Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1: preparation of monomeric peptides and dimers
Firstly, a monomer peptide synthesis process: manual solid phase peptide synthesis operation steps.
1. Swelling resin: amino resin (amino resin for C-terminal amidated sequence) (available from Nankai Synthesis Technologies, Inc., Tianjin) was placed in a reaction vessel, and 15ml/g of methylene chloride (DCM, Dikma Technologies Inc.) was added thereto and shaken for 30min. SYMPHONY type 12-channel polypeptide synthesizer (SYMPHONY model, software version.201, Protein Technologies Inc.).
2. Grafting with the first amino acid: the solvent was removed by suction filtration through a sand core, 3-fold molar addition of the first Fmoc-amino acid at the C-terminus (all Fmoc-amino acids supplied by Suzhou Tianma pharmaceutical group Fine Chemicals, Inc.), 10-fold molar addition of 4-Dimethylaminopyridine (DMAP) and N, N' -Dicyclohexylcarbodiimide (DCC), and finally addition of Dimethylformamide (DMF) (purchased from Dikma Technologies Inc.) for dissolution and shaking for 30min. Blocking with acetic anhydride.
3. Deprotection: DMF was removed, 20% piperidine-DMF solution (15ml/g) was added for 5min, the solvent was removed by filtration, and 20% piperidine-DMF solution (15ml/g) was added for 15 min. Piperidine is supplied by Shanghai chemical company, national drug group.
4. And (3) detection: the solvent was removed by suction. Taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop each, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and turning dark blue to be a positive reaction.
5. Resin washing: two washes with DMF (10ml/g), two washes with methanol (10ml/g) and two washes with DMF (10ml/g) were performed in sequence.
6. Condensation: depending on the specific synthesis conditions, the following methods may be used alone or in combination in the polypeptide synthesis:
the method a comprises the following steps: three times of protective amino acid and three times of 2- (7-azobenzotriazol) -tetramethyluronium hexafluorophosphate (HBTU, Suzhou Tianma pharmaceutical group fine chemicals Co., Ltd.) were dissolved in DMF as little as possible and added to the reaction vessel. Ten times of N-methylmorpholine (NMM, Suzhou Tianma pharmaceutical group, Fine chemical Co., Ltd.) was immediately added to react for 30min, and the detection was negative.
The method b: three times of protected amino acid FMOC-amino acid and three times of 1-hydroxybenzotriazole (HOBt, Suzhou Tianma pharmaceutical group fine chemicals Co., Ltd.) are dissolved by DMF as little as possible, added into a reaction tube, immediately added with three times of N, N' -Diisopropylcarbodiimide (DIC) and reacted for 30min, and the detection shows negative.
7. Resin washing: in this order DMF (10ml/g) was washed once, methanol (10ml/g) was washed twice and DMF (10ml/g) was washed twice.
8. The operation of 2 to 6 steps was repeated, and as shown by the amino acids in Table 1, the corresponding amino acids were sequentially linked from right to left. With K fatty acid modifications, synthesized as follows 9.
9. Synthesis of K { N- ε - [ γ -Glu- (N- α -fatty acid) ] } or K { N- ε - [ γ -Glu- (N- α -fatty diacid) ] }: the protecting group Dde of Fmoc-Lys (Dde) -OH was removed by adding 10ml of 2% hydrazine hydrate and reacting for 30min, the side chain amino group was exposed, washing was performed six times with DMF and methanol alternately, and ninhydrin was detected as blue. 550mg of Fmoc-Glu-OTBU and 250mg of HOBT are weighed, dissolved in DMF, added with 0.3ml of DIC, mixed evenly, added into a reactor to react with lysine side chain amino for 1h, pumped to dry, washed 4 times by DMF, and detected as colorless by ninhydrin. 5ml of 20% piperidine DMF solution was added to the reactor for reaction for 20min to remove the amino protecting group Fmoc of Fmoc-Glu-OTBU, followed by six washes with DMF and methanol alternately, and ninhydrin was detected as blue. Weighing 300mg of fatty acid or fatty diacid and HOBT 250mg, dissolving with DMF, adding 0.3ml of DIC, mixing uniformly, adding into a reactor, reacting for 1h, draining, washing with DMF for 4 times, detecting the ninhydrin to be colorless, washing with methanol for 2 times, and draining.
Synthesis of K { N- ε - [2 × AEEAC- γ -Glu- (N- α -fatty diacid) ] }: after the Dde-Lys (Fmoc) group is removed, 2mM of Fmoc-AEEAC-OH and 2mM of benzotriazole-1-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 45mM of HOBt are added, the mixture is dissolved in DMF, 0.375mM of N, N' -Diisopropylethylamine (DIPEA) is added to the mixture under ice water bath for activation for 3min, and the mixture is added into a reaction kettle for reaction for 2h, and the end point of the experiment is judged by the detection of the ninhydrin method. After the reaction was complete, Fmoc was removed from 20% piperidine-DMF solution (15ml/g) and washed 6 times with DMF. Fmoc-AEEAC-OH, Fmoc-Glu-OtBu and the fatty diacid groups were coupled again in the same way. Reacting with 2% hydrazine hydrate for 30min to remove the sequence lysine protecting group Dde, and grafting on the epsilon amino group of the lysyl side chain through the step 8.
10. The polypeptide after condensation was passed twice through DMF (10ml/g), twice DCM (10ml/g) and twice DMF (10ml/g) and dried by suction for 10 min. Ninhydrin test negative.
11. Removing FMOC protecting group of final N-terminal amino acid of the peptide chain, detecting to be positive, and draining the solution for later use.
12. The resin was washed twice with DMF (10ml/g), twice with methanol (10ml/g), twice with DMF (10ml/g) and twice with DCM (10ml/g) and dried by suction for 10 min.
13. Cleavage of the polypeptide from the resin: preparing cutting fluid (10 ml/g): TFA 94% (J.T. Baker Chemical Company), water 2.5%, ethandithiol (EDT, Sigma-Aldrich Chemistry) 2.5% and trisisopyvalene (TIS, Sigma-Aldrich Chemistry) 1%. Cutting time: and (4) 120 min.
14. Drying and washing: the lysate is blown dry as much as possible with nitrogen, washed six times with ether and then evaporated to dryness at normal temperature.
15. The polypeptides were HPLC purified, identified and stored at-20 ℃ in the dark as follows.
Secondly, the inspection method comprises the following steps:
1. purification of the polypeptide by HPLC: dissolving the crude peptide with pure water or adding a small amount of acetonitrile or other solvents, and purifying according to the following conditions: high performance liquid chromatography (analytical; software Class-VP. service System; manufacturer Japan SHIMADZU) and Venusi MRC-ODS C18 column (30X 250mm, Tianjin Bonna-Agela Technologies). Mobile phase A liquid: 0.1% trifluoroacetic acid aqueous solution, mobile phase B liquid: 0.1% trifluoroacetic acid + 99.9% acetonitrile (purchased from acetonitrile Fisher Scientific). Flow rate: 1.0ml/min, a loading volume of 30. mu.l, a detection wavelength of 220 nm. Elution procedure: 0-5 min: 90% of solution A and 10% of solution B; 5-30 min: 90% solution A/10% solution B → 20% solution A/80% solution B.
2. Finally, the purified effective solution was lyophilized (Freezone Plus 6, L ABCCONCO) to obtain the final product.
3. And (3) identification: a small amount of finished polypeptide is respectively taken and analyzed by HPLC for purity: high performance liquid chromatography (Shimadzu, Japan) and Venusi MRC-ODS C18 column (4.6X150mm, Tianjin Bonna-Agela Technologies). Mobile phase A liquid: 0.1% trifluoroacetic acid in water, mobile phase B liquid: 99.9% acetonitrile + 0.1% trifluoroacetic acid solution, flow rate: 1.0ml/min, a loading volume of 10. mu.l, a detection wavelength of 220 nm. Elution procedure: 0-5 min: 100% of solution A; 5-30 min: 100% solution A → 20% solution A/80% solution B. The purity required to be determined is greater than 95%. See the patent (Chinese patent ZL201410612382.3) granted by us for a specific method.
MS method for identifying molecular weight of polypeptide: adding water into polypeptide with qualified purity for dissolving, adding 5% acetic acid, 8% acetonitrile and 87 water for dissolving, testing electrospray ionization mass spectrometry to determine molecular weight, and the specific method is disclosed in the patent (Chinese patent ZL 201410612382.3).
4. Sealing and packaging the powdery polypeptide, and storing at-20 deg.C in dark.
And thirdly, forming a dimer: monomeric peptides with a single cysteine inside the peptide chain at a concentration of 1mg/ml were incubated overnight at 37 ℃ in aqueous disodium hydrogen phosphate at a pH of 9.5 to form homodimeric peptides. Many poorly soluble dimeric peptides were centrifuged at 4000 rpm for 20 minutes and the pellet was taken as pure dimeric peptide. The dissolved monomeric or dimeric peptide is separated by Sephadex G-25 chromatography (the dimeric fraction is usually the first peak followed by the monomeric or contaminating fraction with physiological saline or NaCl-PB solution as the flow term in a 2X 60cm G-25 column and natural flow rate). The dimeric peptide can be identified by SDS-PAGE electrophoresis or mass spectrometry of the peptide without a thiol reducing agent, and the specific method is shown in an issued patent (Chinese patent ZL 201410612382.3).
Four, GHRH analog peptide monomers and dimers thereof were synthesized by the present research laboratory or entrusted commercial companies, and the inventors confirmed their structures by HPLC purity, ESI or laser flight mass spectrometry, and cysteine oxidation. The GHRH monomeric peptides synthesized by the present invention are shown in table 1, and the amino acid sequences of homodimeric peptides are shown in table 2.
Fifthly, peptide species: the amino acid sequences of GHRH monomer-like peptides synthesized according to the above method are shown in Table 1, and their HPLC purities>95% in hGHRH (1-29) NH2(S1) or hGHRH (1-44) NH2(S2) as a positive control.
TABLE 1 amino acid sequences of novel GHRH monomeric peptides synthesized according to the present invention and their single injection sustained GH-releasing activity
Note: CFA (carbon fat acid) or CFDA (carbon fat diacid) is carbon fatty acid or carbon fatty diacid; the K [ N-epsilon- (gamma-Glu-N-alpha-CFA) ], K [ N-epsilon- (2 xAEEAC-gamma-Glu-N-alpha-CFDA) ] represents fatty acyl or fatty diacid glutamyl modification of lysine K side chain epsilon-amino, and the specific structure is shown as formula 1 or 2.
TABLE 2 amino acid sequences of synthetic GHRH dimer peptides of the present invention and their single injection sustained GH-releasing activity
Note: CFA (carbon fat acid) is carbon fatty acid, and CFDA (carbon fat diacid) is carbon fatty diacid; the K [ N-epsilon- (gamma-Glu-N-alpha-CFA or CFDA) ], K [ N-epsilon- (2 xAEEAC-gamma-Glu-N-alpha-CFDA) ] represents fatty acyl or fatty diacid glutamyl modification of a lysine K side chain epsilon-amino, and the specific structure is shown as a formula 1 or 2. "" indicates a disulfide bond.
Example 2: growth hormone releasing Activity assay
1. Growth Hormone (GH) release assay in mice: kunming mice (female, 18-22g in weight, purchased from the Experimental animals center of TCM university, Guangzhou, n ═ 6). The care and use regimens for all experimental animals were in accordance with the experimental animal guidelines. In the breeding process, mice are bred alternately in light and shade for 12:12 hours at the breeding temperature of 26 +/-1 ℃. Peptide was injected subcutaneously at the hip of Kunming mice at a dose of 0.0133. mu. mol/kg, and blood was collected from the eyeball 1 hour after administration and from the serum at 6000 rpm/30 min after centrifugation, respectively, for GH assay. In the experiment, all the novel monomeric or dimeric peptides were dissolved with T-NaCl-PB solution (detailed in the administration setting), and the negative control group was injected with T-NaCl-PB solution. The murine GH hormone was tested using an ELISA kit (shanghai, combined mai bioengineering, ltd., china). The results are shown in tables 1 and 2.
2. Setting the administration concentration: because many monomeric or dimeric peptides are poorly soluble, the positive, novel monomeric, or dimeric peptides under this test were formulated with T-NaCl-solution (pH6.5) or T-NaCl-PB (pH8.0) solutions: taking a proper amount of pure peptide powder, dropwise adding pure medicinal Tween 80 (Nanjing Will chemical Co., Ltd., China) with the final concentration of 3%, and stirring with a magnetic stirrer at 900 rpm for 5-10 minutes. After the peptide is dispersed in the tween, slowly dropwise adding normal saline (monomer peptide dissolved) or NaCl-PB solution (normal saline solution pH8.0 buffered by disodium hydrogen phosphate, dimer dissolved), continuously stirring until all the set solution is added, continuously and rapidly stirring twice, each time for 20 minutes, and standing for 15 minutes. Centrifuging at 3000 rpm for 10min, and collecting supernatant for use (precipitating to obtain pure peptide, which can be repeatedly dissolved for use). All peptide concentrations were determined by the LOWRY method, with a blank of T-NaCl-PB or T-NaCl solution (NaCl-PB or NaCl solution containing 3% Tween 80).
Our preliminary experiments show that 0.0133. mu. mol/kg1The Y-GHRH peptide induced significant GH release in mice or rat pituitary, so all peptides tested were used at a dose of 0.0133. mu. mol/kg in this test. In the measurement of mouse GH release duration, for the novel GHRH peptide with 1-29 amino acid sequence, hGHRH (1-29) NH was used as the positive control group2(S1); positive drug control hGHRH (1-44) NH was used for novel GHRH peptides of 1-44 amino acid sequence2(S2)。
3. In vitro rat pituitary growth hormone releasing activity: S-D female mice (weight 200 + -20 g, purchased from the university of TCM laboratory center). Rats were subjected to sudden death from dislocation and the pituitary was removed at 30 minutes. After washing with sterilized Lactated Ringer's Buffer (LRB), the pituitary was immediately placed in a 1X 10cm glass tube containing 1ml of LRB,keeping the temperature at 37 ℃. Total heat preservation of all glass tubes for 5h (P)1,P2,I3,I4And I5Representing different periods of 5 hour incubation, each period representing 1 hour), while gently shaking every 5 min. After every 1 hour, all buffer was aspirated for pituitary GH hormone testing, while 1ml of fresh LRB with no or with GHRH-like peptide was added to the pituitary for incubation. In the first 2 hours (P)1And P2) After the preincubation, the subsequent incubation is carried out for 3-6 hours (I)3、I4And I5) In this experiment, the polypeptide is initially increased and a blank LRB solution is used as a control. The net GH release level of the peptide is [ peptide (I)3+I4+I5) -blank (I)3+I4+I5)]. Rat pituitary rat GH hormone was tested using an ELISA kit (shanghai amalimei bioengineering ltd, china). The results are shown in tables 1 and 2.
4. Novel GHRH-like peptide sustained release GH assay in vivo: kunming mice (female, with the weight of 18-22g, n is 6) are injected with peptide in a single subcutaneous way at the hip of the Kunming mice according to the dose of 0.0133 mu mol/kg, 50 microlitres of blood is collected at the inner canthus at the right time 9 points in the morning on different days before and after administration until GH secretion reaches the normal value, and experimental blank control groups are injected with T-NaCl (monomer peptide) or T-NaCl-PB (dimer) solution. The canthus blood 6000 r/heart separation 30min, taking serum to determine GH. The results are shown in tables 1 and 2.
5. And (4) conclusion: (1) for the Y or 2Y series of GHRH peptides, whether in vivo or in vitro, the secretion of pituitary GH is obviously stimulated, so the peptides can be used for treating GH deficiency; (2) for the P or 2P series of GHRH peptides, the in vitro tests clearly stimulated pituitary GH secretion but no significant GH release in vivo, suggesting that such peptides may not act on the pituitary in vivo; (3) for the F or 2F series of GHRH peptides, both in vivo and in vitro, pituitary GH secretion is stimulated to be very low; (4) according to these results, a significantly long-acting GH sustained release time was observed in vivo for the Y or 2Y series of peptides, but not for the P or 2P series of peptides, or the F or 2F series of peptides showed a shorter GH sustained release time at the observed dose because of their low activity; (5) the N-terminal two amino acids of novel GHRH are hydrolyzed according to the hydrolysis mechanism of aminodipeptidase (DPP-4)The GHRH peptides of the present invention having the same sequence should have similar half-lives, such as Y, P and F series having the same sequence or 2Y, 2P and 2F series having similar half-lives, except that the first amino acid at the N-terminus is different. Although the P and 2P series of peptides or the F and 2F series of peptides show no or very low GH values in vivo, their half-lives should be similar to the corresponding Y and 2Y sequences; (6) GHRH (1-29) contains 51% of activity, and has important application value in science and commerce due to convenient and simple synthesis and low cost, and the GHRH preparation method independently embodies the contents. For peptides containing 1-29 or 1-44, (S1) hGHRH (1-29) NH is used2Or (S2) hGHRH (1-44) NH2The standard is made, so that the activity of the peptide is better reflected; (7) the dimers showed at least 2-fold more in vivo duration compared to for the monomeric peptides. According to the position of disulfide bonds, the release time of GH in vivo is Y1 series 2-6 days, Y2 series 5-8 days, Y3 series 2-5 days, Y4 series 3-5 days, Y5 series 2-8 days, Y6 series 3-5 days, Y7 series 3-5 days, Y8 series 2-4 days, 2Y1 series 4-11 days, 2Y2 series 10-15 days, 2Y3 series 4-9 days, 2Y4 series 4-8 days, 2Y5 series 5-17 days, 2Y6 series 6-9 days, 2Y7 series 6-9 days and 2Y8 series 3-7 days, and obviously the necessary long-acting structure comprises Aib →2Ala substitution,18C-18C disulfide bond formation, K20Or K21Fatty acid modification and C-terminal amidation structure, the position of disulfide bond is close to C terminal, and the sustained release time of GH is shorter.
Example 3: treatment of infertility models with dimeric peptides
1. The sterile infertility model test method comprises the following steps: the male rats of 130 Zhonghua hamster or the female rats of 65 Zhonghua hamster (five weeks old, 18-22 g/rat, provided by Sichuan Daoho laboratory animal center) were selected, because preliminary experiments showed that the male rats were significantly less sensitive to peptides than the female rats, the male sterile model n ═ 20, while the female infertility model n ═ 10, because the GHRH of Chinese hamster had the highest 93% homology with human, and were divided into blank control groups (male rats 20, female rats 10) and model rats (male rats 110, female rats 55). All model groups were intraperitoneally injected with 20mg/kg cyclophosphamide (Jiangsu Henry pharmaceuticals, Inc., national drug Standard H32020857, lot number 12032925) once a week for a total of five times. After the injection of cyclophosphamide at the fourth week, the model mice were divided into a model control group (Placebo), a human gonadotropin hMG group and a dimer peptide dose group 2Y-47, 2P-104 or 2F-120 according to the non-differential body weight of the hamster, and each group of male mouse model n-20 or each group of female mouse model n-10 (cyclophosphamide injection causes death of animals, so the number of prepared models was increased). The dosage was weighed and calculated before each group was administered, and the dimer peptide was administered intramuscularly at a dose of 0.0133. mu. mol/kg (dissolved in T-NaCl-PB solution), and the positive control hMG group was administered intramuscularly at 200 units/kg (16.65. mu. mol/kg) (dissolved in T-NaCl-PB solution) (urocortin hMG, gauge intramuscular injection of 400U/mg or 12.012U/. mu. mol, manufactured by Zhuhaili bead pharmaceutical industry). The blank control group and Placebo group were injected with T-NaCl-PB solution once a day until the end of the experiment. At 6-8 weeks, model and normal rats were mated with a cage 1:1 heterosexual, after which only therapeutic drugs were given, and the physiological changes (mental, activity, secretions, etc.) during the estrus of the rats were observed and whether the rats were pregnant or not was observed. At the end of the tenth week, mice were killed by dislocation and pregnant mice were counted by dissection. And (3) pregnancy judgment: the red beaded particles can be seen in the oviduct of the female mouse; newborn mice: it was born ten weeks ago.
2. And (3) carrying out statistics on the pregnancy rate of the hamster: organ gravimetric analysis showed that Placebo group showed significant testicular reduction (P <0.05) compared to the blank control group. Compared with Placebo group, the weight of testis in hMG group and 2Y-47 group is significantly increased (P <0.05 or 0.01), showing that hMG and 2Y-47 significantly stimulate testis action. The testis weights were reduced to different degrees in the 2P-104 or 2F-120 groups compared to the hMG group or 2Y-47 group (FIG. 1). Ovarian gravimetric analysis of female models also showed similar changes.
The hMG, 2Y-47 and 2P-104 groups showed a significant increase in total pregnancy compared to the Placebo or the Placebo group, regardless of the female or male mouse model, and they showed statistical differences from the Placebo or the Placebo group, whereas the 2F-120 group showed a decrease in pregnancy rate and showed no difference from the Placebo or the Placebo group (tables 3 and 4), showing that the reproductive capacity of the adult gerbils was not related to GH.
TABLE 3 female mouse model group pregnancy Rate statistics (Mean + -SD)
P <0.05, a, b vs blank control group, Placebo group comparison, chi-square test.
TABLE 4 Male mouse model group pregnancy Rate statistics (Mean SD)
P <0.05, a, b vs blank control group, Placebo group comparison, chi-square test.
3. And (4) conclusion: (1) an adult 60kg patient is injected with 75-150U of hMG per time per day, the dose ratio of the hMG to the hamster is 9.1 times, the converted hamster dose is averagely 17.05U/kg (1.41968 mu mol/kg), but the hMG has low response to the hamster, 200U/kg (16.65 mu mol/kg) achieves similar pregnancy rate with certain peptides after multiple times of investigation, and the doses of 2Y-47 and 2P-104 are 1/500 of the hMG; (2) in vivo, the central GHRH-GHRH receptor-GH endocrine axis pathway is specific, but testis or ovarian cells present GHRH, GHRH receptors, and GH molecules that do not necessarily induce consistent responses of GH in vivo and in vitro; (3) the high pregnancy rates of 2Y-47 and 2P-104 showed that high GH secretion in vivo was not correlated with pregnancy rates.
Example 4: treatment of model senile dementia with dimer peptides
1. The preparation and treatment method of the senile dementia model comprises the following steps: c57BL/6 mice (SPF grade, male, purchased from Experimental animals center of Guangzhou university of traditional Chinese medicine, 20-25 g/mouse) were light/dark cycle adjusted at 12/12 hours with constant temperature of 23 + -1 deg.C and humidity of 50-60%, and were fed freely with water. After purchase of the animals, the experiment was started 3 days after adaptive feeding.
Mice were randomly divided by body weight into a blank control group, a Placebo group (model control group), a positive control group and a dimer peptide 2Y-47, 2P-104 and 2F 120-administered group, 10 mice per group. The mice of the model control group, the dimer peptide administration group and the positive control group are intragastrically filled with 10mg/kg of aluminum trichloride every day and are simultaneously intraperitoneally injected with 120mg/kg of D-galactose (modeling medicament: aluminum trichloride + D-galactose), the mice of the blank control group are intragastrically filled with distilled water with the same volume every day and are simultaneously intraperitoneally injected with normal saline with the same volume, and the mice of each group are continuously fed with the modeling medicament for 24 weeks. The mice of each group were injected with 0.0133 μmol/kg of dimer peptide (dissolved in T-NaCl-PB solution) subcutaneously on day 1 after the intraperitoneal injection of the model drug in the 13 th week, the blank control group and the model control group were injected with T-NaCl-PB solution subcutaneously, and the positive control group was injected with 38.74mg/kg of donepezil pamoate for injection (diluted with T-NaCl-PB solution) subcutaneously (clinical lot of positive drug 2018L02708, 3mL in specification: 255.4mg, manufactured by Huajiang river, Hai.e., pharmaceutical Co., Ltd.; and the mouse dose was 38.74mg/kg, calculated as 9.1-fold equivalent dose for mice and adults). Each group was administered by subcutaneous injection once every two weeks for 12 weeks, and the molding drugs were administered daily routinely throughout the administration period.
2. Water maze test method: data acquisition and processing of learning and memory of mice was automatically monitored and processed by Morris maze images. The mice are trained in the Morris water maze experiment 3 days before model making and drug administration, each group of mice is trained 3 times per day, the mice are trained to escape from water surface swimming from the starting point of the SMG-2 type square water maze (developed by the institute of medicine of Chinese academy of medical science, China) to reach the terminal stair, and the water maze automatically records the error times and the terminal reaching time (latency) of the mice entering each blind end. Mice were tested for learning and memory the day before and week 12 after treatment, 2 times for each group of mice, Mean error number and Mean latency values were taken, experimental data were Mean ± standard deviation (Mean ± SD), statistical analysis by t-test.
3. As a result: table 5 results show that over the 12 th week of modeling, there was a significant increase in latency and number of errors in each model group (P <0.05) compared to the blank control group, indicating that the model of alzheimer's disease was successful. The Placebo group showed a significant increase (P <0.05 or 0.01) compared to the latency or number of errors in each group before or after dosing. For the latency or number of errors in each group after administration, 2Y-47 and the positive control group showed a significant decrease compared to Placebo, but no significant difference between the 2P-104 and 2F-120 groups. In the above experiments, no experimental phenomena such as weight loss and dysphoria of the mice in the administration group and the mice in the blank control group are found, and the drug has good tolerance to the mice. In conclusion, the 2Y-47 of the invention has better effect, and preliminarily shows that: therapeutic efficacy in Alzheimer's disease is associated with high GH secretion.
Table 5: statistical analysis of memory ability of dementia model mice (Mean + -SD) of each experimental group before and after 12 weeks drug treatment
P <0.05 or 0.01, double greek letter mapping comparative statistical analysis, t-test.
4. Cholinesterase analysis: the cholinesterase assay kit (manufactured by Beijing Jiuqiang Biotechnology corporation) and Hitachi full-automatic biochemical analyzer (model 7180, Japan) were used for assay, and the specifications of the kit and the apparatus were referred to the manufacturer's instructions. The results are shown in FIG. 2: compared with Placebo group, the positive drug and the 2Y-47 group have significant difference (P <0.05), and the animal serum cholinesterase assay shows dose-dependent reduction. Compared with the blank control group, the cholinesterase of Placebo, 2P-104 and 2F-120 groups showed a significant increase. The result shows that 2Y-47 has certain treatment effect on the senile dementia.
Example 5: treatment of diabetes mellitus model II
II, a diabetes model establishing method: c57Bl6/J mice were placed in a standard diet in an environment of SPF rating with free access to water. All experimental operations are conducted according to the ethical and use system guiding principles of experimental animals. After a day of feeding according to the standard diet, 5-week-old C57B16/J male mice were divided into 6 groups: blank control group, Placebo group (T2D model control group), liraglutide group, 2Y-47 low and medium groups (L-2Y-47, M-2Y-47, H-2Y-47). All T2D models were fed 60 kcal% of a high fat diet (D12492, changzhou mouse two biotech limited) until the end of the experiment. The blank control group maintained the standard diet until the end of the experiment. The method for establishing the diabetes model comprises the following steps: after 4 weeks of high-fat feeding, mice were intraperitoneally injected with 75mg/kg streptozotocin (STZ, Sigma chemical Co., USA), 3 days later, re-intraperitoneally injected with a 50mg/kg dose of STZ, and 3 weeks later, diabetic mice had blood glucose equal to or greater than 11 mM. These groups were treated on a high fat diet for an additional 35 days. The liraglutide group was injected subcutaneously with 1.126 nmol/100. mu.L liraglutide, the 2Y-47 group was injected subcutaneously with 3.378, 1.126, 0.375 nmol/100. mu.L (diluted with T-NaCl-PB solution), the blank control and the Placebo group with T-NaCl-PB solution.
2. The results are shown in FIG. 3: after 2Y-47 injection, blood glucose showed no significant change and serum cholinesterase showed a dose-dependent decrease. Compared with the blank control group, the increase of cholinesterase was significant in Placebo, liraglutide and each of the 2Y-47 groups (P < 0.05). Compared with the liraglutide group, the M-and H-2Y-47 groups have no significance, and show that 2Y-47 can reduce cholinesterase and can increase the cognitive function of diabetes.
Example 6: therapeutic effect of dimer of the present invention on hypoimmunity of animals
1. Influence of the drug on the immunity of normal mice: 24 Balb/c male mice were selected, weighing 18-22 g. After 2 days of feeding, the blank control group and the 2Y-47 administration group were randomly divided into 6 groups without weight difference. The 2Y-47 peptide was injected subcutaneously into hip at a single dose of 0.0133 μmol at 12 am, followed by 50 μ l of anticoagulated blood from the intraorbital canthus at 12 pm daily, and blood routine (Sysmex XS-500i and reagents, Japan) was determined until the end of the 18 day experiment. The result of the test in the 2Y-47 group minus the result of the test in the blank control group is the final result.
2. The experimental results are as follows: blood routine analysis shows that after 2Y-47 peptide injection, only the total number and percentage of mononuclear cells are obviously different at different time points, and no influence is shown on other red blood cells, white blood cells or blood platelets and related indexes thereof. On day 1 after administration, each index decreased, probably as a stress response in the animal, and returned to normal the next day. As can be seen in fig. 4, the total number of monocytes was significantly increased at days 2, 7, 14, 15 (P <0.05) compared to the pre-and post-drug day 1 results. Figure 5 shows a significant increase in monocyte percentage at days 2, 7, 14, 15 (P <0.05, 0.01, or 0.001) compared to pre-and/or post-drug day 1 results, with peak results at day 15 and a drop to normal at day 18. These results preliminarily show that 2Y-47 peptide promotes monocyte proliferation and enhances immunity.
3. The preparation and treatment method of the low immunity model comprises the following steps: balb/c mice 60, weighing 18-22g, were randomized into 10 groups (male and female halves) without weight difference between the control blank and model groups (Placebo group, positive control group, L-2Y-47, M-2Y-47 and H-2Y-47). Mice in all model groups were injected with cyclophosphamide 80mg/kg intraperitoneally once a day for 3 consecutive days. After 7 days of the experiment, the therapeutic drugs were administered, and each of the positive control groups was subcutaneously injected with 0.2427mg (0.07808. mu. mol) of thymalfasin (trade name: Thai, 1.6mg specification, approved reference H20030407, product batch 16070106, manufactured by Hainan Shuangcheng pharmaceutical Co., Ltd., dissolved in T-NaCl-PB solution), 2Y-47 groups were subcutaneously injected with 0.2342, 0.07808 and 0.02603. mu. mol, and the blank control group and Placebo group were injected with T-NaCl-PB solution. Each group was injected 2 times per week for 4 weeks. One day before treatment and 4 weeks after treatment, 50 microliters of anticoagulated blood was taken from the inner canthus of the mice, blood routine was determined, and spleen was weighed and H-E stained for mice were sacrificed. Counting red pith: H-E stained sections of three animals per group were taken, 5 sections were taken for each animal and photographed, and the percentage of red marrow cells in the entire field was counted using IPP software, which was the red marrow statistical result. The method is described in Liu Changjiang river and other papers [ Liu Changjiang river and other papers, establishment and evaluation of cyclophosphamide induced mice immunologic hypofunction model, Weifang medical college bulletin 37(1), 4-6,2015 ].
4. And (4) evaluating the results: after the experiment, the analysis of the total number of blood mononuclear cells (FIG. 6) showed that the thymalfasin group showed a significant increase in lymphocytes, but no significant increase in monocytes, a dose-dependent increase in the number of monocytes in the 2Y-47 group, and a significant increase in the H-2Y-47 group (P <0.05), as compared to the Placebo group. Comparison of spleen weights (FIG. 7) showed a significant increase in spleen weight (P <0.05) in thymalfasin and H-2Y-47 animals compared to the placebo group. Spleen H-E staining (FIG. 8A) showed a significant increase in white marrow in thymalfasin groups, and in red marrow in 2Y-47 groups. Spleen red marrow was significantly increased in M-and H-2Y-47 groups (P <0.05) compared to the blank control group and Placebo group (FIG. 8B). These preliminary results show that the 2Y-GHRH sequence represented by 2Y-47 enhances immunity by increasing macrophage numbers in the red marrow region.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Huzhou Nafu biomedical Co., Ltd
<120> preparation and use of novel somatotropin-releasing hormone analogous peptide modification and dimerization
<130> 2.13
<160> 132
<170> PatentIn version 3.3
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Tyr Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Xaa Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
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<221> misc_feature
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Xaa Arg Leu Leu Gln Asp Ile Met Ser Arg
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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<221> misc_feature
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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<221> misc_feature
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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<222> (2)..(2)
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Tyr Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
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<221> misc_feature
<222> (20)..(20)
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
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<221> misc_feature
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<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
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Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
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<221> misc_feature
<222> (29)..(29)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 42
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
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Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Xaa Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
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<221> misc_feature
<222> (38)..(38)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 43
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Xaa Gly Ala Arg Ala Arg Leu
35 40
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<220>
<221> misc_feature
<222> (41)..(41)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 44
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Xaa Ala Arg Leu
35 40
<210> 45
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (43)..(43)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 45
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Xaa Leu
35 40
<210> 46
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε-(γGlu-N-α-18CFA)]
<400> 46
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 47
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 47
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 48
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 48
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 49
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 49
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Lys Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 50
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]-
<400> 50
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 51
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-γGlu-20CFDA]
<400> 51
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 52
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 52
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 53
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 53
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 54
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 54
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 55
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 55
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 56
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 56
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 57
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 57
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 58
<211> 45
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 58
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Lys Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln
20 25 30
Gly Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40 45
<210> 59
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 59
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 60
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 60
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 61
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 61
Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 62
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 62
Tyr Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 63
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 63
Tyr Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Lys Ala Arg Leu
35 40
<210> 64
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 64
Tyr Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 65
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 65
Tyr Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 66
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 66
Tyr Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Lys Ala Arg Leu
35 40
<210> 67
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 67
Tyr Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 68
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 68
Pro Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 69
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 69
Pro Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 70
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 70
Pro Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 71
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 71
Pro Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 72
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 72
Pro Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 73
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 73
Pro Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 74
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 74
Pro Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Arg Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 75
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 75
Pro Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 76
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 76
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 77
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 77
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 78
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 78
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 79
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 79
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 80
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(AEEA)2-γGlu-20CFDA]
<400> 80
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 81
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(AEEA)2-γGlu-20CFDA]
<400> 81
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 82
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 82
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 83
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 83
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 84
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 84
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Arg Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 85
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 85
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 86
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 86
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 87
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 87
Pro Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg
20 25
<210> 88
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 88
Pro Ala Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg
20 25
<210> 89
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 89
Pro Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg
20 25
<210> 90
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 90
Pro Xaa Asp Ala Ile Phe Thr Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg
20 25
<210> 91
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 91
Pro Ala Asp Ala Ile Phe Xaa Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 92
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 92
Pro Ala Asp Ala Ile Phe Xaa Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 93
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 93
Pro Ala Asp Ala Ile Phe Xaa Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 94
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 94
Pro Ala Asp Ala Ile Phe Xaa Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 95
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (22)..(22)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 95
Pro Xaa Asp Ala Ile Phe Xaa Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 96
<211> 29
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 96
Pro Xaa Asp Ala Ile Phe Xaa Asn Cys Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg
20 25
<210> 97
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε-(γGlu-N-α-18CFA)]
<400> 97
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 98
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 98
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 99
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 99
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 100
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 100
Pro Ala Asp Ala Ile Phe Ser Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Xaa Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 101
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (38)..(38)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 101
Pro Ala Asp Ala Ile Phe Ser Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Xaa Gly Ala Arg Ala Arg Leu
35 40
<210> 102
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (41)..(41)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 102
Pro Ala Asp Ala Ile Phe Ser Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Xaa Ala Arg Leu
35 40
<210> 103
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (43)..(43)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 103
Pro Ala Asp Ala Ile Phe Ser Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Xaa Leu
35 40
<210> 104
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε-(γGlu-N-α-18CFA)]
<400> 104
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 105
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε-(AEEA)2-γGlu-20CFDA]
<400> 105
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 106
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-18CFA)]
<400> 106
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 107
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 107
Pro Xaa Asp Ala Ile Phe Ser Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Lys Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 108
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(AEEA)2-γGlu-20CFDA]
<400> 108
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 109
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-γGlu-20CFDA]
<400> 109
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 110
<211> 44
<212> DNA
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 110
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 111
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε-(AEEA)2-γGlu-20CFDA]
<400> 111
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 112
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 112
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 113
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 113
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 114
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 114
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Cys Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 115
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 115
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 116
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 116
Pro Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 117
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 117
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 118
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 118
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 119
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 119
Pro Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Cys Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 120
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 120
Pro Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 121
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 121
Pro Ala Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 122
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 122
Pro Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 123
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa is Hcy
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 123
Pro Xaa Asp Ala Ile Phe Xaa Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Ser Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 124
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε-(γGlu-N-α-18CFA)]
<400> 124
Phe Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 125
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 125
Phe Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 126
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 126
Phe Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 127
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε-(γGlu-N-α-18CFA)]
<400> 127
Phe Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 128
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa is Arg[N-ε- (AEEA)2-γGlu-20CFDA]
<400> 128
Phe Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Xaa Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 129
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(γGlu-N-α-20CFA)]
<400> 129
Phe Xaa Asp Ala Ile Phe Ser Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Lys Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 130
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-(AEEA)2-γGlu-20CFDA]
<400> 130
Phe Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40
<210> 131
<211> 44
<212> PRT
<213> Synthesis
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa is Lys[N-ε-γGlu-20CFDA]
<400> 131
Phe Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln
1 5 10 15
Leu Cys Ala Arg Xaa Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly
20 25 30
Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40