Classifications

• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • 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
  • C12N15/67—General methods for enhancing the expression
  • C12N15/68—Stabilisation of the vector
• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• the present invention relates to cloning vectors , and methods for cloning genes , in particular the 987P f imbrial genes and also to a vaccine containing the f imbriae produced thereby.
• Neonatal diarrhoea in piglets follows the proliferation of certain strains of E. coli in the small intestine. These certain strains synthesise enterotoxins which cause diarrhoea and dehydration which in turn frequently result in death of the piglets. These enterotoxigeni ⁇ strains of E. coli are characterized by a rapid proliferation in the small intestine, which has been attributed to the ability of the bacteria to attach themselves to the intestinal epithelium via fimbriae.
• a number of different fimbrial serotypes of E. coli have been associated with neonatal diarrhoea, the main ones being K88ac, K88ab, K99 and 987P.
• Genes for K88ac, K88ab and K99 are known to be plasmid encoded and have already been cloned.
• the genes coding for 987P fimbriae had not yet been cloned and it had not yet been determined whether these genes are plasmid or chromosomally located. It has been considered that sows can be vaccinated with one or more of the fimbrial antigens so that they produce antibodies thereto which in turn will prevent the enterotoxigenic E.
• fimbrial antigens in quantities sufficient to allow wide scale vaccination of the animals. Isolation of fimbriae from wild-type E. coli is difficult because of low numbers of fimbriae on the E. coll. However, if the genes coding for the fimbriae could be cloned Into a high copy plasmid vector in a suitable host, the expression of the fimbriae could be increased 10-100 fold. These fimbriae can then be isolated in high quantities and the fimbriae so produced would also be free of the wild-type enterotoxins which could produce undesirable side effects upon vaccination.
• the present invention provides a method of construction of a cloning vehicle for cloning large fragments of DNA which method comprises inserting a large fragment of DNA and a partition locus into a high copy plasmid such as pBR or pACYC based vectors.
• Particularly preferred plasmids are pBR322, pBR329 and PACYC184.
• the present invention also provides a method for the construction of a cloning vehicle for the expression of 987P fimbriae.
• Another object is to provide a cloning vector which comprises a plasmid into which has been inserted a DNA segment which contains a partition locus and a DNA segment which contains the genes coding for 987P fimbrial protein.
• the present invention also provides a method for the construction of a cloning vector capable of expressing 987P fimbriae, which method comprises:
• step (ii) Inserting into the plasmid of step (i), a DNA fragment which contains the genes for expression of 987P fimbriae.
• the partition locus most preferably used is the sop locus isolated from the mini F plasmid pML31 as described in Austin and Wierzbicki, PLASMID, 10, 73-81, 1983.
• the invention further provides a transformed microoganism, transformed by a cloning vehicle produced by the method of the invention.
• a very suitable microoganism for use in the present invention is E. coll. It is a preferred feature of the invention that E. coli be transformed by a cloning vehicle capable of expressing 987P fimbrial protein. In particular, it is preferred that E. coli be transformed by pBTA201.
• the invention also provides a vaccine comprising E. coli modified to include the cloning vector of the present invention which is capable of expressing 987P fimbrial protein.
• the vaccine of the present invention may comprise 987P fimbriae isolated from a bacterial culture of the modified bacteria.
• Fig. 1 is a DNA restriction map of the vector of the present invention, pBTA599.
• Fig. 2 is a restriction map pBTA201, the 52 kilobase insert and vector.
• Cloning Vector was constructed by inserting a partition locus from the mini F plasmid pML31 into pBR329.
• Plasmids derived from pBR322 lack the functions required for equal partition. This means that pBR322 is not stably maintained past 50 generations. The partition locus however codes for a mechanism that ensures the accurate partitioning of plasmids at cell division. (Meacock and Cohen, CELL, 20, S29-S42, 1980 and Tucker et. al. CELL 38, 191-201, 1984).
• a partition locus has been shown to be located on the F plasmid of E. coli (Austin and Wierzbicki, PLASMID, 10,
• the digest was loaded onto a 0.8% agarose gel and the 4.1 kb fragment eluted onto NA45 paper. A fragment of approximately 4.2 kb present in the digest coeluted with the 4.1 kb fragment.
• the DNA was extracted from the NA45 paper by heating at 65°C for 60 minutes in TE containing 1M NaCl. The NaCl concentration was reduced to 0.2M, the DNA was phenol and chloroform extracted then finally ethanol precipitated.
• the pBR329 vector was PvuII digested in medium salt buffer, then phosphatased in 0.1M Tris pH8.
• This fragment should contain the partition locus and therefore should not only make pBR329 stable, but also make any recombinant molecules derived from this vector stable, e.g. pBR329 containing large DNA inserts.
• This new vector is called pBTA599 and is as shown in Fig. 1. 1. ANALYSIS OF PBTA599
• pBTA201 60.4kb plasmid
• pBTA360 41.3kb plasmid
• a wild type isolate of 987P was mated with a K12 NxR strain of E coli by a quantitative broth method.
• Log phase cultures of the wild type donor and K12 recipient were mixed in the proportion of 1:10 with the recipient in excess.
• the mating mixture was incubated statically at 37°C for 1 hour then mating pairs were disrupted by vortexing.
• the mating mixture was plated on sheep blood agar + Naladixic Acid. screening for transconjugants was by testing for 987P expression by agglutination using 987P antiserum. A number of positive colonies were isolated. These were shown to contain part of the plasmid content of the wild-type 987P strain. This means that the 987P genes are encoded on plasmid DNA and not on chromosomal DNA.
• Plasmid DNA was isolated from a wild-type isolate of 987P E. coll by a modified method of Hansen and Olsen (J. Bacteriol 135., 227-238, 1978) to ensure high yields of large plasmids.
• Partial BamHI digests of the plasmid DNA were performed to generate DNA fragments larger than 30 kb. The digestion was performed in medium salt buffer with aliquots being removed at 15, 30, 45 and 60 minutes digestion. The 15 minute digestion yielded the required fragment sizes.
• the pBTA599 vector was BamHI digested in medium salt buffer then phosphatased in 0.1M Tris pH 8. The partial 987P digest was ligated to the phosphatased vector in ImM ATP, lOmM Tris pH 8, lOmM MgCl 2 and lOmM DTT with 1 unit of T4-DNA ligase at
• FIG. 2 A restriction map of the 52 Kb insert and vector (PBTA201) is shown in Fig. 2.
• the 987P operon may span a large area of the pBTA201 insert as no subclones from pBTA201 were isolated which still expressed 987P fimbriae.
• 987P VACCINE PRODUCTION Host cells which contain recombinant plasmid expressing 987P fimbriae and assembly genes are maintained as freeze dried vials in the production culture collection. Cells from the storage vials are reconstituted and plated out for single colonies on selective nutrient agar medium. After growth, 987P expressing colonies are identified by sero-agglutination and the most active colonies are used to produce fermenter inocula. The inoculum is used to seed a fermenter which contains a medium comprising tryptone yeast extract salts and carbon source. The fermentation is monitored by standard methods and practices used by those skilled in the art.
• the fimbrial product Upon completion, the fimbrial product is separated from the bacterial cell in situ and the waste biomass is removed by centrifugation. The resultant fimbrial suspension is washed with buffer solutions and concentrated to its final aqueous form using hollow fibre ultrafiltration. The aqueous concentrate is stored with preservatives at 4°C after Quality Control and titre analyses.
• Formulation of the water in oil emulsion vaccine is achieved by aseptically metering in a precise volume of 987P aqueous concentrate along with other sterile antigen components and preservatives.
• the water phase is made up to a volume with sterile water and is equal to that of the sterile oil phase.
• the completed water phase is then added at a pre-determined rate to the oil phase during high speed homogenisation to give the final formulation.
• the vaccine used in the controlled challenge trials contained the four purified fimbrial antigens, K88ac, K88ab,
• Sows were inoculated intramuscularly twice during pregnancy (8 weeks and 1-2 weeks before farrowing) with the vaccine or a placebo.
• Piglets were allowed to suckle naturally before challenge. At between 5 and 13 hours old, they were dosed orally with a mixture of two enterotoxigenic E. coli strains expressing 987P fimbriae. The challenge inoculum contained q 1 x 10 viable bacteria of each strain.

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• 1985
  • 1985-12-03 ES ES549531A patent/ES8705033A1/es not_active Expired
  • 1985-12-04 KR KR860700521A patent/KR870700021A/ko not_active Withdrawn
  • 1985-12-04 DE DE86900017T patent/DE3587473T2/de not_active Expired - Fee Related
  • 1985-12-04 NZ NZ214428A patent/NZ214428A/xx unknown
  • 1985-12-04 EP EP86900017A patent/EP0242359B1/en not_active Expired - Lifetime
  • 1985-12-04 CN CN85109506A patent/CN1017451B/zh not_active Expired
  • 1985-12-04 FI FI863145A patent/FI863145A7/fi not_active Application Discontinuation
  • 1985-12-04 WO PCT/AU1985/000306 patent/WO1986003410A1/en active IP Right Grant
  • 1985-12-04 ZA ZA859297A patent/ZA859297B/xx unknown
  • 1985-12-04 JP JP61500188A patent/JPS62501675A/ja active Pending
  • 1985-12-04 AU AU52097/86A patent/AU596958B2/en not_active Expired
• 1986
  • 1986-08-04 DK DK370186A patent/DK370186A/da not_active Application Discontinuation
• 1994
  • 1994-06-30 SG SG87794A patent/SG87794G/en unknown
  • 1994-07-21 HK HK70594A patent/HK70594A/xx unknown

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