CN101466399A - Stabilized insulin-like growth factor polypeptides - Google Patents

Abstract

The invention relates to stabilized polypeptides having an IGF-I or IGF-2 sequence and an E-peptide sequence, where the natural physiological cleavage of the E-peptide from the IGF is prevented.

Description

Stable insulin-like growth factor polypeptides

Background of invention

Insulin like growth factor (IGF) is the part that cell is used for carrying out with their physiological environment alternative complication system.This complication system (being commonly referred to the insulin like growth factor axle) is made up of the family (IGFBP 1-6) and the dependency IGFBP digestive enzyme (protease) of two cell surface receptors (IGF-1R and IGF-2R), two parts (IGF-1 and IGF-2), six high-affinity igf binding protein matter.This system is not only important to regulating normal physiology, also to many pathological conditions important (Glass, Nat Cell Biol 5:87-90,2003).

The IGF axle has been presented at and has promoted cell proliferation and suppress to work in the cell death (programmed cell death).IGF-1 is mainly secreted by liver because of human growth hormone (hGH) stimulates.In the human body almost each cell (the especially cell in muscle, cartilage, bone, liver, kidney, nerve, skin and the lung) all be subjected to the influence of IGF-1.Except that the Insulin-Like influence, also scalable cell growth of IGF-1.The gene outcome family that IGF-1 and IGF-2 are known as igf binding protein matter regulates.These protein help to regulate the effect of IGF in the mode of complexity, and described mode comprises by prevention and is attached to the effect that suppresses IGF on the IGF receptor and promotes the half-life of IGF in the effect of IGF and the increasing blood flow by helping to be delivered to receptor.There are at least 6 to characterize conjugated protein (IGFBP 1-6).

Its mature form, people IGF-1

(gpetlcgaelvdalqfvcgdrgfyfnkptgygsssrrapqtgivdeccfrscdlrr lemycaplkpaksa; SEQ ID NO:1), being also referred to as somatomedin, is 70 amino acid whose small protein matter, and it has shown can stimulate the growth of cell in culture widely.Mature protein is at first by three known splice variant mRNA codings.The open reading-frame of each mRNA is according to specific I GF-1mRNA coding precursor protein matter, and described precursor protein matter contains the specific E peptide at 70 aminoacid IGF-1 and C-terminal place.These E peptides have been called as Ea (rsvraqrhtdmpktqkevhlknasrgsagnknyrm; SEQ ID NO:2),

Eb (rsvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgk kkeqrreigsrnaecrgkkgk; SEQ ID NO:3) and Ec

(rsvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk; SEQ ID NO:4) peptide, and length range comprises the consensus sequence district from 35 aminoacid to 87 aminoacid and at N-terminal, comprises the variable sequence district at C-terminal.For example, 105 amino acid whose polypeptide of the wild type open reading-frame of IGF-1-Ea coding

(gpetlcgaelvdalqfvcgdrgfyfnkptgygsssrrapqtgivdeccfrscdlrrlemycaplkpaksa?rsvraqrhtdmpktqkevhlknasrgsagnknyrm；SEQ?ID?NO：5)。In physiology's statement, the E peptide is cut down to produce sophisticated 70 amino acid whose IGF-1 of known biologically active from precursor by endogenous proteinase.In specific background, one to three the N-terminal aminoacid of known IGF-1 is cut under physiological condition, produces to have 67-70 amino acid whose active IGF-1.IGF-2 gene expression is characterised in that to have similar feature with processing, except having identified 156 amino acid whose precursors of people IGF-2

(ayrpsetlcggelvdtlqfvcgdrgfyfsrpasrvsrrsrgiveeccfrscdlall etycatpakserdvstpptvlpdnfprypvgkffqydtwkqstqrlrrglpallra rrghvlakeleafreakrhrplialptqdpahggappemasnrk; SEQ ID NO:7) only E peptide

(rdvstpptvlpdnfprypvgkffqydtwkqstqrlrrglpallrarrghvlakeleafreakrhrplialptqdpahggappemasnrk；SEQ?ID?NO：6)。IGF-I and IGF-2 seem to be poor drug candidate, because these protein can be degraded rapidly by the endogenous proteinase among the patients serum.A kind of strategy of having expected is to stablize IGF-1 as medicine by forming complex with its conjugated protein.

Summary of the invention

The present invention is based on such discovery, contain the IGF-1 or the IGF-2 protein tool biological activity and stable when serum exists of its E peptide substantially, produce the IGF-1 or the IGF-2 polypeptide of useful as drug.In compositions of the present invention, for example avoid the normal cutting of E peptide from the IGF-1 by the serine ( position 71 and 72 among the corresponding wild type precursor I GF-1) at sudden change or disappearance arginine at 1 place, E peptide position or 2 places, position.In IGF-2, for example avoid cutting by the arginine at sudden change or disappearance 1 place, E peptide position or the aspartic acid (position 68 and 69 among the corresponding wild type precursor I GF-2) at 2 places, position.This cutting can be avoided or reduce to other modifications of IGF precursor protein matter.

In addition, other modifications of IGF-1 precursor aminoacid sequence can be given extra medicine benefit.For example, polypeptide of the present invention affinity that can show increase the IGF-1 receptor or binding ability that inhibition IGF-1 or the conjugated protein demonstration of IGF-2 are reduced.

For definition and concordance, the amino acid residue of the IGF-1 in the application and the claim or IGF-2 precursor or mature protein numbering is based on the numbering of the wild type precursor protein matter sequence that does not contain signal peptide.

Therefore, the present invention includes the polypeptide that contains people IGF-1 precursor protein matter, wherein reduce from the modification that the E peptide on the IGF-1 cuts by precursor protein matter by protease.The E peptide can be Ea, Eb or Ec peptide.At the N-terminal place of precursor, aminoacid G1, the P2 of precursor protein matter or E3 can be lacked or be suddenlyd change, as R36 (for example, R36A) and R37 (for example, R37A).

For example insert between the amino acid N 95 and T96 of Eb by the aminoacid 93-102 with Ea, precursor protein matter can comprise that also N connects glycosylation consensus sequence NXS/T.Usually, precursor protein matter can comprise covalently bound oligosaccharide to precursor protein matter amino acid side chain (as the arginine side chain of precursor protein matter).

In addition, the residue of precursor protein matter can be replaced by alpha-non-natural amino acid (seed amino acid that for example, comprises acetylene or azido group).This type of alpha-non-natural amino acid can be beneficial to poly-(ethylene glycol) part and be connected on the side chain of precursor protein matter, though typical proteinic Pegylation strategy is known in the art.

Precursor protein matter also can comprise one or more extra E peptides that are connected to precursor protein matter C-terminal.For example, polypeptide can comprise that from the N-terminal to the C-terminal (1) has the IGF-1 precursor protein matter of first Eb peptide, and wherein G1, P1 and E1 are lacked, and one of R36 or R37 or both are suddenlyd change together, R71 and S72 are lacked, and last 7 C-terminal aminoacid of first Eb peptide are lacked; (2) second Eb peptides, wherein last 7 C-terminal aminoacid of R71, S72 and second Eb peptide are lacked; (3) the 3rd Eb peptides, wherein last 7 C-terminal aminoacid of R71, S72 and the 3rd Eb peptide are lacked; (4) the 4th Eb peptides, wherein R71 and S72 are lacked.

Prevent that the effective means that the E peptide is cut from IGF-1 from being R71 or S72 disappearance or sudden change.

Similarly, the present invention includes people IGF-2 precursor protein matter, wherein reduce from of the modification of IGF-2 cutting E peptide by precursor protein matter by protease.Particularly, R68 or D69 disappearance or sudden change can become the effective means of avoiding IGF-2 precursor protein matter protease digestion.

In addition, the E peptide of any IGF-1 can make up with IGF-2, and the E peptide of any IGF-2 can make up with IGF-1 benefit described herein is provided.

The present invention also comprises the method for the treatment of musculoskeletal disease, diabetes, neuronal cell death by the polypeptide of the present invention of administering therapeutic effective dose.Equally, the present invention includes the purposes that polypeptide of the present invention is used for preparing the medicine that is used for the treatment of musculoskeletal disease, diabetes, neuronal cell death or anemia.

In another embodiment, the present invention includes Pegylation IGF-1, it does not contain the E peptide, but has wherein introduced alpha-non-natural amino acid as the Pegylation site.The present invention also comprises and anyly contains alpha-non-natural amino acid disclosed herein and do not contain the Pegylation IGF-1 through modifying of E peptide.

The present invention comprises that also the polypeptide of the present invention of using effective dose is to obtain the veterinary's method and the purposes of desired effects.

Veterinary purpose comprises that (i) improves growth of animal speed and/or degree, (ii) improving them is systemic efficient from food conversion, (iii) improve the milk production of lactogenic animal, (iv) treat animal the become thin symptom (wasting symptom) relevant and (v) treat the lactogenic animal and be used to improve neonate health status with cachexia, wound or other wasting diseasess (consumption disease).

All references list of references or file are incorporated herein by reference hereby.

The accompanying drawing summary

Figure 1A-1C is polypeptide of the present invention and the wild type IGF-1 precursor Western trace of incubation after 0 hour or 16 hours under 37 ℃ of situations that have or do not exist 10% human serum.The expression vector transfection of the multiple IGF-1 construct of coding to the Cos7 cell, and is obtained conditioned medium." 3mut " refers to hIGF-1-E peptide precursor, and it has following three groups of modifications: G1, P2 and E3 disappearance; Arg 37 sports Ala (R37A); With R71 and S72 disappearance.Figure 1A shows the Western trace result (using the antibody of IGF-1) of wild type and 3mut precursor (containing Ea).Figure 1B shows the Western trace result (using the antibody of hIGF-1) of wild type and 3mut precursor (containing Eb).Fig. 1 C shows the Western trace result (using the antibody of hIGF-1) of wild type and 3mut precursor (containing Ec).

Fig. 2 A-2D is for showing the bioactive line chart of multiple IGF-1 polypeptide (" part ").By stimulating the C2C12 sarcoplast to measure biological activity with the Cos7 express polypeptide.Measure total AKT of stimulation back C2C12 cell and the relative quantity of phosphorylation AKT then.Length-R3-IGF-1 is commercial reagent (the production number 1-1271 of Sigma company (Sigma)), and it is made up of sophisticated people IGF-1 aminoacid sequence, and described aminoacid sequence has E3R sudden change and extra 13 amino acid whose N-terminal extension peptides.Fig. 2 A shows the activity of IGF-1-Ea3mut.Fig. 2 B shows the activity of IGF-1-Eb3mut.Fig. 2 C shows the activity of IGF-1-Eab3mut, and described IGF-1-Eab3mut is the 3mut construct, and wherein Ea the 93rd to 102 amino acids is inserted between the 95th and 96 amino acids of Eb.Fig. 2 D shows the activity of IGF-1-Ec3mut.

Fig. 3 A-3D and 4A-4D are for showing by measuring the bonded receptor phosphorylation of response part whether IGF-1 precursor polypeptide of the present invention keeps the selectivity to suitable receptor.Fig. 3 A and 3B have detected the receptor-selective of IGF-1-Ea3mut at IGF-1 receptor (Fig. 3 A) and Insulin receptor INSR (Fig. 3 B).Fig. 3 C and 3D have detected the receptor-selective of IGF-1-Eb3mut at IGF-1 receptor (Fig. 3 C) and Insulin receptor INSR (Fig. 3 D).Fig. 4 A and 4B have detected the receptor-selective of IGF-1-Ec3mut at IGF-1 receptor (Fig. 4 A) and Insulin receptor INSR (Fig. 4 B).Fig. 4 C and 4D have detected the receptor-selective of IGF-1-Eab3mut at IGF-1 receptor (Fig. 4 C) and Insulin receptor INSR (Fig. 4 D)." IGF1-R3 " refers to above-mentioned long R3-IGF-1.The polypeptide of classifying " IGF1Eab " as refers to that Ea the 93rd to 102 amino acids wherein is inserted into the construct between the 95th and 96 amino acids of Eb.

Fig. 5 stimulates behind the C2C12 myotube Western trace of AKT phosphorylation relatively (break up as C2C12 myocyte 3 to 4 days result) for showing different ligands.The many bodies of IGF-1Eb are meant the construct that schematically is shown in Fig. 6 A.

Fig. 6 A and 6B are the diagram of two polypeptide in the polypeptide of the present invention.Fig. 6 A shows to have four groups of IGF-1-Eb precursor polypeptide of modifying: G1, P2 and E3 disappearance; R37 sports A; R71 and S72 disappearance; With last 7 C-terminal aminoacid deletion.In addition, by adding two Eb peptides (but do not have R71 and S72, also do not have last 7 C-terminal aminoacid) and last Eb peptide of interpolation (but not having R71 and S72) prolongs polypeptide in that the C-terminal of polypeptide.This construct is commonly referred to the many bodies of IGF-1-Eb.Fig. 6 B shows to have four groups of IGF-1-Eab precursor polypeptide of modifying: G1, P2 and E3 disappearance; R37 sports A; R71 and S72 disappearance; And Ea the 93rd to 102 amino acids is inserted between the 95th and 96 amino acids of Eb.

Fig. 7 A is people IGF-1 (SEQ ID NO:1) and the sequence alignment of corresponding animal IGF-1.Provided the GenBank accession number of all animal species of being analyzed and their sequence correspondences.G1, P2, E3 to some extent the analyte kind remove in the sterlet (Sterlet) (wherein S2 substitutes P2) and guard.R36 and R37 guard in the species analyzed to some extent.

Fig. 7 B compares the phylogenetic figure of institute's analysis of amino acid sequence for showing with people IGF-1 (SEQ ID NO:1).It below the tree scale of indication per 100 residues of protein sequence " aminoacid replacement " number.Kimura is used for compute distance values apart from general formula, and it is proofreaied and correct from the number of non-NULL bit mismatch and with regard to reticent substituting.The calculating income value is the average number of difference on each site and is between 0 to 1.The complete homogeneity of 0 representative, the no homogeneity of 1 representative.The phylogenetic tree scale uses these on duty with 100.

Fig. 8 A is the sequence alignment of people Ea peptide (SEQ ID NO:2) and multiple animal Ea peptide.Provided the GenBank accession number of all animal species of being analyzed and their sequence correspondences.R71 and S72 guard in the species analyzed to some extent.

Fig. 8 B compares the phylogenetic figure of institute's analysis of amino acid sequence for showing with people IGF-1Ea peptide (SEQ ID NO:2).

Fig. 9 A is the sequence alignment of people Eb peptide (SEQ ID NO:3) and multiple animal Eb peptide.Provided the GenBank accession number of all animal species of being analyzed and their sequence correspondences.R71 and S72 guard in the species analyzed to some extent.

Fig. 9 B compares the phylogenetic figure of institute's analysis of amino acid sequence for showing with people IGF-1Eb peptide (SEQ ID NO:3).

Figure 10 A is the sequence alignment of people Ec peptide (SEQ ID NO:4) and multiple animal Ec peptide.Provided the GenBank accession number of all animal species of being analyzed and their sequence correspondences.R71 and S72 guard in the species analyzed to some extent.

Figure 10 B compares the phylogenetic figure of institute's analysis of amino acid sequence for showing with people IGF-1Ec peptide (SEQ ID NO:4).

Figure 11 A is people IGF-2 (SEQ ID NO:7) and the sequence alignment of corresponding animal IGF-2.Provided the GenBank accession number of all animal species of being analyzed and their sequence correspondences.R68 guard in the species analyzed to some extent; D69 is histidine residues on this position in described chimpanzee except that being conservative chimpanzee.

Figure 11 B compares the phylogenetic figure of institute's analysis of amino acid sequence for showing with people IGF-2 (SEQ ID NO:7).

Figure 12 A is the sequence alignment of people IGF-2E peptide (SEQ ID NO:6) and multiple animal IGF-2E peptide.Provided the GenBank accession number of all animal species of being analyzed and their sequence correspondences.R68 guard in the species analyzed to some extent; D69 is histidine residues on this position in described chimpanzee except that being conservative chimpanzee.

Figure 12 B compares the phylogenetic figure of institute's analysis of amino acid sequence for showing with people IGF-2E peptide (SEQ ID NO:6).

Detailed Description Of The Invention

The present invention relates to substantially contain new IGF-1 and the IGF-2 Precursor Peptide of E peptide, described E Peptide has been modified to prevent, has been reduced or avoided responsible active IGF-1 or IGF-2 to discharge from its E peptide The cutting of typical case's protease. The effect of polypeptide of the present invention is based on such surprising discovery, and namely this type of precursor is many Peptide has biologically active, stable and useful as medicine.

Screen active IGF Precursor Peptide

Can use following mensuration to assess the validity of any polypeptide of the present invention.

Stability polypeptide of the present invention (as in human serum) when endogenous proteinase exists should have enough Stability become active drug. In order to assess stability, the expression vector of coded polypeptide can be turned to Dye the DMEM training that contains 10% hyclone, 100U/ml penicillin and 100 μ g/ml streptomysins Support in the Cos7 cell in the base (ATCC). The culture medium that contains secrete polypeptide can be used for further branch Analyse, or as alternative, the label that obtains easily in the described expression vector codified polypeptide is (such as six groups of ammonia The acidity scale label) be beneficial to express polypeptide in effective purifying Cos7 culture. Yet during preparation, polypeptide Sample normal human serum (Sigma company (Sigma)) or in PBS incubation different time (example Such as 0,1,5,10 and 16 hour), carry out polyacrylamide gel electrophoresis, trace to nitrocellulose On the element and use the one-level antibody of anti-people IGF-1 or IGF-2 and secondary antibody (for example to put together the horseradish mistake The secondary antibody of oxide enzyme) make related protein as seen. Can use many similar traces and detection Technology, some of them can be used fluorescent dye, or even use radionuclide. Precursor band strong Degree should show that with respect to the intensity of IGF-1 or IGF-2 band Precursor Peptide is cut under multiple condition The degree of cutting. 37 ℃ are exposed to 16 hours polypeptide demonstration of the present invention of human serum and do not cut precursor and cutting The ratio of ripe IGF be about 1:2 to 1:0.1, for example about 1:1 to 1:0.5, ratio are especially for approximately 1:1 or about 1:0.5. Usually, described precursor should show at least ratio of 1:1.

AKT phosphorylation polypeptide of the present invention should be kept the ability by the transduction of IGF-1 receptor signal. (IGF-1 and IGF-2 signal transduction all pass through the IGF-1 acceptor). In order to measure this signal transduction energy Whether power can assess the interior target AKT of downstream cell at cell surface response ligand binding and by phosphoric acid Change. In order to analyze the AKT phosphorylation, make the C2C12 sarcoblast hungry in serum free medium, With different ligands it is stimulated then. Cell lysis is also removed by centrifugal. Use respectively PathScan phospho AKT (Ser473) sandwich ELISA kit and PathScan AKT Sandwich ELISA kit (Cell Signaling) by ELISA analyze the AKT phosphorylation and Total AKT level.

IGF-1 receptor-specific polypeptide of the present invention is preferably kept the specificity of IGF-1 acceptor and is answered Be attached on the relevant insulin receptor with low-affinity. In order to assess receptor-specific, express to crossing Add the polypeptide sample in the serum starvation NIH3T3 cell of IGF-1 acceptor or insulin receptor, and make With DuoSet IC people phosphor-IGF-1 acceptor and insulin receptor ELISA kit (peace enlightening System house (R﹠D Systems)) by cell lysis and make lysate carry out ELISA to measure The level of IGF-1 receptor phosphorylation or Phosphorylation of insulin receptor.

Detect in the body in loose mouse model and causing myohypertrophia in order to measure polypeptide of the present invention Background in whether can work to increase the skeletal muscle amount, can make advancing with untreated animal of processing Whether the animal that row motion and mensuration are accepted polypeptide produces more muscle than untreated animal.

Motion model

A model known in the art is based on the use of the Voluntary Wheel with user's deferrable load (consult, such as Konhilas etc., Am J Physiol Heart Circ Physiol 289:H455-H465,2005). Rotating cage has been eliminated physics and the psychology damage of forcing in the training pattern voluntarily Hinder, and thereby be more suitable for (expecting that described healthy individual muscle mass increases in assessment for relative healthy individual Add) drug candidate.

Can use any suitable mouse species. For example, male C57B1/6J mouse can be divided at random Be assigned to experimental group (for example accepting the IGF Precursor Peptide) and control group. Animal stays in separately and contains motion In the cage of training runner; Static control-animal stays in the cage of no runner. At Allen etc., J Appl Physiol 90:1900-1908 has described the training runner in 2001. Briefly, described system Comprise the diameter 11.5cm of stem face width 5.0cm runner (model 6208, Petsmart, Phoenix, AZ), described runner is equipped with the digital magnetic counter (model by the runner rotation-activated BC 600, Sigma Sport, Olney, IL). In addition, each rotor design has the adjustment of allowing to load The resistance mechanical device. This is by being attached to the stainless steel fishing line at the cage top and metal wire being wrapped in solid Fixed pulley (its be fixed in the rotating shaft of cage runner and runner load is not had effect) has come on every side Become. Again with spring and screw metal wire is fixed in the cage top. This design allows intense adjustment The runner of mean allocation load in the runner rotary course. The duration of motion, record each motion animal Run duration and distance value every day. Arbitrarily give the hard rodent food of all animal water and standard. Run voluntarily (exposure of cage runner) of all groups can begin when mean age in about 12 week. According to reality Test group, each the group under different resistances, continued to run 50 days until described animal to about 19 the week ages. By hanging known weight at runner until described runner has slight shift to measure negative on the runner Lotus. All exercise group application of load not on the first when beginning week cage runner. Yet, " zero load " bar Part is actually 2g, and it is determined as keeps runner inertia and the necessary load of friction load. Consider The runner laundering period in 1 week, can be except higher load (it can change after 2 weeks), the interval One week changed the runner load. Anywhere, load range can be from 2g until 12g. Specific Moving period finish after, motion animal and static control-animal are dislocated by neck under inhalation anesthesia immediately Method is carried out euthanasia. Measure body weight, excise fast specific muscle, washing and freezing for later tissue Learn or biochemical measurement.

The loose model of alternative motion also can get for those skilled in the art. Consult, such as Lerman etc., J Appl Physiol 92:2245-2255, the treadmill exercise model of describing in 2002.

The clenbuterol injection model

Clenbuterol is to have the β that promotes growth properties₂Adrenergic agonist, it causes muscle mass Significantly increase. The accurate mechanism of clenbuterol function is still unclear, although proposed muscle protein The matter degraded reduces. Clinically, clenbuterol is as suppressing panting calming medicine, but that it seems mainly to misapply as is strong The body medicine increases the muscle mass of humans and animals.

Inject clenbuterol (3mg/kg, subcutaneous (s.c.)) every day to five mouse, carry out 3,7 or Induce muscle hypertrophy over 14 days. Mouse with the PBS injection is used as negative control. Monitoring every day (order Inspection) described animal is to any bad reaction (being fluffy and disorderly fur, somnolence) for the treatment of. Clenbuterol is controlled Treatment has the mouse of making more to be feared or has more aggressive potentiality, so if mouse lives with colony, Then should especially monitor fighting between them. Mouse is movable, and can normally take food and drink water. Whenever It monitoring mouse is until they were sentenced euthanasia at the 3rd, 7 or 14 day, and collection organization is used to into one Step is analyzed.

Detect in multiple skeletal muscle atrophy model in the body in the muscular atrophy model, can detect this Bright IGF Precursor Peptide is kept the ability of muscle mass under the condition that usually reduces muscle mass. According to hereinafter The instance model of describing, the technical staff can easily design and implement controlled experiment and (comprise the IGF precursor Using and purposes of polypeptide) whether can increase muscle mass to measure this type of polypeptide.

For example, buy the C57B16/2 male mice from the Jackson laboratory.Buy mice, make their about 9 weeks when each experiment beginning.Usually mice lives in containing the miniature isolation cage of normal Rodents food.Weighing mice when each experiment beginning.When each experiment finished, mice was usually by sucking CO ₂, sentenced euthanasia by cervical dislocation then, collect muscular tissue and be used for further processing.The weighing mice is to provide " final body weight ".Collectable skeletal muscle is tibialis anterior, extensor digitorum longus, musculus soleus and gastrocnemius.Its hetero-organization of Shou Jiing is once in a while: the heart, liver,spleen,kidney, testis and brain.Thoroughly all muscle of excision and tissue and in energy measurement weighing to the balance of 0.0001g.To be organized in then in the liquid nitrogen and extract RNA and protein after freezing being used for rapidly, or among the rapid freezing OCT that imbeds on the cork dish (cork disc).Freezing after being used on the cork dish muscle of frozen section immerse by cooled with liquid nitrogen to the condensed isopentane that partly melts shape (thick slush).All samples is stored in-80 ℃.

The dexamethasone treatment

The pharmacological method of muscle consumption is with 20mg/kg peritoneal injection every day dexamethasone in the inducing mouse.Dexamethasone is the synthetic member of hormone glucocorticoids.It works as anti-inflammatory agent and immunosuppressant, and its effectiveness is about 40 times of hydrocortisone.Dexamethasone is used for the treatment of many inflammation and autoimmune disease, for example rheumatoid arthritis.Also can use to offset some side effect of their antineoplastons to carrying out chemotherapeutical cancer patient.Dexamethasone causes the amyotrophy among mice and the people patient.

Land used plug Mi Songjing intraperitoneal (ip) injection mice 3,7 or 14 days.In the end one day, use CO ₂The experimenter is sentenced euthanasia, and collect leg muscle.Monitoring every day (visual inspection) described animal is to any untoward reaction (being fluffy and disorderly fur, somnolence) of treatment.Mice is movable usually, and can normally take food and drink water.Mice with the PBS injection is used as negative control.

Mould is fixed

The physics of multiple muscle group is useless in the atrophy that causes those muscle.Proved that it is the mode that height is useful and tool is repeated of inducing rat and mouse hind leg musculature physical fixation that ankle joint is fixed (" heel of pegging " or casting).

Be used for fixing with different fluorane anesthetized mice.(VET-LITE) ankle joint and knee joint are fixed into 90 degree with lightweight mold materials (casting material) around the joint.Described material soaking is in warm water, then around limb (reserving toe and hip joint freedom).90 degree positions are kept in described joint, until the mold materials drying.Contralateral leg is with comparing.Allow mice to recover and live in the normal miniature isolation cage then from narcotism.Not observed casting and having caused excessively and coerce, and animal moves freely in cage and takes food and drink water.But mice monitoring every day is influenced body weight, activity and excited any adverse events.

In case a kind of mould is applied to mice, monitors animal every day and remain on original position, because chew to guarantee mould.Animal is removable behind anesthesia recovery, drinking-water and feed, and they do not need special straw mattress, cage frame or other auxiliary facilities.

Denervation

Usually, be used for denervation with different fluorane gas anesthesia mice.Use aseptic operation operation (povidone iodine is washed 3 times, uses washing with alcohol at last), in the thigh intermediate section from the sciatic nerve on right side and prescind 2 to 5mm part.Contralateral leg is with comparing.

More particularly, seal skin incision, and allowing before narcotism is recovered, to use the buprenorphine of single dose to inject animal with suture clip.Operation back 3,7 or 14 days is by sucking CO ₂, by cervical dislocation animal is sentenced euthanasia subsequently, and taking-up muscle (gastrocnemius complex, tibialis anterior, extensor digitorum longus, musculus soleus) is used for histology and biochemical analysis.

Consider sciatic nerve by crosscut, make affected limb not actively to induce the skeletal muscle atrophy of relevant muscle.Described animal is also removable behind anesthesia recovery, drinking-water and feed, and they do not need special straw mattress, cage frame or other auxiliary facilities.However, (1 to 2 hour) monitors animal immediately in operation back and the recovery process.In addition, operation back monitoring incision site and general animal health are 3 days.7 to the 10 days removal suture clips in operation back.

Genetic model

The transgenic mice of genetic manipulation also can be used as amyotrophic model.For example, so-called Mini mice (Jackson laboratory, storage number 003258) contains the sudden change of IGF-1 gene knockout, and it causes postnatal growth to slow down unusually, and body weight and size are low.For extraneous information, consult Powell-Braxton etc., Genes Dev 7:2609-2617,1993.In addition, so-called Midi mice (Jackson laboratory, storage number 003259) contains the difference sudden change of IGF-1 gene, and it causes showing as the hypomorph of low adult body weight He other cardiovascular phenotypes.For extraneous information, consult Lembo etc., J Clin Invest 98:2648-2655,1996.

The key of IGF precursor and optional sudden change or modification

Crucial sudden change the present invention part is based on such observation, and the IGF precursor polypeptide that promptly contains its E peptide substantially keeps biological activity and stability under the situation that serum exists.In order to guarantee that the E peptide is not cut by the endogenous proteinase of targeting two bases (dibasic) protease site, usually with any aminoacid deletion, sudden change in two N-terminal, the two base aminoacid of E peptide in the precursor, or hidden.Under the situation of hIGF-1, these two aminoacid are R71 and S72, and under the situation of hIGF-2, these preceding two aminoacid are R68 and D69.

Many kinds are modified and can be prevented cutting:

(1) one or two two bases residue disappearance

(2) make one or two two bases residue sport non-basic amino acid, as alanine

(3) between two base residues, insert one or more non-basic amino acids

(4) near two base residues, put enough glycosylation sites of hidden protease site

(5) as described below, use alpha-non-natural amino acid to replace arbitrary two base residues, be inserted near the two base residues or between carry out site-directed Pegylation.

In addition, residue K68 and K65 seem to work in the cutting of IGF-1/E peptide; Therefore, the sudden change of these residues or disappearance can be incorporated into sensing as described above in the amino acid whose any strategy of two bases.

The sudden change at the N-terminal place of ripe IGF in certain embodiments of the invention, several N-terminal aminoacid deletion or sudden change before the IGF precursor polypeptide.Under the situation of IGF-1, any one in first three N-terminal aminoacid can be lacked or be suddenlyd change, yet under the situation of IGF-2, any one in the first six N-terminal aminoacid can be lacked or suddenly change.Observed some N-terminal aminoacid and carried out natural cutting in vivo, and the introducing of these sudden changes or disappearance makes the interior dependency of polypeptide of the present invention and igf binding protein (IGFBP) reduce to minimum.The interaction of IGF-1 and IGF-2 and IGF-1 receptor is subjected to the adjusting of IGFBP.All 6 IGFBP (particularly IGFBP5) have shown the effect that can suppress IGF, but in some instances, have observed stimulation.At least 99% IGF is usually in conjunction with IGFBP in the circulation.The abundantest IGFBP is IGFBP3 in circulating behind the neonatal period, and it can be with similar affinity in conjunction with IGF-1 and IGF-2.The IGF-1 of the truncate of natural generation (carrying the disappearance of G1, P2 and E3) with the affinity that is lower than natural IGF-1 several times in conjunction with IGFBP3.In addition, in conjunction with important, G6 plays a part similar in the IGF-2 peptide G3 to IGFBP.

Therefore, under the situation of hIGF-1 precursor, any G1, P2 or E3 can lack or suddenly change alone or in combination.When the expectation sudden change, can introduce sudden change to alanine.In another example, under the situation of hIGF-2 precursor, any P4, S5 and E6 can lack or suddenly change alone or in combination.When the expectation sudden change, can introduce sudden change to alanine.

The serine protease cutting that the sudden change IGF-1 at residue 36 and 37 places can be existed in the human serum.R36 or R37 sport A can prevent that IGF-1 this expectation cleavage site place between R36 and R37 from cutting.Under the situation of hIGF-2, R38 sudden change or disappearance can be prevented this harmful cutting.

Glycosylated purposes can be by the half-life in the body of improveing polypeptide of the present invention to the IGF or the E peptide moiety interpolation N connection glycosylation site of precursor when expressing in the mammal that can carry out N connection glycosylation or other eukaryotic cells.External shown people IGF-1Ea at N92 and N100 place by glycosylation connect glycosylation sequences N-X-S/T because these parts of Ea are fit to total N, wherein X can be that the 3rd aminoacid of any aminoacid and triplet is S or T.How consumingly the contiguous aminoacid background that also is known that consensus sequence will influence the glycosylation of agedoite quilt.Therefore, a kind of strategy to Eb or Ec introducing glycosylation site is to insert consensus sequence Ea aminoacid on every side to the part that is roughly the same of Eb or Ec.In following examples, illustrated should strategy detailed enforcement.In any case, can in precursor polypeptide of the present invention, insert any other total N well known by persons skilled in the art and connect glycosylation site (comprising around background amino acid whose).In addition, can connect glycosylation by the O that the specific host of selecting to be used to produce polypeptide is finished polypeptide of the present invention.For example, the yeast strain of using some to be used for the IGF-1 expression causes adding oligosaccharide on serine or threonine.For example consult U.S. Patent number 5,273,966.

Add poly-(ethylene glycol) verified poly-(ethylene glycol) (PEG that is conjugated to; Pegylation) upward useful to the half-life of extended treatment pharmaceutical grade protein.The Pegylation of expecting IGF precursor polypeptide of the present invention can cause similar pharmacy benefit.The method of IGF-1 Pegylation is known in the art.Consult, for example, U.S. Patent application publication 2006/0154865, it has described the beneficial property of lysine list Pegylation IGF-1.This lysine list Pegylation is fit to precursor I GF polypeptide of the present invention.In addition, can finish Pegylation by introducing alpha-non-natural amino acid in any part of polypeptide of the present invention.Can be by Deiters etc., J Am Chem Soc 125:11782-11783,2003; Wang and Schultz, Science 301:964-967,2003; Wang etc., Science 292:498-500,2001; Zhang etc., Science 303:371-373,2004 or U.S. Patent number 7,083,970 in the technology described introduce some alpha-non-natural amino acid.Briefly, some in these expression systems comprise that site-directed mutation is to introduce nonsense codon (as succinum TAG) in the open reading-frame of code book invention polypeptide.Then this type of expression vector is introduced among the host, described host can use for the nonsense codon of introducing tRNA special and that be responsible for selecting alpha-non-natural amino acid.The useful specific alpha-non-natural amino acid of purpose that part is conjugated to polypeptide of the present invention is comprised those aminoacid with acetylene and azido side chain.The IGF precursor polypeptide that contains these amino acids then in protein these selected site by Pegylation.In addition, do not contain this type of Pegylation IGF molecule of E peptide as treatment yet.

Many bodies of E peptide are in some pharmacology's background, and the size that increases peptide or pharmaceutical grade protein is useful to guarantee that medicine is stayed a side or the opposite side of blood brain barrier.Because sophisticated IGF molecule is short relatively peptide, even still connect the E peptide, the size that increases polypeptide of the present invention is useful.A kind of means of doing like this are many bodies that the E peptide is provided at the C-terminal of IGF precursor polypeptide, as what illustrate among some embodiment that describes hereinafter.

The C-terminal disappearance of E peptide suspects that the free cysteine of Eb the 81st position may produce more other effects of low activity medicine in the time of can causing homologous dimerizationization maybe in being present in polypeptide of the present invention.Therefore, the disappearance of C81 or sudden change can be optimized pharmaceutically active among the Eb, and in particular instance, the disappearance of last 7 aminoacid of Eb (being aminoacid 81-87) is useful.

Other sudden changes or be modified at U.S. Patent number 5,077,276; With in the U.S. Patent application publication number 2005/0287151,2006/0211606 and 2006/0166328 other sudden changes or the modification that can be incorporated into the IGF in the IGF precursor polypeptide of the present invention described.

Except people IGF-1 and IGF-2, should explain to the present invention includes all known and unknown non-human animal's precursor I GF-1 or IGF-2 sequences that described sequence contains its E peptide substantially, wherein can avoid or reduce the normal cutting of E peptide according to modification of the present invention.

The preferred type of IGF to be used depends on experimenter's to be treated kind.

Preferably IGF is the species couplings, and for example, when the treatment cow, the preferred type of IGF is cattle IGF.

Though the form of ownership of IGF has effect probably because of high sequence homology in different experimenters, the species coupling will avoid originating from the inductive potential unfavorable immunologic complication to the immunne response of different plant species IGF.

In one embodiment of the invention, provide non-human animal's precursor I GF-1 sequence of modifying.

The IGF-1 sequence that preferably contains its E peptide substantially is wherein according to modify the normal cutting of avoiding or reduced the E peptide from vertebrate the present invention.

For example, this type of sequence is including, but not limited to the sequence from mice, rat, cow, pig, horse, sheep, goat, bird, Canis familiaris L., cat, fish etc., the sequence of no matter natural from any source, synthetic or reorganization.

In another embodiment of the invention, provide non-human animal's precursor I GF-2 sequence of modifying.

The IGF-2 sequence that preferably contains its E peptide substantially is wherein according to modify the normal cutting of avoiding or reduced the E peptide from vertebrate the present invention.

For example, this type of sequence is including, but not limited to the sequence from mice, rat, cow, pig, horse, sheep, goat, bird, Canis familiaris L., cat, fish etc., the sequence of no matter natural from any source, synthetic or reorganization.

The therapeutic use of IGF precursor polypeptide

Indication the present invention comprises that also IGF precursor polypeptide of the present invention is used for the treatment of or prevents purposes in the medicine of musculoskeletal disease in preparation.In addition, the present invention includes IGF precursor polypeptide increases muscle quantities or bone amount in individuality purposes, no matter should individuality whether be in the danger of musculoskeletal disease or suffer from musculoskeletal disease.

Particularly, musculoskeletal disease can be amyotrophy.Amyotrophy has many reasons, comprises the result with glucocorticoid (as hydrocortisone, dexamethasone, betamethasone, prednisone, methylprednisolone or prednisolone) treatment.Amyotrophy also can be the result of the denervated result that causes because of neural wound or degeneration, metabolism or inflammatory neuropathy (for example, Guillian-Barre syndrome, peripheral neurophaty or be exposed to environmental toxin or medicine).In addition, amyotrophy can be adult's motor neuron, infantile spinal muscular atrophy, the hebephrenictype Duchenne-Arandisease, autoimmune motor neuron with many focuses conductor retardance (multifocal conductor block), the paralysis that causes because of apoplexy or spinal cord injury, the skeletal fixationization that causes because of wound, CBR, voluntary inertia (voluntary inactivity), involuntary inertia (involuntary inactivity), metabolic stress or undernutrition, cancer, AIDS, fasting, rhabdomyolysis, thyroid disorders, diabetes, optimum congenital tension force is low excessively, central core disease, thread like body (nemalene) myopathy, myotubular myopathy (centronuclear myopathy), burn, chronic obstructive pulmonary disease, hepatopathy, sepsis, renal failure, congestive heart failure, or old and feeble result.

Musculoskeletal disease also can be a leyden-Mobius myodystrophia, as Duchenne, Becker, myotonia, fascioscapulohumeral, Emery-Deifuss, eye pharynx, omoplate humerus, limb girdle, congenital muscular dystrophy or hereditary distal myopathy.Musculoskeletal disease also can be osteoporosis, fracture, short stature or dwarfism.

Suggestion IGF-1 treatment insulin insensitivity type diabetes are because IGF-1 also can be in conjunction with the heterodimer of IGF-1 receptor and Insulin receptor INSR.Therefore, polypeptide of the present invention can be used for treating diabetes.

IGF-1 is neurophic and improves neuronic survival.Pointed out IGF-1 to can be used for the situation of treatment as seen in motor neuron death in amyotrophic lateral sclerosis (ALS), brain atrophy, aging and the dementia.Therefore, polypeptide of the present invention can be used for treatment and the relevant disease of neuronal death (as ALS, brain atrophy or dementia).

IGF-1 leukocyte increasing group and red blood cell mass, and have additive effect to using erythropoietin.Therefore, polypeptide of the present invention can be used for treating anemia.

Because IGF-1 and IGF-2 are the ubiquity and the essential regulon of the growth of cell division and vertebrates, they can be advantageously used in multiple veterinary's method and strengthen with external source or keep growth of animal.Some examples include, but are not limited to:

(i) speed and/or the degree of raising growth of animal for example strengthen the muscle growth in pig, cattle, poultry and the fish;

(ii) for example in pig, cattle, sheep, poultry and fish, improve them and become systemic efficient (lean meat is to the ratio of fat) from food conversion; With

(iii) improve the milk production in the lactogenic animal (for example milk cattle, sheep, goat).

Other veterinary treatments are used and are included, but are not limited to:

(iv) treat animal (for example companion animals, as Canis familiaris L., cat and horse) the become thin symptom relevant with cachexia, wound or other wasting diseasess; With

(v) treat the lactogenic animal and be used to improve neonatal health status, for example treat lactating sow and be used to improve the neonate performance.

Application process can be sent polypeptide of the present invention by multiple mode (comprise and use the gene delivery carrier).The method that is used for the treatment of delivered substance (as protein or nucleic acid) known in the art can be used for treatment and sends polypeptide of the present invention, for example cell transfecting, gene therapy, directly use, send indirectly with delivery vehicle or pharmaceutically suitable carrier by the reconstitution cell that the nucleic acid that contains coding said polypeptide is provided.

Multiple delivery system is known and can be used for using polypeptide of the present invention, described system for example encapsulated, the microgranule in the liposome, microcapsule, can marking protein reconstitution cell, receptor-mediated endocytosis (consult, for example, Wu and Wu, J Biol Chem 262:4429-4432,1987), make up as the nucleic acid of retrovirus, adeno associated virus, adenovirus, poxvirus (for example, fowlpox virus, particularly bird pox virus) or other carrier parts etc.The method of introducing can be enteral or parenteral, and including, but not limited to Intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, lung, intranasal, ophthalmic, epidural and oral route.Can be by any approach that makes things convenient for, for example by infusion or inject (bolus injection), (for example by epithelium or mucocutaneous linings, oral mucosa, mucous membrane of rectum and intestinal mucosa etc.) absorption use polypeptide, and it can be used jointly with the other biological activating agent.Use can be whole body or partial.In addition, can expect to introduce pharmaceutical composition of the present invention to the central nervous system by any suitable pathways (comprising indoor and intrathecal injection); Can help indoor injection by indoor conduit (for example being attached to the indoor conduit of storage (as ommaya reservoir)).For example also can use pulmonary administration by using inhaler or aerosol apparatus and the preparation that contains spray (aerosolizing agent).

In specific embodiments, can expect regional local application pharmaceutical composition of the present invention to needs treatments; For example and unrestricted, this can by local infusion in the operation process, topical application (for example by injection, by conduit or pass through implant, described implant can be porous, atresia or gel-like material, comprise film, as sialastic film, fiber or commercial skin substitutes) finish.

In another embodiment, activating agent can be with vesicle, and particularly liposome is sent (consulting Langer, Science 249:1527-1533,1990).In another embodiment, activating agent can be sent with controlled release system.In one embodiment, can use pump.In another embodiment, can use polymeric material (consulting Howard etc., J Neurosurg 71:105,1989).In another embodiment, when activating agent wherein of the present invention is the nucleic acid of code book invention polypeptide, can become the part of suitable nucleic acid expression vector by making up described nucleic acid, and for example by using retroviral vector (for example to consult, U.S. Patent number 4,980,286) or pass through direct injection, or by using microparticle bombardment (for example, particle gun; Biolistic, Dupont), or with lipid or cell surface receptor or transfection agents bag quilt, or by with known co-administered (for example the consulting of homeobox sample peptide that enters nuclear, people such as Joliot, Proc.Natl.Acad.Sci.USA 88:1864-1868,1991) wait and use it and make it become intracellular nucleic acid, express to promote its encoded protein matter thereby use described nucleic acid in vivo.Perhaps, nucleic acid can be introduced in the cell and be incorporated into and be used in the host cell DNA expressing by homologous recombination.

The nucleic acid that interior transfection of cell and gene therapy the present invention includes code book invention polypeptide is used for external and the purposes cells in vivo transfection.These nucleic acid can be inserted in many well-known any carriers of carrier that are used for target cell and biological transfection.Nucleic acid can by exsomatize with carrier and neuron target cell interaction and in vivo transfection to cell.Use compositions (for example passing through) to the amount that the experimenter replys with enough initiation treatments to intramuscular injection.

On the other hand, the invention provides the method for target site (being target cell or tissue) in treatment people or other animals, comprise the nucleic acid transfection cell with code book invention polypeptide, wherein said nucleic acid comprises that effectively being connected to the coding target decides inducible promoter on the nucleic acid of fused polypeptide.For the gene therapy method in treatment or the prevention human diseases, for example consult Van Brunt Biotechnology 6:1149-1154,1998.

Conjoint therapy in many embodiments, polypeptide of the present invention can plant added compound with one or more or therapy is co-administered.For example, a large amount of polypeptide can be used jointly with one or more kind treatment chemical compound associatings.Conjoint therapy can comprise simultaneously or alternately use.In addition, described combination can comprise acute or chronic using.Polypeptide of the present invention can be co-administered with anabolic agent such as testosterone or specificity androgen receptor modifier (SARM).The molecule that extra anabolic agent comprises growth hormone (GH) or induces GH to discharge.Ge Ruilin is used in particular for cachectic conjoint therapy, because Ge Ruilin can cause the increase of appetite.Similarly, polypeptide of the present invention can make up with the acceleration anabolism with protein supplements, or makes up with weight increase with physiotherapy or exercise.Any molecule that suppresses myostatin also is the candidate of conjoint therapy.

Pharmaceutical composition the present invention also provides the pharmaceutical composition that comprises IGF precursor protein matter of the present invention and pharmaceutically suitable carrier.Term " pharmaceutically useful " refers to federation management mechanism or government permission, or list in American Pharmacopeia or other generally acknowledged pharmacopeia that is used for the animal or human.Term " carrier " refers to the used diluent of administering therapeutic agent, adjuvant, excipient or carrier.This type of pharmaceutical carrier can be a sterile liquid, and Ru Shui and oil comprise oil, animal, plant or synthetic those that originate from, as Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Semen Sesami wet goods.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glyceryl monostearate, Talcum, sodium chloride, defatted milk powder, glycerol, propylene, ethylene glycol, water, ethanol etc.If desired, compositions also can contain a spot of wetting agent or emulsifying agent, or the pH buffer agent.These compositionss can be taked the form of solution, suspensoid, Emulsion, tablet, pill, capsule, powder, slow releasing preparation etc.Compositions can be mixed with the suppository with conventional binding agent and carrier (as triglyceride).Oral formulations can comprise standard vector, as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.E.W.Martin has described the example of suitable drug carrier in " Remington ' s Pharmaceutical Sciences ".

In some embodiments, according to conventional methods compositions is mixed with the pharmaceutical composition that is suitable in the human vein, using.When needing, described compositions can comprise that also solubilizing agent and local anesthetic (as lignocaine) are to alleviate the pain at injection site place.When using compositions by infusion, described compositions can be made up a prescription with containing aseptic pharmaceutical grade water or brinish infusion bottle.When using compositions, can provide Injectable sterile water or brinish ampoule to make composition before using, can mix by injection.

Polypeptide of the present invention can be mixed with neutrality or salt form.Officinal salt comprises those that free amine group forms, as from those of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., those that form with free carboxy are as from those of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, hydrated ferric oxide., 2-aminopropane., triethylamine, 2-ethylaminoethanol, histidine, procaine etc.

Can be determined at the amount of effective polypeptide of the present invention in disease or the treatment of diseases based on this description by the clinical technology of standard.In addition, can randomly use external test to help identify the optimal dosage scope.The exact dose that uses in the preparation also will depend on the seriousness of route of administration and disease, and should decide according to the judgement of practitioner and each experimenter's situation.Yet the suitable dosage ranges that intravenous is used is generally the reactive compound of the about 20-5000 microgram of every kg body weight.The suitable dosage ranges of intranasal administration is generally about 0.01pg/kg body weight to the 1mg/kg body weight.Can be from from the extrapolated effective dose of dose-effect curve external or the animal model test macro.Particularly, possible dosage can be about 60 to 120 μ g/kg body weight, subcutaneous injection, twice of every day.

Veterinary purpose

Except above-mentioned application process in the people, may consider also that the veterinary uses.

When using to healthy animal with respect to when those animals that suffer from disease are used, described dosage can be different.Those skilled in the art use mensuration known in the art (for example sarcoplast proliferation assay of hereinafter describing (embodiment 79) or breast epithelial tissue are measured (embodiment 80)) easily to carry out the assessment of suitable dosage.The general algoscopy of measuring IGF is known in the art, as those algoscopys among the embodiment 81.

Some species that those skilled in the art will recognize that animal are subjected to the photoperiod effect length to show seasonal fertility.Any embodiment of veterinary's method or purposes can randomly be included in interior special time begin treatment method of animal reproduction cycle to reach desired effects.One skilled in the art will know that and easily to determine reproductive status and cycle, and can make its synchronization by using suitable scheme if desired.

When being used for veterinary's indication, be used for human purposes method except previously mentioned, IGF-1 of the present invention or IGF-2 peptide also can be used as the oral veterinary draught, or the fill-in of the oral or solid feed of animal.

Further describe and unrestricted the present invention by following examples.

Embodiment

Embodiment 1

Make up the DNA expression vector of coding hIGF-1-Ea precursor polypeptide, described polypeptide contains following modification: G1 disappearance, P2 disappearance and E3 disappearance; R37 is mutated into A; And R71 disappearance and S72 disappearance.These sudden changes are called " 3mut " in this disclosure sometimes.This produces following secreted protein sequence:

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：8)

Cos7 cell (deriving from ATCC) is maintained among the DMEM that contains 10% hyclone, 100U/ml penicillin and 100 μ g/ml streptomycins and with dull and stereotyped 1 x 10 of every 10-cm ⁶The density of cell is placed.Use Fugene (Luo Qie company (Roche)) according to manufacturer's description these cell cultures of expression plasmid transfection with 8 μ g.After the transfection 24 hours, washed cell once and in serum-free medium was cultivated 48 hours.Collect supernatant and be stored in-80 ℃.

In order to assess the stability of polypeptide in human serum, will from transfection have the supernatant collected the Cos7 cell of wild type (wt) hIGF-1Ea and hIGF-1Ea3mut 10% human serum (Sigma company (Sigma)) exist or non-existent situation under 37 ℃ of incubations 16 hours.By the 18%SDS-PAGE sample separation, and the goat polyclonal antibody of end user IGF-1 carries out immunoblotting.Result among Figure 1A shows, with the serum incubation after 16 hours wt hIGF-1Ea degrade substantially, and hIGF-1Ea3mut is stable.Light densitometry shows that the ratio of the IGF-1 of uncut IGF-1 and cutting is about 1:6.2, and the ratio of hIGF-1Ea3mut is about 1:0.68, illustrates that these sudden changes produce stable polypeptide.

In order to confirm that hIGF-1Ea3mut can carry out signal transduction by IGF-1R, measure the AKT phosphorylation of the cell that contacts with polypeptide.C2C12 buys in ATCC and maintains in Dulbecco improvement Eagle ' the s culture medium (DMEM) of the high glucose of tool (hero's life sciences company (Invitrogen)) that contains 10% hyclone (AMIMED), 100U/ml penicillin (hero's life sciences company (Invitrogen)), 100 μ g/ml streptomycins (hero's life sciences company (Invitrogen)) and 2mM glutamine (hero's life sciences company (Invitrogen)).In order to analyze the AKT phosphorylation, with the hole 0.15 * 10 of each 6 orifice plate ⁶The density coating C2C12 cell of individual cell was also cultivated 72 hours in growth medium.Make cell in serum-free medium hungry 4 hours, stimulated 30 minutes at 37 ℃ with different ligands then.Also pass through at 4 ℃ with the PhosphoSafe buffer that contains the multiple protein enzyme inhibitor (Cell Signaling) cell lysis, 14,000 * g removed in centrifugal 15 minutes.Use PathScan phospho AKT (Ser473) sandwich ELISA test kit and PathScan AKT sandwich ELISA test kit (Cell Signaling) by elisa assay AKT phosphorylation and total AKT level respectively.The results are summarized among Fig. 2 A of AKT phosphorylation, it shows that hIGF-1Ea3mut can be with the degree activation IGF-1R cellular pathways similar with reorganization IGF-1 to length-R3-IGF-1 positive control reagent.In addition, the data among Fig. 5 show that directly hIGF-1Ea3mut causes the AKT phosphorylation.

Next, in order to guarantee that hIGF-1Ea3mut keeps and the receptor-specific of IGF-1R, in the NIH3T3 culture of crossing expression IGF-1R or Insulin receptor INSR (InsR), add multiple part.These cells with as the same terms of above-mentioned Cos7 cell culture under cultivate.In order to analyze the phosphorylation of IGF-1R and InsR, with the hole 0.2 * 10 of each 6 orifice plate ⁶The density coating NIH3T3-IGF1R of cell and NIH3T3-InsR cell were also cultivated 24 hours in growth medium.Make cell in serum-free medium hungry 18 hours, stimulated 10 minutes at 37 ℃ with different ligands then.Be used for AKT experiment as above-mentioned cell lysis, and use DuoSet IC people phosphor-IGF1R and-InsR ELISA the test kit ((R﹠amp of An Di system house; D Systems)) phosphorylation level by elisa assay IGF-1R and InsR.Be summarized in Fig. 3 A and result among the 3B and show that this IGF-1 precursor polypeptide keeps the specificity of IGF-1 receptor and should be attached on the relevant Insulin receptor INSR with low-affinity.

Embodiment 2

Make up the DNA expression vector of coding hIGF-1-Eb precursor polypeptide, described polypeptide contains following sudden change: G1 disappearance, P2 disappearance and E3 disappearance; R37 is mutated into A; With R71 disappearance and S72 disappearance (that is, described " 3mut ").This produces following secreted protein sequence:

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：9)

Measure polypeptide according to the method for describing among the embodiment 1 above.The use of Figure 1B and light densitometry shows that the ratio of the IGF-1 of uncut IGF-1 and cutting is about 1:9, and the ratio of hIGF-1Eb3mut is about 1:1, illustrates that these modifications produce stable polypeptide.Fig. 2 B shows that hIGF-1Eb3mut can be with the degree activation IGF-1R cellular pathways similar with reorganization IGF-1 to length-R3-IGF-1 positive control reagent.In addition, the data among Fig. 5 show that directly hIGF-1Eb3mut causes the AKT phosphorylation.Be summarized in Fig. 3 C and result among the 3D and show that this IGF-1 precursor polypeptide keeps the specificity of IGF-1 receptor and should be attached on the relevant Insulin receptor INSR with low-affinity.

Embodiment 3

Make up the DNA expression vector of coding hIGF-1-Ec precursor polypeptide, described polypeptide contains following sudden change: G1 disappearance, P2 disappearance and E3 disappearance; R37 is mutated into A; With R71 disappearance and S72 disappearance (that is, described " 3mut ").This produces following secreted protein sequence:

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：10)

Measure polypeptide according to the method for describing among the embodiment 1 above.The use of Fig. 1 C and light densitometry shows that the ratio of the IGF-1 of uncut IGF-1 and cutting is about 1:5, and the ratio of hIGF-1Ec3mut is about 1:0.96, illustrates that these modifications produce stable polypeptide.Fig. 2 D shows that hIGF-1Ec3mut can be with the degree activation IGF-1R cellular pathways similar with reorganization IGF-1 to length-R3-IGF-1 positive control reagent.In addition, the data among Fig. 5 show that directly hIGF-1Ec3mut causes the AKT phosphorylation.Be summarized in Fig. 4 A and result among the 4B and show that this IGF-1 precursor polypeptide keeps the specificity of IGF-1 receptor and should be attached on the relevant Insulin receptor INSR with low-affinity.

Embodiment 4

Make up the DNA expression vector of the chimeric precursor polypeptide of coding hIGF-1-Eab, described polypeptide contains following modification to the hIGF-1-Eb peptide: G1 disappearance, P2 disappearance and E3 disappearance; R37 is mutated into A; R71 disappearance and S72 disappearance (that is, described " 3mut "); And the 93rd to 102 amino acids of between Eb the 95th and 96 amino acids, inserting Ea.The supposition N that described insertion has produced the N92 place connects the glycosylation signal.This produces following secreted protein sequence:

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：11)

Measure polypeptide according to the certain methods of describing among the embodiment 1 above.Fig. 2 C shows that hIGF-1Eab3mut can be with the degree activation IGF-1R cellular pathways similar with reorganization IGF-1 to length-R3-IGF-1 positive control reagent.The result who is summarized among Fig. 4 C and the 4D shows that the specificity that this IGF-1 precursor polypeptide keeps the IGF-1 receptor can not activate Insulin receptor INSR.

Embodiment 5

Make up the DNA expression vector of many bodies of coding hIGF-1-Eb precursor polypeptide, described polypeptide contains last 7 C-terminal aminoacid deletion of following sudden change: G1 disappearance, P2 disappearance, E3 disappearance, R36 disappearance, R37 disappearance, R71 disappearance, S72 disappearance, Eb; With two extra Eb peptides (described two peptides all do not contain R71 and S72 and last 7 C-terminal aminoacid) and the 4th be the C-terminal that last Eb peptide inserts (not containing R71 and S72) this precursor.Fig. 6 A has shown the sketch map of this structure.This produces following secreted protein sequence:

tlcgaelvdalqfvcgdrgfyfnkptgygsssapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaersvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaersvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaersvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：12)

This peptide is carried out AKT phosphorylation assay as describing among the embodiment 1.Fig. 5 shows that the many bodies of this hIGF-1-Eb can carry out signal transduction in the IGF-1R approach.

Embodiment 6

Can express the hIGF-1-Eb precursor polypeptide of the present invention that schematically is shown among Fig. 6 B.This construct contains following modification: G1 disappearance, P2 disappearance, E3 disappearance, R36 disappearance, R37 disappearance, R71 disappearance, S72 disappearance; And between Eb the 95th and 96 amino acids, inserted the 93-102 amino acids of Ea, thereby produced N92 and be connected glycosylation site with N100 position N.This produces following secreted protein sequence:

tlcgaelvdalqfvcgdrgfyfnkptgygsssapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：13)

Embodiment 7

Can express the hIGF-2-E precursor polypeptide of the present invention with following modification: P4 disappearance, S5 lacks and E6 lacks; R38 is mutated into A; And R68 disappearance and D69 disappearance.This produces following secreted protein sequence:

ayrtlcggelvdtlqfvcgdrgfyfsrpasrvsrasrgiveeccfrscdlalletycatpaksevstpptvlpdnfprypvgkffqydtwkqstqrlrrglpallrarrghvlakeleafreakrhrplialptqdpahggappemasnrk(SEQ?ID?NO：14)

Embodiment 8

Can express the hIGF-1-Ea precursor polypeptide of the present invention with following sudden change: G1 disappearance and P2 lacks; E3 is mutated into X, and wherein X is by the alpha-non-natural amino acid of Pegylation; R37 is mutated into A; With R71 disappearance and S72 disappearance.This produces following secreted protein sequence:

Xtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：15)

Embodiment 9-78 (Δ=disappearance)

9)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；R36A；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：16)

10)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；R36A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：17)

10)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；R36A；ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：18)

11)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；R37A；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：19)

12)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；R37A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：20)

13)hIGF-1-Ea：ΔG1，ΔP2，ΔE3，ΔR37；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：21)

14)hIGF-1-Ea：ΔG1，ΔP2，ΔE3，ΔR37；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：22)

15)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；ΔR37；ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：23)

16)hIGF-1-Eb：ΔG1，ΔP2，ΔE3；R36A；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：24)

17)hIGF-1-Eb：ΔG1，ΔP2，ΔE3；R36A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：25)

18)hIGF-1-Eb：ΔG1，ΔP2，ΔE3；R37A；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：26)

19)hIGF-1-Eb：ΔG1，ΔP2，ΔE3；R37A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：27)

20)hIGF-1-Eb：ΔG1，ΔP2，ΔE3；R37A；ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：28)

21)hIGF-1-Eb：ΔG1，ΔP2，ΔE3，ΔR37；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：29)

22)hIGF-1-Eb：ΔG1，ΔP2，ΔE3，ΔR37；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：30)

23)hIGF-1-Eb：ΔG1，ΔP2，ΔE3，ΔR37；ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：31)

24)hIGF-1-Ec：ΔG1，ΔP2，ΔE3；R36A；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：32)

25)hIGF-1-Ec：ΔG1，ΔP2，ΔE3；R36A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：33)

26)hIGF-1-Ec：ΔG1，ΔP2，ΔE3；R36A；ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：34)

27)hIGF-1-Ec：ΔG1，ΔP2，ΔE2；R37A；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：35)

28)hIGF-1-Ec：ΔG1，ΔP2，ΔE3；R37A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：36)

29)hIGF-1-Ec：ΔG1，ΔP2，ΔE3；R37A；ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：37)

30)hIGF-1-Ec：ΔG1，ΔP2，ΔE3，ΔR37，ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：38)

31)hIGF-1-Ec：ΔG1，ΔP2，ΔE3，ΔR37，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：39)

32) hIGF-1-Eab: Δ G1, Δ P2, Δ E3; R36A; Δ R71; Between the aminoacid 95 and 96 of EB, insert Ea aminoacid 93-102 (that is ＂ Eab ＂)

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：40)

33)hIGF-1-Eab：ΔG1，ΔP2，ΔE3；R37A；ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：41)

34)hIGF-1-Eab：ΔG1，ΔP2，ΔE3，ΔR37，ΔR71

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksasvraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：42)

35)hIGF-1-Eab：ΔG1，ΔP2，ΔE3；R36A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：43)

36)hIGF-1-Eab：ΔG1，ΔP2，ΔE3；R37A；ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：44)

37)hIGF-1-Eab：ΔG1，ΔP2，ΔE3，ΔR37，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksarvraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：45)

38)hIGF-1-Eab：ΔG1，ΔP2，ΔE3；R36A；ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：46)

39)hIGF-1-Eab：ΔG1，ΔP2，ΔE3，ΔR37，ΔR71，ΔS72

tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：47)

40)hIGF-1-Ea：ΔP2，ΔE3；R37A；ΔR71，ΔS72

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：48)

41)hIGF-1-Eb：ΔP2，ΔE3；R37A；ΔR71，ΔS72

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：49)

42) the many bodies of hIGF-1-Eb: (Δ G1, Δ P2, Δ E3; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQID?NO：50)

43) the many bodies of hIGF-1-Eb: (Δ P2, Δ E3; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：51)

44)hIGF-1-Ec：ΔP2，ΔE3；R37A；ΔR71，ΔS72

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：52)

45)hIGF-1-Ea：ΔE3；R37A；ΔR71，ΔS72

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：53)

46)hIGF-1-Eb：ΔE3；R37A；ΔR71，ΔS72

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：54)

47) the many bodies of hIGF-1-Eb: (Δ G1, Δ P2, Δ E3; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQID?NO：55)

48) the many bodies of hIGF-1-Eb: (Δ E3; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：56)

49)hIGF-1-Ec：ΔE3；R37A；ΔR71，ΔS72

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：57)

50)hIGF-1-Ea：ΔE3；R37A；ΔR71，ΔS72

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：58)

51)hIGF-1-Eb：ΔE3；R37A；ΔR71，ΔS72

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：59)

52)hIGF-1-Ec：ΔE3；R37A；ΔR71，ΔS72

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：60)

53)hIGF-1-Eab：ΔE3；R37A；ΔR71，ΔS72

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：61)

54) the many bodies of hIGF-1-Eb: (Δ E3; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

gptlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：62)

55)hIGF-1-Ea：E3A；R37A；ΔR71，ΔS72

gpatlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：63)

56)hIGF-1-Eb：E3A；R37A；ΔR71，ΔS72

gpatlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：64)

57)hIGF-1-Ec：E3A；R37A；ΔR71，ΔS72

gpatlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：65)

58)hIGF-1-Eab：E3A；R37A；ΔR71，ΔS72

gpatlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：66)

59) the many bodies of hIGF-1-Eb: (E3A; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

gpatlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：67)

60)hIGF-1-Ea：ΔP2，ΔE3；R37A；ΔR71，ΔS72

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：68)

61)hIGF-1-Eb：ΔP2，ΔE3；R37A；ΔR71，ΔS72

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：69)

62)hIGF-1-Ec：ΔP2，ΔE3；R37A；ΔR71，ΔS72

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：181)

63)hIGF-1-Eab：ΔP2，ΔE3；R37A；ΔR71，ΔS72

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：70)

64) the many bodies of hIGF-1-Eb: (Δ P2, Δ E3; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

gtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：71)

65)hIGF-1-Eb：ΔG1，ΔP2；E3X；R37A；ΔR71，ΔS72

Xtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：72)

66)hIGF-1-Ec：ΔG1，ΔP2；E3X；R37A；ΔR71，ΔS72

Xtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：73)

67)hIGF-1-Eab：ΔG1，ΔP2；E3X；R37A；ΔR71，ΔS72

Xtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：74)

68) the many bodies of hIGF-1-Eb: (Δ G1, Δ P2; E3X; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72)

Xtlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：75)

69)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；R37A；ΔR71；S72X

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksaXvraqrhtdmpktqkevhlknasrgsagnknyrm(SEQ?ID?NO：76)

70)hIGF-1-Eb：ΔG1，ΔP2，ΔE3；R37A；ΔR71；S72X

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksaXvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：77)

71)hIGF-1-Ec：ΔG1，ΔP2，ΔE3；R37A；ΔR71；S72X

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksaXvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk(SEQ?ID?NO：78)

72)hIGF-1-Eab：ΔG1，ΔP2，ΔE3；R37A；ΔR71；S72X

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscd?lrrlemycaplkpaksaXvraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：79)

73) the many bodies of hIGF-1-Eb: (Δ G1, Δ P2, Δ E3; R37A)-Eb (Δ R71; S72X; Δ C-term 7aa)-2xEb (Δ R71, Δ S72, Δ C-term 7aa)-Eb (Δ R71, Δ S72)

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpakXsavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk(SEQ?ID?NO：80)

74)hIGF-1-Ea：ΔG1，ΔP2，ΔE3；R37A；ΔR71，ΔS72；N92X

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlkXasrgsagnknyrm(SEQ?ID?NO：81)

75)hIGF-1-Eb：ΔG1，ΔP2，ΔE3；R37A；ΔR71，ΔS72；C142X

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaeXrgkkgk(SEQ?ID?NO：82)

76)hIGF-1-Eab：ΔG1，ΔP2，ΔE3；R37A；ΔR71，ΔS72；C151X

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnknasrgsagnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaeXrgkkgk(SEQ?ID?NO：83)

78) the many bodies of hIGF-1-Eb (Δ G1, Δ P2, Δ E3; R37A)-3xEb (Δ R71, Δ S72, Δ C-term7aa)-Eb (Δ R71, Δ S72; C71X)

tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaevraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaeXrgkkgk(SEQID?NO：84)

Embodiment 79 sarcoplast proliferation assay

The sarcoplast proliferation assay provides reliable IGF active body external indicator, and is used as the model of the factor that influences embryo sarcoplast and adult satellite cell.The activated factor shows similar in myoblastic primary culture in this system.Increase the sarcoplast in-vitro multiplication by peptide of the present invention and show that it is causing that sarcoplast propagation increases also thereby increase the activity in the final muscle fiber quantity in the uterus.In addition, sarcoplast propagation similarly increase shows that peptide of the present invention can be used for strengthening adult muscle hypertrophy, for example by stimulating the satellite muscle cell proliferation.

Embodiment 80: the breast epithelial tissue is measured

In the lactogenic animal, the amount of breast epithelial tissue is the limiting factor of milk production, because they are cells of galactopoiesis and lactogenic.Use vitro system, can stimulate the milk composition of breeding and produce recruitment from the epithelial cell of animal's mammary gland acquisition by IGF-1 or the IGF-2 that the present invention modifies.Can prove further that the breast epithelial cell that is stimulated proliferation can be implanted in the clean mammary fat pad again and quilt stimulates with propagation and/or galactopoiesis in the lactogenic jenny in a kind of this cell in vitro system.

Embodiment 81: the mensuration of IGF-1 or IGF-2 in blood or other body fluid

Can be by the bent effective dose of measuring the peptide of every dosage parenteral administration of dose-response.For example, can in experimenter's to be treated blood or body fluid, measure the IGF peptide of modification of the present invention to determine dosage.Perhaps, can use the peptide of recruitment and check experimenter's IGF-1 of modification and the serum levels of IGF-2 to the experimenter.Can on mole foundation, calculate the amount of peptide to be used based on the serum levels of the IGF-1 of these modifications or IGF-2.

A kind of method that is used for measuring the suitable dosage of peptide can be measured the IGF peptide of the present invention of biofluid (as body fluid or blood).Can finish (comprising RIA and ELISA) measurement of this level by any means.After measuring the IGF level, use single dose or multiple dose that fluid is contacted with peptide.After this contact procedure, in fluid, remeasure the IGF level.The amount (described effect is that molecule will be applied the effect that is used for) of enough generation expectation effects if fluid IGF level has descended, the dosage of so adjustable whole molecule is to produce maximum effect.This method can be carried out in external or body.Preferably, this method is carried out in vivo, after promptly extracting fluid and measure the IGF level from the experimenter, uses single dose or multiple dose to the peptide of administration this paper (promptly, contact procedure is finished by using to animal), remeasure the IGF level from the fluid that animal is extracted then.

The another kind of method that is used to measure dosage is to use the another kind of detection method of the peptide of the antibody of described peptide or LIFA form.

The interior medicine dynamics of embodiment 82:hIGF-1-Ec3mut

Bull mice (n=3/ group) is accepted intravenous (i.v.) and injects the rhIGF-1 of 1mg/kg and the hIGF-1-Ec3mut of 1.55mg/kg (described in the embodiment 3).Use the back at test material and collected a series of blood sample at 5,15,30 and 60 minutes.Measure the serum-concentration of rhIGF-1 and hIGF-1-Ec3mut by ELISA.This mensuration is special to hIGF-1.

The rhIGF-1 and the hIGF-1-Ec3mut of molar dose such as in the mice medium-sized vein, use.The result is presented at all review time points and goes up than rhIGF-1, and the hIGF-1-Ec3mut protein level is significantly higher, shows that hIGF-1-Ec3mut is more stable in metabolism than the IGF-1 of 70 amino acid longs.

Claims (27)

1. the polypeptide that comprises people IGF-1 precursor protein matter wherein reduces the modification of the cutting of E peptide from the IGF-1 by described precursor protein matter by protease.

2. the described polypeptide of claim 1, wherein said precursor protein matter comprises the Ea peptide.

3. the described polypeptide of claim 1, wherein said precursor protein matter comprises the Eb peptide.

4. the described polypeptide of claim 3, wherein last 7 C-terminal aminoacid deletion of Eb.

5. the described polypeptide of claim 1, wherein said precursor protein matter comprises the Ec peptide.

6. the described polypeptide of any aforementioned claim, the G1 of wherein said precursor protein matter is lacked or is suddenlyd change.

7. the described polypeptide of any aforementioned claim, the P2 of wherein said precursor protein matter is lacked or is suddenlyd change.

8. the described polypeptide of any aforementioned claim, the E3 of wherein said precursor protein matter is lacked or is suddenlyd change.

9. the described polypeptide of any aforementioned claim, the R36 of wherein said precursor protein matter is lacked or is suddenlyd change.

10. the described polypeptide of any aforementioned claim, wherein R36 is mutated into alanine.

11. the described polypeptide of any aforementioned claim, the R37 of wherein said precursor protein matter is lacked or is suddenlyd change.

12. the described polypeptide of any aforementioned claim, wherein R37 is mutated into alanine.

13. the described polypeptide of any aforementioned claim, it also comprises the glycosylation consensus sequence NXS/T that N connects.

14. the described polypeptide of claim 3, it also comprises the amino acid N 95 that is inserted into Eb and the 93-102 amino acids of the Ea between the T96.

15. the described polypeptide of any aforementioned claim, it also comprises covalently bound oligosaccharide to the amino acid side chain of described precursor protein matter.

16. the described polypeptide of claim 15, wherein said oligosaccharide is covalently bound to the arginine side chain of described precursor protein matter.

17. the described polypeptide of any aforementioned claim, the residue of wherein said precursor protein matter is replaced by alpha-non-natural amino acid.

18. the described polypeptide of claim 17, wherein said alpha-non-natural amino acid comprises acetylene or azido group.

19. the described polypeptide of any aforementioned claim, it also comprises the polyalkylene glycol moiety on the side chain that is covalently bound to described precursor protein matter.

20. the described polypeptide of any aforementioned claim, it also comprises the extra E peptide of the C-terminal that is connected to described precursor protein matter.

21. polypeptide, it comprises, from the N-terminal to the C-terminal

IGF-1 precursor protein matter, described precursor protein matter contains first Eb peptide, wherein

G1, P1 and E1 disappearance,

R36 and R37 disappearance,

R71 and S72 disappearance, and

Last 7 C-terminal aminoacid deletion of first Eb peptide;

Second Eb peptide, wherein last 7 C-terminal aminoacid deletion of R71, S72 and second Eb peptide;

The 3rd Eb peptide, wherein last 7 C-terminal aminoacid deletion of R71, S72 and the 3rd Eb peptide; With

The 4th Eb peptide, wherein R71 and S72 disappearance.

22. each described polypeptide in the claim 1 to 20, the R71 of wherein said precursor protein matter or S72 are lacked or are suddenlyd change.

23. comprise the polypeptide of people IGF-2 precursor protein matter, wherein the modification of the cutting of E peptide from the IGF-2 by described precursor protein matter reduced by protease.

24. the described polypeptide of claim 23, the R68 of wherein said precursor protein matter or D69 are lacked or are suddenlyd change.

25. the method for treatment musculoskeletal disease, diabetes, neuronal cell death or anemia, described method comprises the polypeptide to any aforementioned claim of experimenter's administering therapeutic effective dose.

26. each described polypeptide is used to prepare the purposes of the medicine that is used for the treatment of musculoskeletal disease, diabetes, neuronal cell death or anemia in the claim 1 to 24.

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