out-competing the O-antigen encoded by the cloned DNA; the identification of an rfb clone is greatly simplified; a restriction map of the region to be cloned can be determined beforehand by Southern DNA hybridization, and this could permit a better choice of restriction enzyme for cleaving the chromosomal DNA prior to cloning.
  • the probes enabled us to differentiate two 0115 strains, one which was an enterotoxigenic E.coli (ETEC) and another which was associated with septicaemia.
  • ETEC enterotoxigenic E.coli
  • these results illustrate that the probes may also enable us to differentiate organisms which are apparently of the same serotype but produce different diseases. This is extremely important because of course some E.coli serotypes are part of natural human bacterial flora.
  • the DNA probes according to the present invention may be labelled in any suitable manner.
  • a radioactive or non-radioactive label may be used.
  • radioactive isotopes of phosphorus or sulphur e.g. 32 phosphorus and 35 sulphur may be used.
  • non-radioactive labelling is preferred, a biotinylated photo-sensitive label may be selected.
  • a method for preparing a DNA probe which method includes providing a plasmid cloning vector; a first restriction enzyme; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae; partially digesting the plasmid cloning vector with the restriction enzyme; and inserting the fragment of DNA into a restriction site.
  • the restriction site chosen may correspond to the restriction enzyme used or be compatible therewith.
  • the plasmid cloning vector may be of any suitable type* Tne plasmid cloning vector pHC79 has been found to be suitable. Since this plasmid cloning vector includes a BamHI restriction site, a corresponding BamHI or Sau 3A restriction enzyme is preferred.
  • the ligated DNA so formed may be packaged in vitro and transduced into a suitable bacterial strain.
  • an E.coli strain may be used.
  • An E.coli K-12 strain may be used.
  • the E.coli K-12 strain DH1 has been found to be suitable.
  • the method of preparing a DNA probe may further include providing a plurality of restriction enzymes; and digesting the ligated DNA with the restriction enzymes to produce restriction fragments of differing size.
  • the restriction enzymes may include Mlul, Kpn 1,
  • the fragments may be subjected to the further step of filling the protruding ends so formed. This may be undertaken utilizing the Klenow fragment of DNA polymerase 1.
  • the sub clones so formed may include the plasmids pPM 1321, pPM 1322, pPM 1323, pPM 1324, pPM 1325, pPM 1326, pPM 1330 and pPM 2218.
  • the plasmids pPM 1321, pPM 1330 and pPM 1326 are preferred as they have been found to be highly conserved. It is hypothesized that the plasmids encode part of the region that specifies the OlOl serotype and may be associated with any common properties of the O-antigen chains that allow ETEC to be more efficient pathogens or inhabitants of the intestinal tract are likely to be found encoded in this region of the rfb cluster.
  • a method of diagnosing a disease which method includes providing a sample to be tested; and at least one DNA probe including a fragment of DNA which probe detects the presence of the rfb region of an Enterobacteriaceae; contacting the sample with the at least one DNA probe; and _ _
  • the method of diagnosis may in addition provide a method of serotyping the organism responsible for the disease.
  • the method of diagnosis may include providing a series of samples to be tested; and a DNA probe kit including a first DNA probe including a fragment of DNA which detects the presence of the rfb region of an Enterobacteriaceae: and a second DNA probe including a fragment of
  • DNA which detects the presence of the rfb region of an Enterobacteriaceae in a manner different to said first probe such that the first and second probes are serotype specific; contacting each sample with a different DNA probe from the kit; and subjecting the products thereof to a series of assays.
  • the one such sample to be tested may be of any suitable type.
  • a faecal sample may be used.
  • the or each DNA probe is preferably a radioactively or non-radioactively labelled DNA probe, as described above.
  • the product may be assayed in any suitable manner.
  • a Western and/or color blot analysis may be used.
  • the plasmids including restriction fragments of DNA including substantially the whole of the rfb locus of an
  • Enterobacteriacae for example plasmid pPM 1305 may be inserted into a suitable avirulent bacterial carrier strain which may in turn function as a live vaccine.
  • a suitable avirulent bacterial carrier strain which may in turn function as a live vaccine.
  • a method of preparing an avirulent strain of an enterobacterium which method includes providing a sample of a preselected bacterial strain; and a plasmid including a fragment of DNA containing genes encoding for the synthesis of the
  • the plasmid may be plasmid pPM 1305.
  • the preselected bacterial strain may be selected from Salmonella and Shiqella strains. Salmonella tvphimurium. Salmonella typhi and Salmonella dublin have been found to be suitable.
  • Diseases of interest may include those generated by any enterobacterial strain.
  • the avirulent strain of an enterobacterium referred may provide the basis for a vaccine composition.
  • the vaccine composition may be used as a live oral vaccine against enteropathogenic diseases of man or domestic animals.
  • the genes for group- (rfb) and type-antigen biosynthesis of Shiqella flexneri may be introduced into the preselected strain. Further changes may include introduction of the Shi ⁇ ella LPS core biosynthesis genes (rfa) so that the strain expresses the O-antigen of the appropriate Shiqella specificity on the cell surface. Analysis of the LPS by silver staining, ELISA and haemagglutination inhibition may be used to assay the production of the O-antigen. Immunogenicity may be assessed in mice, and rabbits and ultimately monkeys.
  • a vaccine composition useful for effecting immunity against diseases of an Enterobacteriaceae including a bacterial carrier strain; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae, inserted into the chromosome of the bacterial carrier strain.
  • the bacterial carrier strain may be selected from Salmonella and Shiqella strains.
  • the Enterobacteriaceae is preferably an E.coli of the 0101 serotype and the fragment of DNA has a munimum length of approximately 8.9 kilobase pairs, and a maximum length of approximately 11.8 kilobase pairs.
  • the Enterobacteriaceae is an E.coli of the 0157 or 02 serotype.
  • the vaccine composition may be provided in any suitable form.
  • the vaccine composition may be provided in an oral or injectable form.
  • Suitable carriers including aqueous and/or alcoholic carriers may be included.
  • a buffered saline solution may be used.
  • Other recipients and compounding ingredients known per se in the art may be included if desired.
  • a method for the prophylactic treatment of diseases of an Enterobacteriaceae in mammals which method includes providing a mammal to be treated; and a vaccine composition useful for effecting immunity against diseases of an Enterobacteriaceae. including 0 a bacterial carrier strain; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an
  • Enterobacteriaceae inserted into the chromosome of the bacterial carrier strain; and 5 administering to the mammal a prophylactically effective amount of the vaccine composition.
  • a method for the prophylactic treatment of diseases of E.coli of the 0157 serotype in o mammals, including humans which method includes providing a mammal to be treated; and a vaccine composition including a Salmonalla or Shiqella carrier strain; and/or a fragment of DNA derived from plasmid pPM1354, or a digestion derivative thereof; and administering to the mammal a prophylatically effective amount of the vaccine composition.
  • Q E.coli of the 0157 serotype are responsible for haemorrhatic uraemic syndrome and are referred to as Enterohae orratic E.coli and EHEC.
  • Plasmid DNA of single and double digests were analysed in 0.8% and 1.0% agarose gels in TBE buffer. The lines indicate the extent of cloned DNA retained in each of the derivatives of pPM1305. Their ability to mediate synthesis of 0101 O-antigen is indicated.
  • pPM 1305 was initially digested with Mlul and insert DNA ligated into the Mlul sites of pLG339 (Stoker et al. 1982) forming pPM1330.
  • E.coli was digested with restriction endonucleases PstI,
  • the two subclones pPM1321 and pPM1326 from the 0101 rfb clone pPM1305 are believed to represent a highly conserved region of DNA within the rfb gene cluster. These subclones were used as radio-labelled probes to detect restriction fragment length polymorphisms (RFlPs) within 018 and 02 E.coli.
  • RlPs restriction fragment length polymorphisms
  • the restriction maps obtained (D) from using these probes in Southern hybridisation analysis (A, B, C) show the high degree of relatedness between 018 and others not shown on E.coli and the probable close evolutionary relationships.
  • the hybridizations were performed at high stringency; 0.2 x SS C at 65°C.
  • the autoradiography was exposed for 2 days at - 70°C using intensifying screens.
  • Competent cells (0.2ml) were mixed with 1 ug DNA, incubated on ice for 30 min, then heat shocked (42°C) for 2 min. The cells were then allowed to express in nutrient broth for 60 min. at 37°C with shaking and the mixture plated onto the appropriate antibiotic plate.
  • Molecular cloning Molecular cloning
  • genomic DNA from B41 a cosmid library was constructed as previously described (14) .
  • 1-2 ug of genomic DNA partially digested with Sau3A was cloned into the BamHI site of 6 ug alkaline phosphatase treated BamHI digested pHC79 DNA.
  • the ligated DNA was packaged in vitro and transducted into E.coli K-12 strain DHL This procedure usually yielded 1000-2000 ampicillin resistant colonies.
  • Plasmids were transformed into the minicell producing stran DS410. Minicells were purified on sucrose gradients and the plasmid encoded proteins were labelled with -x c t S3-methionine and solubilized in sample buffer as previously described (13) . After electrophoresis in SDS on 11-20% polyacrylamide gels, the dried gel was subjected to autoradiography using Kodak X-omat film at room temperature. Restriction analysis
  • Protuding ends created by cleavage with BamHI and SacI were filled or removed using the Klenow fragment of DNA polymerase I.
  • Klenow fragment Typically, 1.6ug of digested DNA, 2 ul of each dNTP (2mM) and 5 units of Klenow fragment (Pharmacia) were mixed and left at room temperature for 30 min. The reaction was stopped by the addition of lul of -.5mM EDIA and incubation at 65°C for 15 min. Unincorporated dNTPs and enzyme were removed by centrifugation through a Sepharose C1-6B column.
  • Colloidal gold particles (c. 15-20nm) were prepared using the citrate method as described (de Mey and Meormans, 1986) and conjugated with Protein A (Pharmacia) . Immunoqold labelling of whole cells
  • Plasmid DNA was Nick translated with DNA Polymerase i and Southern DNA hybridization analysis (Southern, 1975) was carried out according to a modification of Maniatis et.al. (1982). [ 32 p3 labelled DNA of pPM1305 and its subclones were used to probe suitably digested genomic DNA from a number of 0101 isolates. Autoradiography was carried out at -70°C on Kodak X-omat film, with intensifying screens. The exposure time was 3 days.
  • the cosmid pPM1301 was reduced in size in a two stage process: a partial Hindlll digestion to produce
  • the coding region of the 0101 rfb gene cluster must extend from the region defined by the Sall-Hpal restriction endonuclease sites at (6.0-7.3kb) to the region o between the Sacl-Mlul sites (at 16.2-17.8 kb) ; i.e. a minimum of 8.9 kb and a maximum of 11.8 kb of DNA is required for
  • E.coli isolates tested regardless of the O-serotype. There was no hybridization with eithr V.cholerae, Salmonella or the E.coli K-12 rfb delete strain, QE35 (Sunshine and Kelly, 1971) . Interestingly, this probe reacts strongly with several apparently immunologically unrelated serotypes and the basis for this is being examined.
  • Figure 6 shows that when pPM1322 is used as a probe under conditions of high stringency, this fragment appears to be highly conserved among all isolates.
  • the strain H510a which has a unique LPS banding pattern, while hybridizing with sequences on pPM1322 exhibits a markedly different pattern of fragments, not surpisingly there is no detectable homology with the rfb delete strain Q35 or the E.coli K-12 host strain DHL
  • Figure 7 shows that when pPM1324 is used as a probe under the same conditions as pPM1322, H510a exhibits a unique pattern, whereas 8CE275/6, VAC1676 and VC17651 exhibit identical patterns.
  • Strains B41 and KAT11706 have a fragment in common with the other isolates (except H510a) . No hybridization is seen with strains Q35 or DHl. Interestingly, this probe fails to detect an identical fragment in B41, suggesting that all of the cloned DNA in ⁇ PM1323 and pPM1324 is not contiguous, probably due to the deletion process by which they were generated. Discussion
  • At least six proteins are encoded within the minimal coding region, implicating them in the synthesis and assembly of the O-subunit.
  • Production of the 0101 O-antigen as LPS in an rfb delete strain QE35 implies that only the rfb genes carried by the clones are necessary for its expression and assembly on the E.coli K-12 core (data not shown). Since both structure of 0101 LPS is unknown, and the precise minimal coding region is not defined, it is not possible to say whether all of the proteins expressed in minicells or other additional undetected proteins are required for the synthesis of 0101 O-antigen. Subcloning and deletion analysis suggests that the predicted 9kb required for the _ _
  • 0101 isolates that we have examined appear to exhibit some variability in the LPS, as judged by SDS-PAGE. This difference is not detected serologically with polyvalent antiserum. There are no reports in the literature of 0101 serotype variants, despite the fact that it is a common animal ETEC serotype. It is not yet possible to speculate on the chemical relationships between the different 0101 patterns. The genetic relatedness, as judged by DNA hybridization, one may predict that these differences may simply reflect minor modifications in the O-chain. Current data suggest that multiple 0101 LPS types exist: one type defined by H510a and other types observed in B41, KATH706, 8CE275/6, VAC1676 and VC1751.
  • the DNA hybridization data suggest that the enzymes involved in H510a LPS biosynthesis are less closely related to the other types which share common restriction fragments.
  • the distinct LPS pattern on SDS-PAGE of B41 may reflect the sequence of gene duplication that we have observed in this strain, since the clones of the rfb region of B41, i.e. DHl(pPM1305) and DHl(pPM1330) more closely resemble the LPS patterns of the other 0101 isolates.
  • the 0101 serotype and may be useful in determining the relationships between strains of serotypes other than 0101. Since these probes can hybridize to serologically unrelated serotypes (data not shown; Beger et al. , 1988), they may also form the basis for developing rapi diagnostic probes capable of differentiating disease-causing organisms from the commensal flora of the same serotype. Conventional serotyping has not provided a means of identifying disease-causing organisms. The detection of O-serotype variants by DNA hybridization analysis may provide a more specific method for identifying pathogenic organisms.
  • rfb 0101 genes also provides a basis for the construction of a hybrid vaccine strain in avirulent Salmonella (Stevenson and Manning, 1985), since it has been documented that antibodies to 0101 are more hghly protective against 010LK99/F41 challenge than antibodies to the colonization factors or adhesins K99 or F41 (Duchet-Suchaux, 1986).
  • SPP1 the arrangement of restriction endonuclease generated fragments. Mol. Gen. Genet. 168 : 165-172.

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EP89907053A 1988-06-20 1989-06-20 Serotyping dna probes Withdrawn EP0377711A1 (en)

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AUPI885488 1988-06-20
AU8854/88 1988-06-20

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DE69221120T2 (de) * 1992-04-10 1998-02-26 Schweiz Serum & Impfinst Rekombinanter lebender Impfstoff gegen Gram-negative enterische Pathogene
ATE307200T1 (de) * 1992-07-07 2005-11-15 Fuso Pharmaceutical Ind Sonde zur diagnose einer ansteckenden krankheit verursacht durch staphylococcus epidermidis
US5654417A (en) * 1995-04-14 1997-08-05 Children's Hospital And Medical Center Nucleic acid probes for detecting E. coli O157:H7
WO1998050531A1 (en) * 1997-05-01 1998-11-12 The University Of Sydney Nucleic acid molecules specific for bacterial antigens and uses thereof

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IT1217303B (it) * 1984-02-01 1990-03-22 Enterovax Res Pty Ltd Ceppi batterici particolarmente utili per la produzione di vaccini per il trattamenmto di malattie intestinali e simili e metodo di ingegneria gentica per la produzione dei detti vaccini
EP0250614A1 (en) * 1986-06-23 1988-01-07 Serum Und Impfinstitut Und Institut Zur Erforschung Der Infektionskrankheiten Schweiz. DNA fragments encoding the chromosomal nucleotide sugar synthetases and glycosyl transferases
EP0257837A1 (en) * 1986-08-19 1988-03-02 Enterovax Research Pty. Ltd. Hybrid bacterial strain

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