CN1179780A

CN1179780A - Peptides with growth promotion properties

Info

Publication number: CN1179780A
Application number: CN 96192895
Authority: CN
Inventors: B·L·巴克沃尔特; H－M·希赫; B·S·旺
Original assignee: American Cyanamid Co
Current assignee: Wyeth Holdings LLC
Priority date: 1995-03-31
Filing date: 1996-03-15
Publication date: 1998-04-22

Abstract

The present invention describes peptides which have well-defined secondary structure, preferably a helical conformation, which mimic the corresponding region of porcine somatotropin (pST) and which enhance the activity of pST and promote growth of warm-blooded animals. These peptides compete with pST for binding to the PS-7.6 monoclonal antibody. These peptides contain therein the sequence of amino acids Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa (SEQ ID No. 1), wherein the sequence differs from the native sequence of pST, as well as sequences in which the location of essential amino acids is shifted by three amino acids, representing almost one turn along the helix, which contain therein the sequence of amino acids Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID No. 18) and wherein the sequence differs from the native sequence of pST.

Description

Has the peptide that promotes growth characteristics

Invention field

The present invention relates to elicit the peptide that can improve the Porcine somatotropin activity and promote the antibody of warm-blooded animal growth.Technical background

Porcine somatotropin (pST) is that length is 191 amino acid whose pituitrins, and it has multiple biological activity (document clauses and subclauses 1).This hormone is that the form of the single chain polypeptide arranged with 4 antiparallel alpha-helixs exists (2).Each alpha-helix contains 3.6 amino acid/revolutions.This periodically occurs every 3 or 4 residues in the same one side of spiral.

Domestic animal is used pST can promote its muscle growth, improve feed efficiency, and can reduce fat accumulation (3,4).The interior amount of giving birth to of pST is little, and therefore, people concentrate on striving direction in the preparation of exogenous pST, to use on a large scale on agricultural.But influenced being extensive use of of exogenous pST already in the difficulty of preparing and use.Long-term inject require the pST molecule have stability and pST can be in for some time controllable release.In addition, every day or regularly inject pST frequently and need cost of labor, thus make the strong point of pST become uneconomical.

People have done some effort reducing on these difficulties, comprise that interpolation can strengthen the requirement that the active one-tenth of pST assigns to reduce pST.

Have and report that mono-clonal that cooperates with tethelin and polyclonal antibody can further improve tethelin biological activity (5-9) in vivo.Though the mechanism by the different growth of antibody Jie still imperfectly understands, existing people proposes to come partial interpretation growth phenomenon (10) with the pharmacological kinetics and/or the chorologic variation of tethelin.The somebody proposes, promotion be since antibody the bonded selectivity of growth hormone receptor subclass is regulated due to (5,6,11,12).Report that in addition tethelin is a kind of possible explanation (13) to the bonded improvement of liver body original receptor.It should be noted that these antibody himself do not promote growth when lacking exogenous pST.

Promote a trial of growth of animal to produce the monoclonal antibody that label is PS-7.6 with immunological method, this antibody improves pST biological activity (10) consistently in the rat of cut hypophysis is measured, though when lacking pST, himself do not promote growth.Yet because the required monoclonal antibody and the artificial cost of repeatedly medication of antibody, with the monoclonal antibody of this promotion growth domestic animal being carried out passive immunization is not only economically.

Therefore, need carry out new exploration to promoting growth, to reduce the difficulty that is run in the present scheme.One during these are explored is the peptide in simulation and PS-7.6 monoclonal antibody bonded pST zone.To produce and improve the active PS-7.6 monoclonal antibody of endogenous pST similar antibody on function with animal being carried out active immunity with can be by the epi-position that the PS-7.6 monoclonal antibody is discerned consistent peptide fragment.Therefore, the epi-position that can be discerned by the PS-7.6 monoclonal antibody of evaluation is crucial for the peptide vaccine that design can improve the growth of animal situation.Most of peptides have the one-level conformation of natural protein, but lack the secondary conformation.In solution, the structure of small peptide is conversion (random coil) mutually apace generally.To only in the peptide of small portion, there be the regular hour with the similar conformation of the desired zone of pST.Therefore, the peptide that needs design to have definite secondary conformation.The general introduction of invention

Thereby, an object of the present invention is to design and have clear and definite secondary structure, preferably have the peptide of the helical conformation of simulation pST corresponding zone.

Another object of the present invention is that design is competed with PS-7.6 monoclonal antibody bonded peptide with pST.

Another purpose of the present invention is that exploitation uses these peptides to promote the composition of warm-blooded animal growth.

These purposes of the present invention are finished by the peptide that contains aminoacid sequence Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa (SEQ ID NO:1), and described sequence is different from the native sequences of pST.The present invention also comprises one or more quilts (straight chain) leucine (Nle) metathetical peptide just in first or second leucine or the Xie Ansuan of SEQ ID NO:1.The position that the invention still further relates to indispensable amino acid has been moved and has almost been represented along rotating 3 amino acid whose peptides of spiral, this peptide contains aminoacid sequence Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ IDNO:18), and described sequence is different from the native sequences of pST.By limiting the 3rd amino-acid residue is the peptide that Isoleucine is further modified SEQ ID NO:18, produce aminoacid sequence: Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:19), described sequence is different from the native sequences of pST.

These peptides preferably contain the Serine as helical conformation promotor, in described helical conformation, Serine is near first leucic aminoterminal amino acid among the SEQ ID NO:1 with near the aminoterminal amino acid of first Isoleucine among the SEQ ID NO:19.

Peptide of the present invention and pharmaceutically acceptable adjuvant, diluent or carrier are share, and preparation promotes the composition of warm-blooded animal growth.The simple declaration of accompanying drawing

Fig. 1 describes tryptic digestion thing (swimming lane 1), component #9 (swimming lane 2) and #9-5 (swimming lane 3), they with PS-7.6 monoclonal antibody incubation, with magnetized goat anti-mouse IgG antibody mediated immunity precipitation, through SDS-PAGE electrophoresis, electroblotting survey at anti-pST polyclonal antibody on the pvdf membrane, with rabbit, then with the anti-rabbit igg antibody detection of iodinating donkey, on X-ray film and autoradiogram(ARGM), develop at last.

Fig. 2 describes peptide pST (70-95), pST (64-95) and pST (54-95) (Fig. 2 A) and pST (75-95) and pST (75-90) (Fig. 2 B), they make by synthetic, and by competitive radioimmunoassay (RIA) with regard to they to the PS-7.6 monoclonal antibody and ¹²⁵Interactional inhibition effect is tested between the I-pST.Also cold complete pST is tested and is used as positive control.

Fig. 3 describes the result of radioimmunoassay, and this measures test and radiolabeled pST competition with the combining of PS-7.6 monoclonal antibody, and the comparison thing is: (1) pGH (75-95) peptide, (2) pGH (80-90) peptide and (3) pGH (81-90) peptide.

Fig. 4 describes the segmental amino acid of pST (75-95), and amino-acid residue 75-90 is wherein replaced in regular turn by L-Ala.Test these analogues with competitive RIA.From 5 IC that independently test the effective competition of each residue ₅₀Average.

Fig. 5 describes the spiral projection of the pST (75-95) of pST.Hypographous circle is for discerned crucial amino acid by the PS-7.6 monoclonal antibody.

Fig. 6 describes the result of radioimmunoassay; this measures test and radiolabeled pST competition combining with the PS-7.6 monoclonal antibody; relatively thing is: the peptide (being called " pGH (75-95) " among Fig. 5) that (1) is consistent with the native sequences of the amino acid 75-95 of pST; (2) peptide of SEQ ID NO:2 (being called " peptide-X "); (3), aminoterminal amino wherein by adding ethanoyl the modification type (being called " peptide-X that adds cap ") of adorned peptide-X.

Fig. 7 describes the result of radioimmunoassay, this measures test and radiolabeled pST competition combining with the PS-7.6 monoclonal antibody, relatively thing is: (1) pGH (75-95) peptide and (2) are within the scope of the present invention the peptide (being called " peptide-Y " among Fig. 6) of SEQ ID NO:21 not.

Fig. 8 describes the result of radioimmunoassay, this mensuration test and radiolabeled pST (being called " pGH " among Fig. 5) competition combining with the PS-7.6 monoclonal antibody, relatively thing is: (1) pGH (75-95) peptide, (2) peptide-X, (3) peptide-Y, (4) first leucine is replaced by nor-leucine (Nle), produce peptide-X (being called " Nle80 " among Fig. 7) of SEQID NO:13, (5) second leucines are replaced by nor-leucine (Nle), produce peptide-X (being called " Nle87 ") of SEQ ID NO:14, (6) Xie Ansuan is produced peptide-X (being called " Nle90 ") of SEQ ID NO:15 by nor-leucine (Nle) displacement.

Fig. 9 describes and handles (negative control, article one); With pST individual curing (positive control, second); With pST handle and use the peptide-X that makes by 3 kinds of different piggys carry out immunity before and handle influence that (3 groups of rats, 3-8 bar) grows to the rat of cut hypophysis with pig antibody afterwards.The detailed description of invention

The present invention comprises the design of the peptide in simulation pST spiral zone.These peptides have with the minmal sequence homology of pST so that Toplink keeps spiral structure.These peptide mimicses elicit the antibody that can discern natural pST.

Can be carried out immunity to the target animals kind by the peptide of the epi-position of PS-7.6 monoclonal antibody (producing) identification and produce with the PS-7.6 monoclonal antibody similar and can promote the antibody of growth of animal on function with containing at natural pST.

With being carried out the process useful that active immunity replaces the passive PS-7.6 of using monoclonal antibody to animal by the epi-position that the PS-7.6 monoclonal antibody is discerned.This imagination obtains the support (14) of the up-to-date report of Pell etc., and proofs such as Pell with for the growth hormone fragment (residue 135-154) that promotes related different zones lamb being carried out immunity, can improve the per-cent of thin muscle growth.

In the present invention, limiting tryptic digestion with pST comes mapping with the epi-position of PS-7.6 monoclonal antibody bonded pST in conjunction with reversed-phase liquid chromatography partition method and immuno-precipitation.Pass through the immunoassay of a series of sequential L-Ala analogue of epi-position then, identify key amino acid with the interactional epi-position of PS-7.6 monoclonal antibody.Contain to be that the peptide of epi-position of monoclonal antibody (pST produces at the reorganization) identification of PS-7.6 shows the growth that it can promote the growth-promoting activity of pST and can promote piggy in the rat model of cut hypophysis by label.

Isolate the pST fragment consistent (it contains second spiral (2) of pST) and carry out immunoprecipitation with RPLC with the PS-7.6 monoclonal antibody with amino-acid residue 70-95.Radioimmunoassay (RIA) finds that this fragment is with dose-dependently mode and radiolabeled pST competition combining with the PS-7.6 monoclonal antibody.Yet the serum that obtains from the piggy that crosses with pST (70-95) immunity does not make the level of anti-pST antibody increase significantly.This results suggest, the conformation of pST (70-95) does not meet with true epi-position, or the immunogenicity of peptide is insufficient.

The synthetic some peptides that cover this possible epitope regions of pST (70-95) are also analyzed with competitive RIA.Results suggest, pST (70-95) is the optimal sequence that can be discerned by the PS-7.6 monoclonal antibody.By comparison, very faint and the no return of combining of pST (85-95) and PS-7.6 monoclonal antibody, pST (81-95) be debond fully then.The bonding strength of peptide pGH (80-90) and pGH (75-95) much at one, and pGH (81-90) is very faint with combining of PS-7.6 monoclonal antibody.And, if peptide is too little, as pST (80-95) etc., even then in conjunction with good, because the gathering of certain degree also can appear in the hydrophobicity of each amino-acid residue.For reducing gathering and improving solvability, peptide should be longer.

The design of the peptide among the epitope regions pST (75-95) comprises many factors.Most of peptides have the one-level conformation of natural protein, but lack the secondary conformation.In solution, the structure of small peptide is conversion (random coil) mutually apace generally.To only in the peptide of small portion, there be the regular hour with the similar conformation of the desired zone of pST.

The target of design is helical conformation to be stablized also improve antibody thus to proteinic affinity.There is complementary structure between the binding site of this affinity requirement antibody and the epi-position on the protein.

The amino-acid residue that contacts with antibody combining site is less to the tolerance that replaces.Isoleucine displacement leucine is enough to remove combining of peptide and antibody.Non-key residue can be promoted by desired structure is had or the residue of stabilization is replaced, and in this example, alpha-helix can make peptide simulate the natural protein conformation better.

The strategy that can be used for stablizing alpha-helix has: hydrophobic association (15) between (1) spiral; (2) side-chain charges-coulombic interaction (16); (3) carry out the covalent bonds of side chain by disulphide (17) or lactam bond (18); (4) mix amino-acid residue (19) with high helical propensity; (5) electric charge-spiral bipolar stable (20); (6) end of peptide adds cap (21) and metal ion side chain stable (22).

First leucic key being presented among the following embodiment 1, in this embodiment, first leucine of peptide (SEQ ID NO:2) is replaced by Isoleucine, produces the peptide of SEQ ID NO:21.By comparison, non-contacting residue can be freely by displacement and still combination effectively.Therefore, after the essential residue that will contact antibody was determined, the residue residue of substituted peptide was to obtain stable helical conformation.

The sequential L-Ala of each residue of pST (75-90) replaces and shows residue Leu ⁸⁰, Ile ⁸³And Leu ⁸⁷Add Leu ⁷⁶Or Val ⁹⁰Has keying action to combining (as described in the spiral projection of Fig. 5) with the PS-7.6 monoclonal antibody.Other residues can not made binding affinity that substantial change is arranged by the L-Ala displacement.The zone of amino acid 75-95 comprises second spiral of pST, and the repeat pattern of the i of key amino acid and i+3 (i+7) seems and be consistent with the hydrophobicity sidepiece bonded PS-7.6 monoclonal antibody of spiral.This makes the also available i+3 of the both available i pattern of peptide (almost spiral revolution) pattern synthetic.The basis that the sequence of the epi-position that can be discerned by the PS-7.6 monoclonal antibody and spirane structure provide design to improve the effective peptide vaccine of growth of animal state.

The preparation method of pST and peptide is as follows.Reorganization pST is produced by New York, United States Pearl River city Cyanamid company.Peptide is synthetic with solid phase automatic peptide synthesizer (9600 types, Massachusetts Bedford city Milligen/Biosearch company product).All reagent are available from California Belmont city Peninsula Laboratories company.After dissociating from resin carrier, by preparation property reversed-phased high performace liquid chromatographic (HPLC) all peptides are carried out purifying with C18 chromatographic column (Massachusetts Woburn city Rainin Instrument company product).Property HPLC, FAB mass spectrum and aminoacid component analysis by analysis, the purity of these peptides is all the time more than 97%.In addition, the also available other technologies well known in the art of peptide of the present invention, synthetic or dna nucleotide sequence recombinant expressed etc. the structure in suitable host as the solution phase chemistry.Recombinant expressed can also be the form that is fused to the peptide on the carrier, as in conjunction with the protein of maltose or peptide etc.

In the past, people explored the mapping in the site of identification antibody by synthetic (25) when antigenic enzymic digestion (23,24) or polypeptide are on solid carrier.With the synthetic initial trial of carrying out of polypeptide and unsuccessful, thereby then in this research, carry out the proteolysis degraded (proteolytic degradation) of pST, to finish the epitope mapping of PS-7.6 monoclonal antibody.During beginning, with comprising that the various proteolytic enzyme of Quimotrase, trypsinase and endo-protease Glu-C handle pST at 37 ℃.They all produce little pST fragment (molecular weight＜4kDa).The reason of selecting these small segments is to consider that future is more convenient when solid phase synthesis candidate peptide vaccine.Yet in RIA and Western analytical method, the PS-7.6 monoclonal antibody can not be discerned any one in these small segments.In addition, small peptide generally has the conformation flexibility, produces various change configurations in solution.Its result, the anti-peptide antibody of gained interacts with corresponding natural protein often, and efficient is very low.

The initial experimental result of enzyme liberating shows that the epi-position that can be discerned by the PS-7.6 monoclonal antibody is highstrung at 37 ℃ to proteolytic enzyme.May be secondary/tertiary structure of pST under this temperature a little separate folding, thereby make the critical epitopes zone be exposed to the proteolytic enzyme of total degradation.For limiting the enzymic digestion degree to protect complete PS-7.6 epi-position, especially guard ring and revolution (26), temperature of reaction is limited in 25 ℃, continue 3.

Specifically, by gel filled with the crosslinked ox pancreas trypsin 50 enzymic activity/ml of unit on agarose of 1ml, St.Louis city, Missouri State Sigma company product) in 25 ℃ 200ml phosphate buffered saline buffer incubation 3 days, reorganization pST (100mg) is carried out limited tryptic digestion handles.With linear gradient (the A damping fluid=0.1% TFA/ water of preparation property HPLC (C18 chromatographic column, 21.4mm * 25cm, flow velocity=24ml/min, UV:235nm, Dynamax-300A type, Rainin company product) with the 0-100%B damping fluid in 30 minutes; B damping fluid=0.1% TFA/ acetonitrile) separates the tryptic digestion fragment.Collect all flow points, by RIA Analysis and Identification activeconstituents.Main and the stable tryptic digestion fragment that lacks when being created in 37 ℃ of digestion also is designated as flow point #9.The stripping when 18.89 minutes hold-times of this flow point, it accounts for the yield more than 60% of original pST digest.Find thereafter, it in RIA with ¹²⁵The I-pST competition is with the combination of PS-7.6 monoclonal antibody, and prompting flow point #9 contains the PS-7.6 epi-position.Further separate with the solution reduction flow point #9 of the pH9.5 of dithiothreitol (DTT) in 0.1M bicarbonate of ammonia of 10 molar excess and with HPLC again.When 18.43 minutes hold-times effusive label be flow point of #9-5 continue to show with ¹²⁵The I-pST competition is with PS-7.6 monoclonal antibody bonded ability.

Carry out the combination experiment of immunoprecipitation and Western trace (western blotting), analyze these samples.With the tryptic digestion thing is flow point #9 and #9-5 and PS-7.6 monoclonal antibody 37 ℃ of incubations 60 minutes.Add in the mixture with the magnetic bead (Massachusetts Cambridge city Advanced Magnetics company product) of goat anti-mouse IgG bag quilt and at gentle and incubation 30 minutes in the vibration that remains unchanged.The washing magnetic bead is collected it for several times and with magnet.In SDS-PAGE sample damping fluid, boil protein is separated from magnetic bead, on 16% gel (PAGE, San Diego city, California Novex company product), carry out reductibility Tris-tricine polyacrylamide gel electrophoresis then.The protein component electroblotting that will go out from gel separation in the damping fluid of the pH11 that contains the 10mM 3-of 20% methyl alcohol (cyclohexyl amino)-1-propane sulfonic acid (CAPS) is to poly(vinylidene fluoride) micropore (PVDF) film (Foster City city, California AppliedBiosystems company product).Handle this film with 5% skimmed milk, be exposed to anti-pST polyclonal antibody of rabbit and the anti-rabbit igg antibody of iodinating donkey (Arlington Heights city, this state, Illinois Amersham company product) then successively.At last pvdf membrane is exposed to X-ray film, carries out radioautograph.Be to determine the segmental aminoacid sequence of tryptic digestion, the film trace is carried out of short duration dyeing and bands visible is downcut with the solution that contains 0.1 Coomassie blue, 40% methyl alcohol and 10% acetate.These bands are lined up individual layer and used Hankapiller﹠amp on the upper chuck mould of gas phase sequenator (AppliedBiosystems company product); Hood method (27) is measured phenylthiohydantoin deutero-amino acid.The results are shown in Figure 1.

Very clear, the main component of carrying out limited postdigestive pST with trypsinase is the 15kDa fragment (swimming lane 1) of band 10kDa, 4kDa and 2.5kDa impurity.Yet these submembers separate through HPLC and subsequently in flow point #9 enrichment and become main component (swimming lane 2).At last, the 2.5kDa fragment appears among the flow point #9-5 as unique composition, its prompting, and the epi-position that can be discerned by the PS-7.6 monoclonal antibody is in this flow point (swimming lane 3).To 2.5 sequencing fragments, find that its amino acid 70-95 with pST is consistent then.This segmental chemical structure and immunological characteristic are by solid-phase peptide synthetic (28) conclusive evidence, because the synthetic peptide is singly exempted from combine (Fig. 2) of grand antibody with PS-7.6 with dose-dependently mode and 125I-pST competition in RIA.Active little about 100 times with the competition of the competition specific activity of pST (70-95) itself and unlabelled pST.

For further representing the feature of epi-position exactly, 3 peptides have been synthesized in addition.Method with expansion N-terminal amino-acid residue has prepared peptide pST (54-95) and pST (64-95), and the method that is used in the residue that cuts two ends prepares pST (75-90).Test these peptides with RIA, these peptides show and similarly compete usefulness through the pST of tryptic digestion (70-95) fragment that the residue of prompting between 75 and 90 contains epi-position (Fig. 2).The competitiveness of these peptides is significantly less than the pST of contrast, may be because small peptide inherent conformation is flexible and comprised other discontinuous residues in the antibody recognition part.

Independently competitive RIA has determined Leu ⁸⁰Residue key.As described in Figure 3, peptide pGH (80-90) is almost identical with pGH (75-95) with the bonding strength of PS-7.6 monoclonal antibody, the bonding strength of pGH (81-90) then very a little less than.

The antigenic conformation flexibility of peptide has limited them as antigenic practical application.Yet, can limit the conformation flexibility of peptide by the antigenic secondary structure of simulation natural protein, thereby overcome the restriction in the above-mentioned application.In addition, also know and epi-position in each residue with the contacting of antibody combining site in be essential (29).Because the secondary structure of PS-7.6 monoclonal antibody epi-position is considered to important, has determined the segmental amino acid side chain with the interactional pST of PS-7.6 monoclonal antibody (75-95) by the method for replacing each residue with L-Ala in regular turn.With having the reached main chain conformation that the very big L-Ala that forms the tendency (30) of helical conformation in peptide or protein replaces unlikely change pST (75-95) in regular turn.

This scheme that is called alanine scanning mutagenesis is in the key amino acid of the human growth hormone of evaluation and its acceptor interaction very useful (31).When the interactional possible site of deletion and antibody, L-Ala is assumed that secondary structure is remained unchanged.If contain replacement L-Ala peptide still with antibodies, then be considered to non-contacting residue by metathetical amino acid.

Therefore, synthesized pST (75-90) analogue that a series of L-Ala replace, and estimated them with RIA and competed with PS-7.6 monoclonal antibody bonded ability with pST with solid phase method.If L-Ala displacement causes the emulative IC that obtains equating ₅₀Remarkable increase, then the side chain of residue is considered to crucial.Show by 5 discoveries of independently testing among the Fig. 4 that concludes, be the Leu of hydrophobic amino acid ⁸⁰(in addition referring to Fig. 3), Ile ⁸³And Leu ⁸⁷Identification is crucial to the PS-7.6 monoclonal antibody, and Leu ⁷⁶Or Val ⁹⁰Also be crucial.Though in Fig. 4, the bar value of position 89 is greater than the bar value of position 90, when with total p value check (combined p-value test) the estimation p value of FisherShi, the p value of position 90 is had a mind to free burial ground for the destitute remarkable (p=0.00162), and position 89 free burial ground for the destitute remarkable (p=0.16048) intentionally.Note that Fig. 4 does not comprise and other 5 data of testing complete inconsistent the 6th experiments.

According to the tertiary structure (3) of pST and the projection of second spiral, these 5 Key residues of being represented by hypographous circle among Fig. 5 are positioned at the same one side of spiral, expression and the interactional hydrophobicity band of PS-7.6 monoclonal antibody.

In sum, be the epitope mapping of PS-7.6 monoclonal antibody by limited tryptic digestion to the antibody that promotes growth, and by the key amino acid on the evaluation of the L-Ala replacement in regular turn epi-position.The alpha-helix Projection Display of pST, key amino acid are the hydrophobicity bands that is positioned on the same one side of second spiral.The feature description of epitope sequences and secondary structure provides the antigenic basis of effective peptide of the native conformation of the corresponding epi-position of simulation pST.

According to evaluation, design a series of peptides to key amino acid in the epi-position.In one embodiment of the invention, peptide is selected from the sequence that contains following structural:

Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa(SEQ?IDNO：1)

Described sequence is different from the native sequences of pST, and the example of these peptides comprises:

Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-Asp-Val-Ile-Arg-Arg-Ile (SEQID?NO：2)，

Tyr-Ser-Leu-Asp-Arg-Ile-Ile-Arg-Asp-Leu-Asp-Arg-Val-Ile-Arg-Asp-Ile (SEQID?NO：3)，

Tyr-Ser-Leu-Arg-Arg-Ile-Ile-Arg-Arg-Leu-Arg-Arg-Val-Ile-Arg-Arg-Ile (SEQID?NO：4)，

Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Asp-Asp-Leu-Asp-Asp-Val-Ile-Asp-Asp-Ile (SEQID?NO：5)，

Tyr-Ser-Leu-Asp-Asp-Ile-Ala-Arg-Arg-Leu-Asp-Asp-Val-Ala-Arg-Arg-Leu(SEQ?ID?NO：6)，

Tyr-Ser-Leu-Lys-Ala-Ile-Ala-Glu-Ala-Leu-Lys-Ala-Val-Ala-Glu-Ala (SEQ IDNO：7)，

Tyr-Ser-Leu-Lys-Glu-Ile-Glu-Lys-Leu-Leu-Lys-Glu-Val-Leu-Glu-Lys-Leu(SEQID?NO：8)，

Tyr-Ser-Leu-Asp-Asp-Ile-Ala-Arg-Arg-Leu-Asp-Asp-Val-Ala-Arg-Arg-Ala(SEQ?ID?NO：9)

Tyr-Ser-Leu-Lys-Ile-Ile-Ile-Glu-Ile-Leu-Lys-Ile-Val-Ile-Glu-Ile (SEQ ID NO：10)，

Tyr-Ser-Leu-Lys-Glu-Ile-Glu-Lys-Leu-Leu-Lys-Glu-Val-Leu-Glu-Lys-Leu (SEQID NO:11) and

Tyr-Ser-Leu-Lys-Aib-Ile-Aib-Glu-Aib-Leu-Lys-Aib-Val-Aib-Glu-Aib (SEQ IDNO:12), wherein Aib is the 2-aminoisobutyric acid.

In addition, the one or more of the leucine of SEQ ID NO:1 or Xie Ansuan can be replaced by nor-leucine (Nle), and are specific as follows:

Tyr-Ser-Nle-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-Asp-Val-Ile-Arg-Arg-Ile (SEQID?NO：13)，

Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Nle-Asp-Asp-Val-Ile-Arg-Arg-Ile (SEQID?NO：14)，

Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-Asp-Nle-Ile-Arg-Arg-Ile (SEQID NO:15) and

Tyr-Ser-Nle-Asp-Asp-Ile-Ile-Arg-Arg-Nle-Asp-Asp-Val-Ile-Arg-Arg-Ile (SEQID?NO：16)。

And, can be at the arbitrary end or the two terminal halfcystines that add of the peptide of SEQ ID NO:1.

The example of such peptide is

Cys-Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-Asp-Val-Ile-Arg-Arg-Ile(SEQ?ID?NO：17)

In another embodiment of the present invention, the position of indispensable amino acid residue has been moved and has almost been represented along rotating 3 amino acid of spiral.These peptides are selected from the sequence that contains following structural of the native sequences that is different from pST:

Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val(SEQ?ID?NO：18)

Especially, these peptides comprise as the Isoleucine of the 3rd residue and are selected from the sequence that contains following structural of the native sequences that is different from pST:

Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val(SEQ?ID?NO：19)

The example of such peptide is

Tyr-Ser-Ile-Asp-Asp-Leu-Ile-Arg-Arg-Ile-Asp-Asp-Leu-Ile-Arg-Arg- Val(SEQID?NO：20)

The aminoacid sequence of these peptides is described with method commonly used, that is, first must residue towards N-terminal, last must residue towards C-terminal.The present invention also comprise first must residue towards C-terminal, last must residue towards the same sequence of aminoterminal opposed orientation.Two kinds are oriented in and all have identical spiral projection (referring to Fig. 5) on the side chain and equate on function.

Other peptides in the scope of the invention can easily be prepared with method described herein and condition.

Describe in detail as the following examples, in various experiments, peptide of the present invention is tested.

At first, carry out radioimmunoassay and compete combining with radiolabeled pST with the PS-7.6 monoclonal antibody to determine various peptides (be included within the scope of the invention and outside).As describe in detail among the embodiment 1 with Fig. 6 in show that the peptide of SEQ ID NO:2 (being called " peptide-X ") suppresses interaction between PS-7.6 monoclonal antibody and the radiolabeled pST in the dose-dependently mode stronger than the peptide consistent with the native sequences of the amino acid 75-95 of pST.Aminoterminal amino is not suppressed above-mentioned interaction by the modification shape of ethanoyl metathetical peptide-X (being called " peptide-X that adds cap ").In the experiment that does not show in Fig. 6, the peptide of SEQID NO:8-11 and peptide-X suppress above-mentioned interaction comparably, and SEQ ID NO:3 and 12 suppresses above-mentioned interactional degree and then is lower than peptide-X.

As describe in detail among the embodiment 1 with Fig. 7 in show, the effect of the peptide with sequence Tyr-Ser-Ile-Asp-Asp-Ile-Ile-Arg-Arg-Ile-Asp-Asp-Ile-Arg-Arg-Ile within the scope of the invention (SEQ ID NO:21 is called " peptide-Y ") aspect the above-mentioned interaction of inhibition be not much smaller than pGH (75-95) peptide.Therefore, Isoleucine has been removed and the combining of antibody the replacement of first leucine and Xie Ansuan in the peptide of SEQ ID NO:2.

As describe in detail among the embodiment 1 with Fig. 8 in show, peptide-X and its leucine or Xie Ansuan are being suppressed effect aspect the above-mentioned interaction greater than pGH (75-95) peptide by 3 varients of nor-leucine (Nle) metathetical (SEQ ID NO:13-15), and the effect of peptide-Y is still significantly little.In the experiment that in Fig. 8, does not show, the peptide of SEQ ID NO:16 suppress above-mentioned interactional effect and peptide-X equal.

Then, test the biological activity of these peptides with the rat of cut hypophysis.Because the pituitary gland of these rats is removed with surgical method, therefore, their dysplasias.The rat of cut hypophysis can be used as the useful model (32) of research tethelin to growth result.

As describe among the embodiment 2 with Fig. 9 in show, make peptide-X (SEQ ID NO:2) combine and be emulsified in the Freund's complete adjuvant with Protalbinic acid.Make 3 piggy immunity and allow them accept the Freund's incomplete adjuvant of two booster doses with peptide-X.From these animals, take the blood sample and collecting control serum by same piggy before the injection for the first time.Mix with antibody purification and with pST, injected 5 continuously toward the rat of cut hypophysis then.The rat of only accepting pST grows with the statistics meaningful ways.The rat increment of accepting the antibody that pST and the piggy #1 after the immunity or #2 obtain increases significantly.Though the effect of the antibody that obtains from piggy #3 does not reach the meaningful level of statistics (p=0.08), still have visible growth increment.By comparison, pre-immune antibody is invalid.Result as the rat experiment of cut hypophysis has carried out immunity test with piggy.

Can with peptide individually or best incorporated use on the macromole of output in vivo to playing raising antibody.For example, peptide can be bonded on the keyhole limpet hemocyanin protein such as (KLH).Other macromole in the scope of the invention comprise well known in the art those, as people or bovine serum albumin, Protalbinic acid, myohaemoglobin, beta-galactosidase enzymes, penicillinase, heat shock protein and bacterial toxoid etc.Synthetic molecules such as poly-DL-alanyl-poly-L-Lysine and poly-L-Lysine also are suitable for.

Peptide can be used the ordinary method administration, as subcutaneous injection, intramuscular injection and vein streamer and transdermal or oral administration etc.Preferably peptide (or their binding substances) is combined administration with pharmaceutically acceptable adjuvant, diluent or carrier.Use following dosage regimen good especially: after with peptide administration for the first time, to give the identical peptide of one or more reinforcement amounts at interval with specific time.

As describing in detail among the embodiment 3-5, with peptide of the present invention piggy is carried out immunity and can produce feed efficiency raising, lean meat increase and fatty these desired results of minimizing.

In embodiment 3, table 1, accept the finishing period piggy of the peptide of SEQ ID NO:2 and compare with the piggy of only accepting Protalbinic acid, have a mind to the free burial ground for the destitute in the experimental session feed efficiency and significantly improve.In embodiment 3, table 2, accept the identical piggy of peptide and compare with the piggy of only accepting Protalbinic acid, improvement is arranged on framework characteristic, and lean meat increase and fatty minimizing aspect have a mind to the free burial ground for the destitute and significantly improve.Interesting is that the piggy of accepting peptide does not significantly increase on most of organ weights.By comparison, known pST can increase organ weights.

When in embodiment 5, being subjected to the finishing period piggy of the peptide of SEQ ID NO:2 or the peptide of SEQ ID NO:17 (" Cys-peptide ") to repeat the experiment of embodiment 3 with another winding, two groups of piggys are compared with the piggy of only accepting Protalbinic acid, disagreeable leaf fat is all had a mind to the free burial ground for the destitute and is significantly reduced, and the semitendinosus of piggy of accepting the peptide of SEQ ID NO:2 is had a mind to the free burial ground for the destitute and significantly reduced (referring to table 5 and 6).

In embodiment 4, table 3 and 4, compare with the piggy of only accepting Protalbinic acid, accept peptide growth period piggy at experimental session, lean meat is had a mind to the free burial ground for the destitute significantly to be increased.Compare with the finishing period that stops administration, this is especially remarkable in the administration stage.

For the present invention is more readily understood, list following embodiment.These embodiment but not limit the invention only for illustrative purposes.Embodiment 1 Competitive radioimmunoassay

Carry out radioimmunoassay to determine various peptides and radiolabeled pST competition combining with the PS-7.6 monoclonal antibody.For any given concentration of peptide, the amount with the pST of antibodies in mensuration is more little, and the competitiveness of peptide and antibodies is big approximately.The peptide of the natural pST of simulation will more effectively be replaced pST from the PS-7.6 monoclonal antibody on conformation.The method of radioimmunoassay is as follows.

Every hole bag seals with 2%BSA by 1 μ gPS-7.6 monoclonal antibody and with remaining microtiter well binding site on portable microtiter well.With iodinating pST ( ¹²⁵I-pST, specific activity=180-250 μ Ci/ μ g, Massachusetts Boston city New England Nuclear/DuPont company product) add with competitor with various Dilution ratios.Behind the incubation 60 minutes, it is porose unconjugated to remove thoroughly to clean institute ¹²⁵I-pST, and count in each hole of reflection still associating respectively with the PS-7.6 monoclonal antibody with gamma counter ¹²⁵The remaining radioactivity of the amount of I-pST.

In first experiment of in Fig. 6, describing; carry out radioimmunoassay; test and radiolabeled pST (being called " pGH " among Fig. 6) competition combining with the PS-7.6 monoclonal antibody; relatively thing is: the peptide (being called " pGH (75-95) " among Fig. 6) that (1) is consistent with the native sequences of the amino acid 75-95 of pST; (2) peptide of SEQ ID NO:2 (being called " peptide-X ") and (3) aminoterminal amino are wherein replaced and the modification type (being called " peptide-X that adds cap ") of adorned peptide-X by ethanoyl.

The result shows, is added on pGH (75-95) in the hole and suppresses interaction between the pST of PS-7.6 monoclonal antibody and radioactivity in the dose-dependently mode.In addition, the result also shows, peptide-X is with similarly but slightly strong mode suppresses above-mentioned interaction, and the peptide-X that adds cap then can not accomplish.Not bound by theory, the inefficacy that adds the peptide-X of cap in the competitive assay may be because due to peptide mutually assembles in test soln.

In second experiment in Fig. 7, describing, carry out radioimmunoassay, test and radiolabeled pST competition combining with the PS-7.6 monoclonal antibody, relatively thing is: (1) pGH (75-95) peptide and (2) are within the scope of the present invention the peptide (being called " peptide-Y " among Fig. 7) of SEQ ID NO:21 not.

The result shows, is added on pGH (75-95) in the hole and suppresses interaction between the pST of PS-7.6 monoclonal antibody and radioactivity in the dose-dependently mode.By comparison, the effect of peptide-Y is then significantly little.

In the 3rd experiment of in Fig. 8, describing, carry out radioimmunoassay, test and radiolabeled pST competition combining with the PS-7.6 monoclonal antibody, relatively thing is: (1) pGH (75-95) peptide, (2) peptide-X, (3) peptide-Y, (4) first leucine is replaced by nor-leucine (Nle), produce peptide-X (being called " Nle80 " among Fig. 8) of SEQ ID NO:13, (5) second leucines are replaced by nor-leucine (Nle), peptide-X (being called " Nle87 ") and (6) Xie Ansuan of producing SEQ IDNO:14 are replaced by nor-leucine (Nle), produce peptide-X (being called " Nle90 ") of SEQID NO:15.

The result shows, is added on pGH (75-95) in the hole and suppresses interaction between the pST of PS-7.6 monoclonal antibody and radioactivity in the dose-dependently mode.In addition, find also to comprise that the several peptides in the scope of the invention of peptide-X, Nle80, Nle87 and Nle90 are more effective than pGH (75-95) in this competitive assay.Yet the effect that peptide-X shows is still very little.

Embodiment 2

Has the active enhancing of pST on the Hypox rat of anti-A peptide pig antibody

In the present embodiment, inject a kind of peptide to pig.The serum sample and the pST that will comprise the antibody of anti-this peptide that is produced give the injection of hypox rat together.Antibody strengthens the ability that pST promotes the growth of hypox rat.Test is performed such:

Peptide-X (SEQ ID NO:2) is incorporated into Protalbinic acid and emulsification in Freund's complete adjuvant.Three little pigs is accepted 4 week and 8 weeks of booster dose Freund's incomplete adjuvant then with peptide-X immunity.Strengthen one week of back the last time, get blood on one's body from these animals.Also before injection for the first time, get blood serum in contrast on one's body from same these pigs.Use the ammonium sulfate antibody purification, mix, injected 5 days for hypox continuously with the dosage of 1mg antibody and 5 μ g pST with pST.Three groups of rats are accepted the antibody from different piggys respectively; The 4th group of rat do not accepted injection, and the 5th group of rat only accepted pST.The rat body weight amount of having a net increase of was drawn in Fig. 9 in the 5th day.

Separately with pST handle with the mode with statistical significance stimulate the hypox rat growth ( ^*, p＜0.05).Accepting the rat growth that pST adds the antibody of the #1 that derives from after the immunity or #2 pig obviously increases.Though the effect that derives from the antibody of #3 pig does not reach the level of statistical significance (p=0.08), and the growth-promoting effect that can see is arranged.On the contrary, antibody is then invalid before the immunity.

Embodiment 3

With A peptide immunity finishing period piggy

Present embodiment is set forth the effect of growth velocity, feeding efficiency and raw meat being formed with peptide immunity finishing period piggy of the present invention (from 70-80kg and above final growth phase).

Synthetic peptide (Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-Asp-Val-Ile-Arg-Arg-Ile; SEQ ID NO:2) and be incorporated into Protalbinic acid.In order to contrast, with Protalbinic acid self in conjunction with producing Protalbinic acid antigen.

The hog that the mixing of body weight 70-80kg is raised (new DeKalb terminal crisscross inheritance system) is available from GoldKist Pork, Hillsborough, NC.Pig is the target animals of peptide, and each treatment group needs at least 25 piggys to obtain the statistical significance between processing.Animal is discerned separately with ear's mark of numbering, and places in the swinery of numbering.These piggys accept to add the feed of medicine duomycin 200g/ ton, are shipping back about 1 week of raising.All the other times in experiment are accepted Swine ST food (20% crude protein).Feed and water absorb arbitrarily.

With randomized complete block design animal is assigned in one of two kinds of processing.Animal by original body weight grouping, is carried out following processing:

Group antigen n swinery number

A Protalbinic acid/Protalbinic acid 25 5

B peptide/Protalbinic acid 25 5

Piggy is assigned randomly among the treatment group A or B of same district group.15 piggys begin to handle when the about 75kg of mean body weight every district group.Before first immunisation injection, make pig 1 week of pre-treatment in swinery at least.The Flow-rate adjustment of feed becomes near minimal waste.

Neck subcutaneous injection (SC) comes immune piggy with the complete emulsive peptide of Freund's complete adjuvant/Protalbinic acid binding substances 0.5mg after adopting ear.After 4 weeks at interval, with same amount but the binding substances that contains Freund's incomplete adjuvant carries out booster shots to all piggys.The contrast pig accepts not contain the Protalbinic acid binding substances of peptide.

Observe piggy every day.Any disease or injured animal all obtain suitable animal doctor and take care of.Animal that does not recover in a few days or isolation are removed from experiment with animal to be recovered (lame as being enraged by the companion).

Measure body weight and feed intake weekly, if necessary, when piggy is measured when butchering weight more frequently.

When being 115 ± 2kg, average live hog weight puts to death every hurdle pig.The body measurement comprise freezingly weigh, the weight of respective thickness of back fat, rib eye district, pig height degree and heart, liver, spleen, kidney, leaf fat and semitendinosus.

Use analysis to come analytical data for the method for randomized complete block design through changing.The experimental unit that assessment weight increase and body are measured is a pig one by one.Feed intake and feeding efficiency data use swinery as experimental unit.Average comparison with fisher ' s protected LSD.

Experiment is undertaken by following schedule: all piggys were weighed in first day, and each district's group that mean body weight reaches 75kg begins to handle (comprise the B group is carried out first immunisation).

After 4 weeks of first immunisation and 8 weeks, do booster shots.Measure body weight and the feed intake of piggy weekly.When mean body weight 115 ± 2kg, piggy is put to death then.

This result of experiment sees Table 1 and 2.

Table 1 usefulness peptide active immunity finishing period piggy is to the peptide SEM of the effect project contrast immunity of growth, feed intake and feeding efficiency

Number 5,/25 5/25 body weight (kg) of Protalbinic acid/Protalbinic acid swinery number/piggy

Begin 76.2 76.3 0.3

4 weeks 99.2 98.8 0.8

51.2 51.0 1.3 weightening finishes (g/ days) of final 116.2 117.1 1.0 experiment fates

1-4 week 819 804 26.9

5 weeks-final 743 805 27.5

Ingested in 1 week-final 781 802 18.4 (g/ days)

1-4 week 3,390 3,250 59.7

5 weeks-final 3,316 3,326 93.4

1 week-final 3,357 3,285 54.5 increased weight/ingests

1-4 week 0.241 0.247 0.005

5 weeks-final 0.222 0.242 0.009

1 week-final 0.233 0.244 ^*0.004

^*Compare with control group that there were significant differences, p＜0.05

Table 2 contrasts the peptide SEM of immunity to the effect project that body is measured and organ is weighed with peptide active immunity finishing period piggy

Number 5,/25 5/25 body weight (kg) 82.5 82.2 0.8 getting fat percentage 71.0 70.2 0.3 torso length (cm) 82.6 83.0 0.4 organ weights (g) of Protalbinic acid/Protalbinic acid swinery number/piggy

Heart 370 373 8.6

Liver 1,804 1,829 37.8

Kidney 375 384 8.7

Spleen 161 175 6.6

Leaf fat 1,663 1389 ^*60.9

Semitendinosus 394 434 ^{* *}8.2 fat thickness (cm)

First rib 4.9 4.4 ^*0.14

Last rib 2.9 3.0 0.10

Waist 2.6 2.3 0.12

Back fat 3.5 3.2 0.09

The 6th rib 3.6 3.1 ^*0.11

The tenth rib 3.7 3.2 ^{* *}0.11 regulate the 10th rib fat (cm) 3.3 2.8 ^{* *}0.1 either side of the small of the back district (cm ²) 44.2 44.6 0.8 adjusting either side of the small of the back district (cm ²) 41.6 41.9 0.8 calculate the lean meat (33) of gained

kg 38.1 39.7 ^** 0.4

％ 56.1 57.6 ^* 0.5

^*Compare with control group that there were significant differences, p＜0.05

^*Compare with control group that there were significant differences, p＜0.01

^{* *}Compare with control group that there were significant differences, p＜0.001

Embodiment 4

With A peptide immunity growth period piggy

Use (Princeton, NJ) immunity test of the young growth period piggy (about 15-20kg) of drove repetition embodiment 3 from American Cyanamid Company.When growth phase during, give twice booster shots with 4 weekly intervals at about 50-60kg.Then animal rearing is put to death during to the about 115kg of body weight that will grow up to.This result of experiment sees Table 3 and 4.

Table 3 usefulness peptide active immunity growth period piggy is to the peptide SEM of the effect project contrast immunity of growth, feed intake and feeding efficiency

Number 6,/27 6/26 body weight (kg) of Protalbinic acid/Protalbinic acid swinery number/piggy

Begin 18.5 18.8 0.3

4 weeks 39.8 40.7 0.6

8 weeks 56.3 59.1 ^*0.8

12 weeks 72.9 76.3 ^*1.1

16 weeks 97.6 100.7 1.4

Final 113.0 114.1 1.5 experiment fates, 135.0 131.2+ 1.4 weightening finishes (g/ days)

1-4 week 762.0 782.2 13.4

1-8 week 675.4 718.9 ^*11.6

1-12 week 647.4 683.7 12.3

1-16 week 706.8 730.7 11.6

1 week-final 704.0 728.5 11.6

8 weeks-final 725.1 735.2 16.4

Lean tissue (34) 326.6 343.6 ^*5.8 ingest (g/ days)

1-4 week 1,702 1,632 37.2

1-8 week 1,670 1,631 52.1

1-12 week 1,843 1,857 70.5

1-16 week 2,131 2,187 72.0

1 week-final 2,352 2,401 70.6

8 weeks-final 2,872 3,061 89.2 increased weight/ingest

1-4 week 0.448 0.480 ^*0.011

1-8 week 0.404 0.444 ^*0.013

1-12 week 0.351 0.371 0.010

1-16 week 0.331 0.337 0.011

1 week-final 0.298 0.305 0.010

8 weeks-final 0.251 0.242 0.009

Lean tissue (34) 0.137 0.144 0.005

+ compare with control group that there were significant differences, p＜0.10

^*Compare with control group that there were significant differences, p＜0.05

Table 4 contrasts the peptide SEM of immunity to the effect project that body is measured and organ is weighed with peptide active immunity growth period piggy

Number 6,/27 6/26 body weight (kg) 79.8 80.6 1.1 getting fat percentage 71.1 71.0 0.3 torso length (cm) 83.2 83.5 0.5 organ weights (g) of Protalbinic acid/Protalbinic acid swinery number/pig

Heart 400 439 ^*10.9

Liver 1,796 1,841 33.3

Kidney 387 385 8.5

Spleen 150 158 5.0

Leaf fat 1,441 988 ^*54.7

Semitendinosus 425 461 ^*12.1 fat thickness (cm)

First rib 4.3 4.2 0.16

Last rib 2.2 2.2 0.09

Waist 2.2 2.0 0.09

Back fat 2.9 2.8 0.09

The 6th rib 2.1 2.0+ 0.07

The tenth rib 2.7 2.5 ^*0.08 regulate the 10th rib fat (cm) 2.5 2.2 ^*0.7 either side of the small of the back district (cm ²) 38.2 42.1 ^*0.8 regulate either side of the small of the back district (cm ²) 36.4 40.0 ^{* *}0.7 calculate the lean meat (33) of gained

kg 39.9 41.1 ^** 0.3

％ 56.2 58.5 ^*** 0.4

+ compare with control group that there were significant differences, p＜0.10

^*Compare with control group that there were significant differences, p＜0.05

^*Compare with control group that there were significant differences, p＜0.01

Embodiment 5

With A peptide immunity finishing period piggy

With N-terminal second chain peptide of Gelucystine (SEQ ID NO:17) (Cys-peptide) arranged, repeat the immunity test of embodiment 3, this peptide combines as follows with Protalbinic acid: Protalbinic acid (10mg) is dissolved in the aqueous solution (pH8), with iodoacetic acid acid anhydride (16mg; 20 times are excessive) handle, under room temperature, stirred 2 hours.The iodoacetic acid acid anhydride of ultra-filtration and separation surplus at room temperature reacted modified Protalbinic acid and Cys-peptide 4 hours, and dialysis produces final binding substances.This result of experiment sees Table 5 and 6.It is dead in research process to record 2 piggys of accepting bonded Cys-peptide.

Table 5

With the SEM of peptide active immunity finishing period piggy to the peptide immunity of the effect project contrast immunity of growth, feed intake and feeding efficiency

Number 5,/25 5,/25 5/23 body weight (kg) of Protalbinic acid/Protalbinic acid Cys-peptide swinery number/pig

Begin 76.6 76.7 76.7 0.3

4 weeks 100.8 102.2 102.2 0.9

49.8 49.0 48.4 1.2 weightening finishes (g/ days) of final 119.2 119.2 120.1 1.2 experiment fates

1-4 week 866 909 903 30.2

4 weeks-final 845 811 896 28.7

Ingested in 1 week-final 856 870 902 23.8 (g/ days)

1-4 week 3,180 3,333 3,222 118.7

4 weeks-final 3,430 3,440 3,630 116.2

1 week-final 3,285 3,381 3,389 100.8 increased weight/ingests

1-4 week 0.273 0.273 0.281 0.008

4 weeks-final 0.247 0.236 0.247 0.009

1 week-final 0.261 0.257 0.266 0.008

Table 6 contrasts the SEM of the immunity of immunity to the effect project that body is measured and organ is weighed with peptide active immunity finishing period piggy

Number 5,/25 5,/25 5/23 body weight (kg) 86.0 86.4 85.9 0.9dressing percentage 72.1 72.5 71.5 0.3 torso length (cm) 82.6 82.7 83.5 0.5 organ weights (g) of Protalbinic acid/Protalbinic acid peptide Cys-peptide swinery number/piggy

Heart 397 400 415 8.4

Liver 1,838 1,810 1,885 38.9

Kidney 397 388 411 9.3

Spleen 166 167 183 6.9

Leaf fat 1,569 1291 ^*1313 ^*73.1

Semitendinosus 457 492 ^*467 10.0 fat thicknesses (cm)

First rib 4.5 4.3 4.5 0.13

Last rib 2.6 2.6 2.6 0.10

Waist 2.0 2.0 2.3 0.09

Back fat 3.0 3.0 3.1 0.09

The 6th rib 2.4 2.3 2.3 0.09

The tenth rib 3.0 2.9 2.9 0.13 is regulated the 10th rib fat (cm) 2.6 2.5 2.4 0.1 either side of the small of the back district (cm ²) 46.8 46.7 46.8 1.1 adjusting either side of the small of the back district (cm ²) 43.6 43.6 43.4 1.1 calculate the lean meat (33) of gained

kg 40.6 40.7 40.9 0.5

％ 58.2 58.5 58.6 0.7

^*Compare with control group that there were significant differences, p＜0.05

^*Compare with control group that there were significant differences, p＜0.01 Bibliography

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Sequence table (1) general information: (i) applicant: B.L. Bark Wal spy

H-M. uncommon conspicuous

B.S. prosperous (ii) denomination of invention: have the (iii) sequence number of peptide that promotes growth characteristics: 21 (iv) mailing addresses: (A) address: American Cyanamid Company

(B) street: No. 1, cyanamide square

(C) city: Wei En

(D) state: New Jersey

(E) country: the U.S.

(F) ZIP:07470-8426 (v) computer-reader form

(A) recording medium type: floppy disk

(B) computer: IBM PC compatible type

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIN Release #1.0, Version #1.25 (vi) current application materials:

(A) application number: the U.S.

(B) applying date:

(C) classification: (viii) lawyer/proxy's information

(A) name: A.M. Gordon

(B) number of registration: 30,637

(C) reference/number of putting on record: 32,083-00 (ix) telecom information

(A) phone: 201-831-3244

(B) fax: 201-831-3305 (2) SEQ ID NO:2 information (i) sequence characteristic

(A) length: 15 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity

(ii) molecule type: protein

(iii) suppose: not

(iv) antisense: not

(xi) sequence description: SEQ ID NO:1:

Xaa?Xaa?Leu?Xaa?Xaa?Ile?Xaa?Xaa?Xaa?Leu?Xaa?Xaa?Val?Xaa?Xaa

15 10 15 (2) SEQ ID NO:2 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:2:Tyr Ser Leu Asp Asp Ile Ile Arg Arg Leu Asp Asp Val Ile Arg Arg Ile 15 10 15 (2) SEQ ID NO:3 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:3:Tyr Ser Leu Asp Arg Ile Ile Arg Asp Leu Asp Arg Val Ile Arg Asp Ile 15 10 15 (2) SEQ ID NO:4 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:4:Tyr Ser Leu Arg Arg Ile Ile Arg Arg Leu Arg Arg Val Ile Arg Arg Ile 15 10 15 (2) SEQ ID NO:5 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:5:Tyr Ser Leu Asp Asp Ile Ile Asp Asp Leu Asp Asp Val Ile Asp Asp Ile 15 10 15 (2) SEQ ID NO:6 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:6:Tyr Ser Leu Asp Asp Ile Ala Arg Arg Leu Asp Asp Val Ala Arg Arg Leu 15 10 15 (2) SEQ ID NO:7 information (i) sequence characteristics

(A) length: 16 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:7:Tyr Ser Leu Lys Ala Ile Ala Glu Ala Leu Lys Ala Val Ala Glu Ala 15 10 15 (2) SEQ ID NO:8 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:8:Tyr Ser Leu Lys Glu Ile Glu Lys Leu Leu Lys Glu Val Leu Glu Lys Leu 15 10 15 (2) SEQ ID NO:9 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:9:Tyr Ser Leu Asp Asp Ile Ala Arg Arg Leu Asp Asp Val Ala Arg Arg Ala 15 10 15 (2) SEQ ID NO:10 information (i) sequence characteristics

(A) length: 16 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:10:Tyr Ser Leu Lys Ile Ile Ile Glu Ile Leu Lys Ile Val Ile Glu Ile 15 10 15 (2) SEQ ID NO:11 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:11:Tyr Ser Leu Lys Glu Ile Glu Lys Leu Leu Lys Glu Val Leu Glu Lys Leu 15 10 15 (2) SEQ ID NO:12 information (i) sequence characteristics

(A) length: 16 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (ix) feature not:

(A) title/class symbol: decorating site

(B) position: 5

(C) other information :/mark=Aib/note=" Xaa on 5 is Aib " is feature (ix):

(A) title/class symbol: decorating site

(B) position: 7

(C) other information :/mark=Aib/note=" Xaa on 7 is Aib " is feature (ix):

(A) title/class symbol: decorating site

(B) position: 9

(C) other information :/mark=Aib/note=" Xaa on 9 is Aib " is feature (ix):

(A) title/class symbol: decorating site

(B) position: 12

(C) other information :/mark=Aib/note=" Xaa on 12 is Aib " is feature (ix):

(A) title/class symbol: decorating site

(B) position: 14

(C) other information :/mark=Aib/note=" Xaa on 14 is Aib " is feature (ix):

(A) title/class symbol: decorating site

(B) position: 16

(C) other information :/mark=Aib/note=" Xaa on 16 is Aib " is sequence description (xi): SEQ ID NO:12:Tyr Ser Leu Lys Xaa Ile Xaa Glu Xaa Leu Lys Xaa Val Xaa Glu Xaa 15 10 15 (2) SEQ ID NO:13 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(A) title/class symbol: decorating site

(B) position: 3

(C) other information :/mark=Nle/note=" Xaa on 3 is Nle " is sequence description (xi): SEQ ID NO:13:Tyr Ser Xaa Asp Asp Ile Ile Arg Arg Leu Asp Asp Val Ile Arg Arg Ile 15 10 15 (2) SEQ ID NO:14 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(A) title/class symbol: decorating site

(B) position: 10

(C) other information :/mark=Nle/note=" Xaa on 10 is Nle " is sequence description (xi): SEQ ID NO:14:Tyr Ser Leu Asp Asp Ile Ile Arg Arg Xaa Asp Asp Val Ile Arg Arg Ile 15 10 15 (2) SEQ ID NO:15 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(A) title/class symbol: decorating site

(B) position: 13

(C) other information :/mark=Nle/note=" Xaa on 13 is Nle " is sequence description (xi): SEQ ID NO:15:Tyr Ser Leu Asp Asp Ile Ile Arg Arg Leu Asp Asp Xaa Ile Arg Arg Ile 15 10 15 (2) SEQ ID NO:16 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(A) title/class symbol: decorating site

(B) position: 3

(C) other information :/mark=Nle/note=" Xaa on 3 is Nle " is feature (ix):

(A) title/class symbol: decorating site

(B) position: 10

(C) other information :/mark=Nle/note=" Xaa on 10 is Nle " is sequence description (xi): SEQ ID NO:16:Tyr Ser Xaa Asp Asp Ile Ile Arg Arg Xaa Asp Asp Val Ile Arg Arg Ile 15 10 15 (2) SEQ ID NO:17 information (i) sequence characteristics

(A) length: 18 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:17:Cys Tyr Ser Leu Asp Asp Ile Ile Arg Arg Leu Asp Asp Val Ile Arg Arg 15 10 15 Ile (2) SEQ ID NO:18 information (i) sequence characteristic

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:18:Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Ile Xaa Xaa Leu Xaa Xaa Xaa Val 15 10 15 (2) SEQ ID NO:19 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:19:

Xaa?Xaa?Ile?Xaa?Xaa?Leu?Xaa?Xaa?Xaa?Ile?Xaa?Xaa?Leu?Xaa?Xaa?Xaa?Val

15 10 15 (2) SEQ ID NO:20 information (i) sequence characteristics

(A) length: 17 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description not: SEQ ID NO:20:Tyr Ser Ile Asp Asp Leu Ile Arg Arg Ile Asp Asp Leu Ile Arg Arg Val 15 10 15 (2) SEQ ID NO:21 information (i) sequence characteristics

(A) length: 16 amino acid

(B) type: amino acid

(C) number of share of stock: sub-thread

(D) topological framework: linearity is molecule type (ii): protein is (iii) supposed: (iv) antisense not: (xi) sequence description: SEQ ID NO:21:Tyr Ser Ile Asp Asp Ile Ile Arg Arg Ile Asp Asp Ile Arg Arg Ile 15 10 15 not

Claims

1. peptide comprises aminoacid sequence Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa (SEQ ID NO:1), it is characterized in that described sequence is different from the native sequences of Porcine somatotropin (pST).

2. peptide as claimed in claim 1 is characterized in that, is Serine near first leucic aminoterminal amino acid.

3. peptide as claimed in claim 2 is characterized in that, described peptide is selected from the group of being made up of the peptide of the sequence with SEQ ID NO:2-12 basically.

4. peptide as claimed in claim 1 is characterized in that, at the terminal or two terminal halfcystines that add of peptide.

5. peptide as claimed in claim 4 is characterized in that, described peptide has the sequence that SEQ ID NO:17 describes.

6. peptide,, comprise aminoacid sequence Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:18), it is characterized in that described sequence is different from the native sequences of pST.

7. peptide as claimed in claim 6, it is characterized in that, the 3rd residue is Isoleucine, so that described peptide comprises aminoacid sequence Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:19), described sequence is different from the native sequences of pST.

8. peptide as claimed in claim 7 is characterized in that, is Serine near the aminoterminal amino acid of first Isoleucine.

9. promote the composition of warm-blooded animal growth, it comprises at least one and pharmaceutically acceptable adjuvant, diluent or carrier in claim 1,4,5,6,7 or 8 the peptide.

10. promote the method for warm-blooded animal growth, comprise the composition of warm-blooded animal being used claim 9.