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WO2021083306A1 - Glp-1/gcg双受体激动剂多肽 - Google Patents

Glp-1/gcg双受体激动剂多肽 Download PDF

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WO2021083306A1
WO2021083306A1 PCT/CN2020/125110 CN2020125110W WO2021083306A1 WO 2021083306 A1 WO2021083306 A1 WO 2021083306A1 CN 2020125110 W CN2020125110 W CN 2020125110W WO 2021083306 A1 WO2021083306 A1 WO 2021083306A1
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polypeptide
fusion protein
hec
amino acid
acid sequence
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French (fr)
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蒋鹏
黄丽玫
雷丹
葛驾
肖�琳
周林俊
王慧
凌伊
詹志柱
李锦清
燕江雨
李静
陈小锋
李文佳
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东莞市东阳光生物药研发有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to GLP-1/GCG dual receptor agonist polypeptides, fusion proteins, nucleic acids, constructs, recombinant cells, pharmaceutical compositions and pharmaceutical applications.
  • Obesity is closely related to type 2 diabetes, hyperlipidemia and hypertension.
  • the main role of first-line drugs for the treatment of type 2 diabetes is to reduce blood sugar, but the effect of reducing weight is very limited. How to develop drugs with good blood sugar and weight reduction effects at the same time is the current research and development hotspot in this field.
  • Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted by the intestinal L-cells after eating, which can stimulate the pancreatic ⁇ -cells to secrete insulin, thereby stabilizing the fluctuation of blood glucose after a meal. Its effect of lowering blood sugar is glucose concentration-dependent. While regulating blood sugar, it greatly reduces the risk of hypoglycemia. In recent years, drugs based on GLP-1, such as liraglutide, duraglutide and semaglutide, have gradually occupied a very important position in diabetes drugs. GLP-1 drugs also have the effect of weight loss when lowering blood sugar.
  • GLP-1 acts on the gastrointestinal tract to delay gastric emptying and intestinal peristalsis, and acts on the central nervous system to suppress appetite, so as to reduce food intake.
  • liraglutide has been approved as a weight-loss drug, and its second-generation product, semaglutide, has a better weight-loss effect.
  • semaglutide has a better weight-loss effect.
  • the main effect of this type of drugs is to control sugar and reduce weight, and the effect is limited, and it is still far from meeting clinical needs.
  • Glucagon is a polypeptide hormone secreted by islet ⁇ cells, which can promote glycogen decomposition and gluconeogenesis, and significantly increase blood sugar; at the same time, it can also activate lipase, promote lipolysis, and increase fatty acid oxidation. Increase energy consumption, which has the effect of reducing fat and weight. Because of its physiological role in raising blood sugar, although it can be used in the treatment of hypoglycemia, it limits its application in obesity and weight loss, especially in obese patients with type 2 diabetes.
  • Oxyntomodulin is a dual-receptor agonist that activates the glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR).
  • GLP-1R glucagon-like peptide-1 receptor
  • GCGR glucagon receptor
  • the hypoglycemic effect of its GLP-1 receptor activity can balance the hypoglycemic effect caused by the activation of GCGR, and synergize the effects of the two in reducing weight, so as to achieve a better weight loss effect.
  • OXM oxyntomodulin
  • the present invention proposes a GLP-1/GCG dual receptor agonist polypeptide.
  • the polypeptide having the amino acid sequence shown below: 5 'X 1 SQGT FTSDX 10 SKYLX 15 X 16 X 17 X 18 AX 20 X 21 FX 23 X 24 W L X 27 X 28 X 29 X 30 3 ' ,
  • X 1 is H or Y
  • X 10 is Y or L
  • X 15 is D or E
  • X 16 is M or E
  • X 17 is Q
  • X 18 is R or A
  • X 21 is E or D
  • X 23 is V or I
  • X 24 is Q
  • X 27 is L or M
  • X 28 is A, N or D
  • X 29 is A Or G
  • X 30 is T, PSSGAPPPS or not present.
  • the above-mentioned GLP-1/GCG dual receptor agonist polypeptide according to the embodiment of the present invention has a good effect of reducing blood sugar and weight, and has a long half-life in the body, and is a more effective long-acting weight-reducing and sugar-controlling drug.
  • the above-mentioned polypeptide may further have at least one of the following additional technical features:
  • the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 1-21, SEQ ID NO: 71.
  • the polypeptide has an amino acid sequence shown in SEQ ID NO: 1 to 3.
  • the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 22-34.
  • the present invention proposes a fusion protein.
  • the fusion protein comprises a polypeptide linker peptide fragment and IgG 4 -Fc previously described, the linker peptide disposed between the head and tail polypeptide fragments and IgG 4 -Fc.
  • the fusion protein according to the embodiment of the present invention has a good effect of reducing blood sugar and weight, and has a long half-life in the body, and is an effective long-acting weight-reducing and sugar-controlling drug.
  • the aforementioned fusion protein may further include at least one of the following additional technical features:
  • the N-terminus of the connecting peptide with the C-terminus of the polypeptide linked a connecting peptide coupled to the N-terminus of the C-terminus of the IgG 4 -Fc fragments.
  • the connecting peptide has the amino acid sequence shown in SEQ ID NO:35.
  • the IgG4-Fc fragment has the amino acid sequence shown in SEQ ID NO: 36.
  • the fusion protein has an amino acid sequence shown in SEQ ID NO: 37-70, SEQ ID NO: 72.
  • the present invention proposes a nucleic acid.
  • the nucleic acid encodes the aforementioned polypeptide or the aforementioned fusion protein.
  • the present invention proposes a construct.
  • the construct carries the aforementioned nucleic acid.
  • the aforementioned construct may further include at least one of the following additional technical features:
  • the vector of the construct is pXC17.4.
  • the present invention proposes a recombinant cell.
  • the recombinant cell expresses the aforementioned polypeptide or the aforementioned fusion protein.
  • the present invention proposes a recombinant cell.
  • the recombinant cell contains the aforementioned construct or the genome integrates the aforementioned nucleic acid.
  • the above-mentioned recombinant cell may further include at least one of the following additional technical features:
  • the recombinant cell is a CHO cell.
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition includes the aforementioned polypeptide or the aforementioned fusion protein.
  • the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features:
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes other anti-diabetic drugs
  • the other anti-diabetic drugs include insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha -At least one of a glucosidase inhibitor and an aminodipeptidase IV inhibitor.
  • the present invention provides a protein preparation. According to an embodiment of the present invention, it includes the aforementioned polypeptide or the aforementioned fusion protein.
  • the protein preparation according to the embodiment of the present invention is an effective long-acting weight-reducing and carbohydrate-controlling drug.
  • the present invention proposes the use of the aforementioned polypeptide or the aforementioned fusion protein in the preparation of medicines for the treatment or prevention of metabolic diseases.
  • the metabolic disease includes at least one selected from non-alcoholic fatty liver disease (NAFLD), diabetes, and obesity.
  • NAFLD non-alcoholic fatty liver disease
  • Figure 1 is a blood glucose concentration-time curve at different time points after administration of a sugar-loaded mouse according to an embodiment of the present invention
  • Figure 2 is a graph showing the experimental results of the effect of the fusion protein HEC-12063 on the glucose tolerance of DIO mice according to an embodiment of the present invention
  • Figure 3 is a diagram showing the experimental results of the effect of the fusion protein HEC-12063 on the body weight of DIO mice according to an embodiment of the present invention
  • Figure 4 is an experimental result diagram of the effect of the fusion protein HEC-C046 on the blood glucose of ob/ob mice according to an embodiment of the present invention
  • Figure 5 is a diagram showing the experimental results of the effect of the fusion protein HEC-C046 on the body weight of ob/ob mice according to an embodiment of the present invention
  • Fig. 6 is a graph showing the experimental results of the effect of the fusion protein HEC-C046 on the blood sugar of db/db mice according to an embodiment of the present invention
  • Fig. 7 is a graph showing the experimental results of the effect of the fusion protein HEC-C046 on the body weight of db/db mice according to an embodiment of the present invention.
  • Fig. 8 is a blood glucose concentration-time curve at different time points after administration of a sugar-loaded mouse according to an embodiment of the present invention
  • Figure 9 is a diagram showing the experimental results of the effect of the fusion protein HEC-C079 on the glucose tolerance of DIO mice according to an embodiment of the present invention.
  • Fig. 10 is a graph showing the experimental results of the effect of the fusion protein HEC-C079 on the body weight of DIO mice according to an embodiment of the present invention.
  • the fusion gene is formed by adding the gene sequence of the SUMO tag at the 5'end of the gene encoding the polypeptide.
  • the fusion gene was cloned into a prokaryotic expression vector and induced expression in E. coli cells. After the cells are collected by centrifugation, they are ultrasonically broken and centrifuged to obtain the supernatant, and then purified by nickel column to obtain the fusion protein. Finally, after SUMO protease digestion treatment, the target polypeptide is obtained by reverse-phase purification.
  • the specific process is as follows:
  • the resulting BL21(DE3) strain was constructed, cultured in LB, kanamycin (kanamycin) was added at a final concentration of 50 ⁇ g/mL, and IPTG was used to induce expression for 5h after culture. Take the centrifuged cells, dissolve them with equilibration buffer (20mM Tris-HCl, pH8.0, 150mM NaCl), disrupt the cells with an ultrasonic instrument, and centrifuge the supernatant for purification of the fusion protein. The fusion protein was purified by Ni-NTA affinity chromatography column (GE Healthcare) and eluted with elution buffer (20mM Tris-HCl, pH8.0, 150mM NaCl, 200mM imidazole).
  • Nanomicro UniSil 8-120 C8 Ultra Plus 8um 4.6*250mm is from Suzhou Nanomicro Technology Co., Ltd.
  • GLP-1/GCG dual receptor agonist polypeptide stimulates HEK293 cells expressing GLP-1R or GCGR, and detects cAMP produced by the recipient cells with a cAMP detection kit (Cisbio, 62AM6PEC), establishes a dose-effect curve, and calculates its EC 50 , the results are shown in Table 2 and compared with each other.
  • the GLP-1 / GCG dual receptor agonist polypeptide and IgG4-Fc with linker peptide (GGGGSGGGGSGGGGSA) of (ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG) are fused sequence obtained by double digestion and inserted into the mammalian cell expression vector A series of mutant vectors were constructed between the same restriction sites of, and after being verified by sequencing, the plasmid vectors were extracted with endotoxin-free plasmid extraction kit (OM
  • the amino acid sequence of the GLP-1/GCG receptor dual agonist polypeptide in the vector and the name of the corresponding polypeptide sample are shown in Table 1.
  • the sample name of the fusion protein containing the GLP-1/GCG receptor dual agonist polypeptide constructed in Example 3 is consistent with the sample name of the GLP-1/GCG receptor dual agonist polypeptide obtained in Example 1.
  • the Chinese hamster ovary cells were resuscitated and subcultured. When the density was about 6*10 6 cells/mL, the cells were collected and transfected.
  • the ExpiCHOFectamineTM CHO Transfection Kit (ThermoFisher Scientific) was used. The final concentration of the vector constructed in Example 2 was 1 ⁇ g/mL.
  • reagents such as enhancers were added to maintain the growth of transfected cells. The cell culture solution was harvested when the cell viability dropped to about 80%.
  • the cell culture solution was centrifuged to collect the supernatant, and filtered with a 0.22 ⁇ m filter to remove residual cell debris.
  • the collected cell culture fluid was purified by Protein A chromatography column, the target peak was collected, and then further purified by anion exchange chromatography, and the protein was finally collected by elution with 0.02M PBS.
  • the samples are quantified using a micro-nucleic acid protein analyzer (NanoDrop 2000/2000c Spectrophotometer). The sample was detected by 12% SDS-PAGE electrophoresis, and the electrophoresis pattern showed a single band.
  • the precise molecular weight of the fusion protein determined by mass spectrometry is basically consistent with the theoretical molecular weight.
  • HEK293 cells expressing GLP-1R or GCGR were stimulated by the fusion protein, cAMP detection kit (Cisbio, 62AM6PEC) was used to detect the cAMP produced by the recipient cells, the dose-response curve was established, and the EC 50 was calculated. The results are shown in Table 3. And compare them with each other.
  • the GLP-1/GCG receptor dual agonist sample obtained in the present invention can significantly activate GLP-1R or GCGR receptor cells, among which HEC-12042, HEC-12063 and HEC-C046 are activating GLP-1R or GCGR receptor cells have significant advantages.
  • mice Normal C57BL/6 mice were divided into 6 groups according to blood sugar and body weight (Control, Dulaglutide-7.5nmol/kg, HEC-C046-7.5nmol/kg, HEC-C049-7.5nmol/kg, HEC-C050-7.5 nmol/kg, HEC-12063-7.5nmol/kg), each group of 8 animals, the corresponding vehicle or candidate was given by subcutaneous injection, the animals were fasted 56h after the administration, and the animals in each group were injected intraperitoneally with 2g/kg glucose 72h after the administration , And perform blood glucose testing before and 15, 30, 60, 90 min after sugar administration. Draw the blood glucose concentration-time curve according to the blood glucose values measured at different time points, as shown in Figure 1, calculate the AUC 0 ⁇ 90min Glu of each dose group and the hypoglycemic rate at the peak blood glucose.
  • HEC-12063, HEC-C046, HEC-C049 and HEC-C050 can significantly reduce the blood glucose level of mice with glucose load.
  • mice 8-week-old C57BL/6 mice were fed with a high-fat diet for 4 months and divided into 5 groups according to their body weight (Model, semaglutide 10nmol/kg, HEC-C12063 3nmol/kg, HEC-C12063 10nmol/kg, HEC-C12063 30nmol/kg).
  • the administration was started at the 5th month (the model group was given the corresponding vehicle), semaglutide was administered once a day, and each dose group of HEC-C12063 was administered twice a week for a total of 4 weeks.
  • the animals were weighed before each administration.
  • the IPGTT experiment was performed on the 4th week of administration.
  • HEC-12063 decreased the blood glucose and body weight of DIO mice in a dose-dependent manner. At a dose of 3nmol/kg, it can significantly improve the glucose tolerance of DIO mice and have a certain weight reduction effect; HEC-12063 is at 3nmol/kg. The effect of improving glucose tolerance at the kg dose is better than that of semaglutide-10nmol/kg, and the weight reduction effect of HEC-12063 at the dose of 10nmol/kg is similar to that of semaglutide-10nmol/kg. See Table 5, Table 6 and Figure 2 and Figure 3 for specific data.
  • Table 5 The effect of long-term administration of HEC-12063 on the rate of blood glucose drop in glucose-loaded DIO mice
  • HEC-12063 Long-term administration of HEC-12063 can significantly improve the glucose tolerance of DIO mice and significantly reduce the body weight of DIO mice.
  • Example 9 Effect of candidate HEC-C046 on blood glucose and body weight of ob/ob mice
  • mice 7-week-old ob/ob mice were fed with a high-fat diet for 3 weeks and divided into 3 groups (Model, semaglutide-10nmol/kg, HEC-C046-10nmol/kg group) according to body weight and blood sugar, and started subcutaneously at the 4th week Administration (the model group was given the corresponding vehicle), semaglutide was administered once a day, and HEC-C046 was administered twice a week for 4 weeks. During the administration, the blood glucose and body weight of each group were tested.
  • Example 10 Effect of candidate HEC-C046 on blood glucose and body weight of db/db diabetic mice
  • mice 7-week-old db/db diabetic mice were divided into 4 groups (Model, semaglutide-10nmol/kg, HEC-C046-3nmol/kg, HEC-C046-10nmol/kg group) according to body weight and blood sugar, and were administered subcutaneously (The model group was given the corresponding vehicle), semaglutide was administered once a day, and HEC-C046 was administered twice a week, and the dose of HEC-C046-3nmol/kg was increased to 6nmol/kg on the 17th day of administration. All animals in all groups were given a total of The drug was administered for 4 weeks, and the blood glucose and body weight of each group of animals were tested during the administration period.
  • HEC-C046 can significantly reduce blood sugar and body weight of db/db mice after long-term administration at a dose of 10nmol/kg, and is superior to the effect of Semaglutide at a dose of 10nmol/kg. See Table 9, Table 10 and Figure 6, Figure 7 for specific data.
  • mice Normal C57BL/6 mice were divided into 3 groups according to blood sugar and body weight (Control, Dulaglutide-7.5nmol/kg, HEC-C079-7.5nmol/kg), 8 mice in each group, and the corresponding vehicle or candidate was injected subcutaneously , The animals were fasted 56h after the administration. 72h after the administration, the animals in each group were intraperitoneally injected with 2g/kg glucose, and blood glucose was measured before and 15, 30, 60, and 90 minutes after the administration of the sugar. Draw the blood glucose concentration-time curve according to the blood glucose values measured at different time points, as shown in Figure 8, calculate the AUC 0 ⁇ 90min Glu of each dose group and the hypoglycemic rate at the peak blood glucose.
  • HEC-C079 can significantly reduce the blood glucose level of mice with glucose load.
  • mice C57BL/6 mice aged 7-8 weeks were fed with a high-fat diet for 4 months and then divided into 3 groups (Model, semaglutide 5nmol/kg, HEC-C079 5nmol/kg) according to their body weight. The administration was started at the 5th month (the model group was given the corresponding vehicle), semaglutide was administered once a day, and HEC-C079 was administered twice a week for a total of 4 weeks. The animals were weighed before each administration. The IPGTT experiment was conducted in the 4th week.
  • HEC-C079 can significantly reduce the blood glucose and body weight of DIO mice at a dose of 5nmol/kg; HEC-C079 improves glucose tolerance better than semaglutide-5nmol/kg at a dose of 5nmol/kg, and HEC-C079 at 5nmol/kg The weight reduction effect of dose is similar to that of semaglutide-5nmol/kg. See Tables 12 and 13 and Figure 9 and Figure 10 for specific data.
  • Table 12 Effect of long-term administration of HEC-C079 on the rate of blood glucose drop in DIO mice with glucose load
  • HEC-C079 Long-term administration of HEC-C079 can significantly improve the glucose tolerance of DIO mice and significantly reduce the body weight of DIO mice.

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Abstract

一种GLP-1/GCG双受体激动剂多肽。该多肽具有如下所示的氨基酸序列： 5 'X 1SQGT FTSDX 10 SKYLX 15 X 16X 17X 18AX 20 X 21FX 23X 24W L X 27X 28 X 29X 30 3 '，其中，X 1为H或Y，X 10为Y或L，X 15为D或E，X 16为M或E，X 17为Q，K或R，X 18为R或A，X 20为H或K，X 21为E或D，X 23为V或I，X 24为Q，A，N或E，X 27为L或M，X 28为A，N或D，X 29为A或G，X 30为T，PSSGAPPPS或不存在。上述GLP-1/GCG双受体激动剂多肽，降糖减重效果佳，体内半衰期长，是一种更为有效的长效减重控糖类药物。

Description

GLP-1/GCG双受体激动剂多肽 技术领域
本发明涉及生物医药领域，具体地，本发明涉及GLP-1/GCG双受体激动剂多肽、融合蛋白、核酸、构建体、重组细胞、药物组合物以及制药用途。
背景技术
肥胖与Ⅱ型糖尿病、高血脂和高血压等有着密切的联系。目前一线治疗二型糖尿病的药物主要作用为降糖，而降体重的效果非常有限，如何开发出同时具有良好降糖减重效果的药物是该领域当前的研发热点。
胰高血糖素样肽-1(GLP-1)是一种进食后由肠道L-细胞分泌的多肽激素，能刺激胰岛β细胞分泌胰岛素，从而稳定餐后血糖的波动。其降低血糖的作用具有葡萄糖浓度依赖性，在调节血糖的同时，大大降低了低血糖的风险。近几年基于GLP-1的药物，如利拉鲁肽、度拉鲁肽及索马鲁肽，逐渐在糖尿病药物中占据了非常重要的地位。而GLP-1类药物在降低血糖时还有减肥的效果，其机理为GLP-1作用于胃肠道延缓胃排空和肠道蠕动，以及作用于中枢神经系统抑制食欲等，从而达到减少摄食的目的。目前利拉鲁肽已批准成为减肥药，而其二代产品索马鲁肽减肥效果更佳。但这类药物主要作用在控糖，降低体重的效果有限，仍远未满足临床需求。
胰高血糖素(GCG)是由胰岛α细胞分泌的一种多肽激素，可促进糖原分解和糖异生，使血糖显著升高；同时还可激活脂肪酶，促进脂肪分解，提高脂肪酸氧化，增加能量消耗，从而具有减脂降低体重的作用。由于其有升糖生理作用，虽可用于低血糖方面的治疗，但限制了其在肥胖减肥中的应用，尤其是Ⅱ型糖尿病肥胖者中的应用。
胃泌酸调节素(OXM)是一种激活胰高血糖素样肽-1受体(GLP-1R)和胰高血糖素受体(GCGR)的双受体激动剂。其GLP-1受体活性的降糖作用可平衡因激动GCGR导致的升糖作用，并协同二者在降低体重方面的作用，从而达到更优的减肥效果。已有研究证明OXM在动物模型中具有良好的降糖减重作用，明显优于现有GLP-1类药物，如利拉鲁肽。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
胃泌酸调节素(OXM)存在的一些如稳定性差、受体活性低等的问题，会导致其给药剂量大，同时OXM对GLP-1R与GCGR的活性比值固定，很难达到控糖减重的最佳效果。因此，发明人对OXM进行了优化改造，提高了其稳定性，延长了体内半衰期，并提高了受体活性，降低了给药剂量，同时优化了受体间的平衡达到了最佳的控糖减重效果。
基于上述优化改造结果，在本发明的第一方面，本发明提出了一种GLP-1/GCG双受体激 动剂多肽。根据本发明的实施例，所述多肽具有如下所示的氨基酸序列： 5’X 1SQGT FTSDX 10SKYLX 15X 16X 17X 18AX 20X 21FX 23X 24W L X 27X 28X 29X 30 3’，其中，X 1为H或Y，X 10为Y或L，X 15为D或E，X 16为M或E，X 17为Q，K或R，X 18为R或A，X 20为H或K，X 21为E或D，X 23为V或I，X 24为Q，A，N或E，X 27为L或M，X 28为A，N或D，X 29为A或G，X 30为T，PSSGAPPPS或不存在。根据本发明实施例的上述GLP-1/GCG双受体激动剂多肽，降糖减重效果佳，体内半衰期长，是一种更为有效的长效减重控糖类药物。
根据本发明的实施例，上述多肽还可以进一步具有如下附加技术特征至少之一：
根据本发明的实施例，所述多肽具有如下所示的氨基酸序列： 5’X 1SQGTFTSDX 10SKYLX 15EX 17X 18AK X 21FX 23X 24W LLAGX 30 3’，其中，X 30为PSSGAPPPS或不存在。
根据本发明的实施例，所述多肽具有如下所示的氨基酸序列： 5’HSQGT FTSDY SKYLD X 16X 17X 18AX 20X 21FX 23X 24W LX 27X 28T 3’，其中，X 24为Q，A或N，X 28为N或D。
根据本发明的实施例，所述多肽具有SEQ ID NO:1～21，SEQ ID NO:71任一所示的氨基酸序列。
Figure PCTCN2020125110-appb-000001
Figure PCTCN2020125110-appb-000002
根据本发明的实施例，所述多肽具有SEQ ID NO:1～3所示的氨基酸序列。
Figure PCTCN2020125110-appb-000003
发明人发现，具有SEQ ID NO:1～3所示的氨基酸序列的GLP-1/GCG双受体激动剂多肽在刺激表达GLP-1R或GCGR方面具有显著优势，同时，在体实验研究结果显示，具有SEQ ID NO:1～3所示的氨基酸序列的GLP-1/GCG双受体激动剂多肽在控制体重和降低血糖方面效果显著。
根据本发明的实施例，所述多肽具有SEQ ID NO:22～34任一所示的氨基酸序列。
Figure PCTCN2020125110-appb-000004
在本发明的第二方面，本发明提出了一种融合蛋白。根据本发明的实施例，所述融合蛋白包括前面所述的多肽、连接肽以及IgG 4-Fc片段，所述连接肽设置于所述多肽和IgG 4-Fc片段的首尾之间。根据本发明实施例的融合蛋白降糖减重效果佳，体内半衰期长，是一种有效的长效减重控糖类药物。
根据本发明的实施例，上述融合蛋白还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述连接肽的N端与所述多肽的C端相连，所述连接肽的C端与所述IgG 4-Fc片段的N端相连。
根据本发明的实施例，所述连接肽具有SEQ ID NO:35所示的氨基酸序列。
Figure PCTCN2020125110-appb-000005
根据本发明的实施例，所述IgG4-Fc片段具有SEQ ID NO:36所示的氨基酸序列。
Figure PCTCN2020125110-appb-000006
根据本发明的实施例，所述融合蛋白具有SEQ ID NO:37～70，SEQ ID NO:72所示的氨基酸序列。
Figure PCTCN2020125110-appb-000007
Figure PCTCN2020125110-appb-000008
Figure PCTCN2020125110-appb-000009
Figure PCTCN2020125110-appb-000010
Figure PCTCN2020125110-appb-000011
Figure PCTCN2020125110-appb-000012
在本发明的第三方面，本发明提出了一种核酸。根据本发明的实施例，所述核酸编码前面所述的多肽或前面所述的融合蛋白。
在本发明的第四方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体携带前面所述的核酸。
根据本发明的实施例，上述构建体还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述构建体的载体为pXC17.4。
在本发明的第五方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞表达前面所述的多肽或前面所述的融合蛋白。
在本发明的第六方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞含有前面所述的构建体或基因组中整合有前面所述的核酸。
根据本发明的实施例，上述重组细胞还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述重组细胞为CHO细胞。
在本发明的第七方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括前面所述的多肽或前面所述的融合蛋白。
根据本发明的实施例，上述药物组合物还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述药物组合物进一步包括药物上可接受的载体。
根据本发明的实施例，所述药物组合物进一步包括其他抗糖尿病药物，所述其他抗糖尿病药物包括选自胰岛素类、双胍类、磺酰脲类、罗格列酮或匹格列酮、α-葡萄糖苷酶抑制剂以及氨基二肽酶IV抑制剂的至少之一。
在本发明的第八方面，本发明提出了一种蛋白制剂。根据本发明的实施例，包括前面所述的多肽或前面所述的融合蛋白。根据本发明实施例的蛋白制剂是一种有效的长效减重控糖类药物。
在本发明的第九方面，本发明提出了前面所述的多肽或前面所述的融合蛋白在制备药物中的用途，所述药物用于治疗或预防代谢疾病。
根据本发明的实施例，所述代谢疾病包括选自非酒精性脂肪肝病(NAFLD)、糖尿病、肥胖症的至少之一。
附图说明
图1是根据本发明实施例的糖负荷小鼠给药后不同时间点的血糖浓度-时间曲线；
图2是根据本发明实施例的融合蛋白HEC-12063对DIO小鼠糖耐量的影响实验结果图；
图3是根据本发明实施例的融合蛋白HEC-12063对DIO小鼠体重影响的实验结果图；
图4是根据本发明实施例的融合蛋白HEC-C046对ob/ob小鼠血糖影响的实验结果图；
图5是根据本发明实施例的融合蛋白HEC-C046对ob/ob小鼠体重影响的实验结果图；
图6是根据本发明实施例的融合蛋白HEC-C046对db/db小鼠血糖影响的实验结果图；
图7是根据本发明实施例的融合蛋白HEC-C046对db/db小鼠体重影响的实验结果图；
图8是根据本发明实施例的糖负荷小鼠给药后不同时间点的血糖浓度-时间曲线；
图9是根据本发明实施例的融合蛋白HEC-C079对DIO小鼠糖耐量的影响实验结果图；
图10是根据本发明实施例的融合蛋白HEC-C079对DIO小鼠体重影响的实验结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
实施例1 GLP-1/GCG双受体激动剂多肽的制备方法
通过在编码多肽基因的5’端添加SUMO标签的基因序列，从而形成融合基因。将该融合基因克隆至原核表达载体并在大肠杆菌细胞中进行诱导表达。离心收菌体后，超声破碎并离心取上清，然后通过镍柱纯化获得融合蛋白。最后SUMO蛋白酶酶切处理后，经反相纯化获得目标多肽。具体过程如下：
1.载体构建与引物合成
以pET-28a为表达载体，BL21(DE3)作为宿主，制备多肽的具体步骤如下：
1)设计引物，引物之间互为模板，扩增出多肽基因片段。并以多肽基因片段以及与sumo基因片段为模板，通过融合PCR方法扩增出融合基因片段。
2)构建重组表达载体：融合基因片段，克隆至原核表达载体pET-28a中，转化大肠杆菌BL21(DE3)，挑选重组子，重组表达质粒样品送至广州艾基生物技术有限公司测序验证。
2.表达纯化
构建得到的BL21(DE3)菌株，于LB中培养，加入终浓度为50μg/mL的卡那霉素(kanamycin)，培养后用IPTG诱导表达5h。取离心后的菌体，用平衡缓冲液(20mM Tris-HCl，pH8.0，150mM的NaCl)溶解，超声仪破碎菌体，离心后上清用于融合蛋白纯化。通过Ni-NTA亲和层析柱(GE Healthcare)纯化，用洗脱缓冲液(20mM Tris-HCl，pH8.0，150mM的NaCl，200mM咪唑)洗脱得到融合蛋白。
3.融合蛋白酶切
用平衡缓冲液稀释蛋白液后，按照蛋白量：SUMO蛋白酶为50:1的比例加入SUMO蛋白酶，室温酶切1.5h。
4.多肽的纯化
将酶切后所得的多肽水溶液，加入20％的乙腈。通过粒径8μm的4.6*250mm C8填充柱在AKTA纯化系统进行制备纯化。用20％乙腈/H 2O(含20mM Tris-HCl，pH8.0)为起始，以梯度(1％/min的速度增加乙腈的比例)，流速为1mL/min，将该柱洗脱30分钟，收集含有肽的组分。用液质联用分析分离出的产物。
纳微UniSil 8-120 C8 Ultra Plus 8 um 4.6*250mm来自苏州纳微科技股份有限公司。
实施例2测定GLP-1/GCG双受体激动剂多肽的体外活性
实施例1所获得的GLP-1/GCG双受体激动剂多肽的氨基酸序列以及所对应的多肽样品的样品名称如表1所示。
通过GLP-1/GCG双受体激动剂多肽刺激表达GLP-1R或GCGR的HEK293细胞，用cAMP检测试剂盒(Cisbio，62AM6PEC)检测受体细胞所产生的cAMP，建立量效曲线，计算其EC 50，结果如表2所示，并进行相互比较。
表1：
样品名 氨基酸序列
HEC-1202 HSQGT FTSDY SEYLD SERAR DFVAW LEAGG
HEC-1204 YSQGTFTSDYSKYLDEQAAKEFVNWLLAGGPSSGAPPPS
HEC-1205 HSQGT FTSDY SKYLD MQRAH DFVQW LMNT
HEC-1206 HSQGT FTSDY SKYLD EKRAK EFVQW LMNT
HEC-12041 HSQGTFTSDYSKYLDEQAAKEFVNWLLAGGPSSGAPPPS
HEC-12042 HSQGT FTSDYSKYLDEQAAK EFVNW LLAG
HEC-12043 HSQGT FTSDYSKYLDEQAAK EFVNW LMNT
HEC-12045 HSQGT FTSDY SKYLD EQAAK EFVQW LLAG
HEC-12046 HSQGT FTSDY SKYLD EQAAK DFVQW LLAG
HEC-12047 HSQGT FTSDY SKYLD ERAAK EFVQW LLAG
HEC-12061 HSQGT FTSDY SKYLD EKRAK EFVQW LLAG
HEC-12062 HSQGT FTSDY SKYLD EKRAK EFVAW LLAG
HEC-12063 HSQGT FTSDY SKYLD EKRAK EFIAW LLAG
HEC-12064 HSQGT FTSDY SKYLD EKAAK EFVAW LLAG
HEC-12066 HSQGT FTSDY SKYLD EKRAK EFVAW LMNT
HEC-12067 HSQGT FTSDY SKYLD EKRAK EFIAW LMNT
HEC-12068 HSQGT FTSDY SKYLD EKRAK EFIAW LMDT
HEC-C007 HSQGT FTSDY SKYLD ERAAK DFVQW LMNT
HEC-C008 HSQGT FTSDY SKYLD ERAAK DFVQW LLDT
HEC-C009 HSQGT FTSDY SKYLD EQRAK DFVQW LLDT
HEC-C010 HSQGTFTSDYSKYLDERRAKEFVQWLLDT
HEC-C011 HSQGTFTSDYSKYLDERRAKDFIQW LLDT
HEC-C012 HSQGT FTSDY SKYLD ERRAK DFVAW LLDT
HEC-C044 HSQGT FTSDL SKYLD EKRAK EFIAW LLAG
HEC-C045 HSQGT FTSDY SKYLE EKRAK EFIAW LLAG
HEC-C046 HSQGT FTSDY SKYLD EKRAK EFIEW LLAG
HEC-C047 HSQGTFTSDYSKYLDEKRAKEFIAWLLAGGPSSGAPPPS
HEC-C048 HSQGT FTSDYSKYLDEKRAK EFIAW LLDT
HEC-C049 HSQGT FTSDYSRYLDEKRAK EFIAW LLAG
HEC-C050 HSQGT FTSDYSKYLD EKRAK EFIQW LLAG
HEC-C051 HSQGT FTSDYSKYLD EKRAK DFVQW LLAG
HEC-C052 HSQGT FTSDYSKYLD EQAAK EFIEW LLAG
HEC-C053 HSQGT FTSDYSKYLD EQAAK DFVEW LLAG
HEC-C054 HSQGT FTSDYSKYLD EKAAK EFIEW LLAG
HEC-C055 HSQGT FTSDY SKYLD EKAAK DFVEW LLAG
HEC-C079 HSQGT FTSDY SKYLD EKAAK EFVEW LLAG
表2：
Figure PCTCN2020125110-appb-000013
由表2实验结果可知，本发明所获得的GLP-1/GCG受体双重激动剂多肽样品能够显著激活GLP-1R或GCGR受体细胞。
实施例3融合蛋白载体的构建
采用分子克隆方法，将GLP-1/GCG受体双重激动剂多肽与带有连接肽(GGGGSGGGGSGGGGSA)的IgG4-Fc(ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG)进行融合，所得序列经双酶切后，插入到哺乳动物细胞表达载体的相同酶切位点间，构建一系列突变体载体，经测序验证正确后，采用去内毒素的质粒提取试剂盒(OMEGA)提取质粒载体，-20℃保藏。其中载体中的GLP-1/GCG受体双重激动剂多肽的氨基酸序列以及所对应的多肽样品名称如表1所示。实施例3所构建的包含GLP-1/GCG受体双重激动剂多肽的融合蛋白的样品名称与实施例1所获得的GLP-1/GCG受体双重激动剂多肽的样品名称一致。
实施例4载体转染及在细胞中表达
将中国仓鼠卵巢细胞(CHO)复苏后传代培养，至密度约为6*10 6细胞/mL时收集细胞进行转染，采用ExpiCHOFectamineTM CHO Transfection Kit(ThermoFisher Scientific)，实施例2所构建载体终浓度为1μg/mL。转染第二天加入增强剂等试剂维持转染细胞的生长。细胞活率降至约80％时收获细胞培养液。
实施例5融合蛋白纯化及鉴定
将细胞培养液离心收集上层清液，并用0.22μm滤膜过滤除去残余细胞碎片。对收集的细胞培养液采用Protein A层析柱进行纯化，收集目标峰，再用阴离子交换层析进一步精纯，蛋白最终用0.02M PBS洗脱收集。样品采用微量核酸蛋白测定仪定量(NanoDrop 2000/2000c  Spectrophotometer)。样品经12％SDS-PAGE电泳检测，电泳图谱显示条带单一。通过质谱测定融合蛋白的精确分子量，与理论分子量基本一致。
实施例6测定融合蛋白的体外活性
通过融合蛋白刺激表达GLP-1R或GCGR的HEK293细胞，用cAMP检测试剂盒(Cisbio，62AM6PEC)检测受体细胞所产生的cAMP，建立量效曲线，计算其EC 50，结果如表3所示，并进行相互比较。
表3：
Figure PCTCN2020125110-appb-000014
Figure PCTCN2020125110-appb-000015
由表3实验结果可知，本发明所获得的GLP-1/GCG受体双重激动剂样品能够显著激活GLP-1R或GCGR受体细胞，其中，HEC-12042、HEC-12063以及HEC-C046在激活GLP-1R或GCGR受体细胞方面具有显著优势。
实施例7候选物对正常C57BL/6小鼠糖耐量的影响
实验方法：正常C57BL/6小鼠按照血糖和体重分为6组(Control、Dulaglutide-7.5nmol/kg、HEC-C046-7.5nmol/kg、HEC-C049-7.5nmol/kg、HEC-C050-7.5nmol/kg、HEC-12063-7.5nmol/kg)，每组8只，皮下注射给予相应溶媒或者候选物，给药后56h动物禁食，给药后72h各组动物腹腔注射2g/kg的葡萄糖，并于给糖前及给糖后15、30、60、90min进行血糖检测。根据不同时间点测定的血糖值绘制血糖浓度-时间曲线如图1所示，计算各剂量组AUC 0～90min Glu及血糖峰值时的降糖率。
实验结果：
表4：GCG/GLP-1系列蛋白对糖负荷小鼠血糖的影响
Figure PCTCN2020125110-appb-000016
实验结论：HEC-12063、HEC-C046、HEC-C049与HEC-C050均可显著降低糖负荷小鼠血糖水平。
实施例8候选物HEC-12063对DIO小鼠糖耐量和体重的影响
实验方法：8周龄C57BL/6小鼠高脂饮食喂养4个月后根据体重分为5组(Model、semaglutide 10nmol/kg、HEC-C12063 3nmol/kg、HEC-C12063 10nmol/kg、HEC-C12063 30nmol/kg)。第5个月开始给药(模型组给予相应溶媒)，semaglutide每天给药一次， HEC-C12063各剂量组每周给药两次，共给药4周，每次给药前进行动物体重称量，给药第4周进行IPGTT实验。
实验结果：HEC-12063呈剂量依赖性降低DIO小鼠血糖及体重，在3nmol/kg剂量下即可具有显著的改善DIO小鼠糖耐量作用并具有一定的降体重作用；HEC-12063在3nmol/kg剂量下改善糖耐量作用优于semaglutide-10nmol/kg，HEC-12063在10nmol/kg剂量下降体重作用与semaglutide-10nmol/kg近似。具体数据见表5、表6及图2、图3。
表5：HEC-12063长期给药对糖负荷DIO小鼠血糖下降率的影响
Figure PCTCN2020125110-appb-000017
表6：HEC-12063长期给药对DIO小鼠体重的影响
Figure PCTCN2020125110-appb-000018
实验结论：HEC-12063长期给药可显著改善DIO小鼠糖耐量并显著降低DIO小鼠体重。
实施例9候选物HEC-C046对ob/ob小鼠血糖和体重的影响
实验方法：7周龄ob/ob小鼠高脂饮食喂养3周后根据体重、血糖分为3组(Model、semaglutide-10nmol/kg、HEC-C046-10nmol/kg组)，第4周开始皮下给药(模型组给予相应溶媒)，semaglutide每天给药一次，HEC-C046每周给药两次，共给药4周，给药期间对各组动物血糖及体重进行检测。
实验结果：HEC-C046在10nmol/kg剂量下长期给药可显著降低ob/ob小鼠血糖及体重，且优于Semaglutide在10nmol/kg剂量下的作用。具体数据见表7、表8及图4、图5。
表7：HEC-C046长期给药对ob/ob小鼠血糖的影响
Figure PCTCN2020125110-appb-000019
表8：HEC-C046长期给药对ob/ob小鼠体重的影响
Figure PCTCN2020125110-appb-000020
Figure PCTCN2020125110-appb-000021
实验结论：HEC-C046长期给药对ob/ob小鼠血糖及体重的改善作用优于同剂量下的Semaglutide。
实施例10候选物HEC-C046对db/db糖尿病小鼠血糖和体重的影响
实验方法：7周龄db/db糖尿病小鼠根据体重、血糖分为4组(Model、semaglutide-10nmol/kg、HEC-C046-3nmol/kg、HEC-C046-10nmol/kg组)，皮下给药(模型组给予相应溶媒)，semaglutide每天给药一次，HEC-C046每周给药两次，其中HEC-C046-3nmol/kg于给药第17天剂量提高至6nmol/kg，所有组动物共给药4周，给药期间对各组动物血糖及体重进行检测。
实验结果：HEC-C046在10nmol/kg剂量下长期给药后可显著降低db/db小鼠血糖及体重，且优于Semaglutide在10nmol/kg剂量下的作用。具体数据见表9、表10及图6、图7。
表9：HEC-C046长期给药对db/db小鼠血糖的影响
Figure PCTCN2020125110-appb-000022
表10：HEC-C046长期给药对db/db小鼠体重的影响
Figure PCTCN2020125110-appb-000023
实验结论：HEC-C046长期给药对db/db小鼠血糖及体重的改善作用显著优于semaglutide。
实施例11候选物HEC-C079对正常C57BL/6小鼠糖耐量的影响
实验方法：正常C57BL/6小鼠按照血糖和体重分为3组(Control、Dulaglutide-7.5nmol/kg、HEC-C079-7.5nmol/kg)，每组8只，皮下注射给予相应溶媒或者候选物，给药后56h动物禁食，给药后72h各组动物腹腔注射2g/kg的葡萄糖，并于给糖前及给糖后15、30、60、90min进行血糖检测。根据不同时间点测定的血糖值绘制血糖浓度-时间曲线如图8所示，计算各剂量组AUC 0～90min Glu及血糖峰值时的降糖率。
实验结果：
表11：HEC-C079对糖负荷小鼠血糖的影响
Figure PCTCN2020125110-appb-000024
实验结论：HEC-C079可显著降低糖负荷小鼠血糖水平。
实施例12候选物HEC-C079对DIO小鼠糖耐量和体重的影响
实验方法：7-8周龄C57BL/6小鼠高脂饮食喂养4个月后根据体重分为3组(Model、semaglutide 5nmol/kg、HEC-C079 5nmol/kg)。第5个月开始给药(模型组给予相应溶媒)，semaglutide每天给药一次，HEC-C079每周给药两次，共给药4周，每次给药前进行动物体重称量，给药第4周进行IPGTT实验。
实验结果：HEC-C079在5nmol/kg剂量下可显著降低DIO小鼠血糖及体重；HEC-C079在5nmol/kg剂量下改善糖耐量作用优于semaglutide-5nmol/kg，HEC-C079在5nmol/kg剂量下降体重作用与semaglutide-5nmol/kg近似。具体数据见表12、13及图9、图10。
表12：HEC-C079长期给药对糖负荷DIO小鼠血糖下降率的影响
Figure PCTCN2020125110-appb-000025
表13：HEC-C079长期给药对DIO小鼠体重的影响
Figure PCTCN2020125110-appb-000026
实验结论：HEC-C079长期给药可显著改善DIO小鼠糖耐量并显著降低DIO小鼠体重。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (22)

  1. 一种GLP-1/GCG双受体激动剂多肽，其特征在于，所述多肽具有如下所示的氨基酸序列：
    5’X 1SQGT FTSDX 10 SKYLX 15 X 16X 17X 18AX 20X 21FX 23X 24W L X 27X 28 X 29X 30  3’
    其中，X 1为H或Y，
    X 10为Y或L，
    X 15为D或E，
    X 16为M或E，
    X 17为Q，K或R，
    X 18为R或A，
    X 20为H或K，
    X 21为E或D，
    X 23为V或I，
    X 24为Q，A，N或E，
    X 27为L或M，
    X 28为A，N或D，
    X 29为A或G，
    X 30为T，PSSGAPPPS或不存在。
  2. 根据权利要求1所述的多肽，其特征在于，所述多肽具有如下所示的氨基酸序列：
    5’X 1SQGT FTSDX 10 SKYLX 15 EX 17X 18AK X 21FX 23X 24W LLAGX 30  3’
    其中，X 30为PSSGAPPPS或不存在。
  3. 根据权利要求1所述的多肽，其特征在于，所述多肽具有如下所示的氨基酸序列：
    5’HSQGT FTSDY SKYLD X 16X 17X 18AX 20 X 21FX 23X 24W LX 27X 28T  3’
    其中，X 24为Q，A或N，X 28为N或D。
  4. 根据权利要求2所述的多肽，其特征在于，所述多肽具有SEQ ID NO:1～21，SEQ ID NO:71任一所示的氨基酸序列，优选地，所述多肽具有SEQ ID NO:1～3所示的氨基酸序列。
  5. 根据权利要求3所述的多肽，其特征在于，所述多肽具有SEQ ID NO:22～34任一所示的氨基酸序列。
  6. 一种融合蛋白，其特征在于，包括：权利要求1～5任一项所述的多肽、连接肽以及IgG 4-Fc片段，所述连接肽设置于所述多肽和IgG 4-Fc片段的首尾之间。
  7. 根据权利要求6所述的融合蛋白，其特征在于，所述连接肽的N端与所述多肽的C端相连，所述连接肽的C端与所述IgG 4-Fc片段的N端相连。
  8. 根据权利要求6所述的融合蛋白，其特征在于，所述连接肽具有SEQ ID NO:35所示的氨基酸序列。
  9. 根据权利要求6所述的融合蛋白，其特征在于，所述IgG4-Fc片段具有SEQ ID NO:36所示的氨基酸序列。
  10. 根据权利要求6所述的融合蛋白，其特征在于，所述融合蛋白具有SEQ ID NO:37～70，SEQ ID NO:72所示的氨基酸序列。
  11. 一种核酸，其特征在于，所述核酸编码权利要求1～5任一项所述的多肽或权利要求6～10任一项所述的融合蛋白。
  12. 一种构建体，其特征在于，携带权利要求11所述的核酸。
  13. 根据权利要求12所述的构建体，其特征在于，所述构建体的载体为pXC17.4。
  14. 一种重组细胞，其特征在于，表达权利要求1～5任一项所述的多肽或权利要求6～10任一项所述的融合蛋白。
  15. 一种重组细胞，其特征在于，所述重组细胞含有权利要求12或13所述的构建体或基因组中整合有权利要求11所述的核酸。
  16. 根据权利要求14或15所述的重组细胞，其特征在于，所述重组细胞为CHO细胞。
  17. 一种药物组合物，其特征在于，包括权利要求1～5任一项所述的多肽或权利要求6～10任一项所述的融合蛋白。
  18. 根据权利要求17所述的药物组合物，其特征在于，进一步包括药物上可接受的载体。
  19. 根据权利要求17所述的药物组合物，其特征在于，进一步包括其他抗糖尿病药物，所述其他抗糖尿病药物包括选自胰岛素类、双胍类、磺酰脲类、罗格列酮或匹格列酮、α-葡萄糖苷酶抑制剂以及氨基二肽酶IV抑制剂的至少之一。
  20. 一种蛋白制剂，其特征在于，包括权利要求1～5任一项所述的多肽或权利要求6～10任一项所述的融合蛋白。
  21. 权利要求1～5任一项所述的多肽或权利要求6～10任一项所述的融合蛋白或权利要求20所述的蛋白制剂在制备药物中的用途，所述药物用于治疗或预防代谢疾病。
  22. 根据权利要求21所述的用途，其特征在于，所述代谢疾病包括选自非酒精性脂肪肝病、糖尿病、肥胖症的至少之一。
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