CN114891090B

CN114891090B - Acylated long-acting GLP-1 derivative

Info

Publication number: CN114891090B
Application number: CN202210583262.XA
Authority: CN
Inventors: 曹海燕; 林兆生; 朱志伟; 贾宇琦; 王含; 王冠
Original assignee: Jilin Huisheng Biopharmaceutical Co ltd; Beijing Huizhiheng Biological Technology Co Ltd
Current assignee: Jilin Huisheng Biopharmaceutical Co ltd; Beijing Huizhiheng Biological Technology Co Ltd
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-10-25
Anticipated expiration: 2042-03-11
Also published as: CN114621340A; CN114621340B; CN114891090A

Abstract

The invention relates to an acylated long-acting GLP-1 derivative, in particular to a fatty acid acylation modified long-acting GLP-1 derivative, and also relates to a recombinant engineering bacterium for expressing GLP-1 (7-37) analogues and a construction method thereof. The long-acting GLP-1 derivative has good binding affinity with a GLP-1 receptor and remarkably prolonged action time, and can be used for treating diseases such as diabetes, impaired glucose tolerance, obesity, hypertension, metabolic syndrome, dyslipidemia and the like.

Description

Acylated long-acting GLP-1 derivative

The application is a divisional application of Chinese patent application with the application date of 2022, 03 and 11 months and the application number of 202210235248.0, named as an acylated long-acting GLP-1 derivative.

Technical Field

The invention relates to the field of polypeptide technology and derivatives thereof, in particular to a long-acting GLP-1 (7-37) derivative, a preparation method thereof, a pharmaceutical composition and medical application thereof.

Background

Diabetes is a metabolic disorder disease such as carbohydrate, protein, fat and the like caused by the absolute or relative insulin secretion deficiency and/or insulin utilization disorder, takes hyperglycemia as a main marker, and can be caused by various factors such as heredity, environment and the like. Diabetes is one of three major death diseases of human beings, and the death rate of the diabetes is second to cardiovascular and cerebrovascular diseases and cancers.

Diabetes is largely classified into type 1 diabetes and type 2diabetes, with the majority of patients being type 2diabetes patients (statistically, about 90%). Type 2diabetes (diabetes mellitus type2, T2 DM), old-called non-insulin dependent diabetes mellitus (NIDDM) or adult-onset diabetes (adult-offset diabetes), patients are characterized by hyperglycemia, relative lack of insulin, insulin resistance, etc. At present, clinically used medicaments for treating type 2diabetes mainly comprise biguanides, sulfonylureas, thiazolidinediones, DPP-4 receptor inhibitors, SGLT-2 receptor inhibitors and GLP-1 derivatives. Among them, GLP-1 derivatives have a similar hypoglycemic effect to insulin but almost no hypoglycemic risk, and have a weight-loss effect and a cardiovascular protection function, and thus are becoming major therapeutic drugs and research hotspots for type2 diabetes.

GLP-1 derivatives belong to GLP-1 receptor agonists, and are a class of secretin drugs, secretin is a hormone which enters blood from intestinal cells after food stimulation and can stimulate insulin secretion, the insulin secretion capacity caused by the secretin accounts for about 50-70% of the total insulin secretion amount, and the function of stimulating insulin secretion has the characteristic of glucose concentration dependence.

Glucagon-like peptide1 (GLP-1) is a secretin from intestinal L cells, and has effects of promoting insulin secretion, inhibiting glucagon release, stimulating pancreatic β cell proliferation, inducing pancreatic β cell regeneration, preventing pancreatic β cell apoptosis, improving insulin sensitivity, and increasing glucose utilization. Thus, GLP-1 and its analogs and derivatives play an important role in the development and progression of type 1 and 2 diabetes.

The amino acid sequences of GLP-1 analogues and glucagon are almost half the same, and the analogues also have multiple functions of glucose-dependent insulinotropic secretion and biosynthesis, glucagon secretion inhibition, gastric emptying inhibition and the like (Fugang, gong Min, xu Weiren. Glucagon-like peptide1 and receptor agonist research progress [ J ] Tianjin medicine 2012, 40 (2): 181-184.).

The research results of 10 patients with type 2diabetes with poor blood sugar control conducted by Nauck M, etc. and the patients were respectively given GLP-1 or placebo in a fasting state show that after the patients were infused with GLP-1, the insulin and C peptide levels were significantly increased, the glucagon level was significantly decreased, and the fasting blood sugar level became normal after 4 hours; after the blood sugar level is normal, the patient does not increase the insulin level and the blood sugar level is stable and does not further decrease although GLP-1 is continuously infused, which indicates that GLP-1 is an enterogenic hormone which is released into the blood under the stimulation of nutrient substances (particularly carbohydrate), the insulin secretion promotion effect of the GLP-1 is glucose concentration dependent, and the GLP-1 can play a role of reducing the blood sugar when the blood sugar level is increased, inhibiting the secretion of glucagon, increasing the satiety and reducing the hunger (Normalization of the stimulating hyperglycemic by exogenous glucose-like peptide1 (7-36 amide) in type2 (non-insulin-dependent) diabetes mellitus ([ J ]. Diabetologia, nauck M.,1993,36)). Lancet et al, however, have shown that GLP-1 produces weight loss through a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and ingestion, delay of emptying of gastric contents, etc. In addition, GLP-1 can also act on the central nervous system (especially hypothalamus) to suppress appetite, reduce food intake, thus causing satiety and appetite reduction in human body, and reduce calorie intake, thereby achieving the purpose of reducing weight (Effect of 6-week heart of glucose-lipid peptide1on glucose control, insulin sensitivity, and β -cell function in type2 diabetes.

GLP-1 is rapidly degraded by dipeptidyl peptidase 4 (DPP-4) in vivo, so that the action time of the GLP-1 is greatly limited, and the GLP-1 is difficult to be directly used as a medicament. Therefore, the existing method mainly for improving blood sugar control by GLP-1 mainly takes GLP-1 derivatives which simulate GLP-1 function exogenously and prolong the activity of endogenous GLP-1 as main components.

Currently, among the GLP-1 derivatives that are marketed are exenatide, liraglutide, dulaglutide, lissamide, exenatide microsphere formulations, albiglutide, polyethylene glycol loxapide, and soxhlet Ma Lutai (also known as semaglutide). Wherein, the rope Ma Lutai is a representative in GLP-1 derivative drugs.

Soxh Ma Lutai (Semaglutide) is a long acting GLP-1 derivative developed by Novonide, which only requires once weekly subcutaneous administration and is now approved for sale in many countries. Moreover, norhonode developed an oral formulation of soxhlet Ma Lutai by formulation technology. Structurally, the cable Ma Lutai is obtained by attaching the 26 th Lys position to the side chain of AEEA, glutamic acid and octadecane fatty diacid on GLP-1 (7-37) chain, and substituting the 8 th amino acid with the unnatural amino acid aminoisobutyric acid (Aib) for the original Ala. Compared with liraglutide, the fatty chain of the rope Ma Lutai is longer and the hydrophobicity is increased, but the rope Ma Lutai is modified by short-chain AEEA, and the hydrophilicity is greatly enhanced. After AEEA modification, the modified polypeptide can be tightly combined with albumin to cover DPP-4 enzyme hydrolysis sites, and can also reduce renal excretion, prolong the biological half-life and achieve the effect of long circulation. The combination of different oral hypoglycemic agents has been demonstrated in several clinical trial studies by the cable Ma Lutai to be effective in controlling blood glucose and to enable patients to lose weight, reduce systolic blood pressure and improve pancreatic beta cell function.

Due to the large number of patients with diabetes and obesity, the market is huge and the market demand for related GLP-1 derivatives is still huge. Through long-term research in the laboratory, a series of GLP-1 derivatives with the potential of reducing blood sugar and weight better than that of the rope Ma Lutai are developed. Accordingly, an object of the present invention is to provide a GPL-1 derivative having excellent weight-loss and glucose-lowering ability.

Disclosure of Invention

To solve the above technical problems, or to at least partially solve the above technical problems, the present invention provides a GLP-1 (7-37) polypeptide analogue and a long-acting derivative thereof. The long-acting GPL-1 (7-37) derivative provided by the invention has excellent weight-losing and blood-sugar-reducing capabilities, the weight-losing effect and the blood-sugar-reducing effect of the derivative are obviously superior to those of a rope Ma Lutai, and the derivative has more excellent and wide clinical and market application prospects.

The term GLP-1 (7-37) analog in the present invention refers to a polypeptide obtained by modifying a human native GLP-1 (7-37) amino acid, said modification comprising the removal and/or substitution (substitution) and/or addition (elongation) of one or more amino acid residues, which may be naturally occurring amino acids or artificially synthesized amino acids. The present invention describes the analogs with a simple nomenclature: for example, [ Val8] GLP-1 (7-37) refers to GLP-1 (7-37) analogs in which the histidine naturally occurring at position 8 has been substituted with valinVal.

In the present invention, the term "derivative" with respect to a peptide (e.g., GLP-1 or insulin) means a chemically modified (e.g., covalently modified, etc.) peptide or an analog thereof. Typical modifications are amides, sugars, alkyl, acyl, esters, and the like. An example of a GLP-1 (7-37) derivative is N ^ε26 - ((4S) -4- (hexadecanoylamino) -carboxy-butyryl) [ Arg34, lys26]GLP-1-(7-37)。

In the present invention, the term "aliphatic diacid" includes straight or branched chain aliphatic dicarboxylic acids. Non-limiting examples of aliphatic diacids are succinic acid, adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, and eicosanedioic acid.

In the present invention, the term "pharmaceutically acceptable salt" refers to a salt of a polypeptide or protein that retains the biological activity of the parent.

The term "vector" refers to a vehicle into which nucleotide fragments encoding a protein or polypeptide can be operably inserted to cause expression of the protein or polypeptide. The vector may be used to transform, transduce or transfect a host cell so that it expresses the carried genetic element in the host cell. Examples of vectors include plasmids, artificial chromosomes, bacteriophages, viral particles, and the like. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. The vector may also include materials that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein envelopes.

The term "recombinant expression vector" in the present invention is a nucleic acid molecule encoding a gene, which is expressed in a host cell and contains the necessary elements to control the expression of the gene. Typically, an expression vector comprises a transcription promoter, a gene of interest, and a transcription terminator.

The host cell in the present invention refers to a cell into which a vector comprising a nucleotide sequence fragment encoding a protein or polypeptide of interest can be introduced for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast or higher eukaryote cells.

Thus, in one aspect, the present invention provides a GLP-1 (7-37) analog consisting of a polypeptide having an amino acid sequence represented by the formula:

HX ₈ EGTFTSDVSSYLEEQAAREFIKWLVRRGG；

wherein, X ₈ Selected from V, I, T, L, G or S; preferably, X ₈ Selected from V, I, T.

In another aspect, the invention provides a long-acting GLP-1 (7-37) derivative comprising a fatty acid side chain attached to the K residue of said GLP-1 (7-37) analogue, respectively, preferably via the epsilon amino group on the K residue.

When the GLP-1 (7-37) analog is Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ When GLP-1 (7-37) is adopted, the amino acid sequence of the GLP-1 (7-37) analogue is shown as SEQ ID NO. 1.

When the GLP-1 (7-37) analogue is Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ GLP-1 (7-37) when the compound is GLP-1 (7-37) is shown in SEQ ID NO. 2.

When the GLP-1 (7-37) analogue is Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ When GLP-1 (7-37) is adopted, the amino acid sequence of the GLP-1 (7-37) analogue is shown as SEQ ID NO. 3.

As a preferred embodiment of the present invention, the side chain structure of the fatty acid used in the long-acting GLP-1 (7-37) derivative of the present invention is HOOC (CH) ₂ ) _n CO-wherein n is an integer selected from 10 to 24, more preferably 16 to 20. In particular, the fatty acid side chain may be selected from HOOC (CH) ₂ ) ₁₄ CO-、HOOC(CH ₂ ) ₁₅ CO-、HOOC(CH ₂ ) ₁₆ CO-、HOOC(CH ₂ ) ₁₇ CO-、HOOC(CH ₂ ) ₁₈ CO-、HOOC(CH ₂ ) ₁₉ CO-、HOOC(CH ₂ ) ₂₀ CO-、HOOC(CH ₂ ) ₂₁ CO-or HOOC (CH) ₂ ) ₂₂ CO-, preferably the fatty acid side chain structure is HOOC (CH) ₂ ) ₁₆ CO-。

In a preferred embodiment of the present invention, the fatty acid side chain is linked to the amino acid residue through a linker.

As a preferred embodiment of the present invention, said fatty acid side chain is linked to the epsilon amino group of Lys at position 30 on said GLP-1 (7-37) analog via a linker.

As a preferred embodiment of the present invention, the linker is selected from the group consisting of:

wherein m is an integer from 0 to 6, such as 0, 1, 2, 3, 4, 5, 6, etc., n is an integer from 1 to 3, such as 1, 2, 3, etc., s is an integer from 0 to 3, such as 0, 1, 2, 3, etc., t is an integer from 0 to 4, such as 0, 1, 2, 3, 4, etc., p is an integer from 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, etc.

As a specific embodiment of the present invention, the joint is:

wherein s is 1,n is 1 or 2.

The above-mentioned preferred linker moiety (when n is 1) may be represented by γ -Glu-OEG-OEG according to IUPAC nomenclature; wherein OEG is "2- [2- (2-aminoethoxy) ethoxy]Abbreviation for acetyl ". When HOOC (CH) is selected ₂ ) ₁₆ When CO-is used as a side chain, the combination of the above side chain and linker (acyl group) can be referred to as "[2- (2- [2- (2- [2- (2-) 4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutyrylamino group ] according to IUPAC nomenclature]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group]”。

As a specific embodiment of the present invention, the derivative of the present invention comprises a fatty acid side chain attached to the epsilon amino group of lysine at position 30 of said GLP-1 (7-37) analog, said fatty acid side chain being HOOC (CH) ₂ ) ₁₆ CO-, the fatty acid side chain is linked to the epsilon amino group of lysine at position 30 via-gamma-Glu-OEG-OEG-.

Thus, preferably, the long-acting GLP-1 derivative according to the invention is selected from the group consisting of:

N-ε ³⁰ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G5), the amino acid sequence of which is shown in SEQ ID NO. 1; or

N-ε ³⁰ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino)]Ethoxy) ethoxyBase (C)]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G6), the amino acid sequence of which is shown in SEQ ID No. 2; or

N-ε ³⁰ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G7), the amino acid sequence of which is shown in SEQ ID NO. 3.

In the invention, the inventor finds that the GLP-1 derivative obtained by modifying amino acids at 35 th position and 36 th position of GLP-1 (7-37) analogue polypeptide into amino acids Arg and Gly respectively and then connecting fatty diacid side chains through acylation at specific positions has more excellent blood sugar-reducing and weight-losing activity through partial site change.

On the other hand, the invention provides a recombinant engineering bacterium for highly expressing the GLP-1 analogue, wherein the engineering bacterium is preferably a recombinant escherichia coli engineering bacterium, and more preferably a recombinant escherichia coli BL21 engineering bacterium.

In another aspect, the invention provides a construction method of the recombinant engineering bacteria of the GLP-1 analogue, and the method comprises the following steps: (1) Sequentially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 analogue coding gene sequence in series to prepare a gene expression fragment of the GLP-1 analogue; (2) Inserting the gene expression fragment into a prokaryotic expression plasmid to obtain an expression plasmid of the GLP-1 analogue; (3) And transferring the expression plasmid into escherichia coli to prepare recombinant engineering bacteria for expressing the GLP-1 analogue.

Preferably, the prokaryotic expression plasmid is pET-30a (+), and the gene expression fragment is inserted into the plasmid through NdeI and XhoI sites.

Preferably, the amino acid sequence of the inclusion body promoting sequence is FKFEFKFE, and the EK enzyme cutting sequence is DDDDK.

More preferably, the nucleic acid sequence of the gene expression fragment according to the invention is selected from one of SEQ ID No.4 to 6.

The in vitro binding activity shows that compared with the cable Ma Lutai, the long-acting GLP-1 derivative provided by the invention has better GLP-1R binding affinity. The blood sugar reduction experiment in a diabetes animal model also shows that the long-acting GLP-1 derivative has the blood sugar reduction effect which is obviously superior to that of the cable Ma Lutai. The selection of the mutation site of the invention is proved to bring significant beneficial hypoglycemic activity to GLP-1. In addition, another important role of the GLP-1 derivative is the weight loss effect thereof, and the GLP-1 derivative can be developed into weight loss indication medicines, and the cable Ma Lutai is approved by the FDA in the United states as the weight loss indication medicines. Research results in the obese animal model also show that compared with the rope Ma Lutai, the GLP-1 derivative has a remarkable and beneficial weight loss effect in the diabetic animal model, and does not have the risk of hypoglycemia. Therefore, the long-acting GLP-1 derivative has wider commercial development value compared with the cable Ma Lutai.

In addition, the invention provides a pharmaceutical composition, which comprises the long-acting GLP-1 derivative or the pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials.

In another aspect, the invention provides the use of the long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament for the treatment of diabetes.

In another aspect, the invention provides the use of said long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for the preparation of a weight loss formulation.

Compared with the prior art, the long-acting GLP-1 derivative provided by the invention has the following advantages:

(1) The GLP-1 derivative provided by the invention has more excellent blood sugar reducing capability in diabetics;

(2) Compared with a rope Ma Lutai, the long-acting GLP-1 derivative provided by the invention has more excellent and remarkable weight loss capability and has more excellent weight loss application potential.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive labor.

FIG. 1 is a bar graph of the effect of long-acting GLP-1 derivatives on blood glucose changes in diabetic mouse models: in the figure, for each group of data, a model control group, a cable Ma Lu peptide group, an HS-G5 group, an HS-G6 group and an HS-G7 group are arranged from left to right in sequence;

FIG. 2 is a bar graph of the effect of long-acting GLP-1 derivatives on the rate of weight change in diabetic mouse models; in the figure, a model control group, a cable Ma Lu peptide group, an HS-G5 group, an HS-G6 group and an HS-G7 group are sequentially arranged from left to right;

FIG. 3 is a bar graph of the effect of long-acting GLP-1 derivatives on feeding inhibition in a diabetic mouse model; in the figure, the peptide group Ma Lu, the group HS-G5, the group HS-G6 and the group HS-G7 are arranged from left to right.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.

EXAMPLE 1 preparation of Long-acting GLP-1 derivatives

This example provides various long-acting GLP-1 (7-37) derivatives and methods for their preparation, and in particular, a recombinant engineering bacterium capable of efficiently expressing the derivatives of the present invention is constructed by the following methods (taking HS-G5 as an example):

(1) Construction of coded Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ Expression plasmid for GLP-1 (7-37)

Through a large number of previous researches and experiments, FKFEFKFE is selected as an inclusion body promoting sequence, DDDDK is selected as an EK enzyme digestion sequence, and the inclusion body promoting sequence, the EK enzyme digestion sequence and a GLP-1 analogue coding gene sequence are sequentially fused in series to obtain a gene segment shown as SEQ ID NO. 4; the fragment is inserted into a prokaryotic expression plasmid pET-30a (+) through NdeI and XhoI sites and is subjected to sequencing verification to obtain an expression plasmid called pET-30a (+) -Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ -Glp-1(7-37)。

(2) Construction of expression Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ Recombinant engineering bacterium of (7-37) GLP-1

50 μ L of BL21 competent cells (TransGenBiotech) were thawed on an ice bath, the expression plasmid constructed in step (1) was added and shaken up, 30min on an ice bath, heat shock in a water bath at 42 ℃ for 30s, and then the centrifuge tubes were quickly transferred to an ice bath for 2min without shaking the centrifuge tubes.

Adding 500 mu L of antibiotic-free sterile LB culture medium into a centrifuge tube, uniformly mixing, and culturing at 37 ℃ and 180rpm for 1h to recover bacteria; then, 200. Mu.L of the transformed competent cells were pipetted and applied to a plate of LB agar medium containing kanamycin resistance, the cells were spread out uniformly, the plate was left at 37 ℃ until the liquid was absorbed, the plate was inverted, cultured overnight at 37 ℃, and a single colony in the transformed plate was picked up using an inoculating loop and inoculated in 15mL of a sterile LB medium containing kanamycin antibiotic and cultured overnight at 37 ℃. Adding 500 μ L of overnight culture liquid into 1.5ml sterile centrifuge tube, adding 500 μ L of 50% sterile glycerol, and mixing to obtain glycerol cryopreserved bacteria, and storing at-80 deg.C.

(3) Fermentation expression of recombinant engineering bacteria

Adding 50 μ L glycerol into 50mL 2YT culture medium, freezing to obtain bacterial liquid, adding 50 μ L kanamycin, mixing, placing in a constant temperature oscillator, culturing at 37 deg.C and 200rpm overnight, and measuring OD600 > 5.0 to obtain first-grade seed culture solution.

Taking 40mL of overnight cultured primary seed culture solution, inoculating the overnight cultured primary seed culture solution into 200mL of 2YT culture medium according to the proportion of 1:5, simultaneously adding 200 mu L of kanamycin, uniformly mixing, placing in a constant temperature oscillator, culturing at 37 ℃ and 200rpm for 3h, wherein OD600 is more than 3.0, and obtaining secondary seed culture solution.

Taking 60mL of secondary seed liquid, inoculating the secondary seed liquid into an FDM culture medium (600 mL) according to the proportion of 1.

Centrifuging the collected bacterial liquid for 30min at 8000g to obtain thallus cytoplasm, wherein the thallus cytoplasm is not less than 290g of thallus/L fermentation liquid, and measuring the expression amount of the target protein by using the thallus obtained by centrifugation, wherein the expression amount is not less than 10g/L.

(4) Recombinant Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ Purification of GLP-1 (7-37)

Weighing 100g of the cell paste obtained in the step (3), suspending the cell paste in 500mL of solution (50 mM Tris-HCl, 50mM NaCl, pH 8.0), carrying out ultrasonic treatment in an ultrasonic cell crusher for 30min to crush the cells, centrifuging the obtained homogenate at 13000g for 30min at 4 ℃, collecting a precipitate after the centrifugation is finished, and dissolving the precipitate by using 8M urea to obtain a sample before enzyme digestion.

Concentrating the sample before enzyme digestion by UniPS30-300 (purchased from Suzhou Naichi Microscience and technology Co., ltd.) which is balanced by equilibrium liquid 3 (10 mM ammonium acetate and 20% acetonitrile), eluting by equilibrium liquid 3, then eluting by gradient according to 0-100% eluent (10 mM ammonium acetate and 80% acetonitrile), analyzing by RP-HPLC, and obtaining GLP-1 intermediate product with the purity higher than 70% by the purification process.

The tag sequence was cleaved using EK enzyme: the intermediate product was diluted three times by adding 20mM PB buffer solution of pH7.4, and after adding EK enzyme at 20 ℃ according to EK enzyme: intermediate product = 1.

Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ -fine purity of GLP-1 (7-37): uniPS30-300 (from Sozhou, nami, inc.) equilibrated with equilibration solution 3 (10 mM ammonium acetate, 20% acetonitrile) was concentrated, and after elution with equilibration solution 3, the product was eluted with a gradient of 0-100% eluent (10 mM ammonium acetate, 80% acetonitrile) at a purity of about 90% by RP-HPLC.

0.2M Na was added to the eluted sample ₂ HPO ₄ Adjusting pH to 4.8-5.0,4 deg.C with 1M citric acid to obtain final concentration of 20mM, acid precipitating overnight at 4.8-5363 deg.C, RP-HPLC detecting yield above 90%, centrifuging at 13000g for 30min at 4 deg.C, collecting precipitate, storing at-20 deg.C to obtain Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ -GLP-1 (7-37); and (3) sequencing verification, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

(5) Preparation of long-acting GLP-1 derivatives

Fatty acid modification: val obtained in step (4) ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ Adding water into GLP-1 (7-37) to prepare a 4-6 mg/mL solution, adding 1M sodium hydroxide to adjust the pH value to 11.0-11.5, shaking up to completely dissolve the protein, and quantifying the polypeptide concentration by HPLC; weighing fatty acid powder according to the mole ratio of the polypeptide to octadecanedioic acid mono-tert-butyl ester-glutamic acid (1-tert-butyl ester) -AEEA-AEEA-OSU-3242 to 1:4, dissolving the fatty acid powder in acetonitrile, mixing a polypeptide sample with the fatty acid solution, standing the mixed solution for one hour at 4 ℃, then adding water to dilute the sample by 5 times, adjusting the pH value to 4.8 by using 1M citric acid (or 10% acetic acid) to terminate the reaction, standing the sample at 4 ℃ for acid precipitation for 10min, centrifuging the solution at 13000g and 4 ℃ for 30min after the acid precipitation, and then storing the precipitate at-80 ℃.

Deprotection and purification of fatty acid: to the resulting precipitate was added TFA up to a final peptide concentration of about 10mg/mL, the precipitate was dissolved by shaking, left to stand at room temperature for deprotection for 30min, and then the pH was adjusted to 7.5 to 8.5 by dropping 4M NaOH to terminate the reaction.

Pumping the reaction solution after the reaction termination into UniPS10-300 (purchased from Suzhou Naichi Microscience, inc.) equilibrated with equilibration solution 3 (10 mM ammonium acetate, 20% acetonitrile) at a flow rate of 4mL/min by using a protein purification chromatography system (Seikagaku SDL 100) for concentration, eluting with equilibration solution 3, performing gradient elution with 0-100% eluent (10 mM ammonium acetate, 80% acetonitrile), and collecting the elution peak with a purity of about 90% by RP-HPLC.

Diluting the peak with water by 3 times, adjusting pH to 4.80,4 deg.C, precipitating with acid for 30min, centrifuging, adding PBST buffer (pH 7.0) into the precipitate, redissolving, and freezing at-80 deg.C to obtain N-epsilon ³⁰ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G5).

According to the step (1) in the embodiment, the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 analogue coding gene sequence are sequentially fused in series to respectively obtain the coding Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ GLP-1 (7-37) and Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Arg ³⁵ Gly ³⁶ -GLP-1 (7-37) gene segments, the nucleotide sequences of which are shown in SEQ ID No.5-6, respectively; the rest steps are the same, and HS-G6 and HS-G7 are prepared.

In the examples, the expression of the recombinant engineered bacteria constructed in steps (1) to (4) is shown in table 1:

TABLE 1

Example 2: in vitro cell affinity activity assay

(1) Preparing different long-acting GLP-1 derivative injection

The specific formula is as follows: DMEM blank medium, GLP-1 derivatives: 320nM, 64nM, 12.8nM, 2.56nM, 0.512nM, 0.1024nM, 0.02048nM, 0.004096nM. The positive control group (cable Ma Lutai), the experimental group 1 (HS-G5), the experimental group 2 (HS-G6) and the experimental group 3 (HS-G7) are included.

Selecting HEK293/Luc/GLP1R cells with good culture state, discarding culture solution in the bottle, washing with PBS buffer solution for 1 time, adding 0.05% Trypsin digestive juice for digestion for 3 minutes, adding DMEM basal medium to terminate digestion, and centrifuging to collect cells. DMEM blank medium was used to adjust the cell density to 8.0X 10 ⁵ one/mL, 50. Mu.L/well in 96-well cell culture plates, at 37 ℃ and 5% CO ₂ Incubated under conditions overnight.

In vitro activity of derivatives of GLP-1 analogs was tested using Fire-Lumi luciferase assay kit: preparing a determination culture solution, diluting a sample to 320nM by steps by a DMEM blank culture medium, wherein the single dilution multiple does not exceed 10 times; then, 5-fold serial dilutions were performed in 96-well plates for 8 gradients, with 2 duplicate wells for each dilution.

Removing the cultured cell culture plate from the incubator, adding diluted assay medium to the cell plate at 50. Mu.L/well, and subjecting to 37 ℃ and 5% CO ₂ Incubate under conditions for 6hr. The sample plate was removed from the incubator and allowed to stand at room temperature. Adding 100ul Fire-Lumi detection solution, reacting for 5min, shaking for 10S, and detecting fluorescence intensity. The test data is processed by adopting a four-parameter regression calculation method, and the EC50 value of the sample to be tested can be calculated. The results are shown in table 2:

TABLE 2

Sample (I)	HS-G5	HS-G6	HS-G7	Rope Ma Lutai
					EC50(nM)	0.405	0.399	0.464	0.734

As can be seen from the results in the table, the HS-G5, HS-G6 and HS-G7 of the present invention have lower EC50 values than that of cable Ma Lutai, indicating that they have better binding affinity to human insulin receptor than cable Ma Lutai.

Example 3: study of hypoglycemic Effect in db/db mice

(1) Experimental materials:

the experimental animals are: BKS-Lepr ^REM The number of the/Gpt mice is 25, the week age is 6 weeks old, male.

Experimental pharmaceutical formulation: 1.133mg/mL Na ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, 0.045mg/mL GLP-1 derivative.

(2) The experimental method comprises the following steps:

a. modeling and grouping:

selecting 6-week BKS-Lepr male SPF grade ^REM 25/Gpt mice are randomly grouped according to weight and blood sugar and divided into 5 groups, wherein each group is provided with 5 test animals which are respectively a model control group (solvent), a positive control group (cable Ma Lutai), an experimental group 1 (HS-G5), an experimental group 2 (HS-G6) and an experimental group 3 (HS-G7); wherein the control group of the model was injected with a blank solvent intraperitoneally and subcutaneously.

b. The administration mode comprises the following steps:

the administration was carried out in the manner shown in Table 3, once every 48h, four times, and by intraperitoneal injection.

TABLE 3

c. Detecting the index

Blood glucose value: the blood glucose level before administration and 2h after administration were measured for the first administration, and the blood glucose level 0h before and 2h after each administration was measured for the following three administrations. The 0h before administration is 48h described in table 4 and DAY3, DAY5, DAY7 and DAY9 in fig. 1, which means 48h after the previous administration and is also 0h before the current administration.

(3) Results of the experiment

The statistics of the experimental data are shown in Table 4, the data correspond to a histogram shown in FIG. 1, and in the graph, each group of data sequentially comprises a model control group, a rope Ma Lutai, HS-G5, HS-G6 and HS-G7 from left to right.

Table 4: mean blood glucose condition

Statistical analysis of all data from this experiment was performed using SPSS software. All values are expressed as means ± standard deviation (mean ± SD). For normal distribution data, differences between groups were compared using one-way ANOVA. For all analyses, P <0.05 was considered statistically significant.

From the data results of the experiment (table 4 and fig. 1), it can be seen that the derivatives of the present invention have significantly better hypoglycemic effect than that of rope Ma Lutai when administered for 2h and 48h almost every time; the blood sugar can be controlled within the ideal range in the whole administration period.

Example 4: study of weight loss Effect in db/db mice

(1) Experimental materials:

the product is BKS-Lepr ^REM The mice, 25 in number, 6 weeks old, male, gpt.

(2) Experimental methods

a. Modeling and grouping:

selecting 25 SPF male BKS-LeprRem/Gpt mice at 6 weeks, randomly grouping the mice according to weight and blood sugar, and dividing the mice into 5 groups, wherein each group is provided with 5 test animals which are respectively a model control group (solvent), a positive control group (Soy Ma Lutai), an experiment group 1 (HS-G5), an experiment group 2 (HS-G6) and an experiment group 3 (HS-G7). Wherein the control group of the model was injected with a blank solvent intraperitoneally and subcutaneously.

b. The administration mode comprises the following steps:

the administration was carried out in the manner shown in Table 3, once every 48h, four times, and subcutaneously via intraperitoneal injection.

c. Detection indexes are as follows:

weight: mice were weighed before each dose and 48h after the last dose.

Food intake: a fixed food intake (200 g) was set per cage of mice before the first dose, and then the food intake of the mice was measured 48h after each (including the first) dose.

(3) The experimental results are as follows:

the data of the body weight change of the mice for 9 days were recorded, and the results are shown in table 5, and fig. 2 is a graph of the body weight change rate of the mice after 9 days of administration.

TABLE 5

Sample (I)	Model control group	Positive control group	Experimental group 1	Experimental group 2	Experimental group 3
						DAY1	59.0±7.2	58.6±7.4	58.3±6.3	59.3±4.0	58.5±6.6
DAY3	59.2±6.7	56.9±6.7	54.7±5.9	56.4±5.9	56.2±6.6
						DAY5	59.3±7.1	56.7±6.8	54.8±6.0	55.0±3.5	55.9±6.2
DAY7	59.8±6.6	56.6±6.3	55.1±5.9	55.3±4.1	56.3±6.2
						DAY9	59.1±8.4	56.6±6.8	55.5±5.6	56.1±3.8	56.4±6.2
Change in body weight/g	0.1	-2.0	-2.8	-3.2	-2.1
						Rate of change of body weight/%)	0.17	-3.41	-4.80	-5.40	-3.59

Meanwhile, the food intake of the mice for 9 days was recorded, wherein the food intake inhibition rate = (average of food intake of model control group-average of food intake of administration group)/average of food intake of model control group, and the results are shown in table 6:

TABLE 6

As can be seen from tables 5-6 and figures 2-3 for recording weight loss and food intake data of mice, the long-acting GLP-1 derivative provided by the invention has better weight loss effect and better food intake inhibition than the cable Ma Lutai, and the difference is obvious.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Jilin Huisheng biopharmaceutical Co.,Ltd.

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Claims

1. A gene fragment encoding a fusion polypeptide comprising a GLP-1 (7-37) analogue, characterized in that the nucleic acid sequence of said gene fragment is as shown in any one of SEQ ID No. 4-6.

2. A recombinant expression vector comprising the gene fragment of claim 1.

3. A recombinant engineered bacterium that expresses a GLP-1 (7-37) analog, said recombinant engineered bacterium comprising the recombinant expression vector of claim 2.

4. The recombinant engineering bacteria of claim 3, wherein the recombinant expression vector is a recombinant pET-30a (+) expression vector, and the recombinant engineering bacteria is recombinant BL21 Escherichia coli engineering bacteria.

5. The recombinant engineering bacterium according to claim 4, wherein the recombinant engineering bacterium is constructed by the following method:

(1) Synthesizing the gene fragment of claim 1;

(2) Connecting the gene fragment synthesized in the step (1) to an expression vector pET-30a (+) to construct a recombinant expression vector;

(3) And (3) transferring the recombinant expression vector constructed in the step (2) into escherichia coli BL21 (DE 3) to obtain the recombinant engineering bacteria.

6. The recombinant engineered bacterium of claim 5, wherein step (2) of the method is: and (2) inserting the gene fragment in the step (1) between NdeI and XhoI enzyme cutting sites of an expression vector pET-30a (+), and constructing to obtain the recombinant expression vector.

7. The recombinant engineered bacterium of claim 6, wherein step (3) of the method is: and (3) transforming and introducing the recombinant expression vector into an escherichia coli expression host BL21 (DE 3) through a heat shock method, and screening to obtain the recombinant engineering bacteria.

8. A construction method of the recombinant engineering bacteria of any one of claims 3 to 7, characterized in that the construction method comprises the following steps:

(1) Synthesizing the gene fragment of claim 1;

9. The building method according to claim 8, wherein the step (2) of the building method is: and (2) inserting the gene fragment in the step (1) between NdeI and XhoI enzyme cutting sites of an expression vector pET-30a (+), and constructing to obtain the recombinant expression vector.

10. The construction method according to claim 8 or 9, wherein the step (3) of the construction method is: and (3) transforming and introducing the recombinant expression vector into an escherichia coli expression host BL21 (DE 3) through a heat shock method, and screening to obtain the recombinant engineering bacteria.