CN1296384C - Peptide having preptin functionality - Google Patents

Abstract

The invention relates to a bioactive mammalian peptide. In particular, it relates to a peptide secreted by the pancreatic islet beta -cell that stimulates insulin secretion, termed preptin. Preptin analogs, pharmaceutical compositions which contain preptin or its analogs and their use as medicaments are also provided.

Description

Peptide with preptin function

The present invention relates to a kind of biologically active peptides.Especially, the present invention relates to a kind of peptide that stimulates insulin secretion by pancreas pancreatic excretory.

Background of invention

The pancreas pancreatic mainly is by its secretion of insulin, is playing the part of an important adjusting role in physiology, and Regular Insulin is a kind of peptide hormone (people such as Draznin, (1994)) that middle metabolism is applied deep effect.Second kind of beta cell hormone, i.e. dextrin to insulin secretion and the effect of organizing insulin sensitivity, also helps regulatory function (Cooper, the C (1994) of beta cell by it; People such as Hettiarachchi, (1997)).

In pancreatic, hormone is packaged in the secretion property particle, and by the response to signal such as fuel (for example, glucose, amino acids) or neurohormone sexual stimulus, these hormones have experienced modulability release.These particles contain the compact nucleus that is rich in Regular Insulin and Zn, and have more a spot of insulin C-peptide, dextrin, proinsulin, chromogranin deutero-peptide class, protease and other protein-based (Hutton, J (1989)) in particle matrix.

The present patent application people is current fixedly to be: the pancreas pancreatic is also secreted a kind of modulability peptide.The present patent application people determines that further this peptide strengthens the insulin secretion of glucose mediation.

At this peptide, the present patent application people is called preptin, and the present invention relates to its many aspects widely.

Summary of the invention

Therefore, the invention provides preptin peptide or its analogue in first aspect.

For " preptin ", the present patent application people is meant a kind of 34 amino acid whose peptides, and its sequence is as follows:

Asp?Val?Ser?Thr?R ₁?R ₂?R ₃?Val?Leu?Pro?Asp?R ₄?Phe?Pro?Arg?Tyr?Pro?Val?Gly?Lys

Phe?Phe?R ₅?R ₆?Asp?Thr?Trp?R ₇?Gln?Ser?R ₈?R ₉?Arg?Leu

Wherein:

R ₁Be Ser or Pro;

R ₂Be Gln or Pro;

R ₃Be Ala or Thr;

R ₄Be Asp or Asn;

R ₅Be Gln or Lys;

R ₆Be Tyr or Phe;

R ₇Be Arg or Lys;

R ₈Be Ala or Thr; With

R ₉Be Gly or Gln,

Perhaps its analogue.

In one embodiment, the invention provides people's preptin, its aminoacid sequence is:

Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Set?Thr?Gln?Arg?Leu.

In another embodiment, the invention provides the preptin of rat, its aminoacid sequence is:

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Lys?Phe?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly?Arg?Leu.

In another embodiment, the invention provides the preptin of mouse, its aminoacid sequence is:

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly?Arg?Leu.

These aminoacid sequences are equivalent to the Asp of the proIGF-II E-peptide in every kind of Mammals ₆₉-Leu ₁₀₂

On the other hand, the invention provides the polynucleotide of coding preptin or its analogue.

On the other hand, the invention provides polynucleotide that include encode preptin or its analogue and carrier or clone with expression preptin or described analogue ability.

The present invention also provides the salt of preptin, and it is preferably the acceptable preptin salt of physiology.

On the other hand, the present invention further provides the pharmaceutical composition that comprises preptin or its analogue or preptin salt.

More on the one hand, the invention provides with treatment or prevention is the method that stimulates insulin secretion of purpose, and it comprises that the patient to this treatment of needs or prevention uses preptin or this step of its analogue of significant quantity.

Aspect another, the invention provides preptin or its analogue or its salt and preparing medicine in particular for the purposes in the medicine that stimulates insulin secretion.

On the other hand, the invention provides the method for the insulin secretion of modulation glucose mediation, it comprises preptin, preptin analogue, preptin agonist or this step of preptin antagonist of the patient being used significant quantity.

In other embodiments, the invention provides antibody in conjunction with preptin or its analogue, used the measuring method of these antibody and contained the mensuration test kit of these antibody.

Top general introduction does not comprise full content of the present invention.From following description and from appending claims, with apparent others of the present invention.

Description of drawings

Describe although the present invention has been carried out generality as above, also should be understood that: the present invention includes embodiment, and below these embodiments, provided embodiment.In addition, by reference accompanying drawing wherein, will be better understood the present invention.

Fig. 1 is purifying and the phenogram of preptin.A) mensuration to marker protein matter shows, is located in the continuous gradient of OptiPrep from the organoid of β TC6-F7 cell: granular core (Regular Insulin), particle matrix (dextrin), lysosome (arylsulfatase), plastosome (Oxalacetic transacetase).B) RP-HPLC purifying granule protein matter: collect indicated peak (adding hachure) and be further purified.C) employing MALDI-TOF MS determines purity and the quality (M+H from the main peptide that adds the hachure peak ⁺).D) from adding the peptide of hachure peak purifying, digest after the collection of illustrative plates of RP-HPLC purifying with Lys-C: 1 is NH ₂-terminal fragment, 2 is the COOH-terminal fragment, 3 is indigested peptide.E) check order by Lys-C deutero-peptide in (d), and the structure of the mouse preptin that determines: NH ₂-terminal fragment is a roman, and the COOH-terminal fragment is oblique black matrix, and preptin is located in the fragment of shown mouse proIGF-II E peptide.Structural domain (B, C, A, D, E) to proIGF-II among the figure is all shown.Be positioned at Arg ₆₈The point of contact of identification represent that with black matrix and the two alkaline motif (motif) of inferring shows with septal line.

Fig. 2 is the emiocytosis figure of preptin.A) the RIA typical curve of preptin.B) 24 hours β TC6-F7 conditioned medium and from the granule interior branch of Fig. 1 b, in the RP-HPLC fraction collection part of these two, the RIA of preptin sample immunoreactivity material (PLIM) characterizes.C) from the MALDI-TOF MS of the classification part that contains main PLIM of β TC6-F7 emiocytosis, the peak is corresponding to mouse preptin (M+H ⁺), specific inaccuracy is 0.07%.

Fig. 3 is the effect figure of preptin to insulin secretion.A) preptin mediation from β TC6-F7 emiocytosis Regular Insulin.Curve shows that the concentration of insulin increase has surpassed basal level (when not adding preptin).B) preptin mediation from the isolating pancreas in rat excreting insulin that is poured.Each point is mean value ± sem (compound analysis is for an every curve n=4 pancreas).Area under curve (the second phase secretion of Regular Insulin; P=0.03, unpaired two afterbody t-tests).

Fig. 4 is the figure of immuning tissue of mouse pancreas.The pancreas of collecting from the FVB/n mouse that grows up is cut into slices,, and used immunoperoxidase link coupled goat antirabbit two anti-with phenodin and the dyeing of multi-clone rabbit antiserum(antisera).In the component: a. synalbumin antiserum(antisera) (1: 40); B. anti-preptin antiserum(antisera) (1: 40); C. with d. with synthetic rat preptin c.1mg/ml, d.5mg/ml, 30 minutes anti-preptin antiserum(antisera) (1: 40) of preincubation under the Bar=100 μ m.

Fig. 5 is in the RP-HPLC fraction collection part of rat Langerhans islet or β TC6-F7 particulate fraction, to the RIA sign (standard: Fig. 1 b) of preptin sample immunoreactivity material (PLIM).

Fig. 6 represents to be total to excretory preptin and Regular Insulin from β TC6-F7 cell and isolating rat Langerhans islet.A and b are that being divided into from the preptin of a. β TC6-F7 cell and the isolating rat Langerhans islet of b. and Regular Insulin of glucose mediation secreted.

Fig. 7 is the effect figure of preptin to insulin secretion.A and b represent the gamma globulin of the mouse preptin of a. rabbit Chinese People's Anti-Japanese Military and Political College of purifying and the purity and the quality of the non-immune rabbit gamma globulin of b..1: light chain IgG, M+H ⁺2: complete IgG, M+4H ⁺3: heavy chain IgG, M+H ⁺4: complete IgG, M+2H ⁺Complete IgG, M+H ⁺C be with the gamma globulin of anti-preptin after duration of contact, extent of dilution, temperature and the pH of analog antibody perfusion experiment under the 35 μ g/ml, 37 ℃, the condition of pH7.4 pour into, in 1 minute in conjunction with the ability of preptin.D is the gamma globulin or the contrast (non-immune rabbit gamma globulin) of injecting anti-preptin, glucose is stimulated the isolating effect figure that is poured the insulin secretion of pancreas in rat of (20mM, rectangular wave).Each point is mean value ± s.e.m. (compound analysis is for an every curve n=5 pancreas).Area under curve (the second phase secretion of Regular Insulin; P=0.03, unpaired single afterbody t-test).

Invention is described

Describe as top generality, the present invention relates to a kind of new peptide, and find that it is present in the pancreas pancreatic particle.Determined this peptide, i.e. preptin, the insulin secretion that can stimulate glucose to cause.

In a word, by use a step density gradient centrifugation purifying from the secretory granules of the mouse β TC6-F7 cell of cultivating and to marker protein matter analyze with determine its purity (Fig. 1 a), thus identified preptin.Regular Insulin is used to follow the tracks of the purifying of granular core, can confirm the integrity of membrana granulosa (Fig. 1 a) and be present in dextrin in the particle matrix (Johnson, K (1988)) by mensuration.Then, use reversed-phase HPLC (A ₂₁₄: Fig. 1 b), the grain fraction of solubility is separated.By mass spectrum and NH ₂The order-checking of-end amino acid is determined the identity of peptide.Main peaks contain mouse Regular Insulin-I and-II and C-peptide-I and-(Fig. 1 a) for II.Do not detect any non-beta cell peptide, and dextrin to the plain I of Regular Insulin (1: 23) and mouse islets to the molar ratio of the plain II of mouse islets (1: 3) and the identical (Cooper (1994) of ratio of physiological beta cell; Linde (1989)).

In the main elution peak before next-door neighbour Regular Insulin-I, find to contain a kind of unknown in the past peptide (Fig. 1 b).To homogeneity, its molecular weight is 3950Da (Fig. 1 c) with this peptide purification.To this molecule digestion, then the peptide that obtains is separated (Fig. 1 d) with RP-HPLC with the narrow spectrum proteolytic enzyme of Methionin, carry out complete NH then ₂The order-checking of-terminal protein matter.Complete sequence confirms: this molecule contains 34 amino acid, and it is equivalent to the Asp of mouse proIGF-II E-peptide ₆₉-Leu ₁₀₂(Fig. 1 c).This peptide is exactly the preptin of mouse.

NH at preptin ₂-terminal flank is an Arg point of contact of having discerned, is two alkalescence (Arg-Arg) cutting motif of inferring people such as (, (1985)) Bell (Fig. 1 e) at the terminal flank of its COOH-.These residues high conservative between different plant species, and be likely processing signal after the translation.

Though other investigators show: in cell culture fluid and multiple mammiferous biological fluid, have different proIGF-II E-peptide derived peptide to have (people such as Hylka, (1985); People such as Daughaday, (1992); People such as Liu, (1993)), but they do not identify a kind of peptide suitable with preptin.

The aminoacid sequence of mouse preptin is as follows:

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly?Arg?Leu

The aminoacid sequence that is equal to of the preptin of people and rat is respectively:

Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?ProVal?Gly

Lys Phe Phe Gln Tyr Asp Thr Trp Lys Gln Ser Thr Gln Arg Leu; With

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Lys?Phe?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly?Arg?Leu.

The nucleotide sequence of the polynucleotide of coding preptin is as follows:

gacgtgtcgacccctccgaccgtgcttccggacaacttccccagataccccgtgggcaagttcttccaatatga

Cacctggaagcagtccacccagcgcctg (people)

gacgtgtctacctctcaggccgtacttccggacgacttccccagataccccgtgggcaagttcttcaaattcgac

Acctggagacagtccgcgggacgcctg (rat)

gacgtgtctacctctcaggccgtacttccggacgacttccccagataccccgtgggcaagttcttccaatatgac

Acctggagacagtccgcgggacgcctg (mouse)

Preptin can generate by synthetic or recombination method.For example, when preparing with synthetic method, can use any can the commercial solid phase technique that obtains such as the Merryfield solid phase synthesis process, amino acid is sequentially joined on the amino acid chain that is extending in this method, thereby synthetic preptin (seeing Merryfield, J.Am.Soc.85:2146-2149 (1963)).Automatically the equipment of synthetic peptide also can (Applied Biosystems Inc) locates commercial the acquisition, and can operate according to the specification sheets of manufacturers from supplier such as Perkin Elmer/ Applied Biosystems, Inc..

Preptin also can be inserted in the expression vector by polynucleotide (being generally DNA) sequence with coded protein and in suitable host with its expression, thereby generate with recombination method.Any in the known multiple expression vector of those of ordinary skills can be brought use.After expression vector conversion or any proper host cell of transfection, just can in this cell, realize expressing with the dna molecular that contains the recombinant peptide of encoding.Proper host cell comprises prokaryotic cell prokaryocyte, yeast and higher eucaryotic cells.

Be used to produce the standard technique of reorganization, molecular cloning people such as for example Maniatis: laboratory manual (Molecular Cloning-A Laboratory Mannual, Cold Spring HarbourLaboratories, Cold Spring Harbour, New York (1989)) given description in.

Express carrier and/or the clone of preptin, each have their own effect, and they also become a part of the present invention.

The analogue of preptin and its coded polynucleotide are also within the scope of the invention.Such analogue comprises the functionally equivalent of preptin and above-mentioned polynucleotide.

For preptin itself, its functionally equivalent is included in and can cross reaction takes place with preptin on the immunology and have all proteins with the basic identical function of preptin.Such functionally equivalent can be, for example, contain 6 to 33 amino acid (normally being occurred by the form of brachymemma) with the C-end and comprise preptin one or more avtive spots the fragment of preptin, the substituting of preptin, interpolation or deletion mutant perhaps have the syzygy of other amino acid whose preptin or fragment or mutant.

Form six amino acid of minimal segment, need only them and be successive in the preptin sequence and satisfy functional requirement, just can be from any part of sequence.Certainly, biologically active peptides also may (and conceive clearly) comprises any in those six peptides, perhaps is really or comprises any seven peptides, octapeptide, nonapeptide or decapeptide from the preptin sequence.

Especially preferred such peptide class, they are or comprise six peptides, seven peptides, octapeptide, nonapeptide or decapeptide from people preptin.

The change of residue is possible in the peptide, and this is conceived.For example, the amino acid that adopts routine techniques to replace in the sequence with the amino acid that is equal to is possible.Usually the amino acid group that is equal to known to is:

(a)Ala?Ser?Thr?Pro?Gly；

(b)Asn?Asp?Glu?Gln；

(c)His?Arg?Lys；

(d) Met Glu Ile Val; With

(e)Phe?Tyr?Trp.

As long as resulting peptide can immunological cross-reaction take place with preptin and have the essentially identical function with preptin, also can carry out amino acid whose interpolation and/or disappearance.

The polynucleotide that are equal to comprise the nucleotide sequence of the protein that coding and aforesaid preptin are equal to.The polynucleotide that are equal to also comprise such nucleotide sequence, because the degeneracy of nucleic acid password, they and natural polynucleotide have difference, but these differences do not influence amino acid sequence corresponding.

Can predict whether a certain specific polynucleotide or polypeptide be suitable with top given those polynucleotide or polypeptide according to homology.By utilizing the computerized algorithm that can openly obtain, the sequence of polynucleotide or polypeptide can be compared (align), thereby determine the Nucleotide identity per-cent of a certain specific region with respect to another sequence.Be used to compare and identify that two exemplary algorithm of polynucleotide sequence similarity are BLASTN and fasta algorithm.The similarity of peptide sequence can use the BLASTP algorithm to check.BLASTN and BLASTP software all can the ftp server of NCBI anonymity (ftp: //ncbi.nlm.nih.gov.) on, under/blast/executables/, obtain.When determining according to varient of the present invention, the preferred BLASTN algorithm that uses the 2.0.4 version " on February 24th, 1998 " that is provided with and provides with algorithm by the default parameter described in the file.Comprise the purposes of the BLAST algorithm family of BLASTN and BLASTP, be positioned at URL's Http:// www.ncbi. Nlm.nih.gov/BLAST/newblast.htmlThe web website (website) of NCBI description is arranged, and in people (1997) such as Altschul and Stephen F " BLAST that has vacant position and PSI-BLAST: the Protein Data Bank search utility (Gapped BLAST andPSI-BLAST:a new generation of protein database search programs) of a new generation ", nucleic acids research (Nucleic Acids Res.) these publications of 25:3389-3402, description is arranged also.Computerized algorithm FASTA can be at the ftp website ftp on internet (Internet): //the last acquisition of ftp.virginia.edu.pub/fasta/.When determining according to varient of the present invention, the preferred use 2.0.4 version that is provided with and provides with algorithm by the default parameter described in the file, in February, 1996.The purposes of fasta algorithm, at W R Pearson and D.J.Lipman, " the improvement instrument (Improved Tools for BiologicalAnalysts) that biological sequence analysis person uses ", institute of NAS periodical (Proc.Natl.Acad.Sci.USA), 85:2444-2448 (1988) and W.R.Pearson, " carry out fast and sensitive sequence comparison (Rapid and Sensitive Sequence Comparison withFASTP and FASTA) " with FASTP and FASTA, Enzymology method (Methods in Enzymology), 183:63-98 has given description in (1990).

Analogue of the present invention also comprises the preptin homologue from the species except that people, rat or mouse.Such homologue is by using, and for example, based on the nucleic acid probe of the conserved regions of the preptin polynucleotide of coding people, rat and mouse, can be identified at an easy rate.

Preptin or its analogue also can exist with the purity form of multiple degree.Preferably preptin/ analogue component constitutes at least 50% of prepared product weight, and more preferably at least 80%, more preferably at least 90%, more preferably at least 95% and still more preferably at least 99%.Yet preptin or analogue are in being applied to medicine the time, and general preferred its is in pure or pure substantially form.

When the patient being used preptin or preptin analogue, might use its so pure or pure substantially compound.Yet preptin or preptin analogue also can exist with the form of pharmaceutical composition.Such composition can comprise preptin or preptin analogue, and adds one or more pharmaceutically acceptable carriers and if desired again, randomly other therapeutic ingredient.

The acceptability of carrier is meant that it must be compatible with preptin or preptin analogue, and harmless to subject patient.The desired combined thing also should not include and the inconsistent material of known peptide class.

Can with composition can also unitary dose form exist easily, and can adopt the known any method preparation of pharmaceutical field.All methods include and make activeconstituents this step that combines with carrier, and carrier wherein is made of one or more ancillary components.

The specific form that composition adopted will depend on selected route of administration to a great extent.For example, preptin or preptin analogue can be injected with parenteral route, for example, enter in the blood flow of being treated the patient by intravenous injection.Yet; those skilled in the art welcomes the route of administration variation; they can be intravenously, subcutaneous, intramuscular, intraperitoneal, enteron aisle, through skin, stride mucous membrane, continue the release polymers composition (as; lactide polymer or copolymer particle or implant), perfusion, lung (for example, sucking), nose, mouthful or the like.

At present, preferably be suitable for the composition that parenteral route, especially intravenously are used.Such composition contains the aseptic aqueous solution of preptin or preptin analogue easily.Preferably, solution oozes with the blood etc. of being treated the patient.Preptin or analogue is soluble in water to generate aqueous solution, and then this solution of asepticize just can be prepared such composition easily.At this moment just can be with the packing material form of unit or multiple doses, for example Mi Feng ampoule or bottle transmit composition.

A kind of especially preferred composition is that preptin is in the physiological buffer that is suitable for injecting.

Being suitable for the lasting release property composition (for example, the formulation of biodegradable polymers) that non-enteron aisle is used, also is known in this field.See, for example, United States Patent (USP) 3773919 and 4767628 and PCT publication WO 94/15587.

The form that preptin is converted into salt is also very convenient.Such salt generally is physiologically acceptable salt, and can generate by utilizing any method of technological standard easily.

Especially preferably combine the preptin salt that generates with the negatively charged ion of organic acid by preptin.Such salt includes but not limited to the salt of oxysuccinic acid, acetate, propionic acid, butyric acid, oxalic acid, citric acid, isocitric acid, α-Tong Wuersuan, succsinic acid, fumaric acid and trifluoroacetic acid.

Also the salt that forms like this can be formulated in the pharmaceutical composition, being used for the treatment of property is used in the time of needs.

Below, by with reference to following non-limitative experiment part, some aspects of the present invention are described.

Experiment

First part

Method and material

Cell cultures

With β TC6-F7 mouse pancreas pancreatic, passage number 49-60, in three bottles in 37 ℃ of cultivations, wherein, O ₂To CO ₂Ratio be 95: 5 (volume/volume), the nDMEM (Gibco) that replenishes with 15% heat-killed horse serum and 2.5% foetal calf serum is housed in the bottle, and, be commissioned to train foster again and again in per 5 days by adopting PBS to wash, follow by trypsin 2.5% trypsinase-EDTA) processing.Growing to 70% when converging, each bottle can obtain about 2.0 * 10 ⁸Individual cell.

The particle purifying

Will from 8-12 three bottles, to be in passage number be that the beta cell of 55-60 is gathered in the crops after with trypsin treatment, on average obtains the pure cell of 2.5-4.0ml (1.6-2.4 * 10 ⁹Individual), it is concentrated (1700 * g, 5 minutes), wash twice with PBS then, then with the medium that homogenizes (0.3M sucrose/10mM MES K (σ)/1mM K ₂EGTA/1mM MgSO ₄/ pH6.5) wash once, processing then homogenizes in same media (by 1: 5 volume/volume) on ice.Cell suspending liquid is passed through ball milling (ball-bearing) homogenizer (7.87 * 10 ^-5The cm gap) processing that homogenizes for 20 times is clarified by centrifugal (400 * g, 10 minutes) then, throw out is once homogenized again handle and centrifugal, then with twice supernatant liquor merging (final volume=20ml).By preparing 13% and 31% Optiprep with the medium that homogenizes ^TM(Nycomed) solution (volume/volume), (Auto Densi-flow II is Haakebuchler) in the ultra-clean centrifuge tube (Beckmam) with continuous gradient (31%-13%Optiprep) impouring of 6 * 10ml then.Throw out is tiled in upper strata or bottom, carries out super centrifugal (SW40 T1/160000 * g/16h/4 ℃) then.With RI is the classification part of 1.363-1.368, wherein contains the secretory granules of highest purity, collects, and plastosome and lysosome then are split into RI greater than in 1.371 the classification part.By using radioimmunoassay Regular Insulin (crystalline granular core), dextrin (particle matrix), detect prepared particulate integrity; By the functional examination of arylsulfatase (lysosome) and Oxalacetic transacetase (plastosome), detect purity; And adopt Bicinchoninic acid (Pierce) to measure total protein content.

RP-HPLC

Order is carried out RP-HPLC (A:0.08%TFA volume/volume twice; B:80% acetonitrile+20%A; Applied Biosystems 140B/785A/112A system: Jupiter C18 RP post, 250 * 2.0 millimeters (Phenomonex): 250-300 mul/min; A ₂₁₄), purifying granule protein matter.Before upper prop, at first will secretion property particulate matter centrifugal (16000 * g, 20 minutes).Adopted isocratic elution, and begin order collection, per 30 seconds fraction collection parts in the time of back 19 minutes in 15 minutes of beginning in injection.For protein purification, different slightly gradients has used in order: what RP-HPLC obtained for the first time is semipurified granule protein matter, and RP-HPLC has used milder gradient for the second time, to improve resolving power and purity.

Peptide sequence analysis

Adopt the N-end sequence to measure (the Edman method of automatization; ABI Procise ^TM) and in conjunction with the accurate quality measurement of MALDI-TOF MS, the peptide of purifying is identified.In order to verify complete sequence, use Lys-C (Boehringer Mannheim) that the mouse preptin isolate of purifying is cut, then with the RP-HPLC repurity of resulting peptide fragment.

MALDI-TOF MS mass spectrum

Employing is equipped with 500 megahertz digital oscilloscope (G2030AA, LeCroy) MALDI-TOF MS (G2025A:337 of Hewlett-Packard (Hewlett-Packard) nanometer nitrogen Laser emission light/maximum output 150 little joule/pulse widths 9 nanosecond/90 kilovolt ion-accelerating voltage) adopts to have added recombinant human insulin (Novo Nordisk; M+H ⁺, 5808.66Da; M+2H ⁺, 2904.83) and Somatostatin (Bachem, M+H ⁺, 1638.91; M+Na ⁺, 1660.90) a kind of α-CHC matrix as molecular weight standard, to determine the molecular weight of peptide.Under condition of high vacuum degree (less than 1.0 microtorrs), carry out MS, and adopt outer quality standardization to carry out data gathering (ChemStation:0-20PS method, positive polarity (positivepolarity) is in the 0-20kDa scope) in " single emission " mode.By adopting outer quality stdn interpolation technique, the accurate molecular weight of the peptide of conclusive evidence purifying.

The chemosynthesis of rat preptin

By comparing, the sequence of rat and people's preptin is determined with the known IGF-II of inferring sequence.Presumptive sequence, adopt the Fmoc chemistry, go up, use the FmocLeu-Wang resin initial at peptide synthesizer device (Advanced Chem Tech 396 Robotics Peptide Synthesiser), the preptin of chemosynthesis rat (Auspep Pty, Australia (Australia)).With the peptide deprotection and adopt 92.5%TFA: 2.5% water: 2.5% tri isopropyl silane: the solution of 2.5% dithiothreitol (DTT), downcut peptide thereby handle in 3 hours from resin.By adding diisopropyl ether peptide is precipitated out from TFA solution, then with resolution of precipitate in 30% acetonitrile: in the water, freeze-drying is also used the RP-HPLC purifying.Measure when 47%B (rat preptin eluted) with analysis mode RP-HPLC and confirm purity for greater than 99%, meanwhile, the quality of MALDI-TOF MS conclusive evidence is 3932.4Da ± 0.026%.

The radioimmunoassay of preptin

Adopt the glutaraldehyde single stage method in pH7.0,, then in the NZW rabbit, produce polyclonal antiserum with it with synthetic rat preptin and the coupling of carrier ovalbumin.Adopt chloramine-t method that preptin is carried out the 125I-radio-labeling, use then Sephadex G-10 chromatography (the 50mM phosphoric acid buffer, pH7.5) purifying [ ¹²⁵I] preptin (362 microcuries/microgram).Then, developed the optimization RIA that is used for preptin, wherein by adopting the auxiliary second antibody of PEG B/F to be separated (goat-anti--rabbit method).It is 1: 10000 antiserum(antisera) (surveying periodic whole extent of dilution is 1: 30000) that this method has adopted whole extent of dilution, and the R/T value is 0: 30; The reading of tracer is every pipe 8000 counting/minute (cpm); The incubation time is 24 hours+72 hours; EC ₂₀Value is 344pM preptin; EC ₅₀Be 39pM; Minimal detectable concentration is 11.2 ± 9.8pM; With rodents (rat/mouse) Regular Insulin and dextrin no cross reaction.

The emiocytosis of preptin

At β TC6-F7 cell (passage number ^#52) secretion to preptin in is studied, and β TC6-F7 cell is with 4 * 10 in 24 orifice plates ⁵Individual cells/well is cultivated, and other culture condition is the same.The stimulation of preptin is reached 80% in growth after 3 days implement when converging.Before secreting the research beginning, cell is washed twice in HEPES buffered KRB, follow incubation buffering liquid (0mM glucose in 1 milliliter/hole; 0.1% is dissolved in Fraction V BSA (Sigma) w/w among the HEPES-KRB) in preincubation 1 hour, from every hole, shift out 500 μ l afterwards, and replenish with isopyknic fresh incubation buffering liquid that contains multiple concentration glucose., after 2 hours incubation medium is shifted out at incubation (37 ℃), wash cell three times, use the lysis buffer cracking then with PBS.Then, adopt the RIA that has described, incubation supernatant liquor and cell lysate are carried out Regular Insulin and preptin assay.In the experiment that separates, the time-dependent manner of hormone secretion is also measured.

The sign of excretory preptin sample immunoreactivity material (PLIM)

Since preptin is a kind of cleaved products of the E-peptide of IGF-II, and from serum, isolated other cleaved products (Hylka (1985) that is derived from a smaller area territory in the past; Daughaday (1992); Liu (1993)), so adopt preptin RIA quantitatively can not characterize this fully to be secreted and the characteristic of circulation peptide.Therefore, developed the method that RP-HPLC/preptin RIA combines, with further sign PLIM.With separated plasma aliquot and the 2ml β TC6-F7 conditioned medium of 2ml from people's donor, use the acidifying of 0.1ml 4M acetate respectively, be loaded into then on the C-18Sep Pak (Waters, 1ml volume) that has used 10ml 100% methyl alcohol and 20ml 4% (volume/volume) acetate pre-equilibration.Earlier wash Sep Pak, use the 0.1M acetate elution of bound component of 2ml in 70% methyl alcohol then, by rotary evaporation the final volume of elutriant is decreased to 150 μ l then with 20ml 4% acetate.Then, elutriant by the top described RP-HPLC that carries out, is merged repeatedly the corresponding fraction collection part of chromatography operation.The fraction collection that contains preptin and Regular Insulin is probably partly carried out MALDI-TOF MS.Then, measure damping fluid with preptin the volume of all fraction collection parts is mended to 350 μ l, adopt RIA that preptin and Regular Insulin are analyzed then.Collection of illustrative plates (Fig. 1 b) for material in the immunoreactivity collection of illustrative plates of these secretory products relatively and the particle, 10 μ l partly are diluted to final volume 610 μ l with preptin RIA damping fluid from initial RP-HPLC particle fraction collection, also carry out Regular Insulin and preptin and measure.

The speed of sugar metabolism in isolating rat skeletal muscle

Draw together in the skeletal muscle utilization of beta cell hormone dextrin and Regular Insulin modulation carbohydrate in the periphery tissue pocket.Soleus muscle is separated, incubation and peel off after, used as the model tissue, preptin is changed glucose absorption and the ability that is incorporated in the muscle glycogen is studied.All animal methods of being implemented have all obtained the appropriate permission of animal ethics committee tissue (InstitutionalAnimal Ethics Committee).Male Wistar rat (200 ± 20g) is raised in the controlled room of condition in (20 ℃, 12 little time/dark circulation), and given rat food (Diet 86.NRM Tegel, Auckland) and the arbitrarily drinking-water of standard.With 18 hours rat anesthesia of fasting (vetanarcol 45mg/kg), and adopt cervical dislocation that it is put to death, giving birth to carbon oxygen-KHB (O subsequently ₂With CO ₂Ratio be 95: 5 volume/volume) dissect down soleus muscle, then with its incubation in the nDMEM of Regular Insulin that has replenished multiple concentration and preptin.Muscle longitudinally chosen to open be 3 and wait bars that every final radius is about 1.5mm.Will be [(U) ¹⁴C] (1 millicurie/milliliter Amersham) is diluted in 70% ethanol by 1: 20 (volume/volume) D (+)-glucose, and the gained final concentration is 0.5 microcurie/10 microlitres.With Actrapid ^The insulin human (100 units per ml, Novo Nordisk) of reorganization was diluted among 10 milliliters of nDMEM by 1: 1000.60 μ g rat preptin are dissolved among the 1526 μ l nDMEM, to concentration be 10 μ M, then in nDMEM further the dilution, to obtain the storage liquid of 1 μ M, 100nM, 10nM, 100pM and 1pM.Adopting two different experiment examples, is that (i) stimulation glucose mixes the speed in the glycogen to determine preptin, and still the glucose that (ii) causes as Regular Insulin mixes the antagonist in the glycogen.

The incubation method of preptin antagonist

Four strip of muscle are gone in the bottle, and corotation is gone into 9 bottles, every bottle of preptin (10fM, 100fM that contains living carbon oxygen (carboxygenated) nDMEM, 0 (contrast) or the most effective Regular Insulin (23.7nM) and multiple concentration, 1pM, 0,10pM, 100pM, 1nM or 10nM), be total to 10ml.Then, bottle is being shaken balance in the water-bath (30 ℃, 20 minutes), after this,, adding 10 μ l (0.5 microcurie) D-[(U) by accurate 1.5 minutes intervals ¹⁴C] glucose.Then, under carbogen (carbogen), with strip of muscle 30 ℃ of incubations 120 minutes.Behind the incubation,, strip of muscle is shifted out suck dry moisture from each bottle by 1.5 minutes intervals.With the IQF in liquid nitrogen of these strip of muscle, in preweighted pipe, carry out freeze-drying in 24 hours subsequently, then determine the dry weight of strip of muscle.Then, strip of muscle is dissolved in 250 μ l 60%KOH, 70 ℃ of incubations 45 minutes, spent the night-20 ℃ of precipitations with ice-cold ethanol in the cooling back.Then, by centrifugal (9000 * g, 15 minutes, 0 ℃) preparation glycogen throw out, then with the throw out resuspending, redeposition twice, last sucking-off supernatant liquor is also placed the glycogen throw out and to be carried out finish-drying in 2 hours at 70 ℃.At this moment, can determine the incorporation of 14C by scintillation counting.

Preptin agonist method

Except when strip of muscle is carried out incubation, wherein not the final concentration of insulin-containing (but except the positive control, wherein containing 23.7nM Regular Insulin) and preptin be 0,0.1,1,10 and 100nM outside, all the other all methods are by as above describing.

Preptin is to the effect of insulin secretion

Known Regular Insulin and dextrin autocrine mechanism by inferring, the insulin secretion of modulation beta cell.Therefore, adopt beta cell secretogogue method, preptin is measured the effect of insulin secretion.With β TC6-F7 cell at passage number ^#52 o'clock, in inferior generation, be incubated in 24 orifice plates, and 4 * 10 ⁵Individual cells/well.They were cultivated in nDMEM 3 days, grow to 80% and converge, then wash twice with KRB-HEPES.In the incubation nutrient solution that contains 10mMD (+)-glucose, the preptin final concentration of gained solution is respectively 150,75 with preptin storage liquid serial dilution, and 25,5,1 and 0.1nM.Wash cell then, and will contain the incubation nutrient solution of the preptin of 10mM and multiple final concentration, join in every hole by 1 milliliter/hole.Cell 37 ℃ of incubations 2 hours, is shifted out nutrient solution then.Wash cell three times with PBS, carry out cracking with 500 μ l lysis buffers then.With incubation medium centrifugal (16000 * g, 3 minutes), and supernatant liquor and sedimentary fragment separated.Then, to incubation nutrient solution and lysate by top described Regular Insulin, preptin and the protein analysis of carrying out.

The result

Above-mentioned result of experiment is seen Fig. 1,2 and 3.

Discuss

The preptin of mouse is 34 amino acid whose peptides, and it is equivalent to the Asp of mouse proIGF-II E-peptide ₆₉-Leu ₁₀₂

Carry out integration by the peak area to RP-HPLC, determining the content that preptin exists in particle is 1/8th of Regular Insulin, but the twice of dextrin content (moles/mole).NH at preptin ₂-terminal flank is an Arg point of contact of having discerned, is two alkalescence (Arg-Arg) cutting motif (Bell (1984)) (Fig. 1 e) of inferring at the terminal flank of the COOH-of preptin.These residues high conservative between different plant species, and be likely processing signal after the translation.Many incretogenous precursors are the combinations more than a kind of hormone, wherein have different and usually are tissue-specific proteolysis processing mechanism (Martinez (1989)).Top result shows that proIGF-II is a kind of prohormone that contains more than a kind of peptide hormone product.

IGF-II is a member of Regular Insulin family, and it is regulating growth, differentiation and the metabolism (people (1990) such as DeChiara) of cell.IGF-II is a single polypeptide chain, is derived from BCA and the B structural domain (seeing Fig. 1 e) of proIGF-II, and has synthetic widely in fetus and adult tissue.And the expression of Regular Insulin but almost completely is limited to beta cell.In mammiferous genome, IGF-II gene and insulin gene contiguous (Bell (1985)), and recently in research to the people, identified a kind of VNTR polymorphism in the upstream of INS and IGF-II gene, perhaps this help the otherness of these two kinds of genes to regulate (Ong (1999)).

(Fig. 2 finds preptin and Regular Insulin and dextrin by copurification, thereby confirms that it is a kind of particulate component really a) and with the analysis again of preptin RIA to Fig. 1 a particle purifying collection of illustrative plates to the radioimmunoassay (RIA) of preptin.Carry out RP-HPLC/RIA by particle and β TC6-F7 cell conditioned medium, preptin sample immunoreactivity material (PLIM) is characterized purifying.In the particle and the principal mode of extracellular PLIM, on RP-HPLC all by co-elute (Fig. 2 b).Show to have only a kind of material to have the molecular weight of its molecular weight and mouse preptin identical (Fig. 2 c) to being equivalent to from the mass spectrum that the HPLC purifying substance at the PLIM peak of β TC6-F7 cell is done.RP-HPLC also proves, the principal mode of the PLIM of people and rat plasma and intragranular mouse preptin co-elute.Preptin is divided into from β TC6-F7 cell with Regular Insulin and secretes, and stimulates (Fig. 2 d) to reply glucose, has reached highest level when 1mM or greater concn glucose.

These results confirm that preptin is synthetic in pancreatic, and are packaged in the secretion property particle.In addition, it is divided in glucose dependence mode with Regular Insulin and secretes.

Evidence suggests: secretion of insulin can be comprised Regular Insulin (Kulkarni (1999); Elahi (1982); Argoud (1987)), dextrin (people (1993) such as Waggoner; Silvestre (1996); People such as Degano (1993)) and pancreastatin (Tatemoto (1986)) in the modulation of interior pancreatic hormone.It is generally acknowledged that they are that negative feedback loop by autocrine works, and mediated by combining with special cell surface receptor.Thereby, preptin is studied the effect of insulin secretion.The result who is obtained shows: synthetic rat preptin, with a kind of be the mode that saturability is arranged again of concentration dependent, strengthening glucose (10mM) stimulates Regular Insulin (Fig. 3 is a) from the β TC6-F7 emiocytosis of cultivating.When concentration is 0.1nM and Geng Gao, compare with contrast (not adding preptin), detect the remarkable effect of preptin, and when 75nM, reached maximum effect.Also just under this concentration, dextrin causes the inhibition (people (1993) such as Degano) of insulin secretion.These preptin concentration similar to the concentration of β TC6-F7 emiocytosis (Fig. 2 d), thus they very may be in the physiological pancreas islet in original position betide the beta cell cytolemma near.The concentration dependent that preptin is stimulated insulin secretion and the confirmation of saturability, thus prompting preptin causes these effects by combining with cell surface receptor.

The isolating rat pancreas that is poured is adopted the most effective preptin concentration (75nM), inject synthetic rat preptin, measured the effect (Fig. 3 b) of the insulin secretion that it stimulates glucose (20mM) therein.Compare with contrast (not adding preptin) value, preptin strengthens significantly and (strengthens 30%; P=0.03, the two afterbody t-test of area under curve) second phase secretion (Fig. 3 b) of Regular Insulin.These find that (Fig. 3 is consistent a) with those results that obtain from β TC6-F7 cell.Their promptings: preptin is a kind of physiological regulation of insulin secretion, the preceding feedback autocrine circuit type that it arrives with a kind of new knowledge, strengthen the insulin secretion that glucose stimulates, and perhaps in the retarding effect of other beta cell hormone of contending with, play a role insulin secretion.

Therefore, the present patent application people's viewpoint is: preptin works to replying in the activity of glucose at recovery, initiation and coordination beta cell with a kind of local mode, and the signal that glucose is caused amplifies to the beta cell organ.This effect is similar to preceding Feedback mechanism, the thromboxane A that zymoplasm caused ₂Be released in and just produce this mechanism (Barritt (1992)) in the thrombocyte.

The existence of the mechanism of not expecting before a kind of, by a kind of new pancreatic hormone of this mechanism the insulin secretion that glucose mediates is amplified, the biology of prompting preptin will be very important in type ii diabetes, and type ii diabetes is characterised in that a kind of insulin secretion infringement people (1992) such as () De Fronzo of complexity.In synthetic, the secretion of preptin or the defective aspect the effect, can facilitate the defective of the insulin secretion of glucose mediation in this case, it is perhaps helpful to treatment type ii diabetes or other disease relevant with beta cell minimizing insulin secretion to use preptin.It is to be noted, in the mankind, the series connection repetition number that is positioned at the upstream of adjacent Regular Insulin (INS) and IGF-II gene changes the polymorphism of (VNTR), is regulating this two expression of gene, and with the increase trend correlation of type ii diabetes and these two kinds of diseases of polycystic ovary syndrome.

Second section

Preptin and Regular Insulin are packaged in the islet tissue altogether

In order to study the physiology of preptin,, normal mouse pancreas has been carried out immunohistochemical study by adopting the antiserum(antisera) special to preptin.To adopt the glutaraldehyde single stage method in pH7.0 and ovalbumin coupling (Harlow and Lane) by the synthetic rat preptin of top described (Auspep Pty Ltd) preparation.Adopt New Zealand white rabbit, be used to produce polyclonal antiserum at rat preptin conjugate.

Adopt the second antibody of antiserum(antisera) and employing goat-anti--rabbit immunoperoxidase mark of phenodin and specificity anti-preptin in ground or synalbumin, and all staining agents are carried out 40 times dilute (volume/volume) eventually, the series section from normal adult mice (FVB/n) pancreas is dyeed.Add section earlier with preptin (1 or 5mg/ml) and anti--preptin antiserum(antisera) preincubation 30 minutes, and then with it, to confirm the specificity of preptin immunostaining.

Preptin sample immunoreactivity material (PLIM) and Insulin-Like immunoreactivity material be positioned altogether in the pancreatic (Fig. 4 a, b).Competitiveness studies show that: with preptin antiserum(antisera) and synthetic preptin preincubation, can suppress PLIM dyeing (Fig. 4 b-d) in concentration dependence mode.These research promptings preptin is present in the physiological pancreas pancreatic.

PLIM is present in the normal islet tissue

In order to confirm the PLIM in the normal islet tissue, we have carried out RP-HPLC/RIA to the acidic ethanol extraction thing from isolating rat Langerhans islet.The pancreas pancreas islet is to separate from normal Thirty male rats, and according to presentation method (Wollheim and Sharp (1981); Romanus (1988)) modification basis is to inclusion acidic ethanol extracting.

The results are shown in Figure 5.

Although the level of preptin is than much lower in β TC6-F7 cell, the main peak of PLIM and intragranular preptin are got off by co-elute, and this shows that preptin is the prevailing physiology composition (Fig. 5) of PLIM in normal pancreas islet.These data acknowledgements: having more than from the preptin of β TC6-F7 cell purification is a kind of artifact that is produced by protein cleavage purge process, but preptin just exists with this form and from β TC6-F7 cell and normally rat Langerhans islet secrete out.

Glucose is stimulated reply in preptin and Regular Insulin be divided into and secrete

Because known preptin and Regular Insulin are positioned in the beta cell secretion property particle altogether, secrete so experimentize whether to be divided in the mode of being regulated with definite preptin and Regular Insulin.According to the method for having delivered, adopt β TC6-F7 cell (people (1993) such as Efrat; People such as Knaack (1994)) and isolating rat Langerhans islet people (1987) such as () Gotoh, the peptide secretion that glucose stimulates is studied, and by using specific RIA that preptin and concentration of insulin are measured.

The results are shown in Figure 6.These results show: when β TC6-F7 cell is replied (S Efrat to the glucose (less than 5mM) of inferior physiological concentration, private communication) time, from β TC6-F7 cell (Fig. 6 a) and normal rat Langerhans islet (Fig. 6 b) observe clearly Regular Insulin/preptin and be divided into and secrete pattern.Much lower from the amount ratio of islet tissue (ratio of preptin and Regular Insulin is 1: 500) excretory preptin from β TC6-F7 cell (ratio of preptin and Regular Insulin is 1: 8) excretory level.This observations has been supported HPLC/RIA result, and it shows that the level of preptin in the physiological tissue is more much lower than the preptin level in the beta cell of cultivating.Although the level of preptin is much lower in the physiological tissue, these two models all show clearly: stimulate in order to reply glucose, preptin is divided into from the physiological pancreatic with Regular Insulin and secretes.

Removal endogenous preptin has reduced Regular Insulin significantly and has secreted in order to determine endogenic pancreas preptin possibility role secretion of insulin from the isolating rat pancreas that is poured,-preptin antibody anti-by injecting is to the isolating pancreas model that is poured, endogenic preptin effect is removed, and way is as follows:

With the KHB that has replenished 4% dextran, 0.5%BSA, 3mM arginine, 5.5mM glucose (ultimate density) pancreas is poured into.Use 95%O ₂/ 5%CO ₂Mixture is inflated perfusion liquid, and adopts peristaltic pump that its speed with 2.7ml/min is not had recirculation and inject.Before each 70 minutes perfusion, earlier pancreas is poured into and balance 20 minutes.Entering experiment in the time of 10 minutes,, inject (final concentration at the perfusion liquid gamma globulin is: 35 μ g/ml are in carrier damping fluid (0.1%BSA is in 0.9%NaCl)) by side arm with gamma globulin or the non-immune rabbit gamma globulin of anti--preptin.In addition, in the time of 25 minutes, glucose is injected (final concentration of measuring in perfusion liquid is 20mM) with 20 fens clock times.Collect 1-minute continuous fraction collection part on ice, then it is measured Regular Insulin (RIA).

Adopt Protein A affinity chromatography (Pharmacia Biotech, Hi-Trap Protein A TechRep.Wikstroms, Sweden (1999)), to rabbit anti--gamma globulin or contrast (non-immune rabbit) gamma globulin of rat preptin carry out purifying, to reduce the potential impact from other serum component.Adopt MALDI-TOF MS, composition to these two kinds different gamma globulin classification parts has carried out determining (Fig. 7 a, and under the aforesaid antibody of simulation perfusion experimental conditions, the binding ability of these two kinds different gamma globulin classifications parts is measured b).Under the pancreas experiment condition that is poured, be 20ng/min (Fig. 7 c) by the maximum of the complete bonded preptin of gamma globulin of anti--preptin.

The isolating pancreas that is poured is injected the gamma globulin of anti--preptin or contrast, and it is carried out square wave stimulation (Fig. 7 d) with 20mM glucose.The gamma globulin of anti--preptin makes first and the second phase secretion of Regular Insulin all reduce (the first phase: on average suppress 29% compared with the control, P=0.02, single afterbody t-test significantly; The second phase: on average suppress 26% compared with the control, P=0.03, single afterbody t-test of area under curve).In this experiment, we show: remove endogenic round-robin preptin, can cause the remarkable minimizing of the insulin secretion of glucose mediation.If estimated the concentration quite low (approximately than low 500 times of Regular Insulin) that preptin exists in the physiological pancreas islet, and still can apply remarkable effect to insulin secretion, this result will be very absorbing so.These experimental results are consistent with such prerequisite, and promptly the preptin physiological concentration of pancreas is being played the part of an autocrine role, to improve the insulin secretion of glucose mediation.This effect perhaps with the thromboxane A that causes by zymoplasm ₂Release and the preceding Feedback mechanism similar (Barrit (1992)) that in thrombocyte, causes.

Conclusion

In a word, preptin is a kind of unknown in the past pancreas pancreatic hormone.It is produced by the E-peptide of pro-IGF-II, in the pancreatic particle, exist with significant quantity, be divided into by regulative mode and Regular Insulin to secrete with a kind of, strengthen the insulin secretion that glucose stimulates, and probably by combining with the beta cell surface receptor and in preceding feedback autocrine loop, may working.

Industrial application

Describe as top, the invention provides the analogue of preptin (comprising its people, rat and mouse form) and preptin.Preptin and its analogue are being played the part of a physiological role in stimulating the insulin secretion that is caused by glucose.

Thereby the present invention also provides such certain methods, adopts these methods to modulate the insulin secretion that glucose causes.Such modulation will generally include by using preptin and analogue thereof as mentioned above.But, use the agonist of preptin and antagonist also can realize modulation.

The preptin agonist is exactly to promote or to strengthen the compound of preptin to the effect of insulin secretion.In contrast, the preptin antagonist can or can react with preptin on the contrary with preptin competition exactly, thereby blockades or reduce the compound of preptin to the effect of insulin secretion.

Exist and do not exist under the situation of test compound, the system of insulin secretion effect is being measured, just can identify preptin agonist and preptin antagonist by can measure preptin to those.For example, can use the mensuration system of in the experimental section of this paper, describing.

In the modulation insulin secretion, when needs use preptin agonist or preptin antagonist, can perhaps be formulated as the pharmaceutical composition of aforesaid preptin with agonist/antagonist with pure compound administration.

This patent also provides the immunoreagent in conjunction with preptin.Such immunoreagent (they can be polyclonal antibodies) can produce by the standard technique of using this area, comprising those technology of describing in experimental section.

Also can provide monoclonal antibody.Such antibody generally is by standard program, as described in for example Harlow and the Lane 1988, makes.Briefly say to be exactly to select suitable animal and undertaken by desirable immune programme for children.After one suitable period, the spleen of such animal is extractd, then with single spleen cell usually with the myeloma cell of immortalization, select appropriate condition to merge.After this, with cell clone ground separately, and to each clone supernatant liquor produced that the special suitable antibodies of the desired part of immunogen is tested.

Other suitable technique that is used to prepare antibody comprises lymphocyte perhaps alternatively, also can screen the antibody library that is arranged in phage or similar substrates at the external antigen that is exposed to, for example, and referring to people such as Huse 1989.

In addition, by adopting methods known in the art, can produce recombinant antibodies.For example see United States Patent (USP) 4816567.

Can use with or without the antibody of modifying.Often antibody is connected with a kind of material of detection signal that provides, thereby antagonist carries out mark by mode covalently or non-covalently.Known have multiple different marker and coupling technology, and in the literature they are had report widely.

Thereby, the as above antibody at preptin can be used for monitoring the patient or in the existence of preptin quantitative assay preptin.In such mensuration, can adopt any immunological method easily.Such method comprises immunohistochemical methods assay method, RIA, IRMA and ELISA assay method.

These assay methods can be used for relevant any biological fluid that maybe should contain preptin certainly.Such liquid comprises blood, serum, blood plasma, urine and cerebrospinal fluid.

Also antibody can be included in and measure in the test kit.Such test kit can contain some optional but the composition that brings convenience selects these test kits to the routine work of those skilled in the art's genus in addition.But those in the test kit add component in addition will generally comprise a preptin reference standard, and it can be preptin itself, or a kind of analogue (such as a fragment).

This situation will be gratifying: such as antibody recited above in some cases, by combining with preptin, also can play a role as the antagonist of preptin, thereby disturb the activity of preptin partially or completely.

Hint that as the front discovery of the present patent application people aspect preptin also has the diagnosis implication.For example, in order to cause insulin secretion to suitable level, just need a certain amount of preptin, the preptin that those produced just needs therapeutic intervention than the individuality that the needed preptin that lacks or produced is in lower or non-activity (mutant) state of activity.Thereby diagnosis or method of prognosis are also within the scope of the invention.

In a specific embodiment, diagnosis or method of prognosis will comprise the detection that suddenlys change of the coding gene of preptin and/or preptin mechanism of secretion.For detection, can adopt any in some standard techniques of this area, comprise that strand determines to analyze (Single StrandedConfirmation Analysis) people (1989) such as () Orita or disclosed anti-amplification abruptly-changing system (Amplification RefractoryMutation System) is (ARMS) in No. the 0332435th, european patent application publication.

If detected sudden change, so just might provide correcting scheme.Correcting scheme includes but not limited to gene therapy.In addition, also will use the standard technique of this area.

Other implication and application that the present patent application people identifies preptin, for those skilled in the art will be conspicuous, these people will be understood that: top description just by way of example mode provide, and the present invention is not limited to top description.

Reference

Draznin, B. and LeRoith, D. (write) molecular biology of .II type diabetes. Regular Insulin is to genetic expression and adjusting and to the effect and effect (the Humana Press Inc. of glucose transport, New Jersey (1994)) .Cooper, G.Endocr.Reu.15,163-201 (1994).

Waggoner, P., Chen, C., Worley, J., Dukes, 1. and Oxford, institute of G. NAS periodical (Proc.Natn.Acad.Sci., USA), 90,9145-9149 (1993).

Hettiarachchi, M. waits the people. U.S.'s physiology magazine (Am J.Physiol.), 273, E859-E867 (1997) .Hutton, diabetology magazine (J.Diabetologia), 12,271-281 (1989).

Efrat, people such as S.. diabetes (Diabetes), 42,901-907 (1993).

Knaack, people such as D.. diabetes, 43,1413-1417 (1994).

Linde, people's chromatographic science magazine (J.Chromatgr.) .4642 such as S., 243-254 (1989)

Johnson.K., wait the people. American Journal of Pathology (Am J.Pathol), 130,1-8 (1998).

Bell, G, I. waits the people. nature (Nature), 310,775-777 (1984).

Martinez, J. (writing). peptide hormone prohormone: processing, biologic activity, pharmacy (Peptide hormonesprohormones:processing, biological activity, pharmacology) (Ellis Horwood Ltd, Chichester, 1989).

DeChiara, T., Efstradiadis, A.and Robertson, E. nature, 345,78-79 (1990) .Kulkami, people such as R.. cell (Cell), 96,329-339 (1999).

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DeFronzo, R., Bonadonna, R. and Ferrannini, E.Diabetes Care 15,318-368 (1992).

Hutton, J., Penn, E. and Peshavaria, M. diabetology (Diabetologia) 2,365-373 (1982).

Grodsky, G. and Fanska, R. Enzymology method (Methods Enzymol.), 39,364-372 (1975).

Stempien, M., Fong, N., Rall, L. and Bell, G.DNA, 5,357-361 (1986).

Dull, T., Gray, A.Hayflick, J. and Ullrich, A. nature, 310,777-780 (1984).

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Sequence table

＜110〉GJS cooper

The CM Buchanan

＜120〉peptide

<130>26010?MRB

<140>

<141>

<150>NZ336359

<151>1999-06-18

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<223>Ser?or?Pro?or?an?analog?thereof

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<223>Tyr?or?Phe?or?an?analog?thereof

<220>

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<223>Arg?or?Lys?or?an?analog?thereof

<220>

<221>VARIANT

<222>(31)

<223>Ala?or?Thr?or?an?analog?thereof

<220>

<221>VARIANT

<222>(32)

<223>Gly?or?Gln?or?an?analog?thereof

<400>1

Asp?Val?Ser?Thr?Xaa?Xaa?Xaa?Val?Leu?Pro?Asp?Xaa?Phe?Pro?Arg?Tyr

1 5 10 15

Pro?Val?Gly?Lys?Phe?Phe?Xaa?Xaa?Asp?Thr?Trp?Xaa?Gln?Ser?Xaa?Xaa

20 25 30

Arg?Leu

<210>2

<211>102

<212>DNA

＜213〉homo sapiens

<220>

<221>CDS

<222>(1)..(102)

<400>2

gac?gtg?tcg?acc?cct?ccg?acc?gtg?ctt?ccg?gac?aac?ttc?ccc?aga?tac 48

Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr

1 5 10 15

ccc?gtg?ggc?aag?ttc?ttc?caa?tat?gac?acc?tgg?aag?cag?tcc?acc?cag 96

Pro?Val?Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln

20 25 30

cgc?ctg 102

Arg?Leu

<210>3

<211>34

<212>PRT

＜213〉homo sapiens

<400>3

Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr

1 5 10 15

Pro?Val?Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln

20 25 30

Arg?Leu

<210>4

<211>102

<212>DNA

＜213〉Rattus (Rattus sp.)

<220>

<221>CDS

<222>.(1)..(102)

<400>4

gac?gtg?tct?acc?tct?cag?gcc?gta?ctt?ccg?gac?gac?ttc?ccc?aga?tac 48

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr

1 5 10 15

ccc?gtg?ggc?aag?ttc?ttc?aaa?ttc?gac?acc?tgg?aga?cag?tcc?gcg?gga 96

Pro?Val?Gly?Lys?Phe?Phe?Lys?Phe?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly

20 25 30

cgc?ctg 102

Arg?Leu

<210>5

<211>34

<212>PRT

＜213〉Rattus (Rattus sp.)

<400>5

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr

1 5 10 15

Pro?Val?Gly?Lys?Phe?Phe?Lys?Phe?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly

20 25 30

Arg?Leu

<210>6

<211>102

<212>DNA

＜213〉Mustella (Mus sp.)

<220>

<221>CDS

<222>(1)..(102)

<400>6

gac?gtg?tct?acc?tct?cag?gcc?gta?ctt?ccg?gac?gac?ttc?ccc?aga?tac 48

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr

1 5 10 15

ccc?gtg?ggc?aag?ttc?ttc?caa?tat?gac?acc?tgg?aga?cag?tcc?gcg?gga 96

Pro?Val?Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly

20 25 30

cgc?ctg 102

Arg?Leu

<210>7

<211>34

<212>PRT

＜213〉Mustella (Mus sp.)

<400>7

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr

1 5 10 15

Pro?Val?Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly

20 25 30

Arg?Leu

<210>8

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: provide as an example

<400>8

Ala?Ser?Thr?Pro?Gly

1 5

<210>9

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: provide as an example

<400>9

Asn?Asp?Glu?Gln

1

<210>10

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: provide as an example

<400>10

Met?Glu?Ile?Val

1

Claims (48)

1. biologically active peptides, its aminoacid sequence is as follows:

Asp?Val?Ser?Thr?R ₁?R ₂?R ₃?Val?Leu?Pro?Asp?R ₄?Phe?Pro?Arg?Tyr?Pro?Val?Gly?Lys

Phe?Phe?R ₅?R ₆?Asp?Thr?Trp?R ₇?Gln?Ser?R ₈?R ₉?Arg?Leu

Wherein:

R ₁Be Ser or Pro;

R ₂Be Gln or Pro;

R ₃Be Ala or Thr;

R ₄Be Asp or Asn;

R ₅Be Gln or Lys;

R ₆Be Tyr or Phe;

R ₇Be Arg or Lys;

R ₈Be Ala or Thr; With

R ₉Be Gly or Gln.

2. the people preptin that has following aminoacid sequence:

Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln?Arg?Leu。

3. the rat preptin that has following aminoacid sequence:

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Lys?Phe?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly?Arg?Leu。

4. the mouse preptin that has following aminoacid sequence:

Asp?Val?Ser?Thr?Ser?Gln?Ala?Val?Leu?Pro?Asp?Asp?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Arg?Gln?Ser?Ala?Gly?Arg?Leu。

5. peptide that is selected from people preptin has following aminoacid sequence:

Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln?Arg?Leu

Perhaps its fragment, wherein said fragment is selected from:

(i) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln?Arg；

(ii) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln；

(iii) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr；

(iv) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser；

(v) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln；

(vi) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys；

(vii) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp；

(viii)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr；

(ix) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp；

(x) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr；

(xi) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln；

(xii) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe；

(xiii)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe；

(xiv) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys；

(xv) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly；

(xvi) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val；

(xvii) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro；

(xviii)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr；

(xix) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg；

(xx) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro；

(xxi) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe；

(xxii) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn；

(xxiii)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp；

(xxiv) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro；

(xxv) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu；

(xxvi) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val；

(xxvii) Asp Val Ser Thr Pro Pro Thr; With

(xxviii)Asp?Val?Ser?Thr?Pro?Pro。

6. isolating polynucleotide, it is encoded as each described polypeptide, preptin or its fragment among the claim 1-5.

7. isolating polynucleotide, its coding people's preptin, and it comprises following nucleotide sequence:

gacgtgtcgacccctccgaccgtgcttccggacaacttccccagataccccgtgggcaagttcttccaatatga

cacctggaagcagtccacccagcgcctg。

8. isolating polynucleotide, the preptin of its coding rat, and it comprises following nucleotide sequence:

gacgtgtctacctctcaggccgtacttccggacgacttccccagataccccgtgggcaagttcttcaaattcgac

acctggagaeagtccgcgggacgcctg。

9. isolating polynucleotide, the preptin of its encoding murine, and it comprises following nucleotide sequence:

gacgtgtctacctctcaggccgtacttccggacgacttccccagataccccgtgggcaagttcttccaatatgac

acctggagacagtccgcgggacgcctg。

10. carrier or clone, it comprises polynucleotide any among the claim 7-9, and it can express the peptide by described polynucleotide encoding.

11. carrier as claimed in claim 10 or clone, it comprises the polynucleotide of claim 7.

12. a pharmaceutical composition, it comprises as any described polypeptide, preptin or its fragment among the claim 1-5.

13. a dosage form, it contains just like any described polypeptide, preptin or its fragment among the claim 1-5, and the physiological buffer that is suitable for being applied to the people.

14. dosage form as claimed in claim 13, it is used for drug administration by injection.

15. as claim 13 or 14 described dosage forms, wherein said preptin is as claim 2 or 5 described people preptin or its fragments.

16. as any described polypeptide of claim 1-5, preptin or its segmental preparation, wherein said polypeptide, preptin or its segmental amount are at least 50% weight.

17. the preparation described in claim 16, wherein said polypeptide, preptin or its fragment are at least 80% weight of said preparation.

18. the preparation described in claim 16, wherein said polypeptide, preptin or its fragment are at least 90% weight of said preparation.

19. the preparation described in claim 16, wherein said polypeptide, preptin or its fragment are at least 95% weight of said preparation.

20. the preparation described in claim 16, wherein said polypeptide, preptin or its fragment are at least 99% weight of said preparation.

21. preparation as claimed in claim 16, wherein said polypeptide, preptin or its fragment are pure.

22. any described polypeptide, preptin or its segmental salt among the claim 1-5.

23. salt as claimed in claim 22, it is a kind of physiologically acceptable salt.

24. salt as claimed in claim 23, wherein said polypeptide, preptin or its fragment form by combining with the organic acid negatively charged ion.

25. salt as claimed in claim 24, wherein said salt is selected from the salt of oxysuccinic acid, acetate, propionic acid, butyric acid, oxalic acid, citric acid, isocitric acid, α-Tong Wuersuan, succsinic acid, fumaric acid and trifluoroacetic acid.

26. pharmaceutical composition that comprises as salt as described among the claim 23-25 any one.

27. be used for preparing the purposes of the medicine for the treatment of type ii diabetes as any described polypeptide, preptin or its fragment among the claim 1-5.

28. be used for preparing the purposes of the medicine for the treatment of type ii diabetes as any described salt among the claim 23-25.

29. as any described polypeptide, preptin or its fragment among the claim 1-5 be used for preparing treatment cause or relate to that Regular Insulin is synthetic, the purposes of the medicine of the state of secretion or functional defect.

30. as any described salt among the claim 23-25 be used for preparing treatment cause or relate to that Regular Insulin is synthetic, the purposes of the medicine of the state of secretion or functional defect.

31. any described polypeptide, preptin or its fragment purposes in the medicine of preparation treatment diabetes among the claim 1-5.

32. the purposes of the medicine that any described polypeptide, preptin or its fragment are used for stimulating insulin secretion in preparation among the claim 1-5.

33. the purposes of any described salt in the medicine of preparation treatment diabetes among the claim 23-25.

34. the purposes of the medicine that any described salt is used for stimulating insulin secretion in preparation among the claim 23-25.

35. with as any described polypeptide, preptin or its fragment bonded antibody among the claim 1-5.

36. with as any described polypeptide, preptin or its fragment bonded monoclonal antibody among the claim 1-5.

37. with as claim 2 or 5 described people preptin or its fragment bonded monoclonal antibody.

38. the external immunologic assay method of any described antibody among use such as the claim 35-37.

39. assay method as claimed in claim 38, wherein quantitative assay is carried out in the existence in biological fluid to preptin.

40. assay method as claimed in claim 39, wherein said biological fluid are blood, serum, blood plasma, urine or cerebrospinal fluid.

41. an external immunologic assay method, it uses as any described antibody among the claim 35-37, and it is RIA, IRMA or ELISA.

42. measure test kit for one kind, it comprises as any described antibody among the claim 35-37.

43. mensuration test kit as claimed in claim 42, it comprises as any described antibody and preptin reference standard among the claim 35-37.

44. mensuration test kit as claimed in claim 43, wherein said reference standard are as any described preptin or its fragment among the claim 1-5.

45. a method of identifying the preptin agonist, it comprises step:

Existing and not existing under the situation of material standed for agonist, to measuring by the polypeptide institute inductive insulin secretion degree described in claim 1 of predetermined concentration; And

To having any compound of the insulin secretion effect that improves the preptin mediation,, identified as agonist.

46. a method of identifying the preptin antagonist, it comprises step:

Existing and not existing under the situation of material standed for antagonist, to measuring by the polypeptide institute inductive insulin secretion degree described in claim 1 of predetermined concentration; And

To having any compound of the insulin secretion effect that reduces the preptin mediation,, identified as antagonist.

47. preptin agonist of identifying according to the method for claim 45 or 46 or preptin antagonist are used for regulating the purposes of medicine of the insulin secretion of glucose mediation in preparation.

48. an isolated polypeptide, it has following aminoacid sequence:

Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val?Gly

Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln?Arg?Leu，

Perhaps its fragment, wherein said fragment is selected from:

(i) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln?Arg；

(ii)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln；

(iii)Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr；

(iv) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser；

(v) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln；

(vi) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys；

(vii)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr?Trp；

(viii)Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp?Thr；

(ix) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr?Asp；

(x) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln?Tyr；

(xi) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe?Gln；

(xii)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe?Phe；

(xiii)Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys?Phe；

(xiv)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly?Lys；

(xv) Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val

Gly；

(xvi)?Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val；

And

(xvii)Asp?Val?Ser?Thr?Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro。

Images