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WO1996034958A1 - Peptide intestinal vasoactif - Google Patents

Peptide intestinal vasoactif Download PDF

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Publication number
WO1996034958A1
WO1996034958A1 PCT/CA1996/000280 CA9600280W WO9634958A1 WO 1996034958 A1 WO1996034958 A1 WO 1996034958A1 CA 9600280 W CA9600280 W CA 9600280W WO 9634958 A1 WO9634958 A1 WO 9634958A1
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WO
WIPO (PCT)
Prior art keywords
vasoactive intestinal
vip
peptide
intestinal peptide
nucleotide sequence
Prior art date
Application number
PCT/CA1996/000280
Other languages
English (en)
Inventor
Graham J. M. Cox
Jack Manns
Carolyn Weeks-Levy
Original Assignee
Biostar Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biostar Inc. filed Critical Biostar Inc.
Priority to US08/952,568 priority Critical patent/US6037321A/en
Priority to EP96911888A priority patent/EP0824590A1/fr
Priority to AU54942/96A priority patent/AU5494296A/en
Priority to BR9608118-0A priority patent/BR9608118A/pt
Publication of WO1996034958A1 publication Critical patent/WO1996034958A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to vasoactive intestinal peptide ("VIP”) and cross-reactive peptides.
  • VIP vasoactive intestinal peptide
  • this invention relates to peptides capable of inducing antibodies which neutralize the activity of VIP.
  • This invention also relates to tandemly repeated peptides derived from VIP or from cross-reactive peptides, which can elicit a broad immune response.
  • the present invention is useful for increasing egg production in bird species and for increasing efficiency of feed utilization and rate of gain in food producing animals.
  • VIP has been proven to be a potent releaser of avian prolactin ("PRL") in vivo and in vitro.
  • Prolactin is a hormone produced by the anterior pituitary and it is well established that prolactin can initiate incubation behaviour in birds such as turkeys, bantam hens and many species of wild birds. Incubation behaviour leads to early cessation of egg laying and has a fundamental role in avian reproduction. The incubation behaviour has been of great interest to scientists and producers of hatching eggs and of particular interest in the field of turkey breeding since reproductive efficiency of turkey hens is low in comparison with chickens.
  • active and passive immunization against VIP may be useful for modifying the egg-laying performance of avian species.
  • desired effects are that hens lay eggs for a longer period of time, that there is an increase in the number of eggs laid, and that non-laying hens commence laying eggs.
  • VIP was initially identified for its vasoactive properties in mammals.
  • VIP causes relaxation of isolated gastric and intestinal smooth muscle cells.
  • VIP-induced relaxation is mediated by high-affinity VIP receptors and can be inhibited by VIP antiserum and selective VIP antagonists.
  • Critical affected segments of the gastrointestinal tract occur at the pyloric-duodenal junction, the junction between the small intestine and the large intestine, and the anal sphincter. Immunization of food-producing animals against VIP may result in increased tone of these segments leading to increased efficacy of absorption of food with a consequent increased rate of feed efficiency and rate of weight gain.
  • Immunization against VIP presently entails chemically synthesizing the full length of VIP and conjugating the resulting peptide to a carrier molecule.
  • Commercial application of this non-recombinant technique is expensive and difficult, and moreover conjugation of the VIP with a carrier molecule is highly unpredictable, therefore resulting in low yields of useful antigenic proteins.
  • this technique would require, firstly, a ready source of VIP antigens and, further, antigens which can elicit a broad immune response. These immunogens are not currently available. Thus, it is desirable to develop a recombinant system whereby antigenic fusion proteins are made and the conjugation step is avoided.
  • the present invention relates to a nucleotide sequence comprising (1) a DNA sequence coding for at least one copy of vasoactive intestinal peptide, and/or at least one copy of a cross-reactive
  • Another aspect of the present invention relates to the above nucleotide sequence wherein the at least one copy of vasoactive intestinal peptide and/or
  • tandemly repeated sequences may be the whole nucleotide sequence for VIP or a cross-reactive protein, or fragments of nucleotide
  • nucleotide sequence containing these tandemly repeated nucleotide sequences may be homopolymeric or heteropolymeric.
  • a homopolymeric nucleotide sequence may include tandem repeats of identical whole nucleotide sequences or identical fragments of nucleotide sequences for VIP or a cross- reactive protein.
  • a heteropolymeric nucleotide sequence may include whole nucleotide sequences from different species, or different nucleotide sequences taken from the same or different species.
  • Yet another aspect of the present invention is a recombinant protein comprising (1) at least one peptide derived from (a) vasoactive intestinal peptide and/or (b) a cross-reactive protein and/or ® a fragment derived from vasoactive intestinal peptide and/or a cross-reactive protein, and optionally (2) a carrier molecule, said recombinant protein being capable of producing antibodies which neutralize vasoactive intestinal peptide in vivo .
  • the recombinant protein is a fusion protein of the at least one peptide and the carrier molecule.
  • the recombinant protein may also be a fusion protein of tandem repeats of the peptide derived from VIP or the cross-reactive protein or fragments thereof.
  • the tandemly repeated segment may be one or more identical or different whole VIP's or cross-reactive proteins, or fragments thereof.
  • the recombinant protein containing these tandemly repeated sequences may be homopolymeric or heteropolymeric.
  • a homopolymeric protein may include tandem repeats of identical whole sequences or identical fragments of VIP or a cross-reactive protein.
  • a heteropolymeric protein may include whole sequences of VIP or a cross-reactive protein from different species, or different fragments thereof taken from the same or different species.
  • the recombinant proteins of the present invention are useful in the active and passive immunization against VIP of egg-laying birds and of food-producing animals.
  • This invention also relates to methods of actively or passively immunizing an egg-laying bird or food-producing animal against VIP comprising treating the bird or animal with the above-mentioned fusion . protein or nucleotide sequence.
  • FIGURE 1 is a table depicting the amino acid sequence of VIP from various animal species in which:
  • SEQ ID N0:1 is the amino acid sequence for Chicken VIP
  • SEQ ID NO:2 is the amino acid sequence for Gila Monster VIP
  • SEQ ID NO:3 is the amino acid sequence for Sheep VIP
  • SEQ ID NO:4 is the amino acid sequence for Sheep VIP
  • SEQ ID NO:5 is the amino acid sequence for Rabbit VIP
  • SEQ ID NO:6 is the amino acid sequence for Dog VIP
  • SEQ ID NO:7 is the amino acid sequence for Catshark Dogfish VIP
  • SEQ ID NO:8 is the amino acid sequence for Cod VIP
  • SEQ ID NO: 9 is the amino acid sequence for Cod VIP
  • SEQ ID NO:10 is the amino acid sequence for Mouse VIP
  • SEQ ID NO:11 is the amino acid sequence for Human VIP
  • SEQ ID NO:12 is the amino acid sequence for Pig VIP
  • SEQ ID NO:13 is the amino acid sequence for Goat VIP
  • SEQ ID NO:14 is the amino acid sequence for Guinea Pig VIP
  • FIGURE 2 depicts the amino acid sequences for each of four nested peptides derived from VIP in which: SEQ ID NO:3 is the amino acid sequence for
  • Sheep VIP (representing the consensus amino acid sequence for VIP)
  • SEQ ID NO:15 is the amino acid sequence of the peptide corresponding to VIP's amino acids 1-10
  • SEQ ID NO:16 is the amino acid sequence of the peptide corresponding to VIP's amino acids 7-16
  • SEQ ID NO:17 is the amino acid sequence of the peptide corresponding to VIP's amino acids 13-22,
  • SEQ ID NO:18 is the amino acid sequence of the peptide corresponding to VIP's amino acids 19-28;
  • FIGURE 3 depicts various plasmid DNA constructs, the nucleotide bases of which code for nested peptides derived from VIP and for a cross- reactive peptide derived from pituitary adenylate cyclase activating protein (PACAP) in which:
  • PACAP pituitary adenylate cyclase activating protein
  • SEQ ID NO:3 is the amino acid sequence for Sheep VIP (representing the consensus amino acid sequence for VIP) ,
  • SEQ ID NO:19 is the amino acid sequence of VI, the VIP-derived peptide corresponding to VIP's amino acids 1-12,
  • SEQ ID NO:20 is the amino acid sequence of V2
  • SEQ ID NO:21 is the amino acid sequence of V3
  • SEQ ID NO:22 is the amino acid sequence of V4
  • SEQ ID NO:23 is the amino acid sequence of PI, the PACAP-derived peptide corresponding to VIP's amino acids 14-23 plus four additional PACAP-specific amino acids (the first three and the last one) ;
  • FIGURE 4 depicts the sequences of the oligonucleotides that encode tandem repeats of the four peptides derived from VIP (VI, V2, V3 and V4) and PACAP (PI) exemplified in Figure 3 and SEQ ID NO:3 and 19-23 in which:
  • FIGURE 4a (SEQ ID NO:24) encodes a 150 base pair DNA sequence coding for VI (SEQ ID NO:19),
  • FIGURE 4b (SEQ ID NO:25) encodes a 150 base pair DNA sequence coding for antisense of VI,
  • FIGURE 4c (SEQ ID NO:26) encodes a 150 base pair DNA sequence coding for V2 (SEQ ID NO:20) ,
  • FIGURE 4d (SEQ ID NO:27) encodes a 150 base pair DNA sequence coding for antisense of V2,
  • FIGURE 4e (SEQ ID NO:28) encodes a 162 base pair DNA sequence coding for V3 (SEQ ID NO:21),
  • FIGURE 4f (SEQ ID NO:29) encodes a 162 base pair DNA sequence coding for antisense of V3
  • FIGURE 4g (SEQ ID NO:30) encodes a 150 base pair DNA sequence coding for V4 (SEQ ID NO:22)
  • FIGURE 4h (SEQ ID NO:31) encodes a 150 base pair DNA sequence coding for antisense of V4,
  • FIGURE 4i (SEQ ID NO:32) encodes a 174 base pair DNA sequence coding for the amino acid sequence of PI (SEQ ID NO:23) ,
  • FIGURE 4j (SEQ ID NO:33) encodes a 174 base pair DNA sequence coding for antisense of PI
  • FIGURE 4k (SEQ ID NO:34) encodes a 90 base pair DNA sequence coding for a portion of the amino acid sequence of PI (SEQ ID NO:23)
  • SEQ ID NO:34 encodes a 90 base pair DNA sequence coding for a portion of the amino acid sequence of PI (SEQ ID NO:23)
  • FIGURE 41 (SEQ ID NO:35) encodes a 90 base pair DNA sequence coding for antisense of a portion of
  • FIGURE 4m (SEQ ID NO:36) encodes a 78 base pair DNA sequence coding for a portion of V2 (SEQ ID NO:20)
  • FIGURE 4n (SEQ ID NO:37) encodes a 78 base pair DNA sequence coding for antisense of a portion of V2
  • FIGURE 4o (SEQ ID NO:38) encodes a 84 base pair DNA sequence coding for a portion of V3 (SEQ ID NO:21) ,
  • FIGURE 4p (SEQ ID NO:39) encodes a 84 base pair DNA sequence coding for antisense of a portion of V3,
  • FIGURE 4q (SEQ ID NO:40) encodes a 78 base pair DNA sequence coding for a portion of V4 (SEQ ID NO:22) ,
  • FIGURE 4r (SEQ ID NO:41) encodes a 78 base pair DNA sequence coding for antisense of a portion of V4
  • FIGURE 4s (SEQ ID NO:42) encodes a 78 base pair DNA sequence coding for a portion of VI (SEQ ID NO:19) ,
  • FIGURE 4t (SEQ ID NO:43) encodes a 78 base pair DNA sequence coding for antisense of a portion of VI;
  • FIGURE 5 depicts the immunoreactivity of a fusion protein comprising (1) VIP and SEQ ID NO:3 or a peptide derived from VIP, namely V2 (SEQ ID NO:20) and (2) a carrier molecule, namely leukotoxin, in which bacterial lysates were prepared and subjected to SDS-PAGE and Western blotting as described herein.
  • Nested peptides are derived from the 28 amino acid VIP molecule secreted by various animal species, including, for example, any of the ones listed in Figure 1 and SEQ ID N0S:1-14. For example, three to six nested peptides are prepared, each being 6 to 12 (and preferably 8 to 10) amino acids in length. The nested peptides are then tested to identify those which are capable of raising antibodies which will neutralize VIP in vivo.
  • Oligonucleotides corresponding to each of the nested peptides are synthesized and cloned into a carrier protein gene such as leukotoxin ("Lkt") and the resulting protein encoded by the chimeric gene is tested for its ability to react with antibodies which neutralize the biological effect of VIP in vivo .
  • Lkt leukotoxin
  • each of the individual short peptides are chemically synthesized and then chemically conjugated to a carrier molecule such as ovalbumin. These conjugates are individually tested for their ability to react with antibodies which neutralize the biological effect of VIP in vivo.
  • One or more of the nested peptides having the desired effect is identified and its nucleotide sequence inserted into the DNA coding for a carrier protein such as Lkt.
  • a carrier protein such as Lkt.
  • One copy of the nucleotide sequence may be inserted.
  • tandem repeats of one or more such sequences may be inserted resulting in a multimer.
  • Proteins homologous to VIP such as pituitary adenylate cyclase activating protein ("PACAP"), can be used to accomplish the same objectives in any manner equivalent to that with respect to a peptide derived from VIP.
  • PACAP appears to outcompete VIP for its own receptor and therefore may be a better anti-VIP antigen than VIP itself.
  • PACAP may also have an added advantage in that its presence correlates with innappetance and accordingly it appears to be involved in the control of feeding behaviour.
  • proteins exhibiting structural homology with VIP should also be useful with respect to the present invention. These include proteins such as PHI, PHV(l-42) helodermin and others, summarized in Table 1 of a review article by Jean Christophe in Biochimica et Biophysics Acta (1993) 1154:183-199.
  • the carrier protein serves the function of rendering the fusion product more immunogenic.
  • a carrier protein may not be essential where the tandemly repeated sequences of VIP or fragments thereof form the fusion product.
  • Lkt is but one possible carrier protein, and others can be selected.
  • the carrier protein has a molecular weight in the range of 25 to 100 kDa.
  • the carrier protein such as Lkt
  • Lkt may be placed into a replicable expression vector before or after the insertion of the VIP or related nucleotide sequence(s) .
  • the vector is used to transform a suitable host cell and the transformed host cell is cultured to effect the production of the recombinant fusion protein.
  • the recombinant protein thereby provides a ready source of VIP immunogen useful for producing antibodies which neutralize VIP in vivo without inducing prolactin release.
  • An aspect of the present invention is to provide a VIP immunogen capable of eliciting a broad immune response.
  • the present invention therefore includes a gene construct comprising (preferably different) full length sequences in tandem that may be connected to a carrier protein gene such as the Lkt gene.
  • the VIP gene sequences may be from macaque, gila monster and opossum or any other combination of VIP or cross-reactive protein gene sequences from different species with variances in the amino acid sequence of VIP.
  • the desired gene construct can be made by tandemly ligating VIP cDNA sequences from different species to a DNA coding for a carrier protein.
  • the resulting DNA construct is placed into an expression vector and the vector used to transform a suitable host.
  • the resulting recombinant protein is useful in eliciting a broad immune response in different target species.
  • the invention also relates to a nucleotide sequence which encodes a protein as described variously above, which nucleotide sequence is directly capable of functioning as a vaccine to neutralize the effect of VIP in vivo .
  • injection of DNA encoding a foreign protein into skeletal or cardiac muscle results in the muscle cells taking up the exogenous DNA.
  • Plasmid pAA352 (Canadian Patent No. 2,014,033) serves as the starting point or root vector. It consists of the Lkt gene (approximately 2800 base pairs) in the vector pGH433laci (approximately 4595 base pairs) where the gene is bound by a 5' Bglii vector site and a 3' BamHl vector site.
  • the Lkt gene has been shortened to 1470 base pairs by the excision of the internal BstBl/Nael fragment and its open reading frame maintained by mungbean exonuclease digestion and religation.
  • the resulting plasmid has been designated pSLK (see Figure 3) . This version of the Lkt gene, when expressed in E. coli , results in the production of a 52 kDA protein.
  • VIP is found as a 28 amino acid protein in most mammalian species (see Figure 1 and SEQ ID NOS:1-14) .
  • SEQ ID NOS:1-14 the following gene constructs are synthetically prepared from the consensus sequence (HSDAVFTDNYTRLRKQMAVKKYLNSILN, see SEQ ID NO:3) : VI, amino acids 1-12 and SEQ ID NO:19; V2, amino acids 6-17 and SEQ ID NO:20; V3, amino acids 11-23 and SEQ ID NO:21; and V4, amino acids 17-28 and SEQ ID NO:22, where each of these is synthesized at the nucleotide level in four contiguous copies.
  • PACAP construct PI and SEQ ID NO:23, is prepared based on the region of homology with VIP.
  • This sequence corresponds to amino acids 14-23 of VIP and also has four additional PACAP-specific amino acids (in bold and underlined) SRYRKQMAVKKYLA.
  • the oligonucleotides that encode each of these five peptide-oligomers have been designed such that only the 3' end cloning site is regenerated after subcloning into pSLK at the end of the Lkt gene.
  • the nucleotide sequence of each of these peptide repeats is set out in Figures 4a to 4t and SEQ ID NOS:24-43.
  • oligomers are initially expressed as fusion proteins at the carboxy-terminal end of the Lkt in pSLK.
  • the five resulting constructs have been identified as pSLVl, pSLV2, pSLV3, pSLV4 and pSLPl (see Figure 3) .
  • These constructs encode for, respectively, multimers of the four peptides derived from VIP, namely VI, V2, V3 and V4 (SEQ ID NOS:19-22, respectively), and the peptide derived from PACAP, namely PI (SEQ ID NO:23) .
  • constructs are used to develop the appropriate animal model, using standard immunological testing methods, in other words, a model that is based on the ability to elicit VIP-neutralizing antibody.
  • other constructs can be prepared from non- consensus VIP sequences and from potentially variable different nested sequences, as well as from cross- reactive peptides.
  • Useful constructs are selected based on immunogenicity (biologically relevant antigenicity) , B-cell epitope molarity, and expression levels, etc.
  • Pellets are resuspended in two equal 400 ml volumes of LB broth containing ampicillin which has been prewarmed to 37°C, and the cells are then incubated for 2 hours.
  • Isopropyl-/3,D-thiogalactopyranoside IPTG
  • IPTG Isopropyl-/3,D-thiogalactopyranoside
  • Cells are harvested by centrifugation as above and resuspended in 30 ml of 50 mM Tris-hydrochloride, 25% (w/v) sucrose, pH 8.0, and frozen at -70°C. The frozen cells are then thawed at room temperature and 5 ml of lysozyme (20 mg/ml in 250 mM Tris-HCl at pH 8.0) added. The mixture is vortexed at high speed for 10 seconds and placed on ice for 15 minutes.
  • the cells are then added to 500 ml of lysis buffer (100 mM Tris-HCl, pH 8.0, 50 mM EDTA, 2% Trition X-100) in a 1 L beaker and mixed by stirring with a 2 ml pipette.
  • lysis buffer 100 mM Tris-HCl, pH 8.0, 50 mM EDTA, 2% Trition X-100
  • This solution is placed on ice and sonicated for 30 second bursts five times (with 1 minute cooling time between bursts) using a Braun sonicator and a large probe set at 100 watts of power.
  • Equal volumes of the solution are then placed in Teflon SS34 centrifuge tubes and centrifuged for 20 minutes at 10,000 RPM in a Sorvall SS34 rotor.
  • the pellets are then resuspended in a total of 100 ml of sterile double deionized water by vortexing at high speed and then centrifuging as above. Supernatants are discarded and the pellets are combined in 20 ml of Tris Buffer Solution ("TBS") (10 mM Tris-HCl, 150 mM NaCl, pH 8.0) and frozen at -20°C.
  • TBS Tris Buffer Solution
  • the recombinant inclusion bodies are then solublized by thawing at room temperature and adding 100 ml of 8 M guanidine-HCl in TBS and mixing vigorously with a magnetic stir bar at room temperature for 30 minutes. Solutions are then transferred to a 2 L flask and 1200 ml of TBS added and further mixed for 2 hours.
  • the medium is autoclaved for 20 min at 15 lb/sq. in. on liquid cycle.
  • the medium is allowed to cool and 100ml of a 0.17 M KH 2 P0 4 and 0.72 M K 2 HP0 4 solution is added.
  • This solution is made by dissolving 2.31g of KH 2 P0 4 , 12.54 g of K 2 HP0 4 in a final volume of 100ml of dH 2 0.
  • 30% Acrylamide/Bis solution 146 g of acrylamide and 4 g of N,N' -methylenebisacrylamide are dissolved in double-distilled water ("ddH 2 0") to a final volume of 500 ml.
  • ddH 2 0 double-distilled water
  • Approximately 10 to 15 g of Amberlite MB-3 is added and the resulting solution stirred for 30 min.
  • the solution is then filtered and stored at 4°C in the dark.
  • 1.5 M Tris solution the pH is adjusted to 8.8 with HC1.
  • Tris solution the pH is adjusted to 6.8 with HC1.
  • Sample buffer (IX) 1ml of 0.5 M Tris-HCl pH 6..8, 2ml of 80% Glycerol, 1.6ml of 10% SDS, 0.4ml of 2-mercaptoethanol and 50 ⁇ l of 4% bromophenol blue are mixed together in 26.9ml of ddH 2 0.
  • Coomassie blue stain solution lg of Coomassie blue is dissolved in 400ml of methanol and 100 ml of Acetic acid, the volume is adjusted to 1 litre with dH 2 0.
  • the method comprises inoculating 5ml of Terrific Broth containing 100 ⁇ g/ml Ampicillin ("TB-amp") with a single colony of transformed E. coli which is then grown at 37°C overnight with shaking.
  • 5ml of TB-amp is inoculated with 50 ⁇ l of the above- mentioned overnight culture from single colony and grown at 37°C on a shaker (250 rpm) until a OD 600 0.5-0.6 is reached.
  • 800 ⁇ l of the culture is then transferred to a 1.5 ml centrifuge tube as an uninduced control ("uninduced control”), and 40 ⁇ l of 0.5 M IPTG is added to the test tube ("induced culture”) which is incubated for an additional 2 hours.
  • 500 ⁇ l of the induced culture is transferred to a 1.5ml centrifuge tube. Both the 500 ⁇ l of induced culture and the 800 ⁇ l of uninduced culture are centrifuged for 3 min at 12 000 rpm. The supernatants are discarded and the cells suspended in 100 ⁇ l of IX sample buffer, and can be stored at -20°C.
  • the desired percentage of SDS-PAGE gel is prepared according to separation range (see Table 1 below) .
  • the resolving gel solution can be made according to Table 2 (see below) .
  • the gel (about 4-4.5 ml) is casted, then 100-200 ⁇ l of 1-Butanol (ddH 2 0 saturated) is carefully added on top of the resolving gel.
  • the butanol is washed away with ddH 2 0 when the gel has polymerized and the excessive ddH 2 0 is removed with Whatman filter paper.
  • the stacking gel is then casted according to Table 3 (see below) and the comb inserted.
  • the protein sample is heated at 95°C for 10 min before loading on the SDS-PAGE gels. 5-10 ⁇ l of each sample is loaded into each well. The gel is then run at 170-180 volts until the blue dye front reaches the bottom of the gel. The gel is then stained for 30 min in the staining solution on a shaker. The gel is destained with the destain solution. The destain solution is changed until the background is clear. The gel is then soaked in the gel drying solution for approximately 10 to 30 min, then dried at 37°C overnight between 2 pieces of cellophane membranes wetted with the gel drying solution. Table 1 . Range of molecular weight separation for different % of SDS-PAGE gels
  • FIG. 5 demonstrates the immunoreactivity of a fusion protein comprising (1) VIP (SEQ ID N0:3) or a peptide derived from VIP, namely V2 (SEQ ID NO:20) , and (2) a carrier molecule, namely leukotoxin.
  • VIP SEQ ID N0:3
  • V2 SEQ ID NO:20
  • carrier molecule namely leukotoxin.
  • Frame A of Figure 5 illustrates the results of the Coomassie Brilliant-Blue stained SDS-PAGE minigel.
  • Lane 1 is the profile of molecular weight markers (112, 84, 53, and 35 kDa) .
  • Lanes 2, 4, 6, and 8 are protein profiles from uninduced bacterial cultures while lanes 3, 5, 7, and 9 are from IPTG-induced bacterial cultures.
  • Lanes 2 and 3 are vector-control; lanes 4 and 5 are the recombinant leukotoxin molecule; lanes 6 and 7 are leukotoxin-VIP (V2 derivative - SEQ ID NO:20) fusion protein; lanes 8 and 9 are leukotoxin (full length VIP - SEQ ID NO:3) fusion protein.
  • TBS Tris buffer saline
  • Tris buffer saline with Tween contains 0.05% Tween 20 in TBS.
  • Blocking solution 1% Bovine serum albumin ("BSA”) in TBS.
  • BSA Bovine serum albumin
  • Alkaline Phosphatase (“AP") buffer contains 100 mM NaCl, 5 mM MgCl 2 , and 100 mM Tris,- pH is adjusted to 9.5 with HC1.
  • AP stop buffer contains 10 ml of 1M Tris-HCl pH 7.5 and 10 ml of 0.25 M EDTA pH 8.0. The final volume is adjusted to 500 ml with dH suspend0;
  • NBT Nitro Blue Tetrazolium
  • BCIP 5-Bromo-4-Chloro-3-Indolyl-Phosphate
  • the method comprises running a SDS-PAGE gel as described above for SDS-PAGE minigel.
  • a Whatman paper and a nitrocellulose membrane are then wetted in transfer buffer for 15-30 min.
  • the unstained gel is incubated in 20-30 ml of transfer buffer on a shaker (50 rpm) for approximately 5 to 10 min.
  • the sandwiches are then taken apart and the prestained marker is checked and the gel is stained as usual to determine whether the transfer is complete.
  • the nitrocellulose membrane is washed in two changes of TBS of 5 min each and is then blocked with blocking solution (1% BSA in TBS) for 1 hour on the shaker at room temperature (or at 4-8°C overnight) .
  • the blocking solution is discarded, then approximately 10 to 20 ml of blocking solution containing a 1/100 to 1/2000 dilution of primary antibody (according to the titre) is added to the nitrocellulose membrane which is then incubated at room temperature with shaking for 1 hour (or at 4-8°C overnight) .
  • the antibody solution is discarded and the membrane washed twice with TTBS for 5 min each time.
  • FIG. 5 demonstrates the immunoreactivity of a fusion protein comprising (1) VIP (SEQ ID NO:3) or a peptide derived from VIP, namely V2 (SEQ ID NO:20), and (2) a carrier molecule, namely leukotoxin.
  • VIP SEQ ID NO:3
  • V2 SEQ ID NO:20
  • a carrier molecule namely leukotoxin.
  • Frame B of Figure 5 illustrates the results of this experiment.
  • the primary antibody was raised in rabbit to a VIP-KLH (Vasoactive Intestinal Peptide - Keyhole Limpet Hemocyanin) conjugate.
  • the second layer was a goat anti-rabbit alkaline-phosphatase labelled antibody.
  • the lanes are as described for Frame A above with respect to SDS-PAGE minigels.
  • the immunoreactive recombinant proteins migrate at the expected rate i.e. approximately 55 kDa.
  • the apparently immunoreactive proteins at 85 and 35 kDa are believed to result either from non-specific reactions or be due to FcR-like (Fragment-crystalline Receptor - like) behaviour. These non-specific reactions are observed with most E. coli lysates tested with most antisera that were used.
  • mice Animal Model Development : The above-mentioned fusion proteins are initially tested in mice for their ability to elicit anti-VIP antibody as determined in radioimmunoassay. Vaccine candidates are then similarly tested in commercial fowl and finally in a food/egg production setting.
  • a Nucleotide sequence resulting from the above preparation techniques is injected into, for example, the skeletal breast muscle of a turkey.
  • the muscle cells in the turkey take up the exogenous DNA and effect expression of the encoded peptides derived from VIP or a cross- reactive peptide such as PACAP.
  • the turkey as a result, will be immunized against the biological effect of VIP and therefore exhibit modified egg production.
  • food-producing animals such as beef cattle may be immunized against VIP in a similar fashion, resulting in increased feed efficiency and weight gain.
  • the following example describes the injection of the recombinant peptides resulting from the above preparation techniques into the necks of turkeys, thereby immunizing them against VIP.
  • Eighty turkey hens (eight groups comprising ten hens each) at 16 weeks of age, were injected subcutaneously in the neck at week 0 and week 3 with 100 ⁇ g of antigen. Serum was collected from the wing vein of each bird at weeks 0, 3 and 4. The serum was subjected to radioimmunoassay (RIA) for the presence of anti-VIP antibody.
  • RIA radioimmunoassay
  • the antigens injected in treatment groups 1 through 4 consisted of the multimer recombinant peptides SLV2, SLV3, SLV4, and SLP1 described earlier and illustrated in Figure 3.
  • the antigen injected in treatment group 5 is composed of four full length copies of vasoactive intestinal peptide and a 52 kDa carrier protein, leukotoxin.
  • the antigen in treatment group 6 is composed of the carrier protein, leukotoxin, without any VIP linked via peptide bonds.
  • the antigen in treatment group 7 is composed of VIP chemically conjugated to keyhole limpet hemocyanin (KLH), i.e. not cloned and synthesized as a fusion protein in the same way as the VIP-Lkt fusion product in treatment group 5.
  • KLH keyhole limpet hemocyanin
  • the antigen in treatment group 8 is KLH as a control against group 7.
  • the VIP and PACAP (the hormones) used in the antigens is radioactively labelled by the Chloroamine T method.
  • 1 mCi (lO ⁇ l) 125 Iodine (Amersham IMS.30) is added to 3 ⁇ g of the hormone dissolved in 25 ⁇ l P0 4 buffer.
  • lO ⁇ l of Chloramine T (2 mg/ml dissolved with sodium metabisulphite (0.4 mg/ml) and potassium iodide (10.0 mg/ml) in 0.05M P0 4 buffer is added.
  • the reaction is terminated after exactly 15 seconds by adding 100 ⁇ l of sodium metabisulphite.
  • the mono-iodinated fractions are isolated on a QAE Sephadex A25 column (Pharmacia) .
  • the iodinated hormone is used in a radioimmunoassay (RIA) to calculate the amount of VIP or PACAP hormone bound to antibody in the serum samples.
  • RIA radioimmunoassay
  • the antigens are formulated with 1% thimerosal (to yield 1% final volume) and sterile saline to obtain the desired amount of vaccine having the desired concentration of antigen.
  • the RIA results are summarized in Table 4. With respect to each treatment group, titres of anti-VIP antibodies were calculated. Binding of antigen measured in serum samples is generally considered significant if above 10%. The mean percentage of antigen binding above 10% is given in Table 4. These calculations are based on the analysis of serum samples from those birds which responded to the treatment antigen in each case. The number of birds responding to each treatment by antigen binding greater than 10% are also indicated.
  • the antigen comprising KLH chemically conjugated with VIP provides an expected high rate of antibody response. Antigens consisting solely of KLH or Lkt elicited no antibody response, as expected. Antigens comprising VIP, multimeric fragments of VIP, or PACAP (which has a structural homology with VIP) elicited a significant response.

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Abstract

Peptides capables d'induire des anticorps qui neutralisent l'activité du peptide intestinal vasoactif (VIP). La présente invention concerne en particulier une séquence nucléotidique qui code une protéine de recombinaison comportant (1) au moins un peptide dérivé d'un peptide intestinal vasoactif ou d'une protéine à réaction croisée et éventuellement (2) une molécule porteuse, ladite protéine de recombinaison étant capable de produire des anticorps qui neutralisent le peptide intestinal vasoactif in vivo. La présente invention concerne en outre des peptides à répétition en tandem dérivés du VIP ou de peptides à réaction croisée, qui peuvent provoquer une large réponse immunitaire. Elle est utile pour augmenter la production des ÷ufs chez des espèces aviaires et pour augmenter l'efficacité de l'absorption des aliments et la vitesse de prise de poids d'animaux à viande.
PCT/CA1996/000280 1995-05-03 1996-05-03 Peptide intestinal vasoactif WO1996034958A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US08/952,568 US6037321A (en) 1995-05-03 1996-05-03 Fusion proteins comprising vasoactive intestinal peptide or PACAP
EP96911888A EP0824590A1 (fr) 1995-05-03 1996-05-03 Peptide intestinal vasoactif
AU54942/96A AU5494296A (en) 1995-05-03 1996-05-03 Vasoactive intestinal peptide
BR9608118-0A BR9608118A (pt) 1995-05-03 1996-05-03 Peptìdeo intestinal vasoativo.

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US43310895A 1995-05-03 1995-05-03
US08/433,108 1995-05-03
US51336695A 1995-08-10 1995-08-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107056922A (zh) * 2016-12-24 2017-08-18 山东明鑫集团有限公司 一种具有降血糖作用的pacap多肽类似物的制备方法
JP2023511421A (ja) * 2020-01-23 2023-03-17 ユナイテッド・バイオメディカル・インコーポレーテッド 片頭痛を予防及び治療するための、下垂体アデニル酸シクラーゼ活性化ペプチド（ｐａｃａｐ）を標的とするペプチド免疫原及びその製剤

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* Cited by examiner, † Cited by third party
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WO1992003558A1 (fr) * 1990-08-22 1992-03-05 Andrew Potter Soudures geniques d'interleukine-2-leucotoxine, et leurs utilisations
EP0529487A2 (fr) * 1991-08-22 1993-03-03 Takeda Chemical Industries, Ltd. Dérivés de PACAP ayant une propriété productrice de c-AMP
WO1993008290A1 (fr) * 1991-10-16 1993-04-29 University Of Saskatchewan Immunogenicite renforcee par l'utilisation de chimeres comprenant des leucotoxines
EP0591524A1 (fr) * 1990-10-09 1994-04-13 M & D RESEARCH CO., LTD. Procede pour produire un peptide ou une proteine
WO1994008616A1 (fr) * 1992-09-22 1994-04-28 Regents Of The University Of Minnesota Procede d'augmentation de la production d'×ufs chez des especes aviaires par immunisation active contre un peptide intestinal vasoactif
WO1994019469A1 (fr) * 1993-02-22 1994-09-01 Centre National De La Recherche Scientifique (Cnrs) Proteine de fusion multi-vip et procede de preparation de vip recombinant
WO1996024675A1 (fr) * 1995-02-10 1996-08-15 University Of Saskatchewan CHIMERES GnRH-LEUCOTOXINE

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Publication number Priority date Publication date Assignee Title
WO1992003558A1 (fr) * 1990-08-22 1992-03-05 Andrew Potter Soudures geniques d'interleukine-2-leucotoxine, et leurs utilisations
EP0591524A1 (fr) * 1990-10-09 1994-04-13 M & D RESEARCH CO., LTD. Procede pour produire un peptide ou une proteine
EP0529487A2 (fr) * 1991-08-22 1993-03-03 Takeda Chemical Industries, Ltd. Dérivés de PACAP ayant une propriété productrice de c-AMP
WO1993008290A1 (fr) * 1991-10-16 1993-04-29 University Of Saskatchewan Immunogenicite renforcee par l'utilisation de chimeres comprenant des leucotoxines
WO1994008616A1 (fr) * 1992-09-22 1994-04-28 Regents Of The University Of Minnesota Procede d'augmentation de la production d'×ufs chez des especes aviaires par immunisation active contre un peptide intestinal vasoactif
WO1994019469A1 (fr) * 1993-02-22 1994-09-01 Centre National De La Recherche Scientifique (Cnrs) Proteine de fusion multi-vip et procede de preparation de vip recombinant
WO1996024675A1 (fr) * 1995-02-10 1996-08-15 University Of Saskatchewan CHIMERES GnRH-LEUCOTOXINE

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O.M. YOUNGREN ET AL.: "Active immunization with vasoactive intestinal peptide prevents the secretion of prolactin induced by electrical stimulation of the turkey hypothalamus", GENERAL AND COMPARATIVE ENDOCRINOLOGY, vol. 95, 3 September 1994 (1994-09-03), ACADEMIC PRESS, INC., NEW YORK, US, pages 330 - 336, XP002014871 *
P.J. SHARP ET AL.: "The role of hypothalamic vasoactive intestinal polypeptide in the maintance of prolactin secretion in incubating bantam hens: observations using passive immunization, radioimmunoassay and immunohistochemistry", J. ENDOCRINOLOGY, vol. 122, no. 1, July 1989 (1989-07-01), LONDON, UK, pages 5 - 13, XP000604983 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107056922A (zh) * 2016-12-24 2017-08-18 山东明鑫集团有限公司 一种具有降血糖作用的pacap多肽类似物的制备方法
JP2023511421A (ja) * 2020-01-23 2023-03-17 ユナイテッド・バイオメディカル・インコーポレーテッド 片頭痛を予防及び治療するための、下垂体アデニル酸シクラーゼ活性化ペプチド（ｐａｃａｐ）を標的とするペプチド免疫原及びその製剤

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