WO2005095451A1 - Sequences d'acides amines et de nucleotides du facteur de croissance epidermique de ruminants - Google Patents

Sequences d'acides amines et de nucleotides du facteur de croissance epidermique de ruminants Download PDF

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WO2005095451A1
WO2005095451A1 PCT/AU2005/000464 AU2005000464W WO2005095451A1 WO 2005095451 A1 WO2005095451 A1 WO 2005095451A1 AU 2005000464 W AU2005000464 W AU 2005000464W WO 2005095451 A1 WO2005095451 A1 WO 2005095451A1
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egf
seq
functional
fragment
amino acid
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PCT/AU2005/000464
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Alan George Brownlee
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Commonwealth Scientific And Industrial Research Organisation
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Priority to AU2005229155A priority Critical patent/AU2005229155B2/en
Publication of WO2005095451A1 publication Critical patent/WO2005095451A1/fr

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to an isolated polynucleotide molecule encoding a ruminant epidermal growth factor (EGF) peptide and to the expression of the encoded EGF peptide for use in, biter alia, de-fleecing of wool-fleece bearing animals, particularly sheep.
  • EGF epidermal growth factor
  • EGF epidermal growth factor
  • Human EGF is a peptide of 53 amino acids which includes three intramolecular disulfide bonds, characteristic of EGF peptides, essential for the peptide's biological activity.
  • the active EGF peptide is translated as a long prepro-protein of 1207 amino acids and the EGF peptide is released after proteolytic processing from position 970-1023 of the prepro-protein.
  • human EGF Approximately 70% similarity is observed between human EGF and EGFs that have been previously isolated from other species.
  • human and mouse EGF have 37 amino acids in common across the sequence of 53 amino acids.
  • Six of the common amino acids are cysteine residues (amino acids 6, 14, 20, 31, 33 and 42), which have been found to be absolutely conserved (see Figure 1 showing the amino acid sequences for EGF from various different species) and which form the abovementioned t-hree intramolecular disulfide bonds essential for the peptide's biological activity.
  • the gene encoding the EGF prepro-protein from humans is about 110,000 kilobases in length, and consists of 24 exons, with the encoding sequence for the EGF peptide divided between exons 20 and 21.
  • the gene structure for human EGF has been published as has the full length cDNA sequence for mouse and rat EGF, and a partial cDNA sequence for pig and horse EGF.
  • the Genbank database contains numerous entries for human, mouse and rat EGF cDNAs and the human genome sequence (HGS) contains the full, unannotated gene sequence for the human EGF gene. More recently, the bovine EGF sequence has been cloned, and some precursor sequences for the sheep gene (ie exons 3 to 4 spanning about 400 base pairs of intron sequence) have also been described.
  • EGF EGF-like growth factor
  • EGF has been incorporated with beneficial effect in the culture media for bovine and ovine embryos in vitro and is also -known to be of value for the in vitro culture, manipulation and survival of embryos from other livestock species.
  • EGF inhibits gastric acid secretion and modulates the synthesis of a number of hormones, including the secretion of prolactin from pituitary tumours and chorion gonadotropin from chorion carcinoma cells.
  • EGF is a strong mitogen for many cells of ectodermal, mesodermal and endodermal origin and, in particular, EGF appears to have a role in controlling and stimulating the proliferation of epidermal and epithelial cells, including fibroblasts, kidney epithelial cells, human glial cells, ovary granulosa cells and thyroid cells. EGF also stimulates the proliferation of embryonic cells.
  • EGF is -known to act as a differentiation factor for some cell types, and has been shown to strongly affect the synthesis as well as the turnover of various extracellular matrix proteins including fibronectin, collagens, laminin and glycosaminoglycans. Additionally, EGF increases the turnover and release of calcium from bone tissue, thereby promoting bone resorption, and can also act as a mitogen for the basal cells of the olfactory epithelium that produce olfactory neurones. Further, due to its mitogenic effect on endothelial cells, promoted by thrombin, EGF can also stimulate angiogenesis. Finally, EGF is a strong chemoattractant for fibroblasts and epithelial cells, and, when either alone or acting with other cytokines, has been found to be an important wound healing factor.
  • a further biological effect of EGF is the inhibition of hair growth, first observed when EGF was injected into new born mice, and which has since been administered in sheep to achieve biological de-fleecing (see the abovementioned Australian Patent No 546354).
  • recombinant non-ruminant EGF has been used commercially for this purpose, although it is not administered to pregnant ewes, in particular ewes in the third trimester of pregnancy, because research has indicated a risk of adverse effects in such animals.
  • pregnant ewes must be identified within the flock and subjected to standard shearing.
  • the present applicant considers that this problem may be overcome or alleviated by substituting this non-ruminant EGF with an EGF derived from sheep or another ruminant.
  • the present invention is based on the discovery that while ruminant animals such as sheep do not naturally produce an active EGF peptide, these animals do include within their genomes, ancestral EGF-encoding nucleotide sequences (which are now non-functional) which can be used to generate synthetic, functional ruminant EGF genes.
  • the present applicants have found t-hrough the isolation and sequencing of these remnant nucleotide sequences from sheep, goat, cow and deer and comparing the sequences against those of human and mouse EGF genes, that the sheep, goat and deer sequences for the EGF peptide contain a number of stop codons while the sequence from the cow does not include codons for all of the six cysteine residues that are essential for EGF activity.
  • the present applicant has now been able to modify the isolated sheep sequences to enable the expression of a ruminant EGF.
  • the present applicant has now been able to modify an ancestral ovine EGF- encoding nucleotide sequence by replacing stop codons present in the sequence with functional codons to enable the expression of an ovine EGF.
  • the present invention provides an isolated polynucleotide molecule encoding an epidermal growth factor (EGF) or a functional EGF fragment, wherein said molecule comprises a nucleotide sequence showing at least 50% sequence identity to that set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment.
  • EGF epidermal growth factor
  • the present invention provides an isolated EGF or functional EGF fragment comprising an amino acid sequence showing at least 50% sequence identity to that set forth in SEQ ID No:2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment.
  • the present invention provides a method for producing an EGF or a functional EGF fragment, said method comprising introducing a molecule according to the first aspect into a suitable host cell, and culturing said cell under conditions suitable for the expression of said molecule.
  • the present invention provides a composition suitable for administration to an animal, wherein the composition comprises an EGF or functional EGF fragment according to the second aspect and, optionally, a pharmaceutically- or veterinary- acceptable carrier.
  • the present invention provides a method of de-fleecing a wool fleece-bearing animal, said method comprising administering to an animal an EGF or functional EGF fragment according to the second aspect, or a composition of the fourth aspect, in an amount effective to allow for the removal of the wool fleece without the requirement for shearing.
  • Figure 1 shows a comparison of the amino acid sequences of the EGF peptides of a number of different non-ruminant species and the amino acid sequences of the EGF peptides "encoded" by isolated ancestral ruminant EGF-encoding nucleotide sequences from sheep, goat, cow, Barbary sheep and deer. Stop codons are designated by an asterisk and conserved cysteine residues are indicated. If stop codons are corrected for conserved residues, then the amino acid sequence of the sheep EGF shows no more than about 47% sequence identity with any of the non-ruminant sequences shown in the figure (nb. shows 25 identical residues with the horse sequence which is the closest non-ruminant sequence). On the other hand, when compared with the goat, cow and Barbary sheep, the amino acid sequence of the sheep EGF showed, respectively, about 98%, 81% and 96% sequence identity.
  • Figure 2 shows the sequence of the sheep EGF gene and the deduced peptide sequence where the stop codons have been corrected for the most likely ancestral codon.
  • the first corrected sequence at position 22 is a tyrosine residue (Y) based on the goat EGF-encoding sequence, a cysteine residue was inserted in position 33 based on the conservatism of this residue at this position, and arginine was inserted in position 41 again based on conservatism of this residue at this position.
  • Y tyrosine residue
  • Figure 3 shows an alignment of the nucleotide and inferred amino acid sequence of exons 20 and 21 of the EGF gene. Full intron sequence is not included but the positions are indicated. Sequences for the cow, sheep, Barbary sheep and deer have been derived from the present inventors, the sequences for the pig and human are derived from the Genbank database. The location of the final processed sheep EGF peptide sequence is shown in bold. Stop codons are indicated with an asterisk. The nucleotide sequence from Fallow deer contains a deletion in exon 21 indicated by the dashes. Only the nucleotide and amino acid sequences of the goat EGF encoding portion are included in the alignment. The sheep sequence has been found to be identical in Merino sheep, Wiltshire Horn sheep and Rambouillet sheep.
  • the present applicant has been able to express a ruminant sheep EGF. It is considered that this EGF will be useful for de-fleecing of wool fleece-bearing animals, particularly sheep, and may avoid or reduce the abovementioned problem with recombinant non-ruminant EGF of possibly causing spontaneous abortion in pregnant ewes. More particularly, it is considered that because the ruminant EGF is based on endogenous sequences, the "ruminant EGF" peptide is more likely to be well tolerated by sheep and capable of being administered at substantially lower doses than required for de- fleecing with recombinant non-ruminant EGF. h fact, it is considered that doses as low as 50 to 250 ⁇ g/kg body weight of the ruminant EGF may be suitable for de-fleecing of sheep.
  • the present invention provides an isolated polynucleotide molecule encoding an epidermal growth factor (EGF) or functional EGF fragment, wherein said molecule comprises a nucleotide sequence showing at least 50% sequence identity to that set forth in SEQ ID No:l or a portion thereof which encodes a functional EGF fragment.
  • EGF epidermal growth factor
  • the molecule comprises a nucleotide sequence showing at least 65% sequence identity, more preferably at least 70% sequence identity, still more preferably at least 85%, c— l --.:--.-- .-_ - sequence identity, and even more preferably at least 90%, to that set forth in SEQ ID No:l or a portion thereof which encodes a functional EGF fragment.
  • the molecule comprises a nucleotide sequence showing at least 95% sequence identity to that set forth in SEQ ID No:l or a portion thereof which encodes a functional EGF fragment, or which substantially corresponds to the sequence set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment.
  • An EGF peptide encoded by a polynucleotide molecule according to the present invention may be a hybrid of EGF peptides from two or more species (eg an EGF peptide comprising a hybrid sequence of the amino acid sequences for sheep EGF and one or more other EGF peptides from other ruminant or non-ruminant species). Such a hybrid EGF peptide would retain one or more biological effects of an EGF peptide in an animal.
  • the molecule comprises a nucleotide sequence which substantially corresponds to any one of the sequences set forth in SEQ ID No: 3 (corresponding to goat EGF), SEQ ID No:4 (corresponding to cow EGF), SEQ ID No:5 (corresponding to deer EGF), and SEQ ID No:6 (corresponding to Barbary sheep) or a portion of any one of said sequences which encodes a functional EGF fragment.
  • the present invention also provides an isolated EGF or functional EGF fragment comprising an amino acid sequence showing at least 50% sequence identity to that set forth in SEQ ID No:2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment.
  • the EGF or functional EGF fragment comprises an amino acid sequence showing at least 65% sequence identity, at least 70% sequence identity, still more preferably at least 85% sequence identity, and even more preferably at least 90%, sequence identity to that set forth in SEQ DO No:2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment.
  • the molecule comprises an amino acid sequence showing at least 95% sequence identity to that set forth in SEQ ID No:2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment, or which substantially corresponds to the sequence set forth in SEQ ID No:2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment.
  • the EGF peptide or functional EGF fragment comprises an amino acid sequence which substantially corresponds to any one of the sequences set forth in SEQ ID No:7 (corresponding to goat EGF), SEQ ID No:8 (corresponding to cow EGF), SEQ ID No:9 (corresponding to deer EGF), and SEQ ID No: 10 (corresponding to Barbary sheep) or a portion of any one of said sequences which represents the amino acid sequence of a functional EGF fragment.
  • An EGF peptide according to the present invention may be considered to be a hybrid of EGF peptides from two or more species (eg an EGF peptide comprising a hybrid sequence of the amino acid sequences for sheep EGF and one or more other EGF peptides from other ruminant or non-ruminant species). Such a hybrid EGF peptide would retain one or more biological effects of an EGF peptide in an animal.
  • the present invention also provides an isolated EGF peptide or functional EGF fragment comprising an amino acid sequence which is a hybrid sequence, said hydrid comprising a portion of the sequence from SEQ ID No: 2 and a portion of one or more sequences selected from the group consisting of SEQ ID No:7, SEQ ID No:8, SEQ ID No:9, or SEQ ID No: 10, wherein said hybrid EGF peptide retains one or more of the biological effects of EGF in an animal.
  • Sequence identity percentages referred to herein are to be understood as having been calculated by comparing two nucleotide sequences or two amino acid sequences, as the case may be, using alignment achieved with the Bestfit program with default settings for determining similarity (nb. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences). From the Bestfit alignment, a determination was made of the number of positions with identical nucleotides or amino acids, divided by the total number of nucleotides or amino acids in the respective sequence (ie. 159 in the case of nucleotide sequences and 53 in the case of amino acid sequences), and expressed as a percentage.
  • nucleotide sequences are intended to encompass minor variations in the nucleotide sequences which, due to degeneracy in the DNA code, do not result in a change in the encoded peptide. Further, the term is intended to encompass minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded peptide.
  • substantially corresponding as used herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in biological activity of the EGF peptide or functional EGF fragment. These variations may include conservative amino acid substitutions. The substitutions envisaged are: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, N ⁇ -alkylamino acids.
  • the term "functional EGF fragment” is intended to refer to a fragment of EGF which retains all of the six cysteine residues that are essential for EGF activity and which exhibit activity that is substantially equivalent to the EGF peptide from which it is derived.
  • EGF peptide or functional EGF fragment of the present invention may be produced by protein synthesis methods well known to persons skilled in the art or, more preferably, by recombinant techniques well -known to persons skilled in the art.
  • the present invention also provides a method for producing an EGF or functional EGF fragment, said method comprising introducing a molecule according to the first aspect into a suitable host cell, and culturing said cell under conditions suitable for the expression of said molecule.
  • the present invention also relates to a vector, which comprises the isolated polynucleotide molecule of the present invention, and a host cell which contains said vector.
  • the vector may be, for example, a phage, plasmid, viral or retroviral vector.
  • the polynucleotide molecule of the present invention may be ligated to a vector for propagation in a host cell.
  • a plasmid vector is introduced to a host cell in a precipitate, such as calcium phosphate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell.
  • the polynucleotide molecule should be operatively linked to an appropriate promoter such as phage lambda PL promoter, the E coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs.
  • suitable promoters will be well known to the persons skilled in the art.
  • the vector will preferably include at least one selectable marker. Suitable markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E coli and other bacteria.
  • the host cell may be selected from bacterial cells such as E.
  • coli Streptomyces and S. typhimurium
  • fungal cells such as yeast cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as Chinese hamster ovary (CHO), monkey kidney (COS) cells, human embryonic kidney 293 cells and Bowes melanoma cells
  • Appropriate culture mediums and conditions for host cells of these kinds are well known to persons skilled in the art.
  • the polynucleotide molecule ligated to a vector may comprise a nucleotide sequence encoding an ⁇ GF peptide, a functional ⁇ GF fragment, or an ⁇ GF prepro-protein.
  • the host cell used for expression of the ⁇ GF prepro-protein will preferably be one in which post- translational processing from position 970-1023 of the translated ⁇ GF prepro-protein can occur.
  • the present invention further provides a composition suitable for administration to an animal, wherein the composition comprises an ⁇ GF or functional ⁇ GF fragment according to the second aspect and, optionally, a pharmaceutically- or veterinary-acceptable carrier.
  • pharmaceutically-or veterinary-acceptable carrier is intended to refer to any pharmaceutically-or veterinary-acceptable solvent, suspending agent or vehicle for delivering the ⁇ GF protein to the subject.
  • composition of the present invention may be formulated for administration to an animal through any of the routes well known to persons skilled in the art, in particular subcutaneous administration and oral and nasal administration.
  • the present invention also provides a method of de-fleecing a wool fleece-bearing animal, said method comprising administering to an animal an ⁇ GF or functional ⁇ GF fragment according to the second aspect, or a composition of the fourth aspect, in an amount effective to allow for the removal of the wool fleece without the requirement for shearing.
  • the animal is preferably a sheep, goat, llama or alpaca. More preferably, the animal is a sheep. Most preferably, the animal is a Merino sheep.
  • the method involves administering the ⁇ GF or functional ⁇ GF fragment by subcutaneous administration, for example by injection or through a catheter inserted below the skin.
  • subcutaneous administration may be achieved t-hrough sustained release implant compositions or injectable depot-forming compositions.
  • the EGF or functional EGF fragment will be administered at a dose in the range of 50 to 250 ⁇ g/kg body weight of the animal, or which is sufficient to establish a plasma concentration of the EGF peptide of at least about 10 ng/ml for at least 15 hours.
  • the sheep may be fitted with a wool fleece-retention net such as those described in International Patent Specification No WO 02/43474 and US Patent No 5,447,124 or the present applicant's Australian Patent No 690878.
  • the net retains the wool fleece on the animal until removal is required, and may also assist in the manual removal of the wool fleece.
  • the EGF or functional EGF fragment of the present invention may also suitable for other uses including the promotion of embryo survival in culture.
  • the present invention also extends to an embryo culture medium comprising an EGF or functional EGF fragment according to the present invention.
  • Such media may further comprise antibiotic compounds and other agents as are well known to persons skilled in the art.
  • the present invention also extends to the in vitro culturing of an embryo, comprising incubating said embryo in the presence of an embryo culture medium comprising an EGF or functional EGF fragment according to the present invention.
  • EGF or functional EGF fragment of the present invention may also be suitable for other uses for which EGF peptides are -known to be beneficial.
  • EGF peptides are -known to be beneficial.
  • Examples in this regard include (i) control of mammary gland maturation and development in research studies, (ii) neonatal gut and lung maturation in research studies, (iii) hormone modulation studies, (iv) human models of wound repair which use ruminant animals, and (v) treatment of wounds.
  • Such uses are to be considered to fall within the scope of the present invention.
  • the EGF or functional EGF fragment of the present invention may be used to derive mimetic compounds suitable for the applications described herein.
  • the present invention also extends to such mimetic compounds of the EGF and functional EGF fragment of the second aspect.
  • mimetic compounds may be designed using any of the methods well known in the art for designing mimetics of peptides based upon peptide sequences in the absence of secondary and tertiary structural information (see Kirshenbaum et al., Curr. Opin. Struct. Biol. (1999) 9:530- 535).
  • peptide mimetic compounds may be produced by modifying amino acid side chains to increase the hydrophobicity of defined regions of the peptide (eg substituting hydrogens with methyl groups on aromatic residues of the peptides), substituting amino acid side chains with non-amino acid side chains (eg substituting aromatic residues of the peptides with other aryl groups), and substituting amino- and or carboxy-termini with various substituents (eg substituting aliphatic groups to increase hydrophobicity).
  • mimetic compounds of the EGF or functional EGF fragment of the present invention may be so-called peptoids (ie non- peptides) which include modification of the peptide backbone (ie introducing amide bond surrogates by, for example, replacing the nitrogen atoms in the backbone with carbon atoms), or include N-substituted glycine residues, one or more D-amino acids (in place of L-amino acid(s)) and/or one or more ⁇ -amino acids (in place of ⁇ -amino acids or ⁇ -amino acids).
  • peptoids ie non- peptides
  • modification of the peptide backbone ie introducing amide bond surrogates by, for example, replacing the nitrogen atoms in the backbone with carbon atoms
  • N-substituted glycine residues one or more D-amino acids (in place of L-amino acid(s)) and/or one or more ⁇ -a
  • mimetic compounds of the EGF or functional EGF fragment of the present invention include "retro-inverso peptides" where the peptide bonds are reversed and D-amino acids assembled in reverse order to the order of the L-amino acids in the peptide sequence upon which they are based, and other non-peptide frameworks such as steroids, saccharides, benzazepine 1,3,4- trisubstituted pyrrolidinone, pyridones and pyridopyrazines.
  • non-peptide frameworks such as steroids, saccharides, benzazepine 1,3,4- trisubstituted pyrrolidinone, pyridones and pyridopyrazines.
  • EXAMPLE 1 GENERATION OF A DNA MOLECULE ENCODING SHEEP EGF
  • EGF PCR primers for the EGF gene were constructed from the consensus DNA sequence of human and mouse EGF. These PCR primers were used to amplify a portion of genomic DNA from Merino sheep and the amplified sequence was confirmed, by sequencing, as representing most of exons 14 and 15 and the intervening intron.
  • the native sheep EGF sequence contains stop codons at positions 22, 33 and 41 of the EGFprotein. h order to remove these stop codons, a synthetic gene sequence was generated using an automated synthesiser according to standard methods and the stop codons replaced with functional codons considered to be the most likely ancestral codons. That is, the stop codon at position 22 was replaced with the codon for tyrosine found in the ancestral goat EGF-encoding sequence; the stop codon at position 33 was replaced with a codon for cysteine which is strongly conserved at this position and is essential for EGF activity, and the stop codon at position 41 was replaced with the codon for arginine which is strongly conserved at this position.
  • Figure 2 provides the sequence of the sheep EGF gene and deduced peptide sequence where the stop codons have been corrected for the most likely ancestral codon.
  • BAC clones One of the BAC clones was found to contain most of a gene encoding a sheep EGF prepro- protein and, consequently, major portions of this BAC clone were sequenced, including exons 20 and 21 (human gene designation) encoding the putative sheep EGF peptide. Exon sequences were identified by alignment with the human gene sequence and intron-exon boundaries were located likewise ( Figure 3). It was found that the sequence included three stop codons at positions 22, 33 and 41, thereby explaining why the sheep EGF gene is non-functional. Otherwise, the translated amino acid sequence showed less than 40% sequence identity with the sequence of human EGF (20 of 53 amino acids in common).
  • nucleotide sequences for these EGF-encoding portions are shown in Figure 3.
  • Those of goat, deer and Barbary sheep comprise one or more stop codons (see also Figure 1) and these sequences are similarly amenable to replacement of stop codons with codons that are highly conserved based on sequence alignments.
  • the stop codons at positions 33 and 41 of the EGF nucleotide sequence of Barbary sheep and goat EGF nucleotide sequence can be replaced with the codons for cysteine and arginine respectively.
  • the stop codon at position 47 for the deer EGF sequence can be replaced with the codon for leucine which is strongly conserved at this position.
  • a tryptophan codon can also be inserted at position 49 of the deer EGF sequence.
  • EXAMPLE 2 SYNTHESIS OF SHEEP EGF USING A CELL-FREE SYSTEM
  • RTS 500 Rapid Translation System
  • E. coli ?HY Kit (Roche Molecular Biochemicals) was used for the synthesis of tliree kinds of sheep EGF genes, namely a "native" form (using vector pIVEX 2.3a), a N-terminal Hisx6 form (using vector pIVEX2.4d) and a C-terminal Hisx6 form (using vector pIVEX2.3d) as described in detail in the instruction manual version 2, October 2001, catalogue number 3 269019 (Roche Molecular Biochemicals).
  • the reaction was performed according to the instruction manual in a 1 ml volume, with 10 ⁇ g of vector DNA as template, a reaction temperature of 30°C and a stirrer speed of 120 rpm.
  • pIVEX2.3a (Ncol-Bamffl) was used as the expression vector for native sheep EGF
  • pIYEX2.4d (NcoI-E ⁇ mHI) was used as the expression vector for the ⁇ -terminal Hisx ⁇ fusion
  • pIVEX2.3dMCS (Ndel-Xho ⁇ ) was used as an expression vector for the C-terminal Hisx6 fusion. Details of the plasmid constructs can be found in the instruction manual version 2, October 2001, catalogue number 3 269 019 (Roche Molecular Biochemicals) which can be downloaded at www.roche-applied-science.com/sis/proteinexpression.
  • the in vitro reaction mixture was centrifuged at 12000 rpm for 10 min.
  • the supernatant and pellet were regarded as soluble and insoluble fractions, respectively.
  • These fractions were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions according to standard methods with Real Gel Plate (10-20% gradient gel, BioCraft, Japan).
  • the gels were stained with Coomassie Brilliant Blue (CBB) to identify samples that contained recombinant protein.
  • CBB Coomassie Brilliant Blue
  • Recombinant peptides expressed as insoluble inclusion bodies can be purified under denaturing conditions according to the method described in Pearson R et al., 2000.
  • the recombinant proteins thus purified are in an unfolded state.
  • Prior to use in vaccination they can be renatured using the Renaturation basic kit for proteins according to the manufacturer's instructions (catalogue Number 96827 by Sigma Aldrich) in view of Tsumoto, K et al., 2003 followed by dialysis in 8M urea, 20mM Tris-HCI, pH 8.5, 500 mM NaCl to remove imidazole as described in Pearson R et al., 2000.
  • Cell proliferation assays can be conducted using BaF/3ERX cells, a cell line derived from BaF/3 cells transfected with human EGFR according to the method described in Elleman, T et al., 2001. Briefly, sheep EGF may be added to the cells and cell proliferation measured by
  • EGF EGF may be digested in a small volume of 0.05M phosphate buffer, pH 7 with trypsin (50 g, Worthingtom, TPCK-treated) for 4 hours at 37°C. Subsequently, another 50 mg may be added and the incubation repeated. The digestate can then be dialysed overnight at 10°C in Spectro-Por 6 membrane, and the dialysate freeze dried for storage until required.
  • sheep can be injected inside a back leg with between 50 to 250 ⁇ g/kg of EGF prepared by resolubilising the freeze dried digestate in saline.
  • sheep may be subcutaneously infused with EGF, through the use of a polythene catheter inserted below the skin so that the orifice lays on the right midside region of the body.
  • the freeze-dried EGF can be resolubilised in sterile saline solution and infused using a Harvard pump delivering a volume of 0.030 ml/min.

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Abstract

La présente invention concerne une molécule polynucléotidique isolée codant pour un peptide du facteur de croissance épidermique (EGF) de ruminants, ainsi que l'expression du peptide de l'EGF codé destiné à être utilisé, entre autres, dans le délainage d'animaux porteurs de toison de laine, en particulier de moutons.
PCT/AU2005/000464 2004-03-31 2005-03-31 Sequences d'acides amines et de nucleotides du facteur de croissance epidermique de ruminants WO2005095451A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
AU546354B2 (en) * 1980-08-04 1985-08-29 Commonwealth Scientific And Industrial Research Organisation Egf depilating method
WO2002095029A2 (fr) * 2001-05-18 2002-11-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Sequences d'acide nucleique et de proteine du facteur de croissance epidermique chez les bovins
US20030228612A1 (en) * 2002-04-30 2003-12-11 Kenward Kimberly D. Production of recombinant epidermal growth factor in plants

Patent Citations (3)

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US20030228612A1 (en) * 2002-04-30 2003-12-11 Kenward Kimberly D. Production of recombinant epidermal growth factor in plants

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