Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8103—Exopeptidase (E.C. 3.4.11-19) inhibitors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
  • G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
  • G01N2333/4353—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from nematodes

Definitions

• the present invention relates to novel allergens.
• the present invention is broadly directed to novel nematode allergens (polypeptides) and associated amino acid and nucleotide sequences including functional fragments and variants thereof.
• the invention broadly concerns allergens obtained from, nematodes of the superfamily Trichostrongyloidea.
• the present invention concerns the use of the above molecules to identify animals that are naturally resistant to parasitic nematodes to assist with selective breeding of animals that are innately more disposed to develop immune resistance to, in particular but by no means exclusively, nematode infections in sheep and cattle.
• the molecules of the present invention are most preferably, but not necessarily exclusively, derived from Ostertagia circumcincta (Teladorsagia circumcincta) and Trichostrongylus colubriformis and have application in reducing the impact of these nematodes or similar nematodes on animals as discussed herein.
• Ostertagia circumcincta Teladorsagia circumcincta
• Trichostrongylus colubriformis For ease of reference only the background to the present invention will now be described in relation to Trichostrongylus colubriformis infection. However, it will be appreciated by those skilled in the art that such description is also of general application to most nematodes of the superfamily Trichostrongyloidea.
• Trichostrongylus colubriformis is a nematode parasite which infects the small intestine of sheep and is of significant economic importance in New Zealand and many other countries worldwide. These parasites have a simple life cycle consisting of free-living stages on pasture (egg to infective larvae, L3), and after ingestion, develop through L4-L5-adult in the host gastrointestinal tract. They do not have a tissue migratory phase. T. colubriformis live in mucus covered tunnels eroded on the surface of duodenal and intestinal villi.
• T. colubriformis Various studies have suggested that immunity to T. colubriformis involve the development of Th2-type immune responses involving hypersensitivity type events. These responses include increased mast cell, globule leucocytes and eosinophil numbers in the intestinal mucosa.
• Elevated serum IgE levels have also been associated with immunity to nematode infections
• the release of inflammatory mediators may cause a deterioration of the intestinal microenvironment and consequent expulsion of nematode parasites.
• the inflammatory response may also allow the leakage of immunoglobulins into the intestinal lumen and which may also assist in the expulsion of nematodes from the gastrointestinal tract.
• Antigens that elicit hypersensitivity reactions by binding to IgE are therefore useful in diagnostic assays to identify immune animals with a Th2-type hypersensitivity immune response.
• allergens are therefore useful in diagnostic assays to identify immune animals with a Th2-type hypersensitivity immune response.
• antigens are useful in selective breeding programs for producing animals that are resistant to nematodes.
• Tco-Aspin Trichostrongylus colubriformis
• Tco-Aspin Trichostrongylus colubriformis
• a molecular weight of 31 kDa, pi 5.1 on 2-D electrophoresis gels has been identified by the inventors and its nucleotide and polypeptide sequence determined. It shares a degree of homology with a number of proteins identified as nematode aspartyl protease inhibitors. From Onchocerca volvulus an aspartyl protease inhibitor-like protein has been cloned by a number of investigators.
• Ov33-3 (Lucius, et al., 1988) an immunodominant antigen recognised by 96% of onchocerciasis patients; Oc3.6 (Chandrashekar, et al., 1991 ); OvD5B (Celine Nkenfou, Thesis, "Molecular Cloning of Genes Coding Antigens Specific For Onchocerca volvulus: Evaluation of Expressed Proteins For Use In The Diagnosis Of Onchocerciasis" University of Cameroon (1993).
• allergens for nematode infections in animals, particularly sheep and cattle, and which can be used to diagnose those animals which are naturally inclined to develop immune resistance to nematode infection.
• allergens may be useful in selective breeding programs for animals innately more disposed to develop immune resistance.
• the present invention is broadly directed to novel nematode allergens (polypeptides) and associated amino acid and nucleotide sequences including functional fragments and variants thereof to identify animals that are capable of becoming naturally resistant to nematodes.
• the present invention concerns the use of these molecules to, assist in selective breeding of animals capable of becoming naturally resistant to nematode infections.
• nucleic acid molecules and polypeptides which are the same or functional variants of, aspartyl protease inhibitors: (Tco- Aspin), of Trichostrongylus colubriformis; and/or (Oc-Aspin), of Ostertagia circumcincta; and/or (Hc-Aspin) Haemonchus contortus in identifying animals that are innately more disposed to develop immune resistance to nematodes.
• the inventors have identified a number of IgE binding antigens (allergens) of T. colubriformis, O.circumcinta and H.contortus, using western blotting techniques with purified IgE from various immune animals. Using 2-D electrophoresis, the target allergens have been isolated and identified using mass spectrometry. Recombinant allergen proteins were prepared and tested for their ability to bind IgE from the serum of animals with a history of nematode infection.
• an isolated polypeptide comprising:
• a functional fragment or variant of the polypeptides in a) above wherein the fragment or variant provokes a humoral and/or cellular immunological response in an animal with similar characteristics to that produced by a polypeptide of as outlined in a) above.
• an isolated polypeptide substantially as described above wherein the functional fragment or variant incorporates a B cell or T cell epitope of the polypeptide.
• polypeptide may be derived from a nematode parasite of the superfamily Trichostrongyloidea.
• the polypeptide may also be artificially synthesized for example recombinantly synthesized
• polypeptide and/or nucleic acid molecule may be selected from a nematode parasite from the genera consisting of Trichostrongylus, Cooperia, Dictyocaulus, Haemonchus, Ostertagia or Teladorsagia.
• the nematode parasite maybe of the genera Trichostrongylus or Ostertagia preferably selected from the species consisting of either T. colubriformis or O. circumcincta (Teladorsagia circumcincta).
• nucleic acid molecule wherein the molecule:
• a) comprises a nucleotide sequence as set forth in any one of SEQ ID NOs. 2, 4, 6 or 8;
• b) is a functional fragment or variant of the molecule(s) in a);
• c) is able to hybridise under stringent conditions to the molecule(s) in a) or b); or
• d) is a complement of the molecule(s) defined in a), b) or c); or
• e) is an anti-sense sequence corresponding to any of the sequences in a) - d).
• nucleic acid molecule encoding a polypeptide substantially as described above.
• a vector or construct comprising the nucleic acid molecule of the present invention.
• a host cell which has been transformed with a vector or construct of the present invention.
• an isolated ligand which binds to a polypeptide of the present invention.
• a probe capable of hybridizing under stringent conditions to a nucleic acid molecule of the present invention.
• a probe for a polypeptide substantially as described above.
• a probe for a ligand substantially as described above when said ligand is bound to a polypeptide of the present invention.
• the blood or serum is IgE enriched or IgG depleted using ammonium sulphate precipitation or affinity purification of sheep IgE.
• the exposure step c) may be performed by coating the polypeptide of SEQ ID NO. 1 onto microtiter plates.
• suitable methods for exposing are of course envisaged.
• the monoclonal antibodies of step d) may be mouse monoclonal antibodies.
• antibodies sourced from other animals may also be used without departing from the scope of the present invention.
• the sample may be exposed to the polypeptide via an enzyme-linked immunoassay (ELISA) or other suitable type of assay.
• ELISA enzyme-linked immunoassay
• a method of determining the immune status of an animal comprising the steps of:
• the animal may be selected from sheep, goats, cattle and equines. However, this list should not be seen as limiting as the animal may be any mammal which is prone to infection with nematode parasites.
• the animal may be selected from either cattle or sheep. Most preferably the animal may be a sheep.
• an isolated nucleic acid molecule substantially as described above wherein the molecule is a functional fragment or variant of SEQ ID NO. 6 having at least 94% homology to SEQ ID NO. 6.
• an isolated nucleic acid molecule substantially as described above wherein the molecule is a functional fragment or variant of SEQ ID NO. 2 having at least substantially 70% homology to SEQ ID NO. 2.
• an isolated polypeptide substantially as described above wherein the polypeptide is a functional fragment or variant of SEQ ID NO. 3 having at least 80% homology to SEQ ID NO. 3.
• an isolated nucleic acid molecule substantially as described above wherein the molecule is a functional fragment or variant of SEQ ID NO. 4 having at least substantially 70% homology to SEQ ID NO. 4.
• polypeptide substantially as described above wherein the polypeptide is a functional fragment or variant of SEQ ID NO. 7 having at least 80% homology to SEQ ID NO. 7.
• an isolated nucleic acid molecule substantially as described above wherein the molecule is a functional fragment or variant of SEQ ID NO. 8 having at least 75% homology to SEQ ID NO. 8.
• peptide analogues which include but are not limited to the following:
• Peptidomimetic compounds in which the peptide bond is replaced by a structure more resistant to metabolic degradation. See for example Olson et al, 1993; and
• peptide and peptide analogue includes compounds made up of units which have an amino and carboxy terminus separated in a 1 ,2, 1 ,3, 1 ,4 or larger substitution pattern.
• It also includes compounds that have an amine and carboxyl functional group separated in a 1 ,3 or larger substitution pattern, such as ⁇ -alanine, ⁇ -amino butyric acid, Freidinger lactam (Freidinger et al, 1982), the bicyclic dipeptide (BTD) (Freidinger et al, 1982; Nagai and Sato, 1985), amino-methyl benzoic acid (Smythe and von Itzstein, 1994), and others well known in the art.
• ⁇ -alanine ⁇ -amino butyric acid
• Freidinger lactam (Freidinger et al, 1982)
• BTD bicyclic dipeptide
• amino-methyl benzoic acid Smythe and von Itzstein, 1994
• Statine-like isosteres hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art are also useful for the purposes of the invention.
• a "common” amino acid is a L-amino acid selected from the group consisting of glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine and histidine. These are referred to herein by their conventional three-letter or one-letter abbreviations.
• An “uncommon” amino acid includes, but is not restricted to, one selected from the group consisting of D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), ortho-, meta- or para-aminobenzoic acid, ornithine, citrulline, norleucine, ⁇ -glutamic acid, aminobutyric acid (Abu), and ⁇ - ⁇ disubstituted amino acids.
• polypeptide polypeptide
• peptide and “protein” are all used interchangeably herein to refer to a molecule comprising a chain of one or more amino acids.
• variant refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibit:
• homology selected from substantially 90-99% homology to the sequences contained in the sequence listing and which may include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to said;
• the variant may result from modification of the native nucleotide, or amino acid sequence, by such modifications as; insertion, substitution or deletion of one or more nucleotides or amino acids, or it may be a naturally-occurring variant.
• variant should be taken to include changes (i.e. conservative substitution) to the nucleotide sequences set forth herein which do not alter the amino acid being coded for, due to the degenerate nature of the genetic code.
• variant also includes homologous sequences which hybridise to the sequences of the invention under standard, but most preferably under stringent conditions.
• stringent conditions for determining the degree of homology may refer to:
• low salt concentrations i.e. less than 1M, preferably less than 500mM and most preferably less that 200mM
• high hybridization temperatures i.e. at least 30°C, preferably greater than 37°C and most preferably greater than 50°C
• stringent hybridization conditions can be defined as 2 x SSC at 65°C, or and for example standard hybridization conditions can be defined as 6 x SCC at 55°C, provided always that the variant is capable of diagnosing nematode infection.
• the nucleotide sequence of the native DNA is altered appropriately. This alteration can be effected by synthesis of the DNA or by modification of the native DNA, for example, by site- specific or cassette mutagenesis. Preferably, where portions of cDNA or genomic DNA require sequence modifications, site-specific primer directed mutagenesis is employed, using techniques standard in the art.
• isolated means substantially separated or purified away from contaminating sequences in the cell or organism in which the nucleic acid naturally occurs and includes nucleic acids purified by standard purification techniques as well as nucleic acids prepared by recombinant technology, including PCR technology, and those chemically synthesised.
• the nucleic acid molecule is derived from genomic DNA or the mRNA of the Trichostrongylus colubriformis.
• the nucleic acid molecule may be an RNA, cRNA, genomic DNA or cDNA molecule, and may be single- or double-stranded.
• the nucleic acid molecule may also optionally comprise one or more synthetic, non-natural or altered nucleotide bases, or combinations thereof.
• a fragment of a nucleic acid is a portion of the nucleic acid that is less than full length and comprises at least a minimum sequence capable of hybridising specifically with a nucleic acid molecule according to the present invention (or a sequence complementary thereto) under stringent conditions as defined below.
• a fragment of a polypeptide is a portion of the polypeptide that is less than full length but which still retains a biological function of being capable of diagnosing nematode infection.
• a fragment according to the invention has at least one of the biological activities of the nucleic acid or polypeptide of the invention.
• polypeptides of the invention can be prepared in a variety of ways. For example, they can be produced by isolation from a natural source, by synthesis using any suitable known techniques (such as by stepwise, solid phase, synthesis described by Merryfield (1963), J.Amer.Chem.Soc. Vol 85:2149-2156) or as preferred, through employing DNA techniques.
• the cloning vector may be selected according to the host or host cell to be used.
• Useful vectors will generally have the following characteristics:
• (c) desirably, carry genes for a readily selectable marker such as antibiotic resistance.
• vectors Two major types of vector possessing these characteristics are plasmids and bacterial viruses (bacteriophages or phages).
• bacteriophages or phages include the bacteriophage lambda Uni-ZAPTM XR and the modified plasmid pBAD18 vector, AY2-4. Tight regulation, modulation, and high-level expression are characteristics of vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121-4130).
• the DNA molecules of the invention may be expressed by placing them in operable linkage with suitable control sequences in a replicable expression vector.
• Control sequences may include origins of replication, a promoter, enhancer and transcriptional terminator sequences amongst others.
• the selection of the control sequence to be included in the expression vector is dependent on the type of host or host cell intended to be used for expressing the DNA.
• eucaryotic, yeast, insect or mammalian cells are useful hosts. Also included within the term hosts are plasmid vectors. Suitable procaryotic hosts include E. coll, Bacillus species and various species of Pseudomonas. Commonly used promoters such as ⁇ -lactamase
• lactose (lac) promoter systems are all well known in the art. Any available promoter system compatible with the host of choice can be used. Vectors used in yeast are also available and well known. A suitable example is the 2 micron origin of replication plasmid.
• vectors for use in mammalian cells are also well known.
• Such vectors include well known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences, Herpes simplex viruses, and vectors derived from a combination of plasmid and phage DNA.
• the expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed.
• the control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence.
• useful expression control sequences are the lac system, the trp system, the t ⁇ c system, the trc system, major operator and promoter regions of phage lambda, the glycolytic promoters of yeast acid phosphatase, e.g. Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g. the early and late promoters of SV40, and other sequences known to control the expression of genes of prokaryotic and eucaryotic cells and their viruses or combinations thereof.
• a preferred promoter for use herein is the arabinose promoter (Guzman, L., Belin, D., Carson, M. J. and Beckwith, J.,1995. Ref), however, any suitable promoter is included within the scope of the present invention as would be appreciated by a skilled worker.
• the construction of a vector it is also an advantage to be able to distinguish the vector incorporating the foreign DNA from unmodified vectors by a convenient and rapid assay. Reporter systems useful in such assays include reporter genes, and other detectable labels which produce measurable colour changes, antibiotic resistance and the like.
• the ⁇ -galactosidase reporter gene is used, which gene is detectable by clones exhibiting a blue phenotype on X-gal plates.
• the ⁇ - galactosidase gene may be replaced by a polyhedrin-encoding gene; which gene is detectable by clones exhibiting a white phenotype when stained with X-gal. This blue-white color selection can serve as a useful marker for detecting recombinant vectors.
• ligand refers to any molecule which can bind to another molecule such as a polypeptides or peptide, and should be taken to include, but not be limited to, antibodies, cell surface receptors or phage display molecules.
• phage display molecule refers to a molecule, such as a peptide, protein or antibody; expressed on the cell surface of a bacteriophage or like microorganism, by techniques well known in the art.
• antibody encompasses fragments or analogues of antibodies which retain the ability to bind to a polypeptide of the invention, including but not limited to Fr, F(ab) 2 fragments, scFv molecules and the like.
• the antibody may be polyclonal but is preferably monoclonal.
• the ligand may be a phage display molecule.
• Primer pairs are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, preferably a DNA polymerase. Primer pairs can be used for amplification of a nucleic acid sequence, e.g. by the polymerase chain reaction (PCR) or other nucleic acid amplification methods well known in the art.
• PCR polymerase chain reaction
• PCR-primer pairs can be derived from the sequence of a nucleic acid according to the present invention, for example, by using computer programs intended for that purpose such as Primer (Version 0.5 ® 1991 , Whitehead Institute for Biomedical Research, Cambridge, MA).
• Probes refers to a ligand which is labelled in some way (for example, radioactively, fluorescently or immunologically), and which are used to find and mark a target molecule by binding to the target molecule or a molecule associated therewith.
• the target molecule may be a nucleic acid or polypeptide of the present invention, it may also include a polypeptide of the present invention when complexed (e.g. bound) to another ligand (such as for example an antibody), or the ligand when bound (i.e. associated with) the polypeptide.
• SEQ ID NO. 1 shows the amino acid sequence of T.colubriformis Aspin (Tco-Aspin)
• SEQ ID NO. 2 shows the nucleotide sequence of T.colubriformis Aspin (Tco-Aspin)
• SEQ ID NO. 3 shows the amino acid sequence of a truncated T.colubriformis Aspin (Tco-Aspin*)
• SEQ ID NO. 4 shows the nucleotide sequence of a truncated T.colubriformis Aspin (Tco- Aspin*)
• SEQ ID NO. 5 shows the amino acid sequence of O. circumcincta 0 Aspin (Oc-Aspin ⁇
• SEQ ID NO. 6 shows the nucleotide sequence of O. circumcincta Aspin (Oc-Aspinl SEQ ID NO. 7 shows the amino acid sequence of H.contortus Aspin (Hc-Aspin)
• SEQ ID NO. 8 shows the nucleotide sequence of H.contortus Aspin (Hc-Aspin)
• Figure 1 shows one-dimensional immunoblot analysis of T. colubriformis L3 homogenate proteins probed with time-course IgE samples from sheep during the development of immunity to nematode infection.
• IgE was purified from serum samples taken at various time-points following weaning of lambs in the field out to 18 months of age.
• Arrows indicate major antigen bands detected on immunoblots (I- 42 kDa, II- 31-32 kDa, III- 27-31 kDa, IV- 20-21 kDa, V- 2-14 kDa).
• Figure 2 shows two-dimensional immunoblot analysis of T. colubriformis L3 homogenate proteins. Proteins were subjected to isoelectric focusing (left to right) followed by
• Figure 3 shows two-dimensional gel electrophoresis separation of T. colubriformis L3 homogenate total proteins Coomassie blue stained. Spot corresponding to Tco-
• Aspin is indicated by a circle and Tco-Aspin breakdown product by a square.
• Figure 4 shows the alignment of Tco-Aspin with other members of the putative aspartyl protease inhibitor family.
• Database accession numbers for the previously sequenced members are: (Oo) Ostertagia ostertagi (CAD10783), (Pt) Parelaphostrongylus tenuis (AAG50205), (Ce) Caenorhabditis elegans
• Alignment begins at the putative initiating methionines.
• the conserved residues RDL of putative YVRDLT sequence motif suggested to be critical for inhibitor function are shown by *.
• Invariant cysteine residues are marked by ⁇ .
• Figure 5 shows one dimensional immunoblot of T. colubriformis adultjiomogenate (lane 1 ) and L3 homogenate (lane 2) immunostained with rabbit anti Tco-Aspin sera.
• FIG. 6 shows two-dimensional immunoblot analysis of T. colubriformis L3 homogenate proteins. Proteins were subjected to isoelectric focusing (left to right) followed by SDS-PAGE (top to bottom). Immunoblot was probed with serum from rabbit immunized with purified recombinant Tco-Aspin.
• Figure 7 shows one-dimensional SDS-PAGE gel of affinity purified native Tco-Aspin stained with silver stain and corresponding immunoblot developed with rabbit anti Tco-Aspin sera.
• Figure 8 shows one-dimensional SDS-PAGE gel transferred to PVDF of affinity purified native Tco-Aspin stained with Coomassie blue with segments excised for Edman sequencing (A) and corresponding immunoblot developed with rabbit anti Tco- Aspin sera (B)
• Infective larvae (3 rd stage) of 7 colubriformis (TcL3) were obtained from cultures of faeces taken from monospecifically infected Romney sheep.
• Somatic antigen (TcL3-Homog) was prepared by homogenising exsheathed larvae under liquid nitrogen in a mortar and pestle. Soluble protein was extracted in 5 mM Tris buffer pH 7.6 with protease inhibitor (Complete, Boehringer Mannheim) added. After centrifugation at 3000 rpm for 10 min, the protein concentration of the supernatant was determined by absorbance at 230/260 nm, before being aliquoted and frozen at -70°C.
• TcL3-Homog (-360-1920 ⁇ g) was precipitated in 3600 ⁇ l acetone at -20°C for 30-120 min. After centrifuging at 17000g for 30 min at 4°C, acetone was removed and the precipitate dried. The precipitate was dissolved in -280 ⁇ l of rehydration buffer containing 8 M urea, 3 M thiourea, 4% CHAPS, 40 mM dithiothreitol, 0.5% IPG buffer (pH 3-10 or pH 4-7) and a trace of bromophenol blue.
• the solution was used to rehydrate Immobiline DryStrips (13 or 18 cm, pH 3-10 or pH 4-7) at 30V for 30-60 hr on an IPGphor (Amersham Pharmacia Biotech). Proteins were focused at 20°C according to the following voltage protocol: 120V for 2 hr, 500V for 1 hr, 1000V for 1 hr, 1000-8000V gradient for 30 min, 8000V for 6.0-8.5 hr (Total Vhr were 52000 to 80000 Vhr). After a standard equilibration step, proteins were run in the second dimension on 10 to 18% linear SDS-Page gels. Proteins in the resolved gels were either stained with Copper (Bio-Rad) or microwave assisted Coomassie blue R-250 (Wong 2000) or transferred to nitrocellulose according to the manufacturer's directions (Bio-Rad).
• Proteins transferred to nitrocellulose were initially detected with Ponceau S (Harper & Speicher, 1995) and the membrane marked to assist later identification of proteins in gels for further analysis. All incubations were at room temperature and washing of membranes was with PBS + 0.05% Tween 20, 3 x 5 min. Following blocking with Blotto, nitrocellulose was probed with purified sheep IgE (Shaw et al., 1997) at 10-15 ⁇ g ml "1 overnight. IgE was obtained from sheep infected monospecifically with T colubriformis or field grazing sheep and which showed high levels of immunity as determined by faecal egg count and worm counts (data not shown).
• Membranes were incubated sequentially with mouse monoclonal anti-ovine IgE antibodies (XB6 & YD3 1/15 dilution of culture supernatant) and horseradish peroxidase-conjugated goat anti- mouse IgG gamma chain specific (1/1000) (Sigma Chemical) before detection with 3-Amino-9- ethylcarbozole.
• a protein spot corresponding to a strong IgE binding spot on Western blots with molecular weight 33,000 and pi 5.1 (Tco-Aspin) was analysed to determine amino acid sequence information.
• Protein spots from Coomassie blue stained gels were identified by comparison with IgE stained companion Western blots. Spots were excised, destained and digested with trypsin (Shevchenko et al., 1996). Briefly, spots were destained in 25 mM NH HC0 3 in 50% acetonitrile (ACN). Spots were then dried by centrifugal evaporation and rehydrated in 25mM NH HCO 3 containing 12.5 ⁇ g ml-l trypsin and incubated at 37°C for 16 hr.
• Peptides were then extracted sequentially with 25 mM NH 4 HCO 3 , 50% ACN/0.5% trifluoroacetic acid (TFA)(3 times) and 100% ACN. The combined extracts were dried in a Speedvac, rinsed with milliQ water, then dried again. Extracts were then dissolved in 0.5%TFA. The tryptic digest was cleaned up with ZipTips (Millipore) according to the manufacturer's directions. Peptides were eluted from ZipTips in matrix solution consisting of a saturated solution of ⁇ -Cyano-4-hydroxycinnamic acid in 50% ACN/ 0.5% TFA and spotting directly onto Maldi sample plate.
• TFA trifluoroacetic acid
• Protein spots were submitted to the Australian Proteome Analysis Facility (APAF, Sydney, Australia) for determination of amino acid sequence from selected peptides. Briefly samples undergo a 16-hour tryptic digest at 37°C. The resulting peptides were purified using a ZipTip to concentrate and desalt the sample. The samples were then analysed by ESI-TOF MS/MS using a Micromass Q-TOF MS equipped with a nanospray source, using either flow injection coupled to a Waters CapLC, or manually acquired using borosilicate capillaries for nanospray acquisition. Data was acquired over the m/z range 400-1800Da to select peptides for MS/MS analysis. After peptides were selected, the MS was switched to MS/MS mode and data collected over the m/z range 50-2000Da with variable collision energy settings. Amino acid sequences were then determined from this data.
• APAF Australian Proteome Analysis Facility
• TC41A and TC41 I The protein spot when subjected to QTOF mass spectrometry yielded the sequence of two peptides, TC41A and TC41 I. Also the mature amino terminus of the protein was determined by protein sequencing. Forward and reverse degenerate oligonucleotides were designed and used as primers in polymerase chain reaction (PCR) under permissive conditions. 7. colubriformis total RNA was prepared with Trizol (GibcoBRL) using manufacturers protocols except the initial extraction was performed by grinding larvae in presence of Trizol under liquid Nitrogen. Total RNA was converted to cDNA by standard procedures using Superscriptll (GibcoBRL) and used as the template in subsequent PCR. The primers used in PCR are listed below.
• PCR reactions were performed with 7. colubriformis, H. contortus, and O. circumcincta cDNA with various combinations of these primers, in addition to the nematode spliced leader primer (SL1).
• PCR conditions using degenerate primers was as follows; 95° for 2', followed by 10 cycles of 95° for 30sec, 35° for 45sec, + 1.0° per cycle, 72° for 45sec followed by 30 cycles of 95° for 30sec, 45° for 45sec, + 0.2° per cycle, 72° for 60sec, + 2sec per cycle. Utilizing 7.
• colubriformis cDNA a product of 240bp was observed by agarose gel electrophoresis of products following reactions with the primer combination of SL1 and TC41Arev while all other combinations yielded no detectable PCR product.
• a product of 740bp was observed by agarose gel electrophoresis of products following reactions with the primer combination of SL1 and HO- Aspin 3.1 with the O. circumcincta cDNA template.
• a product of 700bp was observed by agarose gel electrophoresis of products following reactions with the primer combination of Hc- Aspin 5.1 and HO-Aspin 3.1 with the H. contortus template. The 3' end of H.
• contortus Aspin was generated by use of the primers Hc-Aspin 3. RACE and PolyTG in a PCR reaction with the H. contortus cDNA template. PCR products were cloned into pCR2.1 using TA cloning (Invitrogen) and sequenced using ABI 377 automated DNA sequencer (Waikato DNA sequencing facility). Analysis of the 7. colubriformis sequence revealed homology to the 5' end of various nematode protease (pepsin) inhibitor mRNAs. It was also noted that a previously identified EST clone (unpublished) had homology to these protease (pepsin) inhibitors at the 3' end.
• pepsin nematode protease
• Tco-Aspin specific 5' and 3' primers TC41.not3 and TC41.sig.5.nde, each containing restriction endonuclease sites for cloning into the expression vector AY2-4.
• These primers were used to generate the mature Tco-Aspin coding sequence in a PCR reaction using 7. colubriformis cDNA as template.
• the primers TC41.sig.5.nde and Tco- Aspin.3.2. not were used to generate truncated Tco-Aspin (Aspin * ) coding sequence in a PCR reaction using 7. colubriformis cDNA as template.
• the primers Hc.Aspin. ⁇ .Nde and Hc.Aspin.3.Not were used to generate the mature Hc-Aspin coding sequence in a PCR reaction using H. contortus cDNA as template.
• the primers Oc.Aspin. ⁇ .Nde and Oc.Aspin.3.Not were used to generate the mature Oc-Aspin coding sequence in a PCR reaction using O. circumcincta cDNA as template.
• the resultant DNA products were digested b appropriate restriction enzymes, cloned into the expression vector AY2-4 and the cloned insert was sequenced.
• Tco-Aspin/ AY2-4 Bacteria containing either the Tco-Aspin/ AY2-4, Tco-Aspin * / AY2-4, Hc-Aspin/ AY2-4 or Oc- Aspin/ AY2-4 construct were grown in LB broth at 37°C to an optical density (600nm) of 0.8 at which time protein synthesis was induced by the addition of 0.2% L(+)arabinose (BDH). Induction proceeded for 16 hr at 30 at which time the induced bacteria were pelleted and resuspended in 300mM NaCI/ ⁇ OmM P0 4 pH 8.0 buffer containing 1mg/ml lysozyme and incubated on ice for 1 hr.
• optical density 600nm
• BDH L(+)arabinose
• Recombinant protein was immobilized to Ni- NTA resin (Qiagen), washed with 300mM NaCI/ ⁇ OmM P0 4 pH 8.0 buffer containing 20mM imidazole to remove the contaminating E. coli proteins after which the recombinant protein was eluted by the addition of 100mM imidazole. The recombinant protein was subsequently dialyzed versus 300mM NaCI/50mM P0 4 pH 8.0 buffer to remove the imidazole and the protein concentration determined. Recombinant Tco-Aspin was also purified from the insoluble, inclusion body fraction.
• the bacterial pellet following centrifugation of the lysed bacteria was solubilised in 8M urea, 300mM NaCI/50mM P0 4 pH 8.0 buffer and applied to the Ni-NTA column equilibrated in the same buffer. After washing with several column volumes, the column was washed in the same buffer without urea prior to elution in imidazole as above.
• New Zealand white rabbits were immunized with purified 6-his tagged recombinant protein. Immunizing doses consisting of 100 ⁇ g of Tco-Aspin mixed with Montanide ISA ⁇ O at a ratio of 6 parts Montanide: 5 parts aqueous solution were injected intra muscularly and subcutaneously. Four weeks later a second immunizing dose prepared exactly as the first, was administered by the same route. The rabbit was bled by heart puncture under anesthetic 10 days after the second immunization. After clotting for 1 hour at room temperature the blood was centrifuged at 1300g for 15 minutes. The serum was collected and stored at -20°C until needed. This serum is referred to as anti Tco-Aspin.
• IgG was purified from the sera of a rabbit immunised for Tco-Aspin by Protein G sepharose affinity using standard techniques. Purified antibody was bound to NHS-activated sepharose (Amersham Pharmacia) as per the manufacturer's protocols. TcL3-homog was passed through the immobilised rabbit antibody column at 0.5-2.0 ml/min. The column was washed with buffer (20 mM phosphate buffer, 500 mM NaCI, pH 7.0) until a baseline was reached at absorbance of 280 nm. The bound native Tco-Aspin was eluted with either 0.1 M glycine or formic acid pH 3.0. The elutant was neutralised with 1M Tris or ammonium hydrogen carbonate pH 8.0. Eluted fractions were vacuumed dried before analysis by 1-D electrophoresis.
• Antigen-specific lgG1 and IgE were detected by ELISA as described previously (Shaw et al 1998a).
• the optimal concentration of soluble recombinant for coating the plates with Tco-Aspin having the amino acid sequence SEQ ID NO. 1 was determined to be 0.2 and 5 ⁇ g ml "1 in PBS for IgE and lgG1 assays respectively.
• serum samples were obtained from AgResearch's selectively bred Romney sheep lines selected for high (susceptible) or low (resistant) faecal nematode egg count. Samples were also taken at the same time from an unselected control flock. Results were expressed as mean absorbance in O.D. units. For statistical analysis antibody O.D values were log e transformed to normalise the distributions.
• Serum samples were pre-treated by precipitating 600 ⁇ l of serum with ⁇ OO ⁇ l of 76% saturated ammonium sulphate in distilled water. Samples were vortexed for 10 sec, repeated at 1 ⁇ min, and then after 30 min centrifuged in a microcentrifuge (10 min at 13,000 rpm). The supernatant was collected and diluted 1 :1 in distilled water plus 0.1% Tween 20 in preparation for assay.
• Tco-Aspin Native or recombinant Tco-Aspin (0.1-10 ⁇ g) in PBS was injected using 26 gauge needles.
• Tco-Aspin was injected.
• the injection site located on the inside upper rear leg area has minimal wool coverage which aided injection and reaction evaluation.
• 100 ⁇ l of PBS containing Aspin was injected intra-dermally.
• a small distinctive bubble formed at the injection site.
• the time and sheep tag number was then recorded.
• Injection of other animals then continued for around 20 minutes. Reading of reactions was carried in the order of injection.
• the sheep were located, caught, turned over and injection site identified. Reactions were scored on a scale of 0-4 (Table 1 ). A score of 0 or 1 is regarded as negative. A score of 2 or more is regarded as positive.
• the reaction was measured using a vernier caliper by reading the longest diameter of the reaction wheal and its transecting diameter, in millimeters.
• the area of skin test reaction was calculated by multiplying these 2 values (mm 2 )
• TcL3-homogenate was separated electrophoretically, electroblotted and immunostained with affinity purified ovine IgE.
• Previous studies using 1-D SDS-Page gels had identified protein bands at approximate molecule weights of 12-14, 20-21 , 27-31 , 31-32 and 42 kDa as being associated with allergic immunity to 7. colubriformis infections in field grazing sheep ( Figure 1 ).
• TC41A 884.9 [M+2H] 2+ SASEQQE[L/I]TNYEK and TC41 I; 737.46 [M+3K] 3+ GEAEQFL.
• a N- terminal sequence of LTVGTI was obtained by Edman degradation of the 33kDA/pl ⁇ .1 spot following its transfer to a PVDF filter.
• oligonucleotide primers were designed from the internal peptide sequences and used in PCR in combination with each other or with primers corresponding to the nematode splice leader sequence and polyT.
• a PCR product from 7. colubriformis cDNA was obtained using the primers TC41Arev and SL1. This product was TA cloned (InVitroGen) into the plasmid pCR2.1 and the insert was sequenced. Analysis of the sequence revealed that this cDNA was from the same gene as a partial cDNA clone previously sequenced from a 7. colubriformis cDNA library (unpublished).
• the overlapping, combined cDNAs encode a protein (Tco-Aspin) with strong homology to proteins of the aspartyl protease inhibitor family that contains the peptides identified by sequencing of the 33kDa/pl ⁇ .1 spot (see below).
• the primers TC41.sig. ⁇ .nde and TC41.not.3 were synthesized based on DNA sequences at the amino- and carboxyl terminus of the Tco-Aspin coding region. These primers were used in a PCR with a 7. colubriformis cDNA template to obtain a complete copy of the coding DNA for Tco-Aspin.
• the resultant PCR product was sequenced and cloned into the expression vector AY2-4 containing the arabinose promoter, a polyHis domain, and a monoclonal epitope tag.
• the plasmid was transfected into E. coli bacteria and successful transfectants were identified by ampicillin resistance.
• the recombinant bacteria were grown in culture and induced for expression of Tco-Aspin with arabinose.
• Recombinant Tco-Aspin was found in both the soluble protein fraction and in the insoluble, inclusion body fraction following induction.
• the soluble Tco-Aspin was directly purified by immobilised metal affinity chromatography (IMAC). The insoluble fraction was solubilised in a chaotropic agent and then purified by IMAC.
• IMAC immobilised metal affinity chromatography
• This cDNA sequence of 684 base pairs codes for a protein of 228 amino acids (SEQ ID NO. 1 ) with a predicted molecular mass of 2 ⁇ ,414 Da and a calculated pi of ⁇ .31.
• the N-terminus amino acid sequence contains a putative signal peptide (SignalP V1.1; http://www.cbs.dtu.dk/services/SignalP/index.htmn with a suggested cleavage site between residues 1 ⁇ and 16 (alanine-alanine).
• N-terminal sequencing showed that the mature protein's N-terminus is LTVGTI suggesting that further processing removes the amino acids APRQKR.
• the putative mature protein of 21 ⁇ amino acids, without the signal sequence and amino acids APRQKR would have a predicted molecular mass of 23066.77 Da and pi of 4.93. The sequence does not contain predicted N-glycosylation sites.
• suum, PI-3 (Martzen etal, 1990, Kageyama 1998) inhibit the in vitro activity of aspartyl proteases such as pepsin and cathepsin E.
• porcine pepsin we were able to demonstrate a similar inhibitory activity with affinity purified native Tco-Aspin.
• colubriformis DNA coding for this protein it was shown as having homology to a Caenorhabditis elegans hypothetical protein Y5F2A.1. No significant IgE binding could be demonstrated to a recombinant version of this protein.
• Tco- Aspin* truncated Tco-Aspin
• an expression vector was generated expressing a Tco-Aspin peptide lacking the sixty- five amino acids C-terminal to the double lysines at position 164-165 of the protein sequence.
• the molecular weight of this peptide is proportional to that of native Tco-Aspin truncated product (20-21 kDa).
• Optimal protein concentration for specific IgE assays was found to be 0.25 ⁇ g ml "1 for Oc-Aspin and Tco-Aspin* and 5 ⁇ g ml "1 for Hc-Aspin.
• IgE and lgG1 responses to recombinant Tco-Aspin were measured in lambs from lines selectively bred for resistance (R) or susceptibility (S) to nematode parasite infection at AgResearch's Wallaceville Animal Research Centre (Morris et al., 2000). An unselected control (C) line was also maintained. Lambs from all three lines grazed together on worm-infected pasture. Table 2 shows that the R-line lambs had significantly higher levels of anti-Tco-Aspin IgE than did S- or C-line lambs in serum samples taken in January and March when the lambs were approximately four and six months of age.
• the genetic correlation indicates the degree to which genes controlling the expression of one performance measure in an animal are also associated with the expression of another performance measure.
• No significant genetic correlations were found between specific IgE response to Aspin and dag scores, and (not shown) no significant genetic correlations were found between anti-Tco-Aspin lgG1 levels and FEC at four or six months of age, or between anti-Tco-Aspin lgG1 levels and dag scores.
• Table 3 Genetic correlations between measured antibody traits, skin-test traits and log e Faecal Egg Count (FEC), breech soiling score (dag score) or live-weight gain (weaning to January, or January to April).
• Iog e (7co-API-1 IgE Jan+ ⁇ ) -0.44 s.e. 0.12 a -0.51 s.e. 0.12 a 0.18 s.e. 0.16 NS 0.02 s.e. 0.16 NS -0.25 s.e. 0.17 NS -0.15 s.e. 0.18 NS
• Tco-Aspin and perhaps aspartyl protease inhibitors from other gastro-intestinal nematodes might have efficacy in an intradermal skin test aimed at rapidly identifying sheep disposed to develop immune resistance to nematodes under field conditions.
• Table 4 shows that, from an evaluation of the skin test with 218 selection-line animals, those in the R, C and S lines responded significantly differently to the skin test, whether measuring allergic area, using a 0-4 score, or defining a negative/positive criterion for allergy. These three measures were also significantly negatively associated with FEC measured in six-month animals (P ⁇ 0.063 for area, and P ⁇ 0.005, at least for test score (0-4) and negative/positive test outcome). Table 4 also shows that the R-line animals were not significantly different in response from the resilient-line lambs (the latter being bred for their ability to continue growing without the need for anthelmintic treatment when under serious nematode challenge).
• Table 4 Summary of least squares analyses on skin-test traits in ewe lambs, using area measurement, test score, and negative/positive response.
• dags in the breech area is brought about by diarrhoea thought to be associated with inflammatory responses to nematode infections and is particularly evident on some resistant-line sheep (Bisset et al., 2001). We have thus confirmed that it is possible to establish more practical tests to measure IgE levels specific for defined allergens, apart from ELISA, and these may be used effectively as part of a selective breeding program.
• Table 2 (Least squares analysis)_137 born-2001 Resistant (R), Susceptible (S) & Control (C)- line ram lambs.
• Rows 3-5 Skin test results for b'2002 lambs (223 Ewe lambs from all lines of ewe lambs at Wallaceville (Lines 2, 22, 42, 81 , 82 & 85), plus 42 R-line ram lambs, with all FEC data from 1979-2002).
• Table 4 (Least squares analysis) 218 ewe lambs from R, S, C and Resilience lines.
• Coding sequence for Aspins from O. circumcincta (Teladorsagia circumcincta) and H. contortus was obtained and recombinant proteins expressed.
• the coding DNA was inserted into bacterial expression vectors and the corresponding recombinant proteins were produced and purified as described previously for Tco-Aspin.
• the amino acid sequences of the three Aspins are very similar. Homologies with Tco-Aspin for Ostertagia circumcincta are (% identity/% similarity) (87.1%/89.8%) and Haemonchus contortus (84.9%/87.6%)
• Serum samples from Resistant line ram lambs born in 2001 were used to compare the reactivity of IgE to various native and recombinant Aspin preparations (Table 8).
• Tco-Aspin * Native and recombinant Tco-Aspin identified around 58-60% of the resistant line ram lamb as Aspin IgE positive. Of those serum samples positive to native Aspin, recombinant Tco-Aspin and Oc-Aspin identified 96.4% and Hc-Aspin 42.9%. Oc-Aspin identified a further 27.1% serum samples as Aspin IgE positive. The truncated form of Tco-Aspin (Tco-Aspin * ) identified 45.8% of resistant line ram lambs, 71.4% of those animals identified by native Tco-Aspin.
• Tco-Aspin & Tco-Aspin* suggest that most of the IgE epitopes are found on the truncated form of Tco-Aspin.
• recombinant Tco-Aspin is as effective as native 7.
• Recombinant O. circumcincta may be of better use in that it identifies a greater number of sheep as Aspin positive animals.
• H. contortus Aspin is less useful in identifying Aspin positive animals

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