WO1998031793A2 - Immunomodulating properties, amino acid and nucleotide sequence of colostrum derived growth factor - Google Patents

Abstract

There is provided a partial amino acid sequence for the 3' end of porcine colostrum derived growth factor as set out in SEQ ID NO 1. The polynucleotide sequence encoding for that portion of CDGF is also provided and is set out in SEQ ID NO 2. Further, the immunomodulating properties of CDGF have been investigated. It has been found that CDGF is able to stimulate cell proliferation and differentiation and is also able to regulate inflammatory responses.

Description

"Immunomodulating properties, Amino Acid and Nucleotide Sequence of Colostrum Derived Growth Factor"

The present invention relates to novel applications of a growth factor previously found to be present in colostrum, to novel sequence information regarding that growth factor and to an improved process for purifying that factor.

The present invention is concerned with the polypeptide growth factor CDGF (colostrum derived growth factor) described in our WO-A-97/04009. As described in WO-A- 97/04009 CDGF is known to be a polypeptide. (The term "polypeptide", as used herein, refers to any peptide molecule, regardless of molecular size, shape or construction) . CDGF has an apparent molecular weight of over 200kDa and is therefore either bound to a large carrier molecule or is oligomeric.

CDGF has been found to have a molecular weight in the range of 60-80 kDa and is hydrophobic .

CDGF has been found to have the following physical characteristics: 1) Activity abolished by proteolytic enzymes (trypsin) . 2) Essentially heat stable (over 70% activity retained after heating to 100°C for 10 minutes).

3) Stable in 2.4M formic acid.

4) Stable and soluble in 100% of 0.1% trifluoroacetic acid.

5) Activity is retained after treatment with 50mM dithiothreitol .

6) Stable in 50% acetonitrile.

7) Stable and soluble in 50% ethanol.

CDGF may also be identified as providing the following biological effects:

1) Stimulates proliferation of intestinal cells in in vitro cultures.

2) Promotes the differentiation of intestinal cells as measured by lactase activity and protein glycosylation.

3) Stimulates phosphorylation of membrane proteins of microvillar vesicles (MVM vesicles) (as described in the phosphorylation assay in the Examples of WO-A-97/04009) .

4) Stimulation of the Ras/GAP in MVM vesicles.

5) Stimulation of the fos gene. Partial sequence analysis of porcine CDGF has now been completed and the sequence information is set out in SEQ ID Nos. 1, 2 to 9. The nucleotide sequence encoding for the polypeptide of SEQ ID No 1 is set out in SEQ ID No 2.

The present invention therefore provides a polypeptide having an amino acid sequence which comprises the amino acid sequence as set out in SEQ ID No. 1, a functional equivalent or portion thereof.

The term "functional equivalent" as used herein includes modified versions of CDGF which exhibit substantially the same level, or an improved level, of a biological activity as compared to the naturally occurring protein. The modifications to CDGF which fall within this definition include (but are not limited to) versions of CDGF having one or more of the following modifications: amino acids deletions, amino acid insertions and/or amino acid substitutions. Also included within this definition are modifications where whole domains of CDGF are rearranged, deleted or substituted by alternative polypeptides .

The term "portion", as used herein with reference to the sequence of CDGF, refers to at least 50% (preferably at least 60%) of the known sequence.

In a further aspect, the present invention provides a composition, said composition comprising a polypeptide having an amino acid sequence which comprises the amino acid sequence as set out in any one of SEQ ID No. 1, 2 to 9, a functional equivalent or portion thereof.

The present invention also provides the use of the polypeptide of the present invention for pharmaceutical use or as an additive in a nutrient formulation, especially for baby or infant food formulations.

The present invention further provides a polynucleotide comprising a nucleotide sequence as set out in SEQ ID No 2 , or a nucleotide sequence which encodes for a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID Nos 1, 2 to 9, a functional equivalent or portion thereof.

Vectors comprising said polynucleotide and host cells containing them are also part of the invention.

Originally CDGF was only found to be present in colostrum and, in declining amounts, in early milk. Thus, the term "Colostrum Derived Growth Factor" was believed to be a suitable descriptor. However, further investigations indicate that CDGF is present in other body fluids, for example milk, plasma, saliva, intestinal fluids and serum.

Porcine and bovine colostrum are good sources of CDGF.

In our WO-A-97/04009 we described the use of CDGF to promote the growth and/or function of tissues as well as the use of CDGF to combat or alleviate disease states or abnormalities. Thus CDGF was described as having utility for wound healing applications (for example following accidents or surgery) and to combat or alleviate disease conditions where tissue regeneration is required (for example ulcers). CDGF was also described as having utility in combating deleterious side effects of other drug regimes, in particular in decreasing the side effects of chemotherapy or radiotherapy treatment for cancer, especially in combating the influence of such treatments on the gastrointestinal tract. CDGF is believed to be of particular utility for tissue derived from the gastro-intestinal tract, skeletal muscle, liver and glandular tissue.

In more detail, we described that CDGF may be helpful in treating post-operative patients to promote healthy tissue regeneration, and CDGF treatment following the surgical removal of tumours is beneficial.

One of the effects of CDGF is to promote tight junctions between the cells lining the gastrointestinal tract. This is of importance for pre-term neonates , which may possess an immature and "leaky" intestine ( ie the junctions between the cells of the intestine are not sufficiently tight) . This in turn can affect the absorption of nutrients . Likewise neonates which are atopic (and are thus prone to allergy) have also been shown to have immature "leaky" intestines. Administration of CDGF (either as a medicament or as a food supplement) may therefore be advantageous in promoting the maturation of the gastrointestinal tract of such neonates. This has both human and animal application. Examples of animal applications include the use of CDGF to promote early weaning of farm animals (eg pigs), or the use of CDGF to prevent or minimise unwanted side effects of early weaning in animals (eg scour) .

In a related manner, patients requiring total parenteral nutrition (TPN) may benefit from receiving CDGF, either in a separate formulation or as a component of the TPN composition.

With regard to the gastro-intestinal tract, CDGF may: a) Promote the regeneration of damaged tissue.

b) Promote intestinal and body function of pre-term neonates, nutritionally-compromised neonates and adults .

c) Promote the barrier function of the gastro- intestinal tract, thus enhancing disease resistance.

d) Combat intestinal colonisation by micro-organism (especially bacterial pathogens).

It has now been found that CDGF possesses other desirable properties. Specifically, CDGF has immunomodulating properties . Such properties are also found in fragments of CDGF, specifically the polypeptides described above may have these properties and are included by the definition of "functional equivalents or portions thereof" when applied to CDGF.

Thus, the present invention provides the use of the polypeptide CDGF a functional equivalent or portion thereof as an immunomodulator . By the term "immunomodulator" we mean that CDGF alters the activity or production of at least one component of the immune system. Included within this definition is the ability to promote or inhibit antibody production, or differentiation of cellular components (eg lymphocytes) of the immune system.

In another aspect the present invention provides a composition for use as an immunomodulator, said composition comprising CDGF, a functional equivalent or portion thereof as an active ingredient, together with an inert carrier or excipient. In one embodiment the present invention provides a composition for use as an immunostimulator, said composition comprising CDGF, a functional equivalent or portion thereof as an active ingredient, together with an inert carrier or excipient.

In another embodiment the present invention provides a composition for use as an immunoinhibitant, said composition comprising CDGF, a functional equivalent or portion thereof, together with an inert carrier or excipient.

Desirably the compositions of the present invention may be formulated for therapeutic applications (including prophylactic therapeutic applications), and the carrier or excipient should be chosen accordingly.

Alternatively the compositions may be formulated as a food supplement or food substance. For example, the present invention includes baby or infant food formulations and also TPN formulations or invalid food formulations.

The compositions of the present invention may comprise other ingredients. Mention may be made of inter alia, biologically-active ingredients (for example antibiotics; other growth factors such as IGF, EGF, FGF or PDGF including combinations of cytokines with amino acids or polyamines (for example IGF/glutamine combinations); insulin; cytokines for example interleukins or any other agent having a biologically desirable effect), viscosity adjusting agents; osmolarity adjusting agents, buffers, pH adjusting agents, flavourings, stabilisers, colourings, preservatives and the like. Delayed release or controlled release formulations are also included. The compositions of the present invention may be formulated for administration by any suitable means . Particular mention may be made of oral, enteral, parenteral, subcutaneous and nasal routes of administrations. The compositions may be prepared as a spray, solution, suspension, colloid, concentrate, powder, granules, tablets, pressed tablets, capsules (including coated and uncoated tablets and capsules), suppository and the like. Delayed release or controlled release formulations are also included.

Advantageously the compositions will be sterile, and will be suitable for medical use.

In another aspect the present invention provides the use of CDGF, a functional equivalent or portion thereof in the manufacture of a medicament for altering ( for example stimulating or inhibiting) at least one component of the immune system.

In a yet further aspect the present invention provides a method of modifying (eg stimulating or inhibiting) at least one component or response of the immune system of the human or non-human (preferably mammalian) animal body, said method comprising administering a therapeutically effective amount of CDGF, a functional equivalent or portion thereof or a composition comprising such to said body.

The method of treatment described above may be to combat any impairment of the immune system or may be administered prophylactically to stimulate the immune system where it is deemed desirable to combat infection or any other immunological challenge. The invention may be of particular utility in providing a protective effect to neonates, in combatting HIV or AIDS, in promoting lymphocyte proliferation and/or differentiation and in regulating inflammatory responses .

An early example of the identification, purification and activity of CDGF from porcine colostrum is described in detail in the Examples of WO-A-97/04009. This process may be briefly summarised as follows:

a) separation of all components of colostrum having a molecular weight of over 200kDa. (Components having a molecular weight below this cut-off are discarded) ;

b) treatment of the product of step (a) with dithiothreitol and boiling for 10 minutes; and

c) The mixture of step (b) is centrifuged to spin down any precipitated matter; CDGF is located in the supernatant.

In a yet further aspect, the present invention provides an improved process for purifying CDGF. The process of the present invention provides the following steps:

a) separation of all components of colostrum having a molecular weight of over 300kDa. (Components having a molecular weight below this cut-off are discarded) ;

b) treatment of the product of step (a) by reverse phase HPLC (We have found that by using 20-60% acetonitrile over 40 minutes CDGF is located in fraction eluted at 16-18 minutes).

This process is described in more detail in Example 1. Alternatively, using the sequence information now provided, antibodies to specific epitopes can be raised (eg in rabbits) and the purified antibodies used in standard immunoaffinity procedures to obtain pure CDGF.

The present invention will now be further described with reference to the following, non-limiting, Examples and Figures in which:

Fig. 1 The final reverse phase HPLC purification step of CDGF. The column used was Aquapore C₈ reverse phase and solvents were A: 0.1% TFA and B:acetonitrile applied in a linear gradient of 20-60% B over 40 minutes as described in Example 1 with a flow rate of lm/min. UV detection λ:280nm. The symmetrical peak contains purified CDGF.

Fig. 2 HPLC separation of CDGF following Glu-C Digestion. The main trace is shown above a lower (and identical) trace run at lower sensitivity for monitoring purposes. The numbered peaks contain the peptide fragments shown in Table 2. Specifically fragments 1, 2 and 3 were present in peaks 5 and 6; fragment 4 was present in peak 7; fragment 5a was present in peaks 5 and 6 and fragments 5b and 6 were present in peak 6.

Fig. 3 Northern blot showing total and poly(A) + RNA hybridised at low stringency with FI oligonucleotide probe. Arrows indicate specific transcripts.

Fig. 4 Northern blot showing total and poly(A)+ RNA hybridised at low stringency with F2 oligonucleotide probe. Arrows indicate specific transcripts.

Fig. 5 Northern blot showing total and poly(A)+ RNA hybridised at high stringency with F2 oligonucleotide probe. Arrows indicate specific transcripts.

Fig. 6 Northern blot showing total and poly(A) + RNA hybridised at high stringency with F4a oligonucleotide probe. Arrows indicate specific transcripts.

Fig. 7a Northern blot showing total and poly(A)+ RNA hybridised at high stringency with F2 PCR product as probe. Arrows indicate specific transcripts.

Fig. 7b Northern blot showing total and poly(A)+ RNA hybridised at high stringency with FI cDNA as probe. Arrows indicate specific transcripts.

Fig. 8 Shows NF-Kappa B Activation by Electrophoretic Mobility Shift Assay. Left hand column: specific competitor with excess cold oligonucleotide. C: control (media). CT: with cholera toxin. CT + CDGF: with cholera toxin and CDGF.

Fig. 9 Shows lymphocyte transformation in the presence and absence of CDGF in newborn and weaned pigs, measured by incorporation of radiolabelled thymidine. Example 1

Introduction

A protein isolated from porcine colostrum and milk was previously shown to have potent biological activity in the intestine (see WO-A-97/04009 ) . This protein, CDGF, has been shown to interact with receptors expressed on intestinal cells, to activate the Ras/gap second messenger system and to induce the expression of early response genes. Using in vivo and in vitro models colostrum and CDGF have been shown to induce cell proliferation, growth and maturation of the gut, the last defined by measuring intestinal morphology and the precocious expression of brush border enzymes and intestinal glycosylation patterns.

The work described in this Example documents the purification, micro-sequencing and further characterisation of the biological ( immunological ) function of colostrum-derived growth factor.

Purification of colostrum-derived growth factor (CDGF)

Colostrum samples were obtained from sows not later than 8 hours from the onset of farrowing. A total of 4 litres colostrum was used for purification (step a). Acellular, defatted colostrum was then prepared by centrifugation at 27,000g for 30 minutes. For purification of CDGF, defatted acellular colostrum was processed in batches of 64ml. Colostrum was heated for 5 minutes at 100°C and allowed to cool. The supernatant was then recovered by centrifugation at approximately 52,000g for 20 minutes (step b) . Ethanol was added to the supernatant to a final concentration of 50% and the samples allowed to stand on ice for 3 hours. The supernatant was recovered by centrifugation at 52,000g for 20 minutes. The ethanol was then evaporated using a Gyrovap GL concentrator (V.A. Howe, UK) (step c) . Acetonitrile was added to the supernatant to a final concentration of 40% and again the samples stored on ice for 3 hours . The supernatant was recovered by centrifugation and the acetonitrile evaporated as described above (step d) . Proteins with molecular mass at or greater than 300K were separated and concentrated by ultrafiltration using Filtron cartridges (Varian, Cambridge, UK) of molecular cutoff 300K. Samples were washed with ultrapure water and ultrafiltrates discarded (step e) . The concentrates were then applied to 500 mg C18 Bond Elut columns.

Fractionation using C18 Bond Elut columns

Each column was prepared by first pre-wetting with 2 ml acetonitrile (Far U.V. grade, Fisons), followed by 2 ml of 0.1% Trifluoroacetic acid (TFA) . The fraction obtained after ultrafiltration was loaded (under gravity) on the column in 1 ml 0.1% TFA and the activity eluted under constant pressure (approx. 5-lOmm Hg) using a gradient from 20 to 80% acetonitrile/TFA (each % gradient added in 1 ml volumes). Activity was detected between 30 and 50% acetonitrile using both the phosphorylation bioassay and IEC-6 cell proliferation assay (see methods described in WO-A-97/04009 ) . Fractions 40% and 50% were then taken to dryness and stored at 4 C (step f ) .

Purification of CDGF by reverse phase HPLC

CDGF was further purified using 2 stage reverse-phase high performance liquid chromatography (HPLC) . The 40 and 50% Bond Elut fractions were applied to a wide-pore Aquapore C8 reverse phase column (Brownlee, Applied Biosystems, Santa Clara, USA). The sample was eluted at a flow rate of 1 ml/minute in 0.1% TFA in ultrapure water and acetonitrile. The HPLC was run on a linear gradient of 20 to 60% acetonitrile over 40 minutes, eluted peaks monitored at 280 mm and collected in 1 ml fractions. Activity was detected at 16 to 18 minutes retention time (step g) . The activity in tubes 16 to 18 was pooled and applied to the same reverse-phase column using the same flow rate but the gradient comprised of a linear gradient of 15% to 60% acetonitrile in 0.1% ammonium acetate pH 7.4 over 45 minutes. The activity was located in tubes 18 to 20 (step h) . The activity was reapplied to the reverse phase column and run using 0.1% TFA/ACN. The peak of activity was recovered in a symmetrical peak of retention time 17 minutes (step i) . This peak, estimated to be equivalent to 10 μg protein, (calibration against bovine serum albumin at 280 nm) was found to be active and was then submitted for protein sequencing (Figure 1) . The purification scheme is summarised in Table 1.

TABLE 1 CDGF Purification Scheme

Protein Volume Mitogenic Degree of Yield Specific mg/ml ( l) Activity Purification Activity (cpm) /vol (%)

a) Whole Colostrum 200 4100 78,470/lOOμl 0 100 3,923 b) Heated Colostrum 60 1230 62,570/lOOμl 2.66 23.9 10,428 c) Ethanol

Precipitation 42 1230 59,506/lOOμl 3.61 22.8 14,168

Acetonitrile

Precipitation 34 256 58,210/lOOμl 4.36 4.6 17,120

Filtron Size Cn separation and Concentration 13 64 120,396/lOOμl 23.6 2.4 92,612

Bond Elut Separation 1.95 51 77,385/lOOμl 101

1.2 396,846

0.29 ll,100/50μl 195 0.02 765,517 h) RP HPLC TFA/NH₄AC 0.04 12,300/50μl 1,567 0.02 6xl0⁶ i) RP HPLC TFA/ACN 0.01 13,512/50μl 3,568 0.02 14xl0⁶

Note : 1 ) % Yield

(Col) 78,470cpm/(100μl) X 4100ml=3, 217 , 270 , 000=(total activity)

(Purified CDGF) 13,512cpm/(50μl) X 2ml=540, 480=(total activity)

% Yield = 0.02%

2) Specific Activity=activity/unit mg protein

Example 2

Purification of CDGR by affinity based methods.

The affinity of specific antibodies for an immunogenic polypeptide offers a direct route for its isolation from crude sources . Although antibodies to the intact CDGF are not currently available on a commercial basis it is projected that polyclonal antibodies raised against selected CDGF sequences identified from its proteolytic products will be applicable in such a strategy. Thus polypeptides conforming to fragments 1, 2 and 4 have been chemically synthesised using standard methodology and these, conjugated to the carrier protein keyhole limpet haemocyanin are being used to generate polyclonal peptides specific antibodies in rabbits. The antibodies will be isolated from the hyperimmune serum and immobilised on a suitable beaded support by standard procedures. The immobilised anti-peptide antibodies will be employed in a column chromatography configuration to bind such proteins present that contain the sequence epitopes recognised by the antibodies individually or in combination. Example 3

Demonstration of Biological Activity

Phosphorylation bioassay:

Intestinal microvillar membrane (MVM) preparations were solubilised in 20 mM Hepes , 5 Mm MnCl₂, 4mM NaF, lOOμM NaV0₄, lOmM J-glycerophosphate, 1 μg/ml leupeptin, 25 μg/ml Trypsin inhibitor, 25KIU/ml Aprotinin, 0.1M PMSF, 0.05% bacitracin (Buffer B) containing 2% Triton X-100 for 3 hours at 4°C. Solubilised membrane proteins obtained following centrifugation at 27,000g for 30 minutes, were diluted with Buffer B to a protein concentration of 3 mg/ml . Thirty microlitres of solubilised membranes were incubated with CDGF at each stage of purification together with 5μl Buffer B at ambient temperature for 10 minutes and then on ice for a further 15 minutes . Phosphorylation was initiated by the addition of lOμl of 20 μM y-³²P-ATP (20μCi) prepared in Buffer B. The reaction was terminated after 15 minutes at 4°C using 3X Laemmli buffer and boiling for 10 minutes at 100°C. Phosphorylated MVMv were analysed on 7.5% SDS polyacrylamide gels under reducing conditions. Autoradiograms of dried gels were obtained with X-Omat AR film.

Mitogenic Bio-Assay

A cell line isolated from rat intestinal crypt cells, namely IEC 6, was used to detect the mitogenic activity of CDGF during purification. Cells were seeded at 10,000 cells per well of a 24 well Corning plate in 1 ml Dulbecco's Modified Eagle's medium DMEM containing 5% fetal calf serum (FCS), 2mM L-glutamine and penicillin/streptomycin antibiotics. After 16 hours growth, the cells were stepped down into DMEM containing 10 μg/ml transferrin and 0.2 μg/ml sodium selenite for at least 24 hours. The cells were then stimulated with 5% FCS, 10% defatted, acellular colostrum and between 2 and 10% CDGF for 20 hours after which 2 μCi 3H thymidine was added to each well for a further 4 hours. Cells were then washed in Hank's balanced salt solution, fixed in methanol, and precipitated in TCA. Incorporated radioactive thymidine, solubilised in NaOH, was counted using a scintillation counter. CDGF was found to stimulate IEC 6 cells significantly above the controls and, in fact, in some experiments, above the stimulation induced by colostrum (see Mitogenic Activity in Table 1) .

Micro-Sequencing of CDGF

Fifty percent of the peak shown in Figure 1 (approximately 5μg of CDGF) was suspended in 350μl of ammonium bicarbonate buffer, pH 7.8, for sequencing. CDGF was incubated with 2μg of sequencing grade Endoproteinase Glu-C (S.aureus V8 protease) (Promega, UK) in 5μl of ammonium bicarbonate, pH 7.8, and the incubation carried out at 37°C for 2 hours. At 1.5 hours incubation, 5% of the digest was run on a microbore (RP300 column) reverse phase HPLC (Applied Biosystems) using a gradient from 10% to 70% of ACN in aqueous 0.1% TFA, to confirm successful degradation. The total digested sample was then resolved by HPLC and the peptide fragments monitored at 220nm and collected. The peptide fragment isolated in tube 5 ie. Peak 5 (Figure 2) was subjected to automated sequencing on an Applied Biosystems 477A instrument with a 120A on-line phenylthiohydantoin analyser (Hayes et al . 1989). Polybrene (2mg) was loaded on to a glass-fibre filter disc, which was then pre-cycled before loading the digested fragment. After Edman degradation the anilinothiazolinone derivatives cleaved from the peptide were converted automatically into the more stable phenylthiohydantoin forms and separated on an Applied Biosystems PTH C₁₈ column that was developed with a 0-100% (v/v) linear gradient of acetonitrile in aqueous 5% (v/v) solution of tetrahydrofuran. Chromatography was performed at 55°C and the column monitored at 269nm. Peaks 6 and 7 shown in Figure 2 were also sequenced. The sequencing service was provided by Welmet Protein Characterisation Facility, Edinburgh University.

Result

The sequence data obtained was confirmed and augmented and is reported in Table 2. The full sequence obtained linking fragments 2, 1 and 3 is presented in SEQ ID Nol. The sequence of fragments 4, 5a, 5b and 6 are reported separately in SEQ ID Nos 6, 7, 8 and 9 respectively.

TABLE 2 Protein sequence

A further batch of CDGF was prepared for protein sequencing in Example 1 and the sequence data was confirmed and augmented as follows:

CDGF GluC DIGEST SEQUENCE DATA fr 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

L F M Y G G C Q G N A N N F E

r - - -

<3> I L N V P D C/L A T/P G V -

16 17 18 19 20 G/L K G/A/ -

L/D

TABLE 2 continued

fr 5a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

T T A I - R R V V/l/Y - P N P T G/K fr 5b 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

T T A - K S S R/V - N Y P - V/L -

Example 4

Identification of the molecular structure of CDGF

To identify the molecular species (mRNA) described by the above primary sequences, oligonucleotide probes were prepared to the following sequences

(primary sequence) (oligonucleotide probe)

Fl fmyggcqgn tticcytgrcaicciccrtacatraa

(SEQ ID No 10) (SEQ ID NO 11)

F2a fqtppalcqlp ggiarytgrcaiarigciggiggigtytgraa

(SEQ ID No 12) (SEQ ID No 13)

F2b gpckaallryfy taraartayctiariarigcigcyttrcanggncc

(SEQ ID No 14) (SEQ ID No 15)

F4a ilnvpdlatgv tciccigtigciarrtciggiacrttiariat

(SEQ ID No 16) (SEQ ID No 17)

F4b ilnvpdcapgv acicciggigcrcartciggiacrttiardat

(SEQ ID No 18) (SEQ ID No 19)

Probe Fl (SEQ ID No 11) encodes for the primary sequence of SEQ ID No 10 which corresponds to amino acid numbers 2 to 10 of fragment 1, Table 2 (SEQ ID No 3).

Probe F2a (SEQ ID No 13) encodes for the primary sequence of SEQ ID No 12 which corresponds to amino acid numbers 2 to 12 of fragment 2, Table 2 (SEQ ID No 4).

Probe F2b (SEQ ID No 15) encodes for the primary sequence of SEQ ID No 14 which corresponds to amino acid numbers 16 to 27 of fragment 2, Table 2 (SEQ ID No 4).

Probe F4a (SEQ ID No 17) encodes for the primary sequence of SEQ ID No 16 which corresponds to amino acid numbers 4 to 14 of fragment 4, Table 2 (SEQ ID No 6), with L at position No 10 and T at position No 12.

Probe F4b (SEQ ID No 19) encodes for the primary sequence of SEQ ID No 18 which corresponds to amino acid numbers 4 to 14 of fragment 4, Table 2 (SEQ ID No 6), with C at position No 10 and T at position No 12.

Fl, F2a and F2b exhibit homology (50-65% over 20-30 amino acids) with protease inhibitors (eg. bovine colostrum trypsin inhibitor [P00976; note all sequence reference nos are to the Genbank accession no] and alpha-1 microglobulin [P02760]. F4a and F4b exhibits homology (55% over 8 to 13 amino acids) with a known cytokine referred to as human/mouse pre-b cell stimulating factor (PBSF) [accession nos .p48061/I81182 respectively] , neurogenic locus notch protein precursor (Drosophila melanogaster) [accession no.157988] and rat epidermal growth factor, high molecular weight fragment [accession no.S08301].

Note: F4a and F4b were synthesised to cover the L/C option at position 10 and the T/P option at position 12 of the fragment 4 sequence.

To identify the presence of specific transcripts the above oligonucleotide probes were employed in Northern hybridisation protocols using poly (A)+ RNA isolated from porcine lactating mammary tissue and jejunal intestinal tissue. Tissue isolation

Mammary and intestinal tissues were obtained from a lactating sow within 24 hours of farrowing. The sow was anesthetised using halothane/0₂ mix. All procedures were carried out using aseptic technique. An incision was made in the mammary gland and tissue removed from the anterior and posterior regions of the gland. Samples were frozen immediately in liquid nitrogen. The intestines were exposed, following a midline laparotomy, and intestinal tissue from the mid-jejunum collected. Intact intestinal tissue and mucosal scrapings were sampled and frozen immediately in liquid nitrogen.

RNA isolation

Excised mammary and intestinal tissues were stored frozen at -80°C. Total RNA was extracted from frozen pulverized tissues as described by Chomcynski & Sacchi, (1987) and Puissant & Houdebine, (1990). Briefly, the tissue was homogenised, quickly and thoroughly, in 15ml of solution D and then transferred to 50ml Oakridge tubes (Sigma, UK). To the sample, 1.5ml of 2M Na acetate and 15ml phenol were added sequentially and, at each addition, the samples were mixed. The samples were then shaken vigorously with 3 ml of chloroform/isoamyl alcohol (49:1) and left on ice for 15 minutes and then centrifuged for 15 minutes at 4,300g and at 0°C using the Sorvall SS34 rotor. The top phase was removed and the aqueous phase precipitated with 15ml isopropanol and then centrifuged for 1.5 to 2 hours at -20°C. Pellets were re-suspended in 5ml of solution D, followed by 0.5ml Na acetate, pH4.0. The samples were again precipitated with 5ml isopropanol, centrifuged and drained and then re-suspended in 2ml LiCl, centrifuged for 10 minutes at 17,200g and 4°C. The pellets were re-suspended in 2ml TE buffer and extracted with 5ml neutral phenol, 250μl Na acetate, ρH5.2 , and 5ml ethanol and left for 1 hour minimum at -

20°C, pelleted, rinsed with 70% ethanol and dried.

Solution D

250g guanidinium isothiocyanate

312g water

17.6ml 0.75M sodium citrate pH 7.0

7.5ml sarcosyl solution

0.72ml mercaptoethanol solution/50ml solution D

Poly(A)+ RNA isolation

Ambions Poly(A)+ Pure kit was used to purify Poly ι (A) + mRNA. This kit uses Oligo-dT cellulose to bind

Poly(A)+ mRNA. (Ambion Catalogue No. 1915)

Standard solutions were prepared as follows :

Bindinσ soln. Wash soln.

ElutionB

EDTA (0.5M) lOOμl lOOμl 20μl

Tris/HCl pH7.5 (1M) 500 μl 500μl lOOμl

NaCl (5M) 5ml 1ml -

SDS (20%) - 500μl lOOμl

Distilled Water 44ml 48ml 9.8ml For each sample 4ml of binding solution, 30ml of wash solution and 400μl of elution buffer were used. For samples in solution, NaCl was added to a final concentration of 0.45M. The solution was mixed well and transferred to a 15ml RNase-free conical centrifuge tube. Binding buffer was added to a final volume of 4ml. The sample was heated at 65°C for 5 minutes to denature secondary structure and then chilled on ice for 1 minute.

Binding of mRNA to oligo-dT cellulose

lOOmg of oligo-dT cellulose was added to the denatured sample. This is sufficient to extract the poly(A)+ mRNA from 2mg total RNA. The samples were rocked gently at ambient temperature for 1 hour and then centrifuged at 4000g for 3 minutes. The oligo-dt resin was re- suspended in 10ml binding buffer and mixed well, by inversion. The resin was pelleted as described above and the supernatant aspirated and discarded. The wash step was repeated twice to remove non-specifically bound material and rRNA. The resin was then re- suspended in 1ml wash buffer and transfer to a spin column (Ambion, AMS Biotechnology, UK) inserted inside a 2ml microfuge tube (Anachem, UK) . This step was repeated three times and the OD₂₆o of the flow through monitored. The spin column was then placed into a new microfuge tube and 200μl pre-warmed elution buffer added and the samples centrifuged at 4000g at ambient temperature for 30 minutes. The flow-through contains the mRNA. A further 200μl of warm elution buffer was added to the column which was again centrifuged as above for 30 minutes. The RNA was precipitated overnight in 40μl of Na acetate, 2μl of glycogen and 1.1ml of Ethanol at -20°C. The poly(A)+ RNA was pelleted by centrifugation at 12,000g, 4°C for 20 minutes. The pellet was allowed to air dry and finally re-suspended in sterile water to a final concentration of lmg/ml .

RNA fractionation and analysis

Northern hybridisations of total (20μg/lane) or poly(A)+ RNA (lOμg/lane) were performed in Quikhyb hybridisation solution (Stratagene) according to standard protocols following fractionation on formaldehyde-containing 1.5% agarose gels and blotting onto Hybond N membranes (Amersham International, Amersham, Buckinghamshire, UK), (Sambrook, et al . 1989). Hybridisation conditions were as follows:

Preparation of radiolabelled probes

Oligonucleotide probes were radiolabelled using Terminal deoxynucleotide transferase (TdT) (Promega) . Fifty nanograms of oligonucleotide were labelled with 2μl -CTP ( 3000Ci/mmol) (Amersham) and TdT (19 units), following Promega protocols. The average specific activity of labelled probes was 10⁹ cpm/μg.

Fl probes

Pre-hybridised at 60°C for 30 minutes with lmg denatured Salmon sperm DNA. Hybridisation at 60°C for 1 hour followed by stringency washes at either ambient temperature for 15 minutes in 2X SSC/0.1% SDS and for high stringency an additional 30 minutes at 60°C in 0.1 X SSC/0.1% SDS.

F2 and F4 probes

Pre-hybridised at 40°C for 30 minutes with lmg denatured Salmon sperm DNA. Hybridisation at 40 °C for 1 hour followed by two low stringency washes at ambient temperature for 15 minutes in 2X SSC/0.1% SDS.

Result

At low stringency hybridisation conditions probes Fl and F2 detected a number of transcripts of approximately 1.4, 1.8, 2.0 and 2.4kb (Figure 3 and 4). At higher stringency (Figure 5 and 6) probes F2a and F4a recognise single mRNA species which appear to be expressed predominantly in mammary tissue.

Example 5

Reverse Transcription-PCR

Example 4 found that fragments 1, 2 and 4 were expressed predominantly in the mammary tissue and hence reverse transcription PCR was carried out using this tissue.

cDNA for amplification in PCR was made by reverse transcription of poly (A)+ RNA.

500ng of poly (A)+ RNA from mammary tissue was incubated with either 250ng random primers (Promega) or 500ng oligo-dT primer (Promega) at 70°C for 10 minutes to remove secondary structure, then placed on ice. 4μl 5X reaction buffer [250mM Tris-HCl (pH 8.3 at ambient temperature), 50mM KC1, 15mM MgCl₂], 2μl 0.1M DTT, lμl lOmM of a stock solution containing equal amounts of each of all four dNTPs (dATP, dCTP, dGTP and dTTP each at lOmM concentration) were added and the reaction incubated for 2 minutes at 42°C. Forty units Human Placental Ribonuclease Inhibitor (RNasin, Promega) and 200 units MMLV Reverse Transcriptase (Superscript II, Life Technologies) were added. The reaction was incubated at 42°C for 50 minutes and finally stopped by incubation at 70°C for 15 minutes.

For PCR 2μl of the completed reaction was diluted with 13μl sterile water and incubated at 95°C for 5 minutes.

PCR reaction

PCRs were carried out using degenerate oligonucleotide primers to fragments 1, 2 and 4 which are detailed below. Standard PCR reactions were lOOμl volume. All PCR reactions included the reagent Q-solution (QIAgen) which acts to alter the melting pattern of DNA and is used to increase PCR productivity when the DNA to be amplified contains highly GC-rich regions .

When using degenerate oligonucleotide primers a standard reaction, in a final volume of lOOμl, was:

50mM KC1, lOmM Tris-HCl (pH 9 at 25°C), 1.5mM MgCl₂, 0.1% Triton X-100. O.lμM each primer 0.2mM each dNTP 20μl cDNA (diluted and boiled RT reaction product) 20μl Q solution 2.5 units Taq. DNA polymerase (QIAgen)

In all cases the technique known as Hot Start PCR was used. That is the components of the reaction, except enzyme, were assembled in a tube and incubated in a thermal cycler at 95°C for 5 minutes. The temperature was reduced to 70°C and the enzyme added. Forty five cycles of amplification were then carried out. Each cycle consisted of: Step Temperature Time

Denature 95°C 45 minutes Anneal 60°C-45°C Ramp rate 3secs/°C Extension 72°C 2/4 minutes

Note: Extension was 2 minutes for products (<750bp) and 4 minutes for products (>750bp).

After the amplification was complete a final extension step of 10 minutes at 72 °C was carried out.

Reactions using specific primers were carried out in essentially the same way with the exception that annealing was at 50°C for 1 minute.

PCR products were analysed by gel electrophoresis followed by Southern blotting and hybridisation with specific probes, and by cloning and sequencing.

PCR Oligonucleotide primers

Fragment 1 PCR Primers

N

Tyr Gly Gly Cys Gin Gly Asn

1 .5_1 Primer TAY GGN GGN TGY CAR GG

2 .3_1 Primer TTN CCR TGY CAN CCN CC

(Reverse Complement of sequence from Asn to second Gly) .

Fragment 2 PCR Primers F Q T P P A

Phe Gin Thr Pro Pro Ala

3. 5_L Primer TTY CAR ACN CCN CCN GC

R Y F Y

Arg Tyr Phe Tyr

4. 11 Primer MGN TAY TTY TAY

Reverse Complement RTA RAA RTA NGK

5. 1 Nested primer GCC CTG TGC CAG CTC (based on sequence data obtained for fragment 2 with above primers)

Fragment 4 PCR Primers

L N V P D

Leu Asn Val Pro Asp

6. 5_1 Primer YTN AAY GTN CCN GA

D L A T G

Asp Leu Ala Thr Gly 7. 4a Nested Primer GAY YTN GCN ACN GG

D C A P G

Asp Cys Ala Pro Gly

8. 4b Nested primer GAY TGY GCN CCN GG

Oligo-dT PCT Primer

9. 11 Primer GTG GAA TCC TCG AGT CGA C(dT)₁₅

Where: A = deoxyadenosine C = deoxycytidine G = deoxyguanosine T = deoxythymidine

Y = C or T R = A or G M = A or C K = T or G N = A,C,G or T I = inosine (binds to A, C, G or T)

PCR reactions

1. Amplification of fragment 2; primers 1 and 2 were designed to amplify peptide fragment 2.

2. Amplification of fragment 2 to Oligo-dT; the region between fragment 2 and the poly (A)+ RNA was amplified using 3' RACE (Frohman et al . 1988). Two rounds of PCR were undertaken. In the first round primers 3 and 9 were used to amplify specific sequences from cDNA. In the second round the products from the first were amplified with primers 5 and .

3. Amplification of Fragment 4a and 4b to oligo-dT; A similar strategy as described above for fragment 2 was adopted. Primers 6 and 9 were used in round 1 PCR and primers 7 and 9 for 4a and 8 and 9 for 4b in round 2.

4. Amplification of Fragment 1 and 2 to 5 ' end of sequence; 5' RACE was performed exactly according to the Life Technologies, Gibco, protocols described the instruction manual "5' RACE system for rapid amplification of cDNA ends". Primers 2 and 9, were used in round 1, and primers 4 and 9, were used in round 2. All appropriate controls were simultaneously undertaken.

Analysis of PCR products

Gel Electrophoresis

Immediately after PCR, lOμl of reaction mix was analysed by electrophoresis on a 1.5% agarose/Tris,borate, EDTA (TBE) buffer gel. The gel and running buffer contained ethidium bromide at 0.5μg/ml to allow visualisation of DNA under UV light. DNA size markers ( lOObp ladder, Promega) were run alongside the samples for estimation of the size of the PCR products.

Southern Blotting

For Southern blotting gels were denatured in 0.5M NAOH,1.5M NaCl for 1 hour and neutralised in 0.5M Tris- HC1 pH 7.5, 1.5M NaCl before transfer (Southern, 1975). Hybridisation and wash conditions were dependent on the probes used and identical to those described for Northern blots .

In cases were multiple products were obtained from PCR, Southern blotting and hybridisation with specific probes were undertaken to determine the products containing the sequences of interest. Once identified such products were characterised by cloning and sequencing.

Cloning Techniques

PCR products were separated by gel electrophoresis on a 1.5% agarose/TBE gel as described above. Bands of interest were excised from the gel and the DNA eluted using QIAquick Gel extraction kit (QIAgen) . The purified DNA was ligated into the cloning vector pGEM-T (Promega) using the Rapid Ligation Kit (Boehringer Mannheim) and following exactly the manufacturer's protocol. In all cases ligations were carried using both equimolar concentrations of vector and insert, and using a three times molar excess of insert.

Once the ligation was complete, 2μl from a total volume of 20μl was used to transform 50μl JM109 competent cells (JM109, Promega). The transformation was undertaken as described in the manufacturer's protocol.

The transformed cells were plated onto LB-agar plates containing 50μg/ml ampicillin and spread with X- Gal(50μl at 20mg/ml) and isopropyl β-O- thiogalactopyranoside (IPTG) (lOOμl at 24mg/ml) and grown inverted overnight at 37°C. The next day white colonies which contain recombinant DNA were selected and grown for 16 hours in 5ml LB containing ampicillin at 50μg/ml. Recombinant plasmid DNA was prepared from the cells using Wizard SV miniprep kit (Promega) . Manufacturer's instructions were followed precisely.

Results

F2 PCR amplification; A PCR product of the predicted size (78bp) was obtained, cloned into pGem-T vector and sequenced using automated fluorescent technology (Perkin Elmer, ABI Prism) . The following sequence (SEQ ID No 35) was obtained:

ttt caa ace ccc cca gcc ctg tgc cag etc ccc cca gtg ggg ggc ccc tgc aaa gcc tct ttg cgc egg tac ttc tac

Translation=FQTPPALCQLPPVGGPCKASLRRYFY (SEQ ID No 36)

F2 to oligo-dT amplification; A PCR product of approximately 400bp, which hybridised on Southern blots with the F2 amplified product described above, was cloned and sequenced (SEQ ID No 37).

gcc ctσ tσc cag etc ccc cca gtg ggg ggc ccc tgc aaa gcc tct ttg cgc cgt tac ttc tac aac tct acg tec get gag tgt gag etc ttt atg tac ggt ggt tgt cag gga aac gcc aac aat ttt gag ace aca gcg ate tgt egg agg gtc tgc aac ccc cct gac ace aag gta aag aat ggc tga cga gga cac ctg cgc ttc ccc agg cct tga ccg aga get ggt tea get gcc ctt cct gcg tgg ata ccc cgt tct gag ccc atg ttg taa ccc tec ggg act gag tea ata aaa tgg tta aag ttg aaaaaaaaaaaaaaa

(primer sequences are underlined) This sequence gave an open reading frame of 189 bases terminating in a TGA stop codon. An AATAAA polyadenylation signal was identified 18 bases before the poly A, indicating the 3' end of the mRNA.

Translation=ALCQLPPVGGPCKASLRRYFYNSTSAECELFMYGGCQGNANNF ETTAICRRVCNPPDTKVKNG (SEQ ID No 38)

From this data F2, Fl and F3 lie in this orientation in the 3' end or C-terminal region of the RNA and protein respectively.

Although F4a oligonucleotide probe recognised two mRNA transcripts in mammary tissue, namely 1.4kb and lOkb (Figure 6), the F4a/oligo-dT and F4b/oligo-dT PCR reactions did not generate specific products. Note: The 5 ' RACE was performed in order to obtain the linker sequence between F4 and Fl, F2 and F3.

Northern Hybridisation using amplified cDNA probes

Following confirmation of the sequences of the PCR products, these products were then radiolabelled using Hi-prime Kit (Promega) , and used as highly specific- probes in Northern hybridisation protocols, as outlined above. High stringency conditions were employed.

Result

Using the highly-specific cDNA probes at high stringency, a number of transcripts were identified in mammary tissue with probes Fl and F2 (Figures 7a and b) . The sizes of these transcripts were as follows: 0.6kb, 1.2kb, 2.6kb, 6kb and lOkb. Interestingly using these probes 1.2kb and 2.6kb bands were visible in poly(A)+ RNA isolated from intestinal tissue. Also, in mammary tissue the 0.6kb and 6kb bands were particularly prominent. CDGF sequence

The sequence data on CDGF was derived using protein and DNA sequencing. The primary amino acid data revealed homology with two major classes of proteins, namely the protease inhibitor proteins , and a cytokine from the chemokine family. Although Fragment 1 and 2 exhibit homology with proteins such as bovine colostrum trypsin inhibitor, porcine alpha 1 microglobulin, tissue factor pathway inhibitor and amyloid precursor-like protein, the sequence has not been previously described and therefore represents a novel protein. Fragment 3 again represents an unknown sequence but does have homology (34%) to amyloid-precursor proteins (accession no.003157). Using PCR technology we have known shown that fragment 1,2 and 3 belong to a single gene product described in SEQ ID Nol. Fragment 4 contains a sequence which exhibits 61% homology with a cytokine known as pre-B cell stimulating factor (PBSF) or stromal-cell derived factor (SDF) . Similarly, homology was also found with a fragment of epidermal growth factor (EGF) (rat), notch protein (Drosophila) and serrate protein (chicken), the later two families containing EGF repeat motifs. The protein families which share homology with CDGF fragment 4 are intimately involved in the regulation of development, particularly in progenitor cell renewal and differentiation. As with fragments 1, 2 and 3, fragment 4 represents an original sequence (SEQ ID No 6) .

With respect to the sequence data provided there are two protein sequences encoded by two distinct mRNAs . We believe that, although the sequences are derived from two different RNAs, the proteins derived from the mRNAs are subsequently covalently linked as subunits, to form heterodimeric CDGF. An example of this has recently been reported in Proc Natl Acad Sci which describes a novel 155kDa heterodimeric proteinase inhibitor containing transferrin chains (Liang et al.1997). To further support this idea, proteins such as alpha 1-microglobulin, which share strong homology with CDGF, are recognised to form covalent complexes with a number of unrelated proteins (Bergarrd, 1997) and are currently recognised to exhibit ligand binding capacity and carrier function. We believe that CDGF exhibits properties which reflect the nature of both its subunits.

Example 6

Immunomodulatory property of CDGF

CDGF as a regulator of Nuclear Factor-kappaB (NF- kappaB) and pro-inflammatory cytokine release

Colostrum has been long recognised to contain many biologically-active constituents which function to regulate growth and differentiation but also to protect against pathogen invasion. However, a further biologically important function of colostrum relates to its anti-inflammatory activity. Recent data has shown that human colostrum and milk can exert a very potent anti-inflammatory effect on chemically-induced colitis in rats (Grazioso et al.1997). The sequence data and the new data on the biological activity of CDGF suggests that, in addition to its growth/differentiation promoting activity described in WO-A-97/04009, CDGF has anti-inflammatory activity. As postulated for CDGF, IL10, a known cytokine, exhibits both mitogenic and anti-inflammatory activities which are regulated, at the cellular level, via distinct signalling pathways (Crawley et al. 1996). The anti- inflammatory activity of CDGF was demonstrated by investigating the inhibitory potential of CDGF on NF- kappaB activation.

The NF-kappaB family of transcription factors participate in the activation of a diverse range of genes involved in inflammation, immune response, lymphoid differentiation, growth control and development. NF-kappaB has a major role in controlling cytokine and other immuno-regulatory genes . NF-kappaB exists in the cytoplasm as a dimer of DNA-binding subunits bound to an inhibitor Ikappab. Following cellular activation by LPS, or other agents such as cholera toxin which raise cAMP, the inhibitor IkappaB is degraded thereby permitting nuclear translocation of NF-kappaB. NF-kappaB can then bind to target sequences and transactivate the promotors of many cytokines, including pro-inflammatory cytokines, such as TNF . Protease inhibitors are recognised to inhibit NF-kappaB activation and can thus interfere or prevent pathological inflammatory reactions. The sequence data for CDGF indicated a very close homology to protease inhibitors and hence the ability of CDGF to inhibit NF- kappaB activation was investigated.

The current postulate concerning CDGF is that CDGF exhibits bi-functional properties, and so can regulate the growth and differentiation of cells but can also suppress the synthesis and release of inflammatory cytokines. Hence CDGF potentially represents an important immunomodulator. IEC-6 intestinal cells have been previously utilised to studied the growth- promoting property of CDGF. IEC-6 cells were also used to demonstrate the anti-inflammatory function of CDGF. In brief, IEC-6 cells were stimulated with cholera toxin to induce NF kappaB activation and the activation monitored in the presence and absence of CDGF. Nuclear protein extracts were then prepared and the activation status of NF-kappaB examined by electrophoretic mobility shift assay (EMSA) .

Cell Culture Protocol

Cells

Rat intestinal epithelial cell line IEC6 (Passage 31) was purchased from ECACC. Cells were cultured in 75cm² flasks (Corning) and split into 94mm Petri dishes (Greiner) at 4xl0⁵ cells/dish containing lOmls DMEM containing 5% FCS . Cells were grown for 72hrs at 37° C in 5% C0₂ and then each plate was washed twice with 10ml Hank's Balanced Salt Solution (HBSS)and the media replaced with 10ml DMEM containing 5μg transferrin and 0.2μg/ml sodium selenite (DTS) for 24 hours.

Experimental design

Control plates. Each plate washed twice with 10ml HBSS and media replaced with 10ml DTS

Cholera Toxin (CT) plates Each plate washed twice with lOmls HBSS and media replaced with 10ml DTS containing 2μg/ml Cholera Toxin (CT) (Sigma). CT was prepared by adding 58.1μl of a 1.2mg/ml CT solution to 35ml DTS in a 50ml Polypropylene centrifuge tube (Corning) .

Cholera toxin/inhibitor (CT/I) plates 2mls Porcine colostrum (Sow 179 7/8/97) was fractionated using ACA 34 (Biosepra, Cedex, France) in 20mM Hepes/O.IM NaCl, pH 7.05. Fractions were dialysed against distilled H₂0 and lyophilised. Fractions A, B, C and D were mixed in 1ml DMEM and 300μl added to three CT plates . In addition the bond elut preparation and the reverse phase purified protein were tested.

The cells were stimulated for 2 hours, harvested and placed in a 15ml centrifuge tube and centrifuged at 200g for 4 minutes. The media was aspirated off and the cells re-suspended in 2mls of Phosphate Buffered Saline (PBS). The cells were recovered by centrifugation at 200g for 4 minutes.

Preparation of nuclear extracts from cultured cells

The methodology of Johnson et al.1995 (as described below) was followed.

Cells (l-5xl0⁷), pelleted by centrifugation at 200xg for 4 minutes at 4°C as described above, were re-suspended in 1ml of PBS and transferred to eppendorf tubes and again centrifuged at 200g for 5 minutes at 4°C. PBS was aspirated off and the pellet loosened by gentle vortexing. The pellet was then re-suspended in 100μl/10⁷ cells of Sucrose 1 buffer (see below) by gentle pipetting. The lysate was observed microscopically, to confirm lysis and the presence of intact nuclei. The lysate was then centrifuged at 500g for 5 minutes at 4°C to pellet the nuclei. The supernatant (cytoplasm) was transferred to a fresh tube, 0.22 volumes of 5x cytoplasmic extraction buffer were added at 4°C and the samples centrifuged at 12000g for 15 minutes at 4°C. The supernatant was transferred to a fresh tube and stored in 25% glycerol at -80°C.

Washed nuclei were re-suspended in 1ml of Sucrose buffer 1 lacking NP-40 and centrifuged at 500g for 5 minutes at 4°C . The supernatant was aspirated and discarded. Nuclei were re-suspended by gentle pipetting in 20μl/10⁷ cells or 40μl/5xl0⁷ cells in low salt buffer at 4°C. To extract nuclei, a high salt buffer (0.25 or 0.2 volumes of the low salt buffer volume i.e 4μl or 5μl/10⁷ cells) was added and the cells gently mixed by vortexing. The high salt buffer, in 0.25 or 0.2 volumes, was added in aliquots until either 1 volume had been added or the nuclei began to shrink and the viscosity of the solution began to increase. The samples were then incubated at 4°C on a rotary platform with gentle mixing for 20 minutes and then diluted 1:2.5 with diluent (or until the solution was non- viscous) and finally centrifuged at maximum speed of 17,400g for 15 minutes at 4°C . The nuclear extract (supernatant) was then aliquoted into fresh, ice chilled eppendorfs and stored at -80°C.

Measurement of NF-kappaB activity by Electrophoretic Mobility Shift Assay (EMSA) was performed according to the protocol set out in Promega Technical Bulletin no 110.

5' End-Labelling of NF K B Concensus Oligonucleotide was performed according to the protocol set out in Promega cat no E3291.

The following reaction was assembled in a sterile microfuge tube:

NF KB Oligonucleotide ( 1.75pmol/μl) 2μl T4 Polynucleotide Kinase xlO Buffer lμl [y^3ZP]ATP (3,000 Ci/mmol at lOmCi/ l) lμl Nuclease free water 5μl T4 Polynucleotide Kinase (5-10 u/μl) lμl Total volume lOμl The samples were incubated at 37°C for 10 minutes and the reaction terminated by adding lμl of 0.5M EDTA. A volume of 89μl of IxTE buffer was added to the reaction mix. One microlitre of the reaction mix (total counts) was then transferred to 10ml scintillation fluid (Packard Ultama Gold, XR) for determination of percentage incorporation. Free nucleotides were removed using Chromaspin-10 columns (Clonetech) in which the buffer had been replaced by IxTE buffer. One microlitre of the labelled oligonucleotide (minus free nucleotides) was added to 10ml of scintillation fluid and counted in scintillation counter.

Percent incorporation = cpm incorporated x 100 cpm total

Typically, 50% or more of the radioactivity is incorporated in the oligonucleotide.

DNA-protein binding reactions

Each reaction was carried out in a final volume of 8μl by adding lμl of lOx EMSA binding buffer, 4μl of nuclear extract and 3μl water. Each assay also included a negative control reaction containing no extract and a specific competitor reaction containing excess cold oligonucleotide (3.5pmol or lOOx) in addition to extract. Incubation reactions were carried out at ambient temperature for 10 minutes and then 2μl (50,000-200,000 cpm) of y³²P-labelled concensus oligo per reaction was added. The reaction was incubated at ambient temperature for a further 20 minutes, lμl of gel loading buffer was added to each reaction. Note: bromophenol blue may interfere with binding of NF K B to the oligo and should therefore be added only to the negative control reaction. Gel loading buffer lacking BPB should be added to all other reactions .

Electrophoresis of DNA-protein complexes

The reactions were analysed on a 20cm x 20cm 4% non- denaturing acrylamide gel (acrylamide: bisacrylamide ratio of 80:1, 1mm thick. These gels polymerise slowly and were cast and allowed to set overnight prior to use. The gel was pre-run in 0.5xTBE at 100V for 30 minutes and then the samples loaded and the gel run in 0.5xTBE buffer at 100V for 3 h hours, until the BPB front is three quarters of the length of the gel. The gel was transferred onto Whatman 3MM paper, wrapped in cling-film and placed directly against film for the desired exposure time.

Result

The addition of CT to IEC-6 induced a significant activation of NF-kappaB (Figure 8). In the presence of CDGF the stimulation of NF-kappaB was very significantly inhibited. Similar results to those presented in Fig. 8 were found with the bond elut preparation and reverse phase purified protein. This suggests that CDGF may represent a very important immunomodulator during neonatal development. In the neonate, CDGF may serve to regulate potentially damaging inflammatory responses generated as a result of the high antigenic load encountered in the early weeks of life and as a result of the significant proliferative drive triggered by biologically-active molecules, such as CDGF, which may also trigger the autocrine synthesis of pro-inflammatory cytokines. Example 7

Lymphocyte transformation

Porcine lymphocyte transformations were conducted to investigate further the immunomodulatory potential of CDGF .

Method

Ten ml of blood was collected in Heparin coated tubes from newborn and weaned pigs and diluted 1:1 with RPMI (Roswell Park Memorial Institute) 1640 (Sigma, UK). Eight ml of blood was then layered onto 4ml of Histopaque (1.077g) (Sigma, UK) and centrifuged for 30 minutes at 400g . The plasma was then carefully removed without disturbing the interface. The interface was collected with a sterile pipette and diluted in 10ml RPMI 1640 containing 2% fetal calf serum (FCS) (Sigma, UK) and the samples again centrifuged at lOOg for 10 minutes. The supernatant was then discarded and the pellet re-suspended in 5ml RPMI 1640 2%FCS. The pellet was recovered by centrifugation at lOOg for 10 minutes (this step was repeated again) . Lymphocytes were counted using a haemocytometer and the cells adjusted to 5 x lOVml in RPMI 1640 containing 5% FCS. One hundred microlitres of lymphocytes in RPMI 5%FCS were added to each well of a 96 well flat bottom tissue culture plate. Lymphocytes were stimulated with colostrum and CDGF and incubated in 5% C0₂ at 37 °C for 48 hours. Thymidine (Amersham, UK) (0.5μCi) in 50μl RPMI 1640 was added to each well and the plate incubated for a further 16 hours. Lymphocytes were then harvested onto filter paper using a Skatron cell harvester. The paper was dried and added to 9ml scintillant (Ultama Gold XR, Packard) and counted on a Packard β counter for 5 minutes .

Result

CDGF was found to significantly stimulate the proliferation of lymphocytes of weaned pigs (Figure 9). Activation of these cells indicates an important immunological property of CDGF. CDGF at the Bond Elut stage of purification was found to be consistently mitogenic, again with weaned lymphocytes, although the magnitude of the response was found to be variable (Table 3). In some experiments, eg. Experiment D in Table 3 CDGF induced a 20 fold stimulation of pig lymphocytes .

TABLE 3

Porcine Lymphocyte Transformation

Thymidine Incorporation (CPM)

Experiment Control Filtron Bond Elut

Number i i lOul

A 183 4809 731

B 1882 2242 6946

C 717 967 3303

D 3577 NA 71658

Average Filtron 927 2672 (A, B and C)

Average Bond Elut (A, B, C and D) 1590 20660

Test/Control 2.9 12.9 Example 8

CDGF is a potential homologue of SDF

Stromal cell-derived factor (SDF) or pre-B cell growth stimulating factor (PBSF) exhibits homology to CDGF (Fragment 4 sequence). The receptor for SDF/PBSF is fusin or CXCR4 which has recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells (Murakami et al.1997; Haribabu et al.1997). The CXCR4 receptor is a 44kDa protein and is phosphorylated following stimulation with SDF. This stimulation is partially inhibited by the protein kinase C (PKC) inhibitor, staurosporine . The receptor characteristics of SDF ligand are therefore similar to those of CDGF, as described in WO-A-97/04009. CDGF stimulates the phosphorylation of MVM proteins. Notable is the phosphorylation of proteins of approximately 40kDA which is also inhibitable with staurosporine. The potential of CDGF to displace SDF from its receptor site was investigated using radioligand binding assays.

Radioligand binding assays.

For radioligand binding assays, IEC-6 cells (4 X 14Vwell; cell passage, 24) were seeded into 24 well plates (Corning) and allowed to attach and grow for 2 days in DTS supplemented with 5% FCS. Media was then removed and replaced with 400μl of DMEM supplemented with 25mM HEPES and lOmg/ml bovine serum albumin. ¹²⁵I- SDF (2200Ci/mmol) (NEN Life Science Products) was added at 0.2nM to the cells in the presence and absence of CDGF. CDGF was added as two preparations; the product following the Bond Elut (BE/CDGF) separation and as pure protein following reverse phase HPLC (RV/CDGF) . CDGF was added at a concentration of 40nM and 20nM for BE/CDGF and RV/CDGF respectively. Cells were then incubated overnight at 4°C. The cells were harvested in Hepes-buffered DMEM and transferred to Starstedt tubes (Starstedt, Germany) and pelleted by centrifugation at 22,500g for 5 minutes. The media was aspirated off and bound ligand counted on a gamma counter.

Result

BOUND LIGAND ( CPM)

¹²⁵I-SDF +BE/CDGF +RV/CDGF

53 , 570 . 9 30 , 890 . 8 40 , 6 15 . 9

CDGF, both at the Bond Elut stage of purification and as pure preparation, displaced SDF binding from its receptor, by approximately 42% and 24% respectively. The % displacement is expressed relative to the total- bound SDF and not the specific-bound SDF. CDGF may therefore displace up to 60-80% of the specific-bound ligand. These results strongly support the homology between CDGF and SDF.

1 References

2

3 1. Chomcynski, P. and Sacchi, N. 1987. Single step

4 method of RNA isolation by acid guanidinium thiocynate-

5 phenol-chloroform extraction. Anal. Biochem. 162, 156-

6 159. 7

8 2. Crawley, J.B., Williams, L.M., Mander, T., brennan,

9 F.M. and Foxwell, B.M.J. 1996. Interleukin-10

10 stimulation of phosphatidylinositol 3 kinase and p70 S6

11 kinase is required for the proliferative but not anti-

12 inflammatory effects of the cytokine. J. Biol. Chem.

13 27, 16357-16362. 14

15 3. Frohman, M.A. et al, 1988. Rapid production of

16_. full length cDNAs from rare transcripts P.N.A.S (USA)

17 vol 85, pages 8998-9002. 18

19 4. Grazioso, C.F., Werner, A.L. , Ailing, D.W., Bishop,

20 P.R. and Buescher, E.S. 1997. Anti-inflammatory effects

21 of human milk on chemically induced colitis in rats.

22 Pediatr. Res. 5, 639-643. 23

24 5. Haribabu, B., Richardson, R.M., Fisher, I. et

25 al.1997. Regulation of Human Chemokine Recptors CXCR4.

26 J Biol.Chem. 272, 28726-28731. 27

28 6. Hayes, J,D., Kerr, L.A. and Cronshaw, A.D. 1989.

29 Evidence that glutathione S-transferase B,B, and B₂B₂ are

30 the products of separate genes and that their

31 expression in human liver is subject to individual

32 variation. Biochem J. 264, 437-445. 33

34 7. Johnson, D.R. , Levenat, S., and Bale, A.E. 1995.

35 Isolation of intact nucleic for nuclear extract

36 preparation from fragile B-lymphocyte cell line. Biotechniques, 19(2) 192-195.

8. Murakami, T. Nakajima, T. Koyanagi, Y et al . 1997. A small molecule CXCR4 inhibitor that blocks T cell line tropic HIV-1 infection. J Exp . Med. 186, 1389- 1393.

9. Puissant, C. and Houdebine, L.M. 1990. An improvement of the single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Biotech. 8, 148-149.

10. Sambrook, J. , Fritish, E.F., and Miniatis, T. 1989. Molecular cloning, a laboratory manual, second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

11. Southern, E.M 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol, 98, 503.

Composition of Media, Buffers and Solutions

Cell Culture Media

Complete DMEM

Dulbecco's Modified Eagle Medium (Sigma)

Hepes 25mM

L Glutamine 2mM

5% Foetal Calf Serum

Penicillin 100 units/ml

Streptomycin lOOug/ml

DTS

Dulbecco's Modified Eagles Medium (Sigma)

Hepes 25mM

L Glutamine 2mM

Transferrin 5μg/ml

Sodium Selenite 0.2μg/ml

Penicillin 100 units/ml

Streptomycin lOOug/ml

Solutions for nuclear extraction protocol

(Add DTT, PMSF and Nonidet P-40 (NP-40) just prior to use of buffers . )

Stock PMSF 500mM 0.0872g in 1ml Isopropanol

Stock DTT 500mM 0.0771g in 1ml distilled water

Sucrose 1 0.32M Sucrose 10.95g

(100ml) 3mM CaCl₂ 0.0333g 2mM Mg Acetate 0.0429g

O.lmM EDTA 0.00372g lOmM Tris-HCl pH 8.0 0.121g

lmM DTT 200μl of stock solution

0.5mM PMSF lOOμl of stock solution

0.5% NP-40 500μl of stock solution

5x Extraction 0.15M HEPES pH 7.9 3.575g Buffer (100ml) 0.7M KC1 5.222g 15mM MgCl₂ 0.305g

Low Salt 20mM HEPES pH 7.9 0.477g Buffer (100ml) 25% Glycerol 25ml 1.5mM MgCl₂ 0.0305g 0.02M KC1 0.149g 0.2mM EDTA 0.00744g

0.5mM DTT lOOμl of stock solution

0.5mM PMSF lOOμl of stock solution

High Salt 20mM HEPES pH 7.9 0.477g Buffer (100ml) 25% Glycerol 25ml

1.5mM MgCl₂ 0.0305g

0.8M KC1 5.964g

1% NP-40 lOOOμl of stock solution 0.5mM DTT lOOμl of stock solution 0.5mM PMSF lOOμl of stock solution

Diluent (100ml) 25mM HEPES pH 7. 9 0.596g

25% Glycerol 25ml

0. lmM EDTA 0.00372g

0.5mM DTT lOOμl of stock solution

0.5mM PMSF lOOμl of stock solution

Glycerol Storage50mM Tris-HCl pH 8. 0 0.606g

Buffer (100ml) 40% Glycerol 40ml

5mM MgC12 O.lOlg

0. lmM EDTA 0.00372g

lmM DTT 200μl of stock solution

0.5mM PMSF lOOμl of stock solution

Solutions for EMSA Protocol

Gel Shift Binding 10X Buffer Gel Loading Buffer

40%Glycerol 250mM Tris-HCl, pH 7.5 lOmM MgC12 40% Glycerol

5mM EDTA 0.2% Bromophenol

Blue

5mM DTT

500mM NaCl lOOmM Tris_HCl, pH 7.5

0.5mg/ml poly

(dI-dC)«poly(dI- -dC 1 TBE 10X Buffer (1L) TE Buffer

107.80g Tris base lOmM Tris-HCl, pH

8.0 approx 55g Boric acid lmM EDTA

7 . 44g Disodium EDTA» 2H₂0

Add components in the order listed to approx. 800ml. Add slightly less than the total amount boric acid. Dissolve, adjust pH to 8.3 with boric acid and make up to 1L.

Polγacxγlamlde gel formulation (70ml)

TBE 5x buffer 7ml

2% Bis-acrylamide 1.75ml

40% Acrylamide 7ml

80% Glycerol 2.19ml

Distilled water 51.5ml

Temed 35ml 10% Ammonium Persulphate 525ml

Sequence Listing

b) SEQ ID Nol

LFQTPPALCQLPPVGGPCKASLRRYFYNSTSAECELFMYGGCQGNANNFE

TTAICRRVCNPPDTKVKNG

b) SEQ ID No2

TTTCAAACCCCCCCAGCCCTGTGCCAGCTCCCCCCAGTGGGGGGCCCCTGCAAAG CCTCTTTGCGCCGATACTTCTACAACTCTACGTCCGCTGAGTGTGAGCTCTTTAT GTACGGTGGTTGTCAGGGAAACGCCAACAATTTTGAGACCACAGCGATCTGTCGG AGGGTCTGCAACCCCCCTGACACCAAGGTAAAGAATGGCTGACGAGGACACCTGC GCTTCCCCAGGCCTTGACCGAGAGCTGGTTCAGCTGCCCTTCCTGCGTGGATACC CCGTTCTGAGCCCATGTTGTAACCCTCCGGGACTGAGTCAATAAAATGGTTAAAG TTGAAAAAAAAAAAAA

Claims

Claims

1. A polypeptide having an amino acid sequence which comprises the amino acid sequence as set out in any one of SEQ ID Nos. 1, 2 to 9, a functional equivalent or portion thereof.

2. A composition comprising a polypeptide as claimed in Claim 1.

3. The use of the polypeptide as claimed in Claim 1 as a pharmaceutical or as an additive in a nutrient formulation.

4. A composition as claimed in Claim 2 in the form of a baby food formulation, TPN formulation or invalid food formulation.

5. A composition as claimed in Claim 4 in the form of a baby or infant food formulation.

6. A polynucleotide comprising a nucleotide sequence as set out in SEQ ID No 2.

7. A polynucleotide comprising a nucleotide sequence which encodes for a polypeptide of Claim 1.

8. A vector comprising a polynucleotide as claimed in either one of Claims 6 and 7.

9. A host cell comprising a polynucleotide as claimed in either one of Claims 6 and 7, or a vector as claimed in Claim 8.

10. Use of CDGF, a functional equivalent or portion thereof, or of a polypeptide as claimed in Claim 1 as an immunomodulator.

11. Use of CDGF, a functional equivalent or portion thereof, or of a polypeptide as claimed in Claim 1 in the manufacture of a medicament for modifying at least one component of the immune system.

12. An immunomodulating composition, said composition comprising CDGF, a functional equivalent or portion thereof, or of a polypeptide as claimed in Claim 1 as an active ingredient, together with an inert carrier or excipient.

13. A composition as claimed in Claim 12 wherein said active ingredient acts as an immunostimulant.

14. A composition as claimed in Claim 12 wherein said active ingredient acts as an immunoinhibitant .

15. A composition as claimed in Claim 12 wherein said active ingredient regulates inflammation.

16. A composition as claimed in Claim 12 wherein said active ingredient promotes cell proliferation and/or differentiation.

17. A composition as claimed in Claim 16 wherein said cells are lymphocytes.

18. A method of modifying at least one component or response of the immune system of the human or non-human animal body, said method comprising administering a therapeutically effective amount of CDGF, a functional equivalent or portion thereof, or of a polypeptide as claimed in Claim 1 to said body.

19. A method as claimed in Claim 18 wherein the immune system component modified is a lymphocyte.

20. A process for purifying CDGF, said process comprising the following steps:

a) separation of all components of colostrum having a molecular weight of over 300kDa; and

b) treatment of the product of step (a) by reverse phase HPLC.

21. A process for purifying CDGF, said process comprising raising antibodies to a polypeptide as claimed in Claim 1 and using said antibodies in immunoaffinity techniques.