CN111269312A

CN111269312A - Heterologous fusion protein

Info

Publication number: CN111269312A
Application number: CN201811471370.8A
Authority: CN
Inventors: 刘忠
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2018-12-04
Filing date: 2018-12-04
Publication date: 2020-06-12
Anticipated expiration: 2038-12-04
Also published as: CN111269312B

Abstract

The present invention provides for the replacement of a particular fusion protein at multiple positions in the GLP-1 portion and Fc portion of the molecule. Wherein, the Ser at the 11 th site, the Val at the 85 th site, the Val at the 86 th site, the Ser at the 87 th site and the Val at the 91 st site of the Fc are replaced, and the terminal Lys is deleted, thereby overcoming the problem of potential immunogenicity related to the application of GLP-1-Fc fusion to a great extent, and the results of in vitro and in vivo activity experiments also prove that the activity of the GLP-1 analogue disclosed by the invention is obviously higher than that of the existing GLP-1 analogue. Therefore, the analogs can be used to better prevent, prevent or reduce diabetes and obesity and/or complications.

Description

Heterologous fusion protein

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a heterologous fusion protein.

Background

Diabetes Mellitus (DM) is a complex metabolic disease with elevated blood sugar caused by endocrine metabolism disorder due to insufficient insulin secretion or synthesis inhibition in vivo, and is mainly divided into type 1 diabetes mellitus (T1 DM), type2diabetes mellitus (T2 diabetes mellitus, T2DM) and other types of diabetes mellitus, according to statistics, the type2diabetes mellitus accounts for a large proportion, which accounts for more than 90% of the total number of patients with diabetes mellitus, and the disease period is more than 35-40 years old, and more serious complications appear in the later period of onset.

Glucagon-like peptide (GLP) is an intestinal hormone secreted by human body, and is formed by cracking Glucagon molecules through intestinal proteolytic enzyme to generate two Glucagon-like peptides GLP-1 and GLP-2. Among them, GLP-1 has two active forms, i.e., GLP-1(7-37) and GLP-1(7-36) amide forms, and has the same effect of promoting insulin secretion in vivo, so it is also called insulinotropic peptide (Incretin or insulinotropic peptide) (Negar Sarrazadeh et al, Pharmaceutical Sciences, Vol.96, 1925-1954 (2007)).

GLP-1(7-37)：

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly GlnAla Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly。

GLP-1 agonist is taken as a kind of polypeptide drug, and can only be injected for administration at present, and in order to improve the compliance of patients, sustained-release injection and non-injection administration become main directions for the research and development of the kind of drugs. Structural modification is adopted to cover DPP-IV enzyme sites and prolong the biological half-life, which is the most mainstream method at present. The structural modification comprises two types of chained high-molecular protein and polyethylene glycol modification, wherein Abiglutide and Dulaglutide which are used once a week belong to the typical former type, and although a plurality of polyethylene glycol modified protein or polypeptide medicaments are available on the market, the polyethylene glycol modified GLP-1 analogue starts later, and only the somaglutide enters the third clinical stage. Certainly, some companies can prepare the sustained-release microspheres by means of preparations to realize once a week, and a typical product is Bydureon. There are also companies that directly avoid the disadvantages of injection administration, develop products such as implants, oral administration, transdermal administration and inhalation, wherein oral administration is the most ideal administration route, but need to solve the problems of stomach acid and enzyme damage and absorption of GLP-1 analogue in intestines and stomach, so as to improve bioavailability and reduce individual difference, the current companies that orally take GLP-1 analogue are the Oramed company and the emisphene company, wherein NN9924 of Nound is an oral soma peptide, and the Iseland has reported the exenatide enteric-coated tablet in China.

Amidated GLP-1 derivative Liraglutide (T, DiabetesCare, 2007, 30: 1608-1610) developed by Novo Nordisk of Denmark has Lys at position 34 replaced by Arg as compared with native GLP-1, and a fatty acid side chain having 16 carbons is linked to Lys at position 26 of GLP-1 via glutamyl. Liraglutide can form a non-covalent linked derivative with albumin in plasma, retains the function of GLP-1, antagonizes the degradation of DPP IV, has a half-life prolonged to 11-15 hours, and is suitable for once-a-day administration (Elbrond B, Diabetes Care, 2002, 25: 1398-1404). The C-terminal lysine at position 37 in another GLP-1 analog CJC-1131 under development can form covalent linkage with cysteine residue 34 in albumin in plasma after in vitro modification, and can also achieve the goal of once-a-day administration (Kim, JG, Diabetes, 2003, 52: 751-759). Therefore, GLP-1 and exendin-4 can better retain the biological activity through the GLP-1 analogue obtained by mutating (1 or a plurality of amino acids), deleting or adding amino acids to the C terminal by a chemical or genetic engineering means.

Exendin-4：

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu GluAla Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser。

Exendin-4 is a GLP-1 analogue isolated from lizard saliva and consisting of 39 amino acids, the amino acid sequence of which has sequence similarity with several members of the GLP family and 53% homology with human GLP-1. Because the second position of the N terminal is Ala (GLP-1 is Gly), the N terminal is not easy to be degraded by DPP-IV enzyme, and has longer half life and stronger biological activity. The chemical synthesized product of Exendin-4 is named Exenatide (trade name Byetta), and is jointly developed by Amylin and Lilly in 1995, and approved by FDA to be marketed in 4 months in 2005.

The expression and activity of human GLP-1(7-36) in vivo are strictly regulated, and when the second bit Ala at the N terminal of the human GLP-1(7-36) is hydrolyzed by dipeptidyl peptidase (DPP), inactive GLP-1 (9-36) is formed, and the metabolite is also an in vivo natural antagonist of GLP-1R. Therefore, the natural human active GLP-1 has short half-life in vivo and the metabolism rate of 2 min; in addition, under physiological conditions, GLP-1 is mainly excreted through the kidney, and the clinical application of the human GLP-1 is limited. The second amino acid Gly of non-human-derived artificially synthesized Exenatide is different from Ala of human GLP-1, and can effectively resist the degradation of dipeptide acyl peptidase; the C-terminal rigid (PSSGAPPPS) amino acid sequence of Exenatide can increase the stability of polypeptide, and the blood sugar reducing capability of Exenatide in vivo is about 1000 times stronger than that of GLP-1.

Exenatide peptide secondary structure: the N end of Exenatide is irregularly curled, amino acid residues with opposite charge side chains are alternately arranged on the same side surface of the middle part of Exenatide, a helix is formed through a salt bridge or a polar hydrogen bond, and the C end of Exenatide is hydrophilic Trp-Cage. The interaction mechanism of Exenatide with the GLP-1 receptor has been studied clearly.

A series of different structural designs have been developed to extend the structural half-life of GLP-1 analogs and enhance biological activity. CN1384755A discloses a novel Exendin agonist preparation and an administration method thereof, and discloses a compound structure and a preparation method of Exenatide. CN102532303A discloses the modification of amino group of lysine or amino group of N-terminal histidine residue in Exenatide with methoxypolyethylene glycol. CN101980725B discloses fatty acid-PEG-Exenatide. CN105753963A discloses Exenatide analogs with single-point or multi-point amino acid mutations. CN102397558A discloses the substitution of certain amino acids in Exenatide with cysteine, and the modification with PEG or PEG with methyl substituted end.

Although many efforts are made in the development of GLP-1 analogue long-acting drugs, the GLP-1 analogue on the market at present has poor stability and low drug effect, and innovative modification and research of GLP-1 analogue are still a very important topic as the structure and activity basis of long-acting drug development.

Various approaches have been taken to prolong the clearance half-life of the GLP-1 peptide or to reduce clearance of the peptide from the body while maintaining biological activity. One approach involves fusing a GLP-1 peptide to an immunoglobulin Fc moiety. Immunoglobulins generally have a long circulating half-life in vivo. For example, IgG molecules have a half-life in humans of up to 23 days. The immunoglobulin Fc portion is part of the reason for this in vivo stability. While retaining the biological activity of the GLP-1 molecule, GLP-1-Fc fusion proteins have the advantage of retaining the biological activity of the GLP-1 molecule due to the stability provided by the Fc portion of the immunoglobulin.

However, the problem of immunogenicity of many fusion proteins when repeatedly administered over long periods of time is still prevalent.

Proteins, including therapeutic proteins, are generally immunogenic, in part because the product of protein endocytosis and proteolysis by antigen presenting cells binds the peptide to molecules called the Major Histocompatibility Complex (MHC), which then present the peptide to T cells. Antigen peptide-MHC complexes on the surface of Antigen Presenting Cells (APC) stimulate T cells to proliferate, differentiate and release cytokines. At the same time, B-cell differentiation and antibody production are induced, which may further limit the utility of the therapeutic protein by clearance of the therapeutic protein. Thus, antigenic peptides derived from therapeutic proteins are capable of eliciting a range of unwanted immune responses. The utility of therapeutic proteins is limited by the neutralization of antibodies, and because T-cell and B-cell responses can cause inflammatory and allergic reactions in patients, their induction is often detrimental.

Disclosure of Invention

The present invention provides a heterologous fusion protein that provides multiple positional substitutions in the Fc portion, thereby largely overcoming potential immunogenicity problems associated with administration of Fc fusions and improving stability. The therapeutic peptide bound by the heterologous fusion protein may be some therapeutic peptide capable of being fused to Fc, such as exenatide, liraglutide, lissamide, somaglutide, ldeglira, albiglutide, dulaglutide, ITCA650, LAEx4, Lixilan, and the like.

The invention also provides an improved GLP-1 analogue, and the results of in vivo and in vitro activity experiments prove that the GLP-1 analogue has obviously higher activity than the existing GLP-1 analogue. Therefore, the analogues of the present invention can be used for better prevention, prevention or reduction of diabetes and obesity and/or complications.

The GLP-1 analogue and the immunoglobulin Fc form a fusion protein through a joint, the immunoglobulin Fc part is obtained by modifying the Fc fragment of human IgG4, and the sequence number of the Fc fragment is SEQ ID NO: 1, the specific sequence is as follows:

Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Xaall-Cys-Pro-Ala-Pro-

Glu-Phe-Leu-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-

Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-

Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-

Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-

Glu-Glu-Gln-Phe-Asn-Ser-Thr-Tyr-Arg-Xaa85-Xaa86-Xaa87-Val-Leu-Thr-

Xaa91-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-

Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-

Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-

Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-

Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-

Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-

Leu-Asp--Ser-Asp--Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val--

Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-

Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-

Leu-Ser-Leu-Gly，

wherein Ser at position 11, Val at position 85, Val at position 86, Ser at position 87 and Val at position 91 are replaced, and terminal Lys is deleted, and the method specifically comprises the following steps:

xaa at position 11 is Pro;

xaa at position 85 is Ala or Phe;

xaa at position 86 is Phe or Glu;

xaa at position 87 is Glu, Phe or Leu;

xaa at position 91 is Gly.

Sequence 1 is abbreviated as:

in a preferred embodiment, it is preferably SEQ ID NO: 2, sequence 2:

substitution at position 11 with Pro;

xaa at position 85 is Ala;

xaa at position 86 is Phe;

xaa at position 87 is Glu;

xaa at position 91 is Gly.

Sequence 2 abbreviation:

in a preferred embodiment, it is preferably SEQ ID NO: 3, sequence 3:

substitution at position 11 with Pro;

xaa at position 85 is Phe;

xaa at position 86 is Glu;

xaa at position 87 is Phe;

xaa at position 91 is Gly.

Sequence 3 abbreviation:

the fusion protein, the therapeutic peptide of which is a GLP-1 analog, wherein the C-terminal glycine residue of the GLP-1 analog is fused to the N-terminal alanine residue of the Fc portion via a peptide linker, wherein the peptide linker is SEQ ID NO: 4, in particular to Gly-Ala-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.

The peptide linker may also be selected from the following sequences:

a) Gly-Gly-Gly-Gly-Gly-Ser-Lys-Lys-Lys-Lys-Ser-Gly-Gly-Gly-Gly-Ser, such as SEQ ID NO: 5 is shown in the specification;

b) Gly-Ala-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser, as shown in SEQ ID NO: 6 is shown in the specification;

c) Gly-Gly-Gly-Gly-Gly-Ser-Lys-Lys-Lys-Lys-Ser-Gly-Gly-Gly-Gly-Gly-Ser, as shown in SEQ ID NO: 7 is shown in the specification;

d)Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ IDNO：8)。

in the fusion protein, the GLP-1 analogue is obtained by modifying GLP-1(7-37), and in order to conveniently renumber the GLP-1 analogue, the original 7-bit His is numbered as 1 bit, and the sequence is SEQ ID NO: 9 is sequence 9:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Xaa-Leu-Val-Lys-Gly-Xaa-Xaa；

xaa at the 25 th position is β Trp or β Phe or β His, and β th position in a sequence table is subject to the specification;

xaa at position 30 is Pro or Val or Glu or Ala;

xaa at the 31 th position is Lys-Lys-Lys-Gln-Gln-NH₂；Lys-Lys-Pro-Phe-Phe-Pro-Cys-NH₂；Lys-Lys-Ser-Cys-NH₂；Gly-Gly-Lys-Lys-Cys-NH₂。

In a preferred embodiment, the nucleic acid sequence of SEQ id no: 10, sequence 10:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-β Phe-Leu-Val-Lys-Gly-Ala-Gly-Gly-Lys-Lys-Cys-NH₂。

in a preferred embodiment, the nucleic acid sequence of SEQ id no: 11, sequence 11:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-β Phe-Leu-Val-Lys-Gly-Pro-Lys-Lys-Pro-Phe-Phe-Pro-Cys-NH₂。

in a preferred embodiment, the nucleic acid sequence of SEQ id no: 12, sequence 12:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-β Phe-Leu-Val-Lys-Gly-Glu-Gly-Gly-Lys-Lys-Cys-NH₂。

in a preferred embodiment, the nucleic acid sequence of SEQ id no: 13, sequence 13:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-β Phe-Leu-Val-Lys-Gly-Val-Gly-Gly-Lys-Lys-Cys-NH₂。

in a preferred embodiment, the nucleic acid sequence of SEQ id no: 14, sequence 14:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-βPhe-Leu-Val-Lys-Gly-Val-Lys-Lys-Lys-Gln-Gln-NH₂。

in a preferred embodiment, the nucleic acid sequence of SEQ id no: 15, sequence 15:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-β His-Leu-Val-Lys-Gly-Ala-Gly-Gly-Lys-Lys-Cys-NH₂。

in a preferred embodiment, the nucleic acid sequence of SEQ id no: 16, sequence 16:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-β Trp-Leu-Val-Lys-Gly-Ala-Lys-Lys-Ser-Cys-NH₂。

in a preferred embodiment, the fusion protein, wherein the sequence of the GLP-1 analog is SEQ ID NO: 10.

the fusion protein is a sequence 10-linker sequence 4-sequence 2, is named as HIP-1, and has a sequence number of SEQ ID NO: the sequence of the 17 specific protein is as follows:

the fusion protein is a sequence 10-linker sequence 4-sequence 3, named as HIP-2, and has a sequence number of SEQ ID NO: the sequence of 18 specific proteins is:

in a preferred embodiment, the fusion protein, wherein the sequence of the GLP-1 analog is SEQ ID NO: 11.

the fusion protein is a sequence 11-linker sequence 4-sequence 2, named as HIP-3, and has a sequence number of SEQ ID NO: the 19 specific protein sequence is:

the fusion protein is a sequence 11-linker sequence 4-sequence 3, named as HIP-4, and has a sequence number of SEQ ID NO: 20 the specific protein sequence is:

in a preferred embodiment, the fusion protein, wherein the sequence of the GLP-1 analog is SEQ ID NO: 12.

the fusion protein is a sequence 12-linker sequence 4-sequence 2, named as HIP-5, and has a sequence number of SEQ ID NO: the sequence of the 21 specific protein is as follows:

the fusion protein is a sequence 12-linker sequence 4-sequence 3, named as HIP-6, and has a sequence number of SEQ ID NO: the sequence of the 22 specific protein is as follows:

in a preferred embodiment, the fusion protein, wherein the sequence of the GLP-1 analog is SEQ ID NO: 13.

the fusion protein is a sequence 13-linker sequence 4-sequence 2, is named as HIP-7, and has a sequence number of SEQ ID NO: the 23 specific protein sequence is:

the fusion protein is a sequence 13-linker sequence 4-sequence 2, is named as HIP-8, and has a sequence number of SEQ ID NO: the sequence of the 24 specific proteins is:

in a preferred embodiment, the fusion protein, wherein the sequence of the GLP-1 analog is SEQ ID NO: 14.

the fusion protein is a sequence 14-linker sequence 4-sequence 2, is named as HIP-9, and has a sequence number of SEQ ID NO: 25 the specific protein sequence is:

the fusion protein is a sequence 14-linker sequence 4-sequence 3, is named as HIP-10, and has a sequence number of SEQID NO: 26 the specific protein sequence is:

in a preferred embodiment, the fusion protein, wherein the sequence of the GLP-1 analog is SEQ ID NO: 15.

the fusion protein is a sequence 15-linker sequence 4-sequence 2, is named as HIP-11, and has a sequence number of SEQID NO: 27 the specific protein sequence is:

in a preferred embodiment, the fusion protein, wherein the sequence of the GLP-1 analog is SEQ ID NO: 16.

the fusion protein is a sequence 16-linker sequence 4-sequence 2, is named as HIP-12, and has a sequence number of SEQID NO: 28 specific protein sequences are:

among the above sequences, the Glp-1 analogue sequences 10-16 before Fc fusion show better stability than other Glp-1 analogues, wherein the sequences 10 and 11 are better; the half-life can be further prolonged after the fusion with Fc, wherein Fc sequence 2 is better than sequence 3.

The fusion protein, wherein the GLP-1 analogue can also be exenatide, liraglutide, linatide, soxhlet peptide, Ideglira, albiglutide, dolaglutide, ITCA650, LAEx4, Lixilan and the like.

The Fc part of the fusion protein can be obtained by a solid-phase synthesis method and can also be obtained by eukaryotic cell expression; the GLP-1 analog moiety can be obtained by solid phase synthesis methods.

Solid phase synthesis method:

when the C terminal is carboxyl, the Wang resin is selected for chemical solid phase synthesis of two polypeptides. After the synthesis is finished, the obtained polypeptide resin with the side chain protecting group is cracked, and the C terminal is broken from the resin to form carboxyl. When the C-terminal of the polypeptide is amide, Fmoc-PAL-PEG-PS resin is selected for chemical solid-phase synthesis of the polypeptide. After the synthesis is finished, the obtained polypeptide resin with the side chain protecting group is cracked, and the C terminal is broken from the resin to form amide. The above solid phase synthesis was carried out on a polypeptide synthesizer.

The C-terminal cysteine was first attached to the resin, and then one amino acid was carried out from the C-terminus to the N-terminus. After the synthesis is complete, the Glp-1 analogue polypeptide is purified by reverse phase HPLC chromatography (Waters, C18 preparative column) after deprotection and cleavage from the resin. The purification conditions are that phase A contains 0.5% (V/V) acetic acid water solution, phase B contains 80% acetonitrile and 0.5% (V/V) acetic acid water solution, and gradient elution is carried out by 0-100% B solution. Collecting target polypeptide, and lyophilizing to obtain dry powder. The molecular weight of GLP-1 of sequence 10 is 4288.69 by mass spectrometry, which is consistent with the theoretical value.

In one embodiment, the modified GLP-1 analogs of the invention are prepared as follows:

when Xaa at position 31 is Lys-Lys-Lys-Gln-Gln-NH 2: solid phase synthesis was chosen and, further, Fmoc method was used. As the C end of the polypeptide is an amide group, the Fmoc method can use Fmoc-Rink resin which is characterized in that stable amide bonds are formed by reacting carboxyl of Fmoc-Gln (Trt) -OH with carrier amino, and a crude product can be obtained by cracking after the peptide grafting.

Gly-Gly-Lys-Lys-Cys-NH at position 31₂In this case, a solid-phase synthesis method is selected, and further, Fmoc method is used. As the C end of the polypeptide is an amido group, the Fmoc method can use Fmoc-Rink resin, the principle is that the carboxyl group of Fmoc-Cys (Trt) -OH reacts with the amino group of the carrier to form a stable amide bond, and a crude product can be obtained by cracking after the peptide grafting.

Fmoc-Rink resin (100-200 meshes, the crosslinking degree is 1-2%, and the substitution value is 0.3-0.6 mmol NH/g) is selected as a starting material, and the molar ratio of the protected amino acid to the Fmoc-Rink resin is 2: 1 (or 3: 1). The starting Fmoc-Rink resin (substitution 0.436mmol/g) was reacted with 20% piperidine (PIP/DMF) as a deprotecting agent at room temperature for 25 min, filtered with suction and the resin was washed 6 times with DMF (or DCM). Dissolving equimolar amounts of protected amino acids Fmoc-AAn and HOBt in DMF, cooling at-5 deg.C for more than 30min, slowly adding N, N-diisopropylcarbodiimide solution in dichloromethane to the protected amino acid DMF solution, reacting at-5-5 deg.C for 30min under stirring, and adding to the deprotected resin.

After 3 hours of reaction of the deprotected resin with the activated protected amino acid, the condensation reaction was monitored with ninhydrin test: the resin is blue or dark blue when the condensation reaction is incomplete; the resin is colorless or yellowish when the condensation reaction is complete. After monitoring the complete condensation of the condensation, the resin was washed 6 times with DMF. The condensation of the activated amino acids is carried out in sequence according to the polypeptide sequence. After completion of the peptide-joining reaction, the resin was washed with DMF and then shrunk with methanol, and the resin was dried under reduced pressure.

The dried peptide resin was added to TFA cleavage reagent (in mL: g, TFA: H) at 13mL/g peptide resin dosage₂O, TIS and DTT are equal to 90: 2.5: 5), and after reaction at 0-5 ℃ for 30min, the mixture is stirred and reacted for 2-3h at room temperature. The reaction mixture was filtered, the filtrate was collected and concentrated to 25% of the initial volume at 35 ℃ under reduced pressure. Adding the concentrated solution into anhydrous ether frozen below-10 deg.C, shaking, precipitating at below-10 deg.C for 60 min, filtering, collecting filter cake, washing the filter cake with anhydrous ether for 3-5 times, and drying the filter cake under reduced pressure to constant weight to obtain polypeptide crude product.

Taking the crude polypeptide, dissolving the crude polypeptide in 10% acetic acid water solution for purification. Purification was performed using high performance liquid preparative chromatography with a mobile phase system of 0.1% TFA/water to 0.1% TFA/acetonitrile. The purification method comprises the following steps of using a chromatographic column with 10-micron reversed phase C18 as a chromatographic packing, ultraviolet detection wavelength of 215nm and 50mm x 300mm, enabling the flow rate to be 60mL/min, feeding 2.0-3.0g of sample each time, adopting a gradient system for elution, circularly feeding and purifying, collecting a main peak, and detecting the purity by using an analytical liquid phase, wherein the purity of the purified main peak is more than 98%. The prepared eluent is decompressed and concentrated under the condition of water bath at the temperature of 30-37 ℃ to obtain a concentrated solution of the primary purified material.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm and a flow rate of 30-60 ml min < -1 >. Collecting the target fraction, concentrating the collected fraction at a temperature below 35 deg.C under reduced pressure until the fraction does not flow out, lyophilizing the rest fraction to obtain refined product, and vacuum lyophilizing to obtain pure GLP-1 analogue.

The cell expression method can refer to CN 104277112A, firstly, the coding gene of the heterologous fusion protein is constructed; then cloning the gene to a eukaryotic expression vector to obtain a eukaryotic expression vector capable of expressing the fusion protein; then the expression vector is used for transfecting host cells to enable the host cells to express the recombinant fusion protein, and then the recombinant fusion protein is obtained through separation and purification. The eukaryotic expression vector can be PET32 and the like. The host cell can be FreeStyle 293F, yeast cell, CHO cell, NSO cell, etc.

Among them, wild-type human IgG4 protein can be obtained from various sources. For example, a cDNA library can be prepared from cells expressing the mRNA of interest at detectable levels to obtain these proteins. Libraries can be screened using probes designed using published DNA or protein sequences for a particular protein of interest. For example, in Adams et al, (1980) Biochemistry 19: 2711-2719; goughet et al, (1980) Biochemistry 19: 2702-2710; dolby et al, (1980) proc.natl.acad.sci.usa 77: 6027-6031; rice et al, (1982) proc.natl.acad.sci.usa 79: 7862-7862; falkner et al, (1982) Nature 298: 286-; and Morrison et al, (1984) ann. rev. immunol.2: 239-, 256, describe immunoglobulin light or heavy chain constant regions.

The cDNA or genomic library can be screened with selected probes using standard procedures, for example, as described in Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold spring harbor Laboratory Press, NY (1989). An alternative method for isolating genes encoding immunoglobulin proteins is to use the PCR method [ Sambrook et al, supra; dieffenbach et al, PCR Primer: a Laboratory Manual, Cold spring harbor Laboratory Press, NY (1995) ]. PCR primers can be designed based on published sequences.

The full-length wild-type sequence cloned from a particular library can generally be used as a template for generating an IgG4Fc analog fragment of the invention, wherein the IgG4Fc analog fragment retains the ability to confer a longer plasma half-life to a GLP-1 analog that is part of the fusion protein. PCR techniques can be used to generate fragments of IgG4Fc analogs using primers designed to hybridize to sequences corresponding to the desired ends of the fragments. PCR primers can also be designed to generate restriction enzyme sites to facilitate cloning into an expression vector.

The DNA encoding the GLP-1 analogs of the present invention can be produced by a variety of different methods, including those cloning methods and chemically synthesized DNA as described above.

The sequence number is SEQ ID NO: 17 is a fusion protein consisting of the sequence of SEQ ID NO: 10 by peptide linker SEQ ID NO: 4 and SEQ ID NO: 2, preferably the fusion protein obtained by ligation of the polynucleotide sequence numbered SEQ id no: 29, the specific sequence is:

preferably, the amino acid sequence of the signal peptide used in the present invention is as shown in SEQ ID NO: 30, the specific sequence is as follows:

the polynucleotide sequence of the signal peptide is shown as SEQ ID NO: 31, the specific sequence is as follows:

host cells are transfected or transformed with the expression or cloning vectors described herein for fusion protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. The skilled person can select culture conditions such as medium, temperature, pH, etc. without undue experimentation. In general, the methods can be described in Mammalian cell Biotechnology: principles, methods and Practical techniques for maximizing cell culture productivity were found in the Practical Approach, m.butler eds (IRL Press, 1991) and Sambrook et al, supra. Transfection methods are known to the skilled worker, for example CaPO4 and electroporation. General aspects of mammalian cell host system transformation are described in U.S. Pat. No.4,399,216. Generally according to van Solingen et al, J Bact.130 (2): 946-7(1977) and Hsiao et al, proc.natl.acad.sci.usa 76 (8): 3829-33 (1979). However, other methods of introducing DNA into cells may be used, such as nuclear microinjection, electroporation, bacterial fusion with protoplasts of intact cells, or polycations, such as 1, 5-dimethyl-1, 5-diaza-undecamethylene polymethylenebromide (polybrene) or polymithine. For various techniques for transforming mammalian cells, see Keown et al, methods Enzymology 185: 527-37(1990) and Mansour et al, Nature 336 (6197): 348-52(1988).

Suitable host cells for cloning or expressing the nucleic acids (e.g., DNA) in the vectors herein include FreeStyle 293F, yeast cells, CHO cells, NSO cells, and the like.

Eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the fusion protein vector. Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Schizosaccharomyces pombe) [ beacon and number, Nature 290: 140-3 (1981); EP 139,383 published on 2.5.1995 ]; muyveromyces hosts [ U.S. patent No.4,943,529; fleer et al, Bio/Technology9 (10): 968-75(1991) such as Kluyveromyces lactis (K.lactis) (MW98-8C, CBS683, CBS4574) [ de Louvhouse et al, J.Bacteriol.154 (2): 737-42 (1983); kluyveromyces fragilis (k.fiagalis) (ATCC 12, 424), kluyveromyces bulgaricus (k.bulgaricus) (ATCC 16, 045), kluyveromyces vachelli (K wickeramii) (ATCC24, 178), K walltii (ATCC 56, 500), kluyveromyces drosophilus (k.drosophilum) (ATCC 36.906) [ Van den Berg et al, Bio/Technology 8 (2): 135-9(1990) ]; thermomoierans and kluyveromyces marxianus (k. marxianus); yarrowia (EP402,226); pichia pastoris (Pichia pastoris) (EP 183,070) [ Sreekrishna et al, j.basicpicrobiol.28 (4): 265-78 (1988); candida genus (Candida); trichoderma reesia (EP 244,234); neurospora crassa (Neurospora crassa) [ Case et al, Proc. Natl. Acad Sci. USA 76 (10): 5259-63 (1979); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis (EP394,538), published in 1990 at 10 months and 31 days; and filamentous fungi such as Neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium (WO 91/00357, published 1/10 1991) and Aspergillus (Aspergillus) hosts, such as Aspergillus nidulans (a. nidulans) [ Balance et al, biochem. biophysis. res. comm.112 (1): 284-9 (1983); tilburn et al, Gene 26 (2-3): 205-21 (1983); yelton et al, proc.natl.acad.sci.usa 81 (5): 1470-4(1984) and aspergillus niger (a. niger) [ Kelly and Hynes, EMBO j.4 (2): 475-9(1985)]. The methylotrophic yeast is selected from Hansenula (Hansenula), Candida (Candida), Kloeckera (Kloeckera), Pichia (Pichia), Saccharomyces, Torulopsis (Torulopsis), and Rhodotorula (Rhodotorula). A list of exemplary specific species of such yeasts can be found in C.Antony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for expressing the fusion proteins of the invention are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Trichoplusia ni (Spodoptera) Sp, Trichoplusia ni high5, and plant cells. Examples of useful mammalian host cell lines include NSO myeloma cells, Chinese Hamster Ovary (CHO) cells, SP2, and COS cells. More specific examples include monkey kidney CVl line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line [293 or subcloned 293 cells grown in suspension culture, Graham et al, j.gen virol, 36 (1): 59-74 (1977); chinese hamster ovary cells/-DHFR [ CHO, Urlaub and Chasin, proc.natl.acad.sci.usa, 77 (7): 4216-20 (1980); mouse support cells [ TM4, Mather, biol. reprod.23 (1): 243-52 (1980); human lung cells (w138.atccccl 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumor (MMT060562, ATCC CCL 51).

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Expression and cloning vectors will generally contain a selection gene, also referred to as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., neomycin, methotrexate, or tetracycline), (b) complement autotrophic deficiencies, or (c) supplement key nutrients not available from complex media (e.g., the gene encoding bacillus D-alanine racemase).

Examples of suitable selectable markers for mammalian cells are those which are capable of identifying cells competent to take up the nucleic acid encoding the fusion protein, such as DHFR or thymidine kinase. When wild-type DHFR is used, suitable host cells are e.g. [ Urlaub and Chasin, proc.natl.acad.sci.usa, 77 (7): 4216-20(1980) describe CHO cell lines deficient in DHFR activity that were prepared and propagated. Suitable selection genes for use in yeast are the trpl gene present in the yeast plasmid YRp7 [ Stinchcomb et al, Nature 282 (5734): 39-43 (1979); kingsman et al, Gene 7 (2): 14l-52 (1979); tschumper et al, Gene 10 (2): 157-66(1980)]. The Trp1 gene provides a selectable marker for yeast mutants lacking the ability to grow in tryptophan, such as atccno.44076 or PEPC1 [ Jones, Genetics 85: 23-33(1977)].

Expression and cloning vectors typically contain a promoter operably linked to the fusion protein-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Examples of suitable promoter sequences for use with yeast hosts include 3-phosphoglycerate kinase [ Hitzeman et al, j.biol.chem.255 (24): 12073-80(1980) or other glycolytic enzymes [ Hess et al, j.adv.enzyme reg.7: 149 (1968); holland, Biochemistry 17 (23): 4900-7(1978), for example enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters are the promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization, which are inducible promoters with the additional advantage of transcription controlled by growth conditions. Suitable vectors and promoters for yeast expression are further described in EP 73,657. For example, transcription of fusion protein-encoding mRNA from vectors in mammalian host cells can be controlled by promoters from viral genomes, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis b virus, and simian virus 40(SV 40); heterologous mammalian promoters, such as the actin promoter or immunoglobulin promoter, and heat shock promoters, provided that these promoters are compatible with the host cell system.

Transcription of a polynucleotide encoding a fusion protein by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA that act on a promoter to increase its transcription, usually about 10 to 300 bp. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, a-keto protein (ketoprotein) and insulin). However, one will typically use eukaryotic viral promoters. Examples include the SV40 enhancer (bp 100-270) posterior to the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer posterior to the origin of replication, and the adenovirus enhancer. The enhancer may be spliced into the vector at the 5 ' or 3 ' position of the fusion protein coding sequence, but is preferably located at the 5 ' position of the promoter.

Expression vectors for eukaryotic host cells (nucleated cells of yeast, fungi, insect, plant, animal, human, or other multicellular organisms) will also contain sequences necessary for transcription termination and stabilization of the mRNA. These sequences can be obtained generally from the 5 'and occasionally the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the fusion protein.

Various forms of fusion proteins can be recovered from the culture medium or host cell lysate. If membrane bound, it may be released from the membrane using a suitable detergent solution (e.g.Triton-X100) or enzymatic cleavage. The cells used in the expression of the fusion protein can be disrupted by a variety of physical or chemical methods, such as cyclic freeze-thawing, sonication, mechanical disruption, or cell lysis reagents.

Once the fusion protein of the invention is expressed in a suitable host cell, the analog can be isolated and purified. The following steps are representative of suitable purification steps: separating carboxymethyl cellulose by classification; gel filtration such as Sephadex G-75; anion exchange resins such as DEAE or Mono-Q; cation exchange such as CM or Mono-S; a metal chelating column to bind an epitope tag form of the polypeptide; reversed phase HPLC; carrying out chromatographic focusing; silica gel; ethanol precipitation; and ammonium sulfate precipitation.

A variety of protein purification Methods can be used, such Methods are well known in the art and are described, for example, in Deutscher, Methods in Enzymology 182: 83-9(1990) and Scopes, protein purification: principlsand Practice, Springer-Verlag, NY (1982). The purification step chosen depends on the production method used and on the nature of the particular fusion protein produced. For example, fusion proteins comprising an Fc fragment can be efficiently purified using protein A or protein G affinity matrices. The fusion protein may be eluted from the affinity matrix using low or high pH buffers. Mild elution conditions will help to prevent irreversible denaturation of the fusion protein.

The Fc portion contains substitutions at positions 11, 85, 86, 87, 91 with a deletion of the terminal lys. The substituted analogs reduce the immunogenicity of the fusion protein.

The GLP-1 analogue can be prepared into a dimer by a method for example, GLP-1 analogue polypeptide with the purity of more than 98% is dissolved in 50mmol/LTris-HCl (pH8.5) buffer, and the final concentration of the polypeptide is 1-2.0 mg/ml. The reaction was carried out overnight at 4 ℃ or at room temperature for 4-6 hours, 90% of the GLP-1 analog was analyzed by RP-HPLC to form a dimer, the reaction was terminated by adding 1% TFA, and the dimer was separated by purification using a C18 column.

The invention uses double activated polyethylene glycol molecule as conjugate, and reduces the conjugate and free sulfhydryl at C end of analog polypeptide into covalent polyethylene glycol coupled homodimer under certain reaction condition: GLP-1-Cys-PEG-Cys- -GLP-1.

The dual activated polyethylene glycol molecules (PEG) include dual activated maleic acid PEG, characterized as MAL-PEG-MAL; or comprise a di-activated sulfhydryl PEG characterized as SH-PEG-SH. Or comprise di-activated ortho-dipyridyl disulfide (PEG) characterized as OPSS-PEG-OPSS.

The polyethylene glycol molecules used in the homodimers of insulinotropic peptide analogs of the present invention can be small or large molecules with molecular weights ranging from 500 to 40,000 daltons. The significant prolongation of the in vivo half-life of the insulinotropic hormone secretagogue peptide analogue dimer can be achieved by using a PEG molecule with a molecular weight of more than 5000 as a dimer conjugate. The preferred PEG molecule size is between 10KD-40 KD.

The fusion protein of the invention contains an Fc portion derived from human IgG4 but includes multiple substitutions compared to the wild-type human sequence. The Fc portion consists of the two heavy chain constant regions of the antibody bound by non-covalent interactions and disulfide bonds. The Fc portion may comprise a hinge region and may be via CH₂And CH₃The domain extends to the C-terminus of the antibody. The Fc portion may also comprise one or more glycosylation sites.

Native Glp-1 (7-37):

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

the GLP-1 analog portion of the fusion protein of the invention comprises substitutions at positions 31, 36, and 37 (i.e., substitutions at positions 25, 30, and 31 of the new numbering of the invention) as compared to native Glp-1 (7-37).

The GLP-1 analogue can be artificially and chemically synthesized by adopting an Fmoc method, and the C terminal of the GLP-1 analogue is amidated.

The GLP-1 analogs of the present invention may be salified, including a variety of inorganic or organic salts, such as hydrochloride, phosphate, sulfate, maleate, oxalate, citrate, and lactate; salt formation with certain inorganic and organic bases, such as sodium hydroxide and N-methyl-glucosamine.

The GLP-1 analogs of the invention can be administered as a single drug or can be administered in combination with other drugs, or as a pharmaceutically acceptable carrier, or to provide a modifiable active precursor for long-acting polypeptide drug development. The application of the GLP-1 analogue or the pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier can be used. The "pharmaceutically acceptable carrier" does not destroy the pharmaceutical activity of the compounds of the present invention and their useful salts, while the effective amount thereof is non-toxic to humans. The "pharmaceutically acceptable carrier" can be used without limitation: ion exchange materials, aluminum stearate, lecithin, serum proteins for pharmaceutical preparations, saturated vegetable fatty acids, cellulosic substances, ethylene-polyoxyethylene-block polymers, cyclodextrins or chemically modified derivatives or other soluble derivatives thereof, and the like.

Other pharmaceutically acceptable excipients such as fillers such as anhydrous lactose, starch, lactose beads and glucose, binders such as microcrystalline cellulose, disintegrants such as croscarmellose sodium, croscarmellose starch, low-substituted hydroxypropylcellulose, lubricants such as magnesium stearate, absorption enhancers, excipients, solubilizers and colorants may also be added to the pharmaceutical composition of the present invention.

The above GLP-1 analogues or pharmaceutically acceptable salts thereof and pharmaceutical compositions of the present invention can be administered by enteral or parenteral routes. Parenteral routes of administration include subcutaneous, intradermal, intramuscular, nasal, mucosal administration or inhalation. The preparation can be developed into injection, cream, ointment, patch, aerosol, etc.

The GLP-1 analogue or the fusion protein thereof or the pharmaceutically acceptable salt thereof and the pharmaceutical composition can be used for single-drug or combined-drug treatment of related diseases, and are within the scope understood by a person skilled in the art.

The fusion protein can be used for producing a medicament for treating non-insulin-dependent diabetes mellitus and also can be used for producing a medicament for treating obesity or inducing weight loss in an overweight subject.

The present invention provides explanations of some terms to facilitate better understanding of the technical aspects of the present invention.

A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, either naturally occurring or synthetic. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

The "Fc" region contains the CH comprising antibody₂And CH₃Two heavy chain fragments of a domain. Two heavy chain fragments consisting of two or more disulfide bonds and passing through CH₃The hydrophobic interaction of the domains remains together.

A "protein" is a macromolecule comprising one or more polypeptide chains. Proteins may also contain non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituent groups may be added to the protein in the cell in which the protein is produced, and the groups will vary from cell type to cell type. Proteins are defined herein below in terms of their amino acid backbone structure; substituents such as the subject carbohydrate groups are generally not specified, but may still be present.

"heterologous peptide" a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.

A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or phage, that is capable of autonomous replication in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that permit insertion of nucleic acid molecules in a specific manner without loss of the essential biological function of the vector, as well as nucleotide sequences encoding marker genes suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes generally include genes that provide tetracycline resistance or ampicillin resistance. Cloning vectors, which have a relaxed replicon and can carry a foreign gene and replicate and amplify in host cells, are vectors used for cloning and amplifying DNA fragments (genes).

"expression vector" is a vector having the basic elements of a cloning vector (ori, Ampr, Mcs, etc.) and further having DNA sequences necessary for transcription/translation; is a bacterium used for engineering production, the introduced target gene can be expressed in the bacterium to produce the required product, the introduced gene is produced by cloning vector, the expression vector has higher protein expression efficiency, generally because of having strong promoter.

A "recombinant host" is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or an expression vector.

An "integrated transformant" is a recombinant host cell in which heterologous DNA is integrated into the genomic DNA of the cell.

A "fusion protein" is a hybrid protein expressed from a nucleic acid molecule comprising the nucleotide sequences of at least two genes.

"immunoglobulin moiety" refers to a polypeptide comprising an immunoglobulin constant region. For example: the immunoglobulin moiety may comprise a heavy chain constant region.

"expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression includes transcription of the structural gene into mRNA and translation of the mRNA into one or more polypeptides.

Detailed Description

The present invention will be described in more detail with reference to examples. It will be apparent to those skilled in the art from this disclosure that these examples are provided only for illustrating the present invention more specifically and are not intended to limit the scope of the present invention. Examples are exemplified by HIP-1 to 12. All the materials used, not specifically mentioned, are commercially available.

Some common abbreviations in the present invention have the following meanings:

abbreviations	Means of
		Resin	Resin composition
Fmoc	Fmoc group
		HOBt	1-hydroxybenzotriazoles
DCM	Methylene dichloride
		DMF	N, N-dimethylformamide
DIC	N, N' -diisopropylcarbodiimide
		Rink Amide-MBHA Resin	Rink amide-MBHA
MeOH	Methanol
		TFA	Trifluoroacetic acid
TIS	Tri-isopropyl silane
		DTT	Dithiothreitol
-Trt	Trityl radical
		-tBu	Tert-butyl radical
-Pbf	2,2, 4, 6, 7-pentamethylbenzofuran-5-sulfonyl
		-Boc	Tert-butyloxycarbonyl radical
-OtBu	Tert-butyl ester radical

Reference example 1 preparation of Fmoc-L- β -Phe-OH

Fmoc-L- β -Phe-OH, Fmoc-L- β -His (Trt) -OH and Fmoc-L- β -Trp-OH are prepared by Synthetic Communications, vol.32, nb.4, (2002) and p.651-657.

The synthetic route of Fmoc-L- β -Phe-OH is as follows:

preparation of Fmoc-L- β -Phe-OH:

TsC1(0.21g, 1.1mmol) and pyridine (0.08ml, 1mmol) were added to a solution of Fmoc-Phe-OH (0.39g, 1mmol) in THF (5ml), stirred at 0 ℃ for 15min, saturated CH was added to the reaction mixture₂N₂Dry CH of₂Cl₂(20mL) the solution was stirred at 0 ℃ for lh. The progress of the reaction was checked by TLC and IR. After the reaction is completed, acetic acid is added dropwise to decompose excess CH₂N₂. Sequentially with NaHCO₃(25 ml. times.3), 5% HCl (25 ml. times.3) and NaCl wash mixture, anhydrous Na₂SO₄Drying, distilling under reduced pressure, and collecting the residue with CH₂Cl₂Recrystallization from hexane gave crystals of intermediate II.

Intermediate II (1mmol) was dissolved in a solution of 1, 4-dioxane (10ml) and water (5ml), silver benzoate (5.7mg, 0.025mmol) was added and refluxed at 70 ℃ for 6 hours. Filtering, and distilling the filtrate under reduced pressure. Dissolving the residue in saturated Na₂CO₃The mixture was stirred for 20 minutes in an aqueous solution (20 ml). The mixture was washed with diethyl ether (20 ml. times.3). The pH of the aqueous phase was adjusted to 2 and extracted with ethyl acetate (20 ml. times.3). The extracts were combined, washed with water (20 ml. times.2) and the organic phase with anhydrous Na₂SO₄Drying and distilling under reduced pressure. The residue is substituted by CH₂Cl₂Recrystallizing in hexane to obtain Fmoc-L- β -Phe-OH.

Reference example 2 preparation of sequence 10

Sequence 10: molecular weight 3660.1;

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-βPhe-Leu-Val-Lys-Gly-Ala-Gly-Gly-Lys-Lys-Cys-NH₂。

this example provides a method for producing a polypeptide of sequence 10.

The preparation method of the sequence 10 polypeptide comprises the following steps: adopting amino resin with Fmoc protection, after removing Fmoc protecting group on the amino resin, synthesizing sequence 10 polypeptide peptide resin, removing resin and side chain protecting group to obtain sequence 10 polypeptide crude product, and preparing and purifying the sequence 10 polypeptide crude product to obtain the sequence 10 polypeptide refined product.

(1) Preparation of sequence 10 polypeptide peptide resin:

rink Amide-MBHA Resin (5.0g, 0.42mmol/g, 2.1mmol) and 40ml CH were added to a 100ml solid phase reactor₂Cl₂Swelling the resin for 30min, removing CH₂Cl₂Removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution, after 10min, pumping out the reaction solution, removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution again, after 10min, pumping out the reaction solution, and washing the resin by using DMF (6 x 40ml) for later use; simultaneously placing 3.7g Fmoc-Cys (Trt) -OH (6.3mmol) and 0.9g HOBt (6.6mmol) in a 100ml flask, adding 40ml DMF for dissolving, cooling to 0-5 ℃ in ice bath, dropwise adding 1.0ml DIC (6.6mmol), reacting for 5min after the addition, transferring the reaction liquid to the obtained Fmoc-removed amino resin, and adding N₂After bubbling and mixing, condensation reaction was carried out for 3 hours, and then the reaction solution was taken out and the resin was washed with DMF (6X 40 ml).

Repeating the deprotection and condensation steps, sequentially coupling Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- β -L-Phe-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Tyr) -OH, Fmoc-Glu (Thr) and Fmoc-Glu-OH, sequentially, performing a coupling reaction, wherein after removing the protective groups of Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-.

(2) Preparation of crude polypeptide of sequence 10:

adding the amino resin loaded peptide resin of sequence 10 polypeptide prepared above into 150ml TFA/TIS/H₂Removing resin and side chain protecting group from O (v/v/v ═ 95: 2.5) mixed solution, suction filtering after 2.5H, collecting filtrate, and using TFA/TIS/H₂Washing resin with O (volume ratio of 95: 2.5) mixed solution (10ml), filtering, combining filtrates, concentrating under reduced pressure at 35 deg.C until no fraction flows out, slowly pouring the concentrated solution into 3L cold diethyl ether, stirring, precipitating white precipitate, filtering, grinding filter cake, washing with cold diethyl ether for 3 times, vacuum drying at 35 deg.C for 5 hr to obtain crude product of sequence 10 polypeptide 7.0g, yield: 91.0%, HPLC purity: 56.2 percent.

(3) Preparation of refined product of polypeptide of sequence 10:

7.0g of crude sequence 10 polypeptide are dissolved in 20ml of 10% aqueous acetic acid, filtered through a 0.45 μm membrane and purified by preparative HPLC: loading a C18 column, wherein the loading is performed for 1.8-2.2 g each time, the wavelength is 214nm, gradient elution is performed for 5min on (10% acetonitrile-H2O (containing 1% TFA) and the like, gradient elution is performed for 35min on 10% to 80% acetonitrile-H2O (containing 1% TFA), the flow rate is 50ml/min, a gradient system is adopted for elution, circulation sample injection purification is performed, a main peak is collected and the purity is detected by an analytical liquid phase, the purity of the purified main peak is more than 98%, and the prepared eluent is subjected to reduced pressure concentration under the water bath condition of 30-35 ℃ to obtain a primary purified material concentrated solution.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm at a flow rate of 30-60 ml/min^-1. Collecting target fraction, concentrating the collected fraction at water temperature below 35 deg.C under reduced pressure until the fraction does not flow out, lyophilizing the rest fraction to obtain refined product, and vacuum lyophilizing to obtain refined product of sequence 10 polypeptide 3.1g, with total yield of 40.5% and HPLC purity of 99.1%.

Reference example 3 preparation of sequence 11

Sequence 11: molecular weight 4059.6;

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-βPhe-Leu-Val-Lys-Gly-Pro-Lys-Lys-Pro-Phe-Phe-Pro-Cys-NH₂。

this example provides a method for producing a polypeptide of sequence 11.

The preparation method of the sequence 11 polypeptide comprises the following steps: adopting amino resin with Fmoc protection, after removing Fmoc protecting group on the amino resin, synthesizing sequence 11 polypeptide peptide resin, removing resin and side chain protecting group to obtain sequence 11 polypeptide crude product, and preparing and purifying the sequence 11 polypeptide crude product to obtain the sequence 11 polypeptide refined product.

(1) Preparation of sequence 11 polypeptide peptide resin:

rink Amide-MBHA Resin (5.0g, 0.42mmol/g, 2.1mmol) and 40ml CH were added to a 100ml solid phase reactor₂Cl₂Swelling the resin for 30min, removing CH₂Cl₂Removing Fmoc protecting group on the resin with 30ml of 20% piperidine/DMF solution, after 10min, removing the reaction solution, removing Fmoc protecting group on the resin with 30ml of 20% piperidine/DMF solution again, after 10min, removing the reaction solution, washing the resin with DMF (6X 40ml)And is ready for use; simultaneously placing 3.7g Fmoc-Cys (Trt) -OH (6.3mmol) and 0.9g HOBt (6.6mmol) in a 100ml flask, adding 40ml DMF for dissolving, cooling to 0-5 ℃ in ice bath, dropwise adding 1.0ml DIC (6.6mmol), reacting for 5min after the addition, transferring the reaction liquid to the obtained Fmoc-removed amino resin, and adding N₂After bubbling and mixing, condensation reaction was carried out for 3 hours, and then the reaction solution was taken out and the resin was washed with DMF (6X 40 ml).

Repeating the deprotection and condensation steps, sequentially coupling Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- β -L-Phe-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gln (t) -OH, Fmoc-Gly-OH, Fmoc-Glu (Glu-Glu) (OH, Fmoc-Leu-OH, Fmoc-Tyr-Ala-OH, Fmoc-Thr-OH, Fmoc-Thr-Thru-NH-OH, Fmoc-Ser-Lys (Boc) -OH, Fmoc-Boc-L-OH, Fmoc-L-OH, Fmoc-L-OH, Fmoc-L-OH, Fmoc-T-OH, Fmoc-N (T-OH, Fmoc-T-OH, Fmoc-T-OH, F.

(2) Preparation of crude polypeptide of sequence 11:

adding the amino resin loaded peptide resin of the sequence 11 polypeptide prepared in the above into 150ml TFA/TIS/H₂Removing resin and side chain protecting group from O (v/v/v ═ 95: 2.5) mixed solution, suction filtering after 2.5H, collecting filtrate, and using TFA/TIS/H₂Washing resin with mixed solution (10ml) of O (volume ratio of 95: 2.5), vacuum filtering, mixing filtrates, concentrating under reduced pressure at 35 deg.C until no distillate flows out, slowly pouring the concentrated solution into 3L cold diethyl etherStirring to separate out white precipitate, filtering, crushing the filter cake, washing with cold ether for 3 times, and vacuum drying at 35 deg.c for 5 hr to obtain crude polypeptide of sequence 11 in 7.3g and yield: 85.3%, HPLC purity: 51.7 percent.

(3) Preparation of refined product of polypeptide of sequence 11:

7.3g of crude sequence 11 polypeptide are dissolved in 20ml of 10% aqueous acetic acid, filtered through a 0.45 μm membrane and purified by preparative HPLC: loading a C18 column, wherein the loading is performed for 1.8-2.2 g each time, the wavelength is 214nm, gradient elution is performed for 5min on (10% acetonitrile-H2O (containing 1% TFA) and the like, gradient elution is performed for 35min on 10% to 80% acetonitrile-H2O (containing 1% TFA), the flow rate is 50ml/min, a gradient system is adopted for elution, circulation sample injection purification is performed, a main peak is collected and the purity is detected by an analytical liquid phase, the purity of the purified main peak is more than 98%, and the prepared eluent is subjected to reduced pressure concentration under the water bath condition of 30-35 ℃ to obtain a primary purified material concentrated solution.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm at a flow rate of 30-60 ml/min^-1. Collecting target fraction, concentrating the collected fraction at water temperature below 35 deg.C under reduced pressure until the fraction does not flow out, lyophilizing the rest fraction to obtain refined product, and vacuum lyophilizing to obtain refined product of sequence 11 polypeptide 3.0g, with total yield of 35.6% and HPLC purity of 99.3%.

Reference example 4 preparation of sequence 12

Sequence 12: molecular weight 3717;

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-βPhe-Leu-Val-Lys-Gly-Glu-Gly-Gly-Lys-Lys-Cys-NH₂。

this example provides a method for producing a polypeptide of sequence 12.

Preparation method of the sequence 12 polypeptide: adopting amino resin with Fmoc protection, after removing Fmoc protecting group on the amino resin, synthesizing sequence 12 polypeptide peptide resin, removing resin and side chain protecting group to obtain sequence 12 polypeptide crude product, and preparing and purifying the sequence 12 polypeptide crude product to obtain a sequence 12 polypeptide refined product.

(1) Preparation of sequence 12 polypeptide peptide resin:

Repeating the deprotection and condensation steps, sequentially coupling Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- β -L-Phe-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu Otbu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu-Otbu) -OH, Fmoc-Leu-OH, Fmoc-Ty-Tyr-OH, Fmoc-Glu-OH, Fmoc-Thr-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu (Boc-Lys-OH, Fmoc-Gly-Ala-OH, Fmoc-Ala-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu, Fmoc-Glu-OH, Fmoc-Glu.

(2) Preparation of crude polypeptide of sequence 12:

adding the amino resin-loaded peptide resin of sequence 12 prepared above into 150ml TFA/TIS/H₂Removing resin and side chain protecting group from O (v/v/v ═ 95: 2.5) mixed solution, suction filtering after 2.5H, collecting filtrate, and using TFA/TIS/H₂Washing resin with O (volume ratio of 95: 2.5) mixed solution (10ml), filtering, combining filtrates, concentrating under reduced pressure at 35 ℃ until the fraction does not flow out, slowly pouring the concentrated solution into 3L cold diethyl ether, stirring, separating out white precipitate, filtering, grinding the filter cake, washing with cold diethyl ether for 3 times, and vacuum drying at 35 ℃ for 5 hours to obtain 7.0g crude polypeptide of sequence 12, yield: 89.6%, HPLC purity: 55.5 percent.

(3) Preparation of refined product of polypeptide of sequence 12:

7.0g of crude sequence 12 polypeptide are dissolved in 20ml of 10% aqueous acetic acid, filtered through a 0.45 μm membrane and purified by preparative HPLC: loading a C18 column, wherein 2.0-2.5 g of the C18 column is loaded each time, the wavelength is 214nm, gradient elution is performed for 5min on (10% acetonitrile-H2O (containing 1% TFA) and the like, gradient elution is performed for 35min on 10% to 80% acetonitrile-H2O (containing 1% TFA), the flow rate is 50ml/min, gradient system elution is adopted, circulating sample injection purification is performed, a main peak is collected and the purity is detected by an analytical liquid phase, the purity of the purified main peak is more than 98%, and the prepared eluent is subjected to reduced pressure concentration under the water bath condition of 30-35 ℃ to obtain a primary purified material concentrated solution.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm at a flow rate of 30-60 ml/min^-1. Collecting target fraction, concentrating the collected fraction at water temperature below 35 deg.C under reduced pressure until the fraction does not flow out, lyophilizing the rest fraction to obtain refined product, and vacuum lyophilizing to obtain refined product of sequence 12 polypeptide 3.1g, with total yield of 39.8% and HPLC purity of 98.8%.

Reference example 5 preparation of sequence 13

Sequence 13: molecular weight 3687;

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-βPhe-Leu-Val-Lys-Gly-Val-Gly-Gly-Lys-Lys-Cys-NH₂。

this example provides a method for producing a polypeptide of sequence 13.

The preparation method of the sequence 13 polypeptide comprises the following steps: adopting amino resin with Fmoc protection, after removing Fmoc protecting group on the amino resin, synthesizing sequence 13 polypeptide peptide resin, removing resin and side chain protecting group to obtain sequence 13 polypeptide crude product, and preparing and purifying the sequence 13 polypeptide crude product to obtain the sequence 13 polypeptide refined product.

(1) Preparation of sequence 13 polypeptide peptide resin:

a100 ml solid phase reactor was charged with Rinkamide-MBHAResin resin (5.0g, 0.42mmol/g, 2.1mmol) and 40ml CH₂Cl₂Swelling the resin for 30min, removing CH₂Cl₂Removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution, after 10min, pumping out the reaction solution, removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution again, after 10min, pumping out the reaction solution, and washing the resin by using DMF (6 x 40ml) for later use; simultaneously placing 3.7g Fmoc-Cys (Trt) -OH (6.3mmol) and 0.9g HOBt (6.6mmol) in a 100ml flask, adding 40ml DMF for dissolving, cooling to 0-5 ℃ in ice bath, dropwise adding 1.0ml DIC (6.6mmol), reacting for 5min after the addition, transferring the reaction liquid to the obtained Fmoc-removed amino resin, and adding N₂After bubbling and mixing, condensation reaction was carried out for 3 hours, and then the reaction solution was taken out and the resin was washed with DMF (6X 40 ml).

Repeating the deprotection and condensation steps, sequentially coupling Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- β -L-Phe-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Tyr) -OH, Fmoc-Glu (Thru) -OH, Fmoc-Glu-OH, Fmoc-Glu (Boc-Glu) and Fmoc-Glu (20ml) to obtain a solution, drying the peptide, removing peptide (DMF) and the peptide, after the peptide-Glu-HCl/DMF-HCl is removed by a reaction (DMF) and the peptide-HCl, the peptide-HCl, the peptide is removed by a reaction step (DMF) and the amino-HCl is performed by a reaction step (DMF) and the steps are performed by a reaction (20 ml-HCl, wherein the peptide-HCl is performed by a reaction step (20-HCl, the amino-HCl-.

(2) Preparation of crude sequence 13 polypeptide:

adding the amino resin loaded polypeptide peptide resin of sequence 13 prepared in the above into 150ml TFA/TIS/H₂Removing resin and side chain protecting group from O (v/v/v ═ 95: 2.5) mixed solution, suction filtering after 2.5H, collecting filtrate, and using TFA/TIS/H₂Washing resin with O (volume ratio of 95: 2.5) mixed solution (10ml), filtering, combining filtrates, concentrating under reduced pressure at 35 ℃ until the fraction does not flow out, slowly pouring the concentrated solution into 3L cold diethyl ether, stirring, separating out white precipitate, filtering, grinding the filter cake, washing with cold diethyl ether for 3 times, and vacuum-drying at 35 ℃ for 5 hours to obtain crude polypeptide of sequence 13 (6.9 g, yield): 88.9%, HPLC purity: 55.8 percent.

(3) Preparation of refined product of polypeptide of sequence 13:

6.9g of crude sequence 13 polypeptide are dissolved in 20ml of 10% aqueous acetic acid, filtered through a 0.45 μm membrane and purified by preparative HPLC: c18 column is selected for sampling, each time the sample is 2.0-2.5 g, the wavelength is 214nm, (10% acetonitrile-H)₂Gradient elution of O (1% TFA) for 5min, 10% to 80% acetonitrile-H₂Gradient eluting O (containing 1% TFA) for 35min at flow rate of 50ml/min, eluting with gradient system, circularly injecting sample, purifying, collecting main peak, and detecting purity with analytical liquid phase, wherein the purity of purified main peak should be greater than 98%. The prepared eluent is decompressed and concentrated under the condition of water bath at the temperature of 30-35 ℃ to obtain a concentrated solution of a primary purified material.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm at a flow rate of 30-60 ml/min^-1. Collecting the target fraction, and concentrating the collected fraction at a temperature below 35 deg.C under reduced pressureAnd (3) the distillate does not flow out any more, and the rest distillate is freeze-dried to obtain a refined product, and the refined product is freeze-dried in vacuum to obtain 3.0g of the refined product of the sequence 13 polypeptide, wherein the total yield is 38.5 percent, and the HPLC purity is 98.6 percent.

Reference example 6 preparation of sequence 14

Sequence 14: molecular weight 3982;

this example provides a method for producing a polypeptide of sequence 14.

The preparation method of the sequence 14 polypeptide comprises the following steps: adopting amino resin with Fmoc protection, after removing Fmoc protecting group on the amino resin, synthesizing sequence 14 polypeptide peptide resin, removing resin and side chain protecting group to obtain sequence 14 polypeptide crude product, and preparing and purifying the sequence 14 polypeptide crude product to obtain a sequence 14 polypeptide refined product.

(1) Preparation of sequence 14 polypeptide peptide resin:

a100 ml solid phase reactor was charged with Rinkamide-MBHAResin resin (5.0g, 0.42mmol/g, 2.1mmol) and 40ml CH₂Cl₂Swelling the resin for 30min, removing CH₂Cl₂Removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution, after 10min, pumping out the reaction solution, removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution again, after 10min, pumping out the reaction solution, and washing the resin by using DMF (6 x 40ml) for later use; simultaneously, 3.8g of Fmoc-Gln (Trt) -OH (6.3mmol) and 0.9g of HOBt (6.6mmol) are placed in a 100ml flask, 40ml of DMF is added for dissolution, the temperature is reduced to 0-5 ℃ in an ice bath, 1.0ml of DIC (6.6mmol) is added dropwise, reaction is carried out for 5min after the addition is finished, the reaction liquid is transferred to the obtained amino resin with Fmoc removed, and N is₂After bubbling and mixing, condensation reaction was carried out for 3 hours, and then the reaction solution was taken out and the resin was washed with DMF (6X 40 ml).

Repeating the deprotection and condensation steps, sequentially coupling Fmoc-Gln (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- β -L-Phe-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-Glu-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Glu-OH, Fmoc-Gln (Tyr) -OH, Fmoc-Thr-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-.

(2) Preparation of crude polypeptide of sequence 14:

adding the amino resin-loaded peptide resin of the sequence 14 polypeptide prepared in the above into 150ml TFA/TIS/H₂Removing resin and side chain protecting group from O (v/v/v ═ 95: 2.5) mixed solution, suction filtering after 2.5H, collecting filtrate, and using TFA/TIS/H₂Washing resin with O (volume ratio of 95: 2.5) mixed solution (10ml), filtering, combining filtrates, concentrating under reduced pressure at 35 ℃ until the fraction does not flow out, slowly pouring the concentrated solution into 3L cold diethyl ether, stirring, separating out white precipitate, filtering, grinding the filter cake, washing with cold diethyl ether for 3 times, and vacuum-drying at 35 ℃ for 5 hours to obtain 7.1g crude polypeptide of sequence 14, yield: 85.3%, HPLC purity: 52.1 percent.

(3) Preparation of refined product of polypeptide of sequence 14:

7.1g of crude sequence 14 polypeptide are dissolved in 20ml of 10% aqueous acetic acid, filtered through a 0.45 μm membrane and purified by preparative HPLC: c18 column is selected for sampling, each time the sample is 2.0-2.5 g, the wavelength is 214nm, (10% acetonitrile-H)₂Gradient elution of O (1% TFA) for 5min, 10% to 80% acetonitrile-H₂Gradient eluting with O (containing 1% TFA) for 35min at 50ml/min, and circulatingAnd (3) sampling and purifying, collecting a main peak, and detecting the purity by using an analysis liquid phase, wherein the purity of the purified main peak is more than 98%. The prepared eluent is decompressed and concentrated under the condition of water bath at the temperature of 30-35 ℃ to obtain a concentrated solution of a primary purified material.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm at a flow rate of 30-60 ml/min^-1. Collecting target fraction, concentrating the collected fraction at water temperature below 35 deg.C under reduced pressure until the fraction does not flow out, lyophilizing the rest fraction to obtain refined product, and vacuum lyophilizing to obtain refined product of sequence 14 polypeptide 3.0g, with total yield of 36.2% and HPLC purity of 98.2%.

Reference example 7 preparation of sequence 15

Sequence 15: molecular weight 3650.1;

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-βHis-Leu-Val-Lys-Gly-Ala-Gly-Gly-Lys-Lys-Cys-NH2。

this example provides a method for producing a polypeptide of sequence 15.

The preparation method of the sequence 15 polypeptide comprises the following steps: adopting amino resin with Fmoc protection, after removing Fmoc protecting group on the amino resin, synthesizing sequence 15 polypeptide peptide resin, removing resin and side chain protecting group to obtain sequence 15 polypeptide crude product, and preparing and purifying the sequence 15 polypeptide crude product to obtain the sequence 15 polypeptide refined product.

(1) Preparation of sequence 15 polypeptide peptide resin:

rink Amide-MBHA Resin (5.0g, 0.42mmol/g, 2.1mmol) and 40ml CH were added to a 100ml solid phase reactor₂Cl₂Swelling the resin for 30min, removing CH₂Cl₂Removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution, after 10min, pumping out the reaction solution, removing the Fmoc protecting group on the resin by using 30ml of 20% piperidine/DMF solution again, after 10min, pumping out the reaction solution, and washing the resin by using DMF (6 x 40ml) for later use; 3.7g Fmoc-Cys (Trt) -OH (6.3mmol) and 0.9g HOBt (6.6mmol) are placed in a 100ml flask, 40ml DMF is added for dissolution, the temperature is reduced to 0-5 ℃ in ice bath, and 1.0ml DIC (6.6 ml DIC) is added dropwisemmol), reacting for 5min after the addition is finished, transferring the reaction liquid to the Fmoc-removed amino resin obtained above, and N₂After bubbling and mixing, condensation reaction was carried out for 3 hours, and then the reaction solution was taken out and the resin was washed with DMF (6X 40m 1).

Repeating the deprotection and condensation steps, sequentially coupling Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- β -L-His (Trt) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu-Ou tbOH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu-Ou, Fmoc-Leu-OH, Fmoc-Tyr-OH, Fmoc-Glu-Ou, Fmoc-OH, Fmoc-Throc-Thr-OH, Fmoc-Glu-NH-OH, Fmoc-Glu-OH, Fmoc-Glu-DMF, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu-OH, Fmoc-Glu.

(2) Preparation of crude polypeptide of sequence 15:

adding the amino resin loaded peptide resin of the sequence 15 polypeptide prepared in the above into 150ml TFA/TIS/H₂Removing resin and side chain protecting group from O (v/v/v ═ 95: 2.5) mixed solution, suction filtering after 2.5H, collecting filtrate, and using TFA/TIS/H₂Washing resin with O (volume ratio of 95: 2.5) mixed solution (10ml), filtering, combining filtrates, concentrating under reduced pressure at 35 ℃ until the fraction does not flow out, slowly pouring the concentrated solution into 3L cold diethyl ether, stirring, separating out white precipitate, filtering, grinding the filter cake, washing with cold diethyl ether for 3 times, and vacuum-drying at 35 ℃ for 5 hours to obtain crude polypeptide of sequence 15 (6.9 g, yield): 90.5%, HPLC purity: 57.6 percent.

(3) Preparation of refined product of polypeptide of sequence 15:

6.9g of crude sequence 15 polypeptide are dissolved in 20ml of 10% aqueous acetic acid, filtered through a 0.45 μm membrane and purified by preparative HPLC: c18 column is selected for sampling, each time the sample is 2.0-2.5 g, the wavelength is 214nm, (10% acetonitrile-H)₂Gradient elution of O (1% TFA) for 5min, 10% to 80% acetonitrile-H₂Gradient eluting O (containing 1% TFA) for 35min at flow rate of 50ml/min, eluting with gradient system, circularly injecting sample, purifying, collecting main peak, and detecting purity with analytical liquid phase, wherein the purity of purified main peak should be greater than 98%. The prepared eluent is decompressed and concentrated under the condition of water bath at the temperature of 30-35 ℃ to obtain a concentrated solution of a primary purified material.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm at a flow rate of 30-60 ml/min^-1. Collecting target fraction, concentrating the collected fraction at water temperature below 35 deg.C under reduced pressure until the fraction does not flow out, lyophilizing the rest fraction to obtain refined product, and vacuum lyophilizing to obtain refined product of sequence 15 polypeptide 3.2g, with total yield of 41.6% and HPLC purity of 99.0%.

Reference example 8 preparation of sequence 16

Sequence 16: molecular weight 3671;

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-βTrp-Leu-Val-Lys-Gly-Ala-Lys-Lys-Ser-Cys-NH2。

this example provides a method for producing a polypeptide of sequence 16.

The preparation method of the sequence 16 polypeptide comprises the following steps: adopting amino resin with Fmoc protection, after removing Fmoc protecting group on the amino resin, synthesizing sequence 16 polypeptide peptide resin, removing resin and side chain protecting group to obtain sequence 16 polypeptide crude product, and preparing and purifying the sequence 16 polypeptide crude product to obtain the sequence 16 polypeptide refined product.

(1) Preparation of sequence 16 polypeptide peptide resin:

Repeating the deprotection and condensation steps, sequentially coupling Fmoc-Ser (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- β -L-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gln (t) -OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Tyr) OH, Fmoc-Ala-OH, Fmoc-Gln (t) -OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Tyr) and Thru-OH, after the deprotection and condensation steps, the peptide-Glu-peptide-Glu-peptide-Glu-peptide-Glu-peptide-Ala-OH, the peptide-Ala-OH, the peptide-Ala-OH.

(2) Preparation of crude polypeptide of sequence 16:

the amino resin-loaded peptide resin of sequence 16 prepared above was added to 150ml TFA/TIS/H₂Removal of O (v/v/v ═ 95: 2.5) mixed solutionFiltering the resin and the side chain protecting group after 2.5H, collecting filtrate, and using TFA/TIS/H₂Washing resin with O (volume ratio of 95: 2.5) mixed solution (10ml), filtering, combining filtrates, concentrating under reduced pressure at 35 ℃ until the fraction does not flow out, slowly pouring the concentrated solution into 3L cold diethyl ether, stirring, separating out white precipitate, filtering, grinding the filter cake, washing with cold diethyl ether for 3 times, and vacuum-drying at 35 ℃ for 5 hours to obtain crude polypeptide of sequence 16 (6.8 g, yield): 88.7%, HPLC purity: 55.6 percent.

(3) Preparation of refined product of polypeptide of sequence 16:

6.8g of crude sequence 16 polypeptide are dissolved in 20ml of 10% aqueous acetic acid, filtered through a 0.45 μm membrane and purified by preparative HPLC: c18 column is selected for sampling, each time the sample is 2.0-2.5 g, the wavelength is 214nm, (10% acetonitrile-H)₂Gradient elution of O (1% TFA) for 5min, 10% to 80% acetonitrile-H₂Gradient eluting O (containing 1% TFA) for 35min at flow rate of 50ml/min, eluting with gradient system, circularly injecting sample, purifying, collecting main peak, and detecting purity with analytical liquid phase, wherein the purity of purified main peak should be greater than 98%. The prepared eluent is decompressed and concentrated under the condition of water bath at the temperature of 30-35 ℃ to obtain a concentrated solution of a primary purified material.

And (3) carrying out secondary desalting purification on the concentrated solution of the material collected by the primary purification under the conditions of: mobile phase a phase: purifying the water; phase B: acetonitrile solution, detection wavelength: 215nm at a flow rate of 30-60 ml/min^-1. Collecting target fraction, concentrating the collected fraction at water temperature below 35 deg.C under reduced pressure until the fraction does not flow out, lyophilizing the rest fraction to obtain refined product, and vacuum lyophilizing to obtain refined product of sequence 16 polypeptide 3.0g, with total yield 38.6% and HPLC purity 99.0%.

Validation example 1. in vitro activity or potency of a GLP-l compound fusion protein of the invention was determined.

The potency of analogues of GLP-1 was determined by stimulating the formation of cyclic AMP (cAMP) in media containing membranes expressing the human GLP-1 receptor.

Purified plasma membranes from the stably transfected cell line BHK467-12A (tk-ts 13) expressing the human GLP-1 receptor were stimulated with the GLP-1 analogue or derivative of interest, and cAMP-producing titers were measured with the AlphaScreen cAMP assay kit from Perkinelmer Life Sciences. The rationale for the AlphaScreen assay is the competition between endogenous cAMP and exogenously added biotin-cAMP. Capture of cAMP was achieved by specific antibody conjugated to acceptor beads.

Cell culture and membrane preparation: stably transfected cell lines and high expressing clones were selected for screening. Cells were plated in DMEM, 5% FCS, 1% Pen/Strep (penicillin/streptomycin) and 0.5mg/ml selection marker G418 at 5% CO₂And (5) growing.

Cells at 2X approximately 80% confluence were washed with PBS, harvested with Versene (an aqueous solution of ethylenediaminetetraacetic acid tetrasodium salt), centrifuged at 1000rpm for 5 minutes, and the supernatant removed. The additional steps were all performed on ice. The cell pellet was homogenized in 10ml of buffer 1(20mM Na-HEPES, 10mM EDTA, pH 7.4) by Ultrathurax for 20-30 seconds, centrifuged at 20,000rpm for 15 minutes, and the pellet was resuspended in 10ml of buffer 2(20mM Na-HEPES, 0.1mM EDTA, pH 7.4). The suspension was homogenized for 20-30 seconds and centrifuged at 20,000rpm for 15 minutes. The homogenization and centrifugation were repeated again for the suspension in buffer 2, and the membrane was resuspended in buffer 2. Protein concentration was determined and the membranes were stored at-80 ℃ until use.

The assay was performed in a flat bottom 1/2-area 96-well plate. The final volume was 50. mu.l per well.

The solutions and reagents were as follows:

AlphaScreen cAMP assay kit from Perkin Elmer Life Sciences; contains anti-cAMP acceptor beads (10U/. mu.l), streptavidin donor beads (10U/. mu.l) and biotinylated-cAMP (133U/. mu.l).

AlphaScreen buffer, pH 7.4: 50mM TRIS-HCl; 5mM HEPES; 10mM MgCl2, 6H 2O; 150mM NaCl; 0.01% tween. The following were added to AlphaScreen buffer (to final concentrations) before use: BSA: 0.1 percent; IBMX: 0.5 mM; ATP: 1 mM; GTP: luM are provided.

cAMP standard (dilution factor in assay 5): cAMP solution: mu.L of 5 mMcAMP-stock + 495. mu. LAlphaScreen buffer.

Preparation of cAMP Standard and GLP-1 analogs or derivatives to be tested in AlphaScreen bufferSuitable dilution series of organisms, for example the following 8 concentrations of GLP-1 compound: 10^-7、10^-8、10^-9、10^-10、10^-11、10^-12、10^-13And 10^-14M and series 10 of e.g. cAMP^-6To 3X 10^-11。

Membrane/acceptor beads: hGLP-1/BHK 467-12A membrane was used; 6 μ g/well corresponds to 0.6mg/ml (the amount of membrane used per well can vary).

"without film": acceptor beads in AlphaScreen buffer (Final 15. mu.g/ml)

"6 μ g/pore membrane": membrane + acceptor beads in AlphaScreen buffer (final 15 μ g/ml).

Add 10. mu.l "no membrane" to cAMP standards (duplicate per well), positive and negative controls.

Add 10. mu.l "6. mu.g/well membrane" to GLP-1 and analogues (duplicate/triplicate per well)

Positive control: 10. mu.l "No Membrane" + 10. mu.l AlphaScreen buffer

Negative control: 10 μ l "No Membrane" +10 μ l cAMP stock solution (50 μ M)

Because the beads are sensitive to direct light, any treatment is in darkness (as dark as possible) or in green. All dilutions were made on ice.

The operation method comprises the following steps:

preparing an AlphaScreen buffer solution;

dissolving and diluting the GLP-1/analog/cAMP standard in AlphaScreen buffer;

a donor bead solution was prepared and incubated at R.T for 30 minutes;

cAMP/GLP-l/analog was added to the plate: 10. mu.l per well;

membrane/acceptor bead solutions were prepared and added to the plates: 10. mu.l per well;

addition of donor beads: 30. mu.l per well;

plates were wrapped in aluminum foil and incubated in a shaker at RT for 3 hours (very slow);

counting on AlphaScreen-each plate was preincubated for 3 minutes before counting in AlphaScreen;

from GrCalculation of EC by aph-Pad Prism software (version 5)₅₀[pM]The values, results are shown in Table 1.

Dula glycopeptide EC₅₀EC value of 13.712nM, HIP-1₅₀The value was 0.102 nM.

TABLE 1 in vitro Activity of the respective fusion proteins EC₅₀Value comparison

Fusion proteins	EC50(nM)
		HIP-1	0.102
HIP-2	0.334
		HIP-3	0.517
HIP-4	1.358
		HIP-5	1.158
HIP-6	1.155
		HIP-7	1.289
HIP-8	1.988
		HIP-9	1.772
HIP-10	2.142
		HIP-11	2.228
HIP-12	2.145
		Dolaglutide	13.712

Demonstration of the Effect of the linker on the Activity of the fused GLP-1 analog peptide of example 2

Comparison of peptide linker SEQ ID NO: 4-8 on the activity. The fusion peptides of the different linkers were tested for activity at the human GLP-1 receptor according to the cAMP assay method described in the above example. The fusion molecules tested were able to activate the human GLP-1 receptor in a cAMP cell-based assay. Linker sequences 4, 6 and 7 were slightly better than 5 and 8 in this assay.

Verification of blood glucose Change in db/db diabetic mice injected with a Single dose in example 3

Male diabetic db/db mice, 8 weeks old, with a body weight of 42 + -2 g, were randomly divided into 6 groups of 6 mice per group by body weight. The test group is injected subcutaneously according to the dosage of 1.5mg/kg, the positive group is injected with 1.5mg/kg of dolauda, the experiment 1 group is injected with 1.5mg/kg of HIP-1-12, and the model group is injected with PBS buffer solution with the same volume dosage (10 mE/kg). Each group of animals was subjected to intraperitoneal injection of 200. mu.l of a 40% glucose solution for each of pre-administration (0h), post-administration (1 h, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 180h, 192h and 204h, respectively, and then blood glucose level RBG (blood glucose meter measurement) was measured by tail vein bleeding 30 and 60 minutes after administration of glucose. Blood glucose data are expressed as mean ± standard deviation (means ± SD) and analyzed using SPSS18.0 statistical software. Normal distribution, wherein the mean difference among multiple groups is subjected to one-factor variance analysis, the homogeneity of variance is detected by LSD, and the irregularity of variance is detected by Dunnett-T3; the non-normality distribution adopts a non-parameter test, and P < 0.05 represents that the statistical difference is significant.

Under the same dosage, the experimental group and the positive control drug, the dolacilin, have the function of reducing blood sugar. Through t-test, the random blood sugar values of animals in HIP-1-12 groups and the dolalupeptide group are remarkably reduced in comparison with a model group within 0 h-6 h, and P is less than 0.01, which shows that the onset time of the animals in vivo is similar, and the blood sugar reducing effect in a short time is similar; in addition, it is known from the change of mouse RBG values within 9 days after administration that the blood sugar-reducing effect of the dolastatin can be maintained to 3.5 days, the blood sugar value of the dolastatin has no statistical difference compared with that of the model group at 100 hours after administration, while the blood sugar level of the mice of HIP-1-12 groups can be maintained to 240 hours after administration, namely the blood sugar level of the mice at 11 days still has statistical difference (P < 0.05) compared with that of the model group, wherein the HIP-l group can be maintained to 276 hours after administration, and the HIP-9 group can be maintained to 240 hours after administration. The stability of other fusion proteins of the invention is also higher than that of dulaglutide. Therefore, the fusion protein has high stability and is expected to be developed into a long-acting GLP-l receptor agonist which is administrated once per week or longer.

TABLE 2 comparison of duration of time for which each fusion protein maintains hypoglycemic effects

Fusion proteins	Duration (h) to maintain hypoglycemic Effect
		HIP-1	276
HIP-2	270
		HIP-3	272
HIP-4	266
		HIP-5	269
HIP-6	263
		HIP-7	263
HIP-8	260
		HIP-9	240
HIP-10	248
		HIP-11	242
HIP-12	242
		Dolaglutide	100

Verification example 4 db/db diabetic mice were given HIP-1-12 with random blood glucose and HbA1c content changes for 10 weeks

Male db/db SPF-grade mice (purchased from shanghai schleck laboratory animals ltd), 8 weeks old, after 1 week of adaptive breeding, 30 db/db mice were randomly assigned to 5 groups (n-6) according to Random Blood Glucose (RBG): the model group, the dolaglutide group and the HIP-1-12 are administrated according to low (0.75mg/kg), medium (1.5mg/kg) and high (3mg/kg) doses. Each administration group was given the corresponding dose of the drug solution by subcutaneous injection, and the model group was given the PBS buffer by subcutaneous injection in a volume of 10 ml/kg. Animals of each group were dosed once a week for 10 weeks, and RBG values of mice of each group at different blood sampling time points after dosing were measured with a glucometer (an accurate glucometer, product of changsanno biosensing gmbh) respectively, and data was recorded. Setting blood sampling time points: pre-first dose (0d), post-dose 7d, 14d, 2ld, 28d, 35d, 42d, 49d, 56d, 63d and 70 d. At 70d, each group of mice was fasted for 14 hours and then bled from the orbit, and then immediately the glycated hemoglobin assay kit (immunoturbidimetry) and its kit were used to measure the glycated hemoglobin (HbA1c) content of whole blood using a H700 specific protein analyzer, and the results were expressed as the percentage (%) of HbAlc to total hemoglobin.

Data are expressed as means ± standard deviation (means ± SD) and analyzed using SPSS18.0 statistical software. Normal distribution, single-factor analysis of variance for mean difference among multiple groups, Dunnett-t test for homogeneity of variance, and Dunnett-t3 test for heterogeneity of variance; the non-normality distribution adopts a non-parameter test, and P < 0.05 represents that the statistical difference is significant.

The change trend research of random blood sugar values of mice of each group after continuous administration for 10 weeks shows that the blood sugar values of the mice of the HIP-1-12 high, medium and low dose groups are reduced to a certain degree relative to the blood sugar value of the model group, and the blood sugar reducing activity of the mice shows dose dependence. The HIP-1-12 can be prompted to effectively and continuously control the blood sugar level of db/db diabetic mice. Moreover, the blood sugar reducing effects of the first administration and the last administration of the HIP-1-12 are similar, which indicates that the effect of reducing the receptor sensitivity caused by the long-term administration of the HIP-1-12 does not occur.

Glycated hemoglobin (HbA1c) is a product of the combination of blood glucose and hemoglobin of red blood cells, and is proportional to the level of blood glucose. Because the life of the red blood cells in the blood circulation is about 120 days, the glycosylated hemoglobin can reflect the total level of the blood sugar 4-12 weeks before blood taking, and the defect that the fasting blood sugar only reflects the instantaneous blood sugar is overcome. Therefore, HbA1c is the most important evaluation index for long-term blood glucose control and is also an important basis for clinical decision on whether to change the treatment scheme. The HbA1c test result in the embodiment can stably and reliably reflect the blood sugar control condition of the mouse 2-3 months before blood drawing. The results of measurement of HbAlc content of each group of mice after 10 weeks of continuous administration are shown in Table 3 below.

TABLE 3 effect of HIP-1 on random blood glucose in db/db mice (means + -SD, n ═ 6)

Note: p < 0.05 for each dose group compared to model group; p < 0.01. Units mmol/L.

Moreover, HlP-1 the proportion of HbAlc that decreased to normal levels (HbAlc < 7%) in the treated group of subjects at 0.75mg once weekly was 14% higher than that of dulaglutide at 0.75mg once weekly. The HIP-1-12 effect is approximate.

Sequence listing

<110> Lunan pharmaceutical group, Inc

<120> a heterologous fusion protein

<160>31

<170>SIPOSequenceListing 1.0

<210>1

<211>229

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(11)..(11)

<223>11 th X is Pro

<220>

<221>UNSURE

<222>(85)..(85)

<223>85 th X is Ala or Phe

<220>

<221>UNSURE

<222>(86)..(86)

<223> X at position 86 is Phe or Glu

<220>

<221>UNSURE

<222>(87)..(87)

<223> X at position 87 is Glu, Phe or Leu

<220>

<221>UNSURE

<222>(91)..(91)

<223> X at position 91 is Gly

<220>

<221>UNSURE

<222>(230)..(230)

<223> 230X deletions

<400>1

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Xaa Cys Pro Ala Pro Glu

1 5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

65 70 75 80

Ser Thr Tyr Arg Xaa Xaa Xaa Val Leu Thr Xaa Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

100 105 110

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

130 135 140

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

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

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Leu Gly

225

<210>2

<211>229

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

65 70 75 80

Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

100 105 110

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

130 135 140

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

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

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Leu Gly

225

<210>3

<211>229

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

65 70 75 80

Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

100 105 110

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

130 135 140

Gln Val Ser Leu Thr Cys Leu Val LysGly Phe Tyr Pro Ser Asp Ile

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

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

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Leu Gly

225

<210>4

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser

<210>5

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>5

Gly Gly Gly Gly Ser Lys Lys Lys Lys Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>6

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser Gly Gly Gly Gly Ser

20

<210>7

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Gly Gly Gly Gly Ser Lys Lys Lys Lys Ser Gly Gly Gly Gly Ser Lys

1 5 10 15

Lys Lys Lys Ser Gly Gly Gly Gly Ser

20 25

<210>8

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>9

<211>31

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> Xaa at position 25 is Trp or Phe or His

<220>

<221>UNSURE

<222>(30)..(30)

<223> Xaa at position 30 is Pro or Val or Glu or Ala

<220>

<221>UNSURE

<222>(31)..(31)

<223> Xaa at position 31 is Lys-Lys-Lys-Gln-Gln-NH2 or Lys-Lys-Pro-Phe-Phe-Pro-Cys-NH2 or Lys-Lys-Ser-Cys-NH2 or Gly-Gly-Lys-Lys-Cys-NH2

<400>9

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys Gly Xaa Xaa

20 25 30

<210>10

<211>35

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> Xaa at position 25 is β Phe

<400>10

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys Gly Ala Gly Gly

20 25 30

Lys Lys Cys

35

<210>11

<211>35

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> Xaa at position 25 is β Phe

<400>11

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Pro Lys Lys Pro Phe

20 25 30

Phe Pro Cys

35

<210>12

<211>35

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> Xaa at position 25 is β Phe

<400>12

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys Gly Glu Gly Gly

20 25 30

Lys Lys Cys

35

<210>13

<211>35

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> Xaa at position 25 is β Phe

<400>13

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys Gly Val Gly Gly

20 25 30

Lys Lys Cys

35

<210>14

<211>35

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> Xaa at position 25 is β Phe

<400>14

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys Gly Val Lys Lys

20 25 30

Lys Gln Gln

35

<210>15

<211>35

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> position 25 Xaa is β His

<400>15

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys Gly Ala Gly Gly

20 25 30

Leu Leu Cys

35

<210>16

<211>34

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(25)..(25)

<223> position 25 Xaa is β Trp

<400>16

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys Gly Ala Lys Lys

20 25 30

Ser Cys

<210>17

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>17

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Ala Gly Gly

20 25 30

Lys Lys Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 5560

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>18

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>18

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Ala Gly Gly

20 25 30

Lys Lys Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>19

<211>280

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>19

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Ala Lys Lys

20 25 30

Ser Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

35 40 45

Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

50 55 60

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

65 70 75 80

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

85 90 95

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

100 105 110

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

115 120 125

Gln Phe Asn Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His

130 135 140

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

145 150 155 160

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

165 170 175

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

180 185 190

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

195 200 205

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

210 215 220

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

225 230 235 240

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu GlyAsn Val

245 250 255

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

260 265 270

Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>20

<211>280

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>20

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Ala Lys Lys

20 25 30

Ser Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

35 40 45

Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

50 55 60

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

65 70 75 80

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

85 90 95

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

100 105 110

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

115 120 125

Gln Phe Asn Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu His

130 135 140

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

145 150 155 160

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

165 170 175

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

180 185 190

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

195 200 205

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

210 215 220

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

225 230 235 240

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

245 250 255

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

260 265 270

Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>21

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>21

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Glu Gly Gly

20 25 30

Lys Lys Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>22

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>22

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Glu Gly Gly

20 25 30

Lys Lys Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>23

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>23

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Val Gly Gly

20 25 30

Lys Lys Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>24

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>24

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Val Gly Gly

20 25 30

Lys Lys Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>25

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>25

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Val Lys Lys

20 25 30

Lys Gln Gln Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>26

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>26

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Val Lys Lys

20 25 30

Lys Gln Gln Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Phe Glu Phe Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>27

<211>281

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>27

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala His Leu Val Lys Gly Ala Gly Gly

20 25 30

Lys Lys Cys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Phe Asn Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

180 185 190

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>28

<211>280

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>28

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Ala Lys Lys

20 25 30

SerCys Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

35 40 45

Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

50 55 60

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

65 70 75 80

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

85 90 95

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

100 105 110

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

115 120 125

Gln Phe Asn Ser Thr Tyr Arg Ala Phe Glu Val Leu Thr Gly Leu His

130 135 140

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

145 150 155 160

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

165 170 175

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

180 185 190

Thr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

195 200 205

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

210 215 220

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

225 230 235 240

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

245 250 255

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

260 265 270

Lys Ser Leu Ser Leu Ser Leu Gly

275 280

<210>29

<211>843

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

cacgccgaag gaaccttcac ctcagatgtg tcatcatacc tcgaaggcga agccgccaag 60

gaattcatcg cattcctcgt gaaaggagca ggaggaaaga agtgcggcgc gggcggcggc 120

ggctcgggag gaggaggatc gggaggagga ggatcggccg aatcgaaata tggtcccccc 180

tgtccaccat gtcccgctcc cgaatttctt ggtggtcctt ctgtttttct ttttccccca 240

aaaccgaaag atactcttat gatttccggc actccagagg ttacttgtgt tgttgtggac 300

gcctcacagg aggatccaga ggcccaattc aattggtatg tcgatggcgt cgaagtccac 360

aacgccaaaa ccaaaccccg cgaagagcag ttcaattcta cttatcgtgc cttcgaagtc 420

cttactggtc tccatcaaga ttggcttaat ggcaaggaat acaaatgcaa ggtctccaac 480

aaaggcctcc cctcatcaat cgaaaaaacc atctcgaaag ccaaaggcca accccgcgag 540

ccccaggtct atactctccc cccctctcaa gaagagatga ccaaaaacca ggcctcactc 600

acgtgcctcg tcaaaggatt ctacccgtcc gatattgccg tcgagtggga atcaaacgga 660

gagccagaga acaactacaa aaccacccca ccggtgctcg attcagatgg ctcatttttc 720

ctctactcac gtctcactgt cgacaaatcc cgctggcagg aaggaaacgt cttttcatgt 780

tcagtcatgc acgaagccct ccacaatcat tatacccaaa aatcgctgtc actcatgttg 840

gga 843

<210>30

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>30

Met Gly Lys Leu Ile Phe Trp Leu Val Phe Trp Leu Thr Ile Phe Trp

1 5 10 15

Leu Gly Ser Ala

20

<210>31

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

atgggcaaac tcatattctg gcttgtgttt tggctgacta ttttttggct tggatcagct 60

Claims

1. An immunoglobulin Fc fragment comprising SEQ ID NO: 1, the specific sequence is as follows:

Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Xaal1-Cys-Pro-Ala-Pro-

Glu-Phe-Leu-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-

Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-

Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-

Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-

Glu-Glu-Gln-Phe-Asn-Ser-Thr-Tyr-Arg-Xaa85-Xaa86-Xaa87-Val-Leu-Thr-

Xaa91-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-

Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-

Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-

Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-

Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-

Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-

Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-

Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-

Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-

Leu-Ser-Leu-Gly，

xaa at position 11 is Pro;

xaa at position 85 is Ala or Phe;

xaa at position 86 is Phe or Glu;

xaa at position 87 is Glu, Phe or Leu;

xaa at position 91 is GlV.

2. A heterologous fusion protein comprising a therapeutic peptide and an immunoglobulin Fc portion as set forth in claim 1.

3. The fusion protein of claim 2, wherein the therapeutic peptide is a GLP-1 analog having a C-terminal glycine residue fused to an N-terminal alanine residue of an Fc portion by a peptide linker, wherein the peptide linker is SEQ ID NO: 4, in particular to Gly-Ala-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.

4. The fusion protein of claim 3, wherein the peptide linker is further selected from the group consisting of:

a) Gly-Gly-Gly-Gly-Gly-Ser-Lys-Lys-Lys-Lys-Ser-Gly-Gly-Gly-Gly-Ser, as shown in SEQ ID NO: 5 is shown in the specification;

d) Gly-Gly-Gly-Gly-Gly-Ser, and is shown in SEQ ID NO: shown in fig. 8.

5. The fusion protein of claim 3 or 4, wherein the GLP-1 analog consists of the amino acid sequence of SEQ ID NO: sequence 9 consists of seq id no:

xaa at position 25 is β Trp or β Phe or β His;

xaa at position 30 is Pro or Val or Glu or Ala;

6. A GLP-1 analogue, the sequence of which is shown in SEQ ID NO: 9, the method is as follows.

7. A peptide linker having the sequence set forth in SEQ ID NO: 4 or 5 or 6 or 7 or 8.

8. Use of a fusion protein according to any one of claims 1 to 4 for the manufacture of a medicament.

9. Use of a fusion protein according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of non-insulin dependent diabetes mellitus.

10. Use of a fusion protein according to any one of claims 1 to 4 for the manufacture of a medicament for treating obesity or inducing weight loss in an overweight subject.

11. The fusion protein of claim 4, wherein the GLP-1 analog is also exenatide, liraglutide, linagliptin, somagluteptide, Ideglira, albigluteptide, dulaglutide, ITCA650, LAEx4, Lixilan.