CN114874314B

CN114874314B - Recombinant engineering bacterium for highly expressing GLP-1 analogue and construction method thereof

Info

Publication number: CN114874314B
Application number: CN202210578109.8A
Authority: CN
Inventors: 曹海燕; 林兆生; 朱志伟; 王建宇; 辛瑞; 曹丙洲
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: 2021-12-28
Filing date: 2022-01-30
Publication date: 2022-11-11
Anticipated expiration: 2042-01-30
Also published as: CN114796462B; CN115850438B; WO2023124847A1; CN114874314A; CN115850438A; CN114796462A; CN114621339A; CN114621339B

Abstract

The invention relates to a GLP-1 polypeptide analogue, a long-acting GLP-1 derivative of the polypeptide analogue modified by fatty acid acylation. The invention also relates to a recombinant engineering bacterium for expressing the GLP-1 (7-37) analogue and a construction method thereof. The long-acting GLP-1 derivative obtained by modifying the GLP-1 (7-37) analogue has good binding affinity with a GLP-1 receptor (GLP-1R), and compared with other GLP-1 derivatives on the market, the long-acting GLP-1 derivative has more obvious and excellent blood sugar reducing capability and weight losing capability.

Description

Recombinant engineering bacterium for highly expressing GLP-1 analogue and construction method thereof

The application is a divisional application of Chinese patent application with the application date of 2022, 30.01 month and the application number of 202210113945.9, named as 'a long-acting GLP-1 derivative'.

Technical Field

The invention relates to the field of polypeptide technology and derivatives thereof, relates to a long-acting GLP-1 derivative, and particularly relates to a recombinant engineering bacterium for highly expressing GLP-1 (7-37) analogues and a construction method thereof.

Background

Diabetes is a group of metabolic disorders such as carbohydrates, proteins, fats and the like caused by the absolute or relative insufficient secretion and/or utilization disorder of insulin, 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 type 2, 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 risk of hypoglycemia, and have both weight loss effect and cardiovascular protection function, and thus are becoming the main therapeutic drugs and research hotspots for type 2 diabetes.

Glucagon-like peptide 1 (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 glucagon analogues have multiple functions such as glucose-dependent insulinotropic secretion and biosynthesis, glucagon secretion inhibition, gastric emptying inhibition and the like (Fujian, gong Min, xu Weiren glucagon-like peptide 1 and receptor agonist research progress [ J ] Tianjin medicine, 2012, 40 (2): 181-184.).

In the literature, the authors studied 10 patients with type 2diabetes with poor glycemic control and administered GLP-1 or placebo, respectively, in the fasting state in a normalysis of stimulating hyperglycemic by y exogenous glucose-like peptide 1 (7-36 amide) in type 2 (non-insulin-dependent) diabetes patients ([ J ]. Diabetologia, navck m.,1993,36), and the results showed that patients with insulin and C-peptide levels significantly increased, glucagon levels significantly decreased, and fasting blood glucose levels became normal after 4 hours after infusion of GLP-1; after the blood sugar level is normal, the insulin level of a patient can not rise any more although GLP-1 is continuously infused, the blood sugar level is also kept stable and does not further drop, which shows that GLP-1 is used as an enterogenous hormone which is released into blood under the stimulation of nutrient substances (particularly carbohydrate), the insulin secretion promotion effect of the hormone is glucose concentration dependent, and the hormone can play a role in reducing blood sugar when the blood sugar rises, inhibit the secretion of glucagon, increase satiety and reduce hunger, thereby achieving the effect of reducing blood sugar. In contrast, in the literature Effect of 6-week core of glucose-like peptide 1on glycine control, insulin sensitivity, and β -cell function in type 2diabetes, ([ J ]. Lancet, zander, mette, madsbad, et al, 2002), researchers have shown that GLP-1 produces weight-reducing effects via a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and feeding, and delay of gastric emptying. In addition, GLP-1 can also act on central nervous system (especially hypothalamus) to suppress appetite, reduce food intake, thereby causing satiety and appetite decrease, and reducing calorie intake, thereby reducing weight.

At present, GLP-1 derivatives are mainly marketed as exenatide, liraglutide, dulaglutide, lisanatide, exenatide microsphere preparation, albiglutide, polyethylene glycol loxapide and soxhlet Ma Lutai (also named as semaglutide). Wherein, the rope Ma Lutai is a representative in GLP-1 derivative drugs.

Soo Ma Lutai (Semaglutide) is a long acting GLP-1 derivative developed by Novonide, which requires subcutaneous administration only once a week and is currently marketed 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 cable Ma Lutai is longer, and the hydrophobicity is increased, but the cable Ma Lutai is modified by short-chain AEEA, so that the hydrophilicity is greatly enhanced. After AEEA modification, the modified protein 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.

Therefore, an object of the present invention is to provide a GPL-1 derivative and a preparation thereof having excellent weight-loss and blood-glucose-lowering ability.

Disclosure of Invention

To solve the above technical problem or to at least partly solve the above technical problem, 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 reduction and blood sugar reduction capabilities, the weight reduction effect can even reach nearly twice that of a rope Ma Lutai, and the blood sugar reduction effect is also obviously better than that of a rope Ma Lutai.

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

HX ₈ EGTFTSDVSSYLE _E QAAX ₂₆ EFIAWLVX ₃₄ X ₃₅ X ₃₆ G；

wherein X ₈ Selected from V, I, T, L, G or S, X ₂₆ Is R or K, X ₃₄ Is R or K, X ₃₅ Is G or R, X ₃₆ Is G or R, wherein X ₂₆ And X ₃₄ Only one of them is K;

in a second aspect, the present invention provides a long-acting GLP-1 (7-37) derivative comprising fatty acid side chains attached to the K residues of said GLP-1 (3-37) analogues, respectively, preferably via the epsilon amino group on the K residue.

When the GLP-1 (7-37) analog is Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ 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 Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ When GLP-1 (7-37) is adopted, the amino acid sequence of the GLP-1 (7-37) analogue is shown as SEQ ID NO. 2.

When the GLP-1 (7-37) analogue is Ile ⁸ Glu ²² Arg ²⁶ Lys 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.

When the GLP-1 (7-37) analogue is Thr ⁸ Glu ²² Arg ²⁶ Lys 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. 4.

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-where 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 residue via a linker.

As a preferred embodiment of the present invention, the fatty acid side chain is linked to position X on the GLP-1 (3-37) analog via a linker ₂₆ Or X ₃₄ The epsilon amino group of Lys is linked.

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.

As for the above-mentioned preferred linker moiety (when n is 1), it may be represented by γ -Glu-OEG-OEG wherein OEG represents a group-NH (CH) according to IUPAC nomenclature ₂ ) ₂ O(CH ₂ ) ₂ CO-i.e. "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, said derivative of the present invention comprises a fatty acid side chain attached to the epsilon amino group of lysine in position 34 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 34 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 ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ ]GLP-1 (7-37) (abbreviated HS-G1); or

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ ]GLP-1 (7-37) (abbreviated HS-G2); or

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G3); or

N-ε ³⁴ - [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 ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G4).

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 acylating specific positions has more excellent blood sugar reducing and weight reducing activities through partial site change.

In a third aspect, 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 a fourth 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 serially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 analogue coding gene sequence 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 the 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, or the prokaryotic expression plasmid is pET-28a (+) and the gene expression fragment is inserted into the plasmid through NcoI and XhoI sites.

Preferably, the amino acid sequence of the inclusion body promoting sequence is FKFEFKFE, and the EK enzyme digestion 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.5 to 8.

The in vitro binding activity shows that compared with the cable Ma Lutai, the long-acting GLP-1 derivative provided by the invention maintains GLP-1R binding affinity equivalent to that of the cable Ma Lutai. However, the blood sugar reduction experiment in a diabetes animal model shows that the long-acting GLP-1 derivative has the blood sugar reduction effect which is obviously better than that of the cable Ma Lutai, and particularly the blood sugar reduction effects of HS-G3 and HS-G4 are particularly outstanding. Another important role of GLP-1 derivatives is their weight loss effect, which can be developed as weight loss indication drugs, and the cable Ma Lutai has been approved by the FDA in the united states as a weight loss indication drug. Research results in the obese animal model show that compared with the rope Ma Lutai, the GLP-1 derivative (especially HS-G3 and HS-G4) has approximately two or more times weight loss effect in a normal animal model and a diabetic animal model, and does not have hypoglycemia risk, so that the long-acting GLP-1 derivative has wider commercial development value.

In a fifth aspect, the invention provides a pharmaceutical composition comprising said long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In a sixth aspect, the invention provides the application of the long-acting GLP-1 derivative or the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof in preparing a medicament for treating diabetes.

In a seventh 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 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 the cable Ma Lutai, the long-acting GLP-1 derivative provided by the invention has more excellent and remarkable weight loss capability for normal people and diabetic patients, and has better 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 of the present invention or the technical solutions in the prior art, 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 that other drawings can be obtained according to the drawings without inventive exercise.

FIG. 1 is a bar graph of the effect of long-acting GLP-1 derivatives on blood glucose changes in db/db mice:

in the figure, for each group of data, from left to right, a model control group, a cable Ma Lu peptide group, an HS-G1 group, an HS-G2 group, an HS-G3 group and an HS-G4 group are sequentially arranged;

FIG. 2 is a bar graph of the effect of long acting GLP-1 derivatives on the rate of change of body weight in db/db mice;

in the figure, a model control group, a cable Ma Lu peptide group, an HS-G1 group, an HS-G2 group, an HS-G3 group and an HS-G4 group are arranged from left to right in sequence;

FIG. 3 is a bar graph of the effect of long-acting GLP-1 derivatives on the rate of change of body weight in normal mice;

in the figure, from left to right, the model control group, the cable Ma Lu peptide group, the HS-G3 group and the HS-G4 group are shown in sequence.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, aspects of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments 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-G1 as an example):

(1) Construction of the code Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Expression plasmid for GLP-1 (7-37)

Through a large amount of researches and experiments, FKFEFKFE is finally 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 fragment shown as SEQ ID NO. 5; inserting the fragment into prokaryotic expression plasmid pET-28a (+) through NcoI and XhoI sites, and sequencing to verify to obtain expression plasmid named pET-28a (+) -Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -Glp-1(7-37)。

(2) Construction of expression Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Recombinant engineering bacterium of (7-37) GLP-1

BL21 competent cells (TransGenBiotech) were thawed (50. Mu.L) on an ice bath, the expression plasmid constructed in step (1) was added, shaken gently, and placed in an ice bath for 30min. Then carrying out water bath heat shock at 42 ℃ for 30s, and then quickly transferring the centrifugal tube into an ice bath for placing for 2min without shaking the centrifugal tube;

adding 500. Mu.L of sterile LB medium (containing no antibiotic) into a centrifuge tube, mixing uniformly, culturing at 37 ℃ for 1h at 180rpm to resuscitate the bacteria, then sucking 200. Mu.L of transformed competent cells, adding the cells to a plate containing kanamycin-resistant LB agar medium, spreading the cells uniformly, placing the plate at 37 ℃ until the liquid is absorbed, inverting the plate, culturing overnight at 37 ℃, the next day, picking a monoclonal colony in the transformation plate by using an inoculating loop, inoculating into 15mL of sterile LB medium (containing antibiotic), and culturing overnight at 30 ℃. Adding 500 μ L of overnight culture liquid into a sterile 1.5ml 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 mu L of glycerol into 50mL of 2YT culture medium for freezing and storing bacterial liquid, simultaneously adding 50 mu L of kanamycin, uniformly mixing, placing in a constant temperature oscillator, culturing at 37 ℃ and 200rpm overnight to obtain a first-grade seed culture solution, and measuring OD600 to be more than 5.0;

taking 40mL of first-level seed culture solution for overnight culture, inoculating the first-level 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, putting the mixture into a constant temperature oscillator, culturing for 3h at 37 ℃ and 200rpm, wherein OD600 is more than 3.0, and obtaining second-level 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;

the expressed bacteria liquid is centrifuged for 30min at 8000g to obtain thallus cytoplasm, the thallus yield is about 300g of thallus/L of fermentation liquid, and the thallus obtained by centrifugation is subjected to target protein expression amount measurement, and the expression amount is not less than 10g/L.

(4) Recombinant Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ 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, pH8.0), carrying out ultrasonic treatment for 30min by using an ultrasonic cell crusher to crush the cells, centrifuging the obtained homogenate for 30min at 13000g at 4 ℃, collecting precipitates after the centrifugation is finished, and dissolving the precipitates by using 8M urea to obtain a sample before enzyme digestion;

concentrating a 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, 20% acetonitrile) in advance, eluting by the equilibrium liquid 3, then carrying out gradient elution by 0-100% eluent (10 mM ammonium acetate, 80% acetonitrile), and analyzing by SDS-PAGE, wherein the purity of the GLP-1 intermediate product generated by the purification process is higher than 70%;

the tag sequence was cleaved using EK enzyme: adding 20mM PB buffer solution with pH =7.4 into the intermediate product to dilute the intermediate product three times, adding EK enzyme according to EK enzyme: intermediate product =1:15 at 20 ℃, uniformly mixing, performing enzyme digestion overnight, and analyzing the enzyme cleavage rate by SDS-PAGE to be approximately 80%;

Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -fine purity of GLP-1 (7-37): concentrating UniPS30-300 (purchased from Suzhou Naichi Microscience, inc.) equilibrated with equilibration solution 3 (10 mM ammonium acetate, 20% acetonitrile), eluting with equilibration solution 3, and gradient-eluting with 0-100% eluent (10 mM ammonium acetate, 80% acetonitrile), wherein the purity of the eluate is about 90% by SDS-PAGE;

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 20mM to allow acid precipitation overnight, performing SDS-PAGE detection at yield above 90%, centrifuging at 13000g for 30min at 4 deg.C, collecting precipitate, and storing at-20 deg.C to obtain Ile ⁸ Glu ²² Arg ²⁶ Lys5 ³⁴ -GLP-1(7-37)。

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

Fatty acid modification: ile obtained in step (4) ⁸ Glu ²² Arg ²⁶ Lys ³⁴ 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 polypeptide to octadecanedioic acid mono-tert-butyl ester-glutamic acid (1-tert-butyl ester) -AEEA-AEEA-OSU-5363 and 1:4, dissolving the fatty acid powder in acetonitrile, mixing a polypeptide sample with a fatty acid solution, standing the mixed solution at 4 ℃ for one hour, 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 at 4 ℃ for acid precipitation for 10min, centrifuging at 13000g and 4 ℃ for 30min after acid precipitation, and then storing the precipitate at-80 ℃;

and (3) deprotection and purification of fatty acid: adding TFA to the obtained precipitate until the final concentration of the polypeptide is about 10mg/mL, shaking to dissolve the precipitate, standing at room temperature for deprotection for 30min, and then dropping 4M NaOH to adjust the pH value to 7.5-8.5 to terminate the reaction;

pumping the reaction solution after the reaction termination into UniPS10-300 (purchased from Suzhou Na micro-technology Co., ltd.) equilibrated with an 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, after the equilibration solution 3 is washed by water, carrying out gradient elution according to a 0-100% eluent (10 mM ammonium acetate, 80% acetonitrile), collecting an elution peak, and detecting the purity of the elution peak to be about 90% by RP-HPLC;

diluting the elution peak with water by 3 times, adjusting pH to 4.80,4 deg.C by acid precipitation for 30min, adding PBST buffer solution with pH of 7.0 into the precipitate after centrifugation for redissolving, and freezing at-80 deg.C to obtain N-epsilon ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ ]GLP-1 (7-37) (abbreviated as HS-G1).

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 obtain the gene containing the coding Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1(7-37)、Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ GLP-1 (7-37) and Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ -GLP-1 (7-37) gene segments, the nucleotide sequences of which are shown in SEQ ID No.6-8, respectively; wherein the content of the first and second substances,

Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ GLP-1 (7-37) was prepared in exactly the same manner as in steps (1) to (5) ³⁴ - [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 ³⁴ ]GLP-1 (7-37) (abbreviated HS-G2);

Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ GLP-1 (7-37) and Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ GLP-1 (7-37), which is obtained by inserting the above fragment into a prokaryotic expression plasmid pET-30a (+) and verifying the sequencing according to the substantially same method as that of the steps (1) to (5) except that NdeI and XhoI sites are selected, and finally obtaining expression plasmids (the other steps are the same) by: n-epsilon ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G3), and, N- ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37) (abbreviated as HS-G4).

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: 1.133mg/mL Na ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, GLP-1 derivatives: 1200ng/mL, 300ng/mL, 75ng/mL, 18.75ng/mL, 4.6875ng/mL, 1.172ng/mL, 0.293ng/mL, 0.073ng/mL.

Selecting RIN-m5F cells in good culture state, discarding culture solution in bottle, washing with Versene solution (ethylenediamine tetraacetic acid solution) for 1 time, adding 0.05% (v/v) TRYPSIN digestive juice, adding RPMI1640 basic culture solution to stop digestion, centrifuging to collect cells, adjusting cell density to 6.7 × 10 with RPMI1640 basic culture solution ⁵ one/mL, 100. 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 using cAMP detection kit (R & D): preparing a determination culture solution, diluting a sample to 1200ng/mL step by using the determination culture solution, wherein the single dilution multiple does not exceed 10 times; after that, 4-fold serial dilutions were performed in 96-well plates for 8 gradients, with 2 duplicate wells for each dilution.

Taking out the cultured cell culture plate from the incubator, discarding the old culture solution, and blotting the old culture solution on filter paper(ii) a Adding the diluted solution to a cell plate at 100. Mu.L per well, at 37 ℃ 5% ₂ The drug was allowed to act for 3.5min under conditions, the sample solution on the cell culture plate was decanted, blotted dry on filter paper, and diluted cell lysate (1 × Lysis Buffer) was added to the cell culture plate, 120 μ L per well, frozen at-80 ℃ and thawed by shaking at room temperature.

(2) And (3) cAMP content detection:

the desired strips were lined up in a 96-well plate rack and primary antibodies were incubated: adding 50 microliter of primary antibody solution into each well, sealing the plate by using a sealing plate membrane, and putting the plate into a microplate oscillator to incubate for 1 hour at room temperature.

Washing: discarding the liquid, spin-drying, adding 300 μ L of washing buffer solution into each well, placing into a microplate oscillator, shaking for 1min, discarding, repeating the above steps for 4 times, and drying on filter paper.

Loading and secondary antibody: adding 50 mu L of secondary antibody into each hole, taking 100 mu L of cell lysate from each hole in the cell plate, transferring the cell lysate into the holes of the enzyme label plate, sealing the plate by a sealing plate membrane, uniformly mixing, putting into a micropore plate oscillator, and incubating for 2 hours at room temperature.

Color development: and (3) uniformly mixing the color development liquid A and the color development liquid B in equal amount, adding 200 mu L of mixed liquid into each hole, putting the mixed liquid into a micropore plate oscillator, and performing light-proof color development for 30 minutes.

And (4) terminating: the reaction was stopped by adding 100. Mu.L of stop solution to each well.

And (3) determination: placing the sample in an enzyme-labeling instrument, and detecting the OD value of each hole by using 570nm as reference wavelength and 450nm as detection wavelength; completing detection within 30min, and drawing a cAMP analysis curve graph to calculate an EC50 value; the test data were processed using a four-parameter regression calculation method, and the EC50 value of the sample to be tested was calculated, with the results shown in table 2:

TABLE 2

Sample (I)	HS-G1	HS-G2	HS-G3	HS-G4	Rope Ma Lutai
						EC50(nM)	4.814	9.269	3.842	2.463	1.892

As can be seen from the results in the table, HS-G3 and HS-G4 of the present invention have lower EC50 values compared to HS-G1 and HS-G2, which are closer to the EC50 value of cable Ma Lutai, indicating that they have binding affinity to the receptor comparable to cable Ma Lutai.

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

(1) Experimental materials:

the experimental animals are BKS-LeprREM/Gpt mice, the number of the experimental animals is 30, the week age is 6 weeks old, and the experimental animals are 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 30 SPF male 6-week BKS-LeprRem/Gpt mice, randomly grouping the mice according to weight and blood sugar, dividing the mice into 6 groups, setting 5 test animals in each group, and respectively setting the test animals as a model control group (solvent), a positive control group (So Ma Lutai), an experiment group 1 (HS-G1), an experiment group 2 (HS-G2), an experiment group 3 (HS-G3) and an experiment group 4 (HS-G4); 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: measuring the blood glucose value before administration and 2h after administration at the first administration, and measuring the blood glucose value 0h before administration and 2h after administration at the next three administrations;

the 0h before re-administration is 48h described in table 4 and DAY3, DAY5, DAY7 and DAY9 in fig. 1, which means 48h after the previous administration and also 0h before re-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 cable Ma Lutai, HS-G1, HS-G2, HS-G3 and HS-G4 from left to right;

table 4: mean blood glucose condition

Statistical analysis of all data for 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; wherein, the HS-G3 has the most obvious effect and the best blood sugar reducing effect, and is also superior to HS-G1 with similar sequence; while the blood sugar reducing effect of HS-G4 is better than that of the rope Ma Lutai and is also better than that of HS-G2. Therefore, the GLP-1 derivative has very excellent hypoglycemic effect.

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

(1) Experimental materials:

the product is BKS-LeprREM/Gpt mice, the number of which is 30, the age of 6 weeks and 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) Experimental methods

a. Modeling and grouping:

30 SPF male 6-week BKS-LeprRem/Gpt mice are selected, randomly grouped according to weight and blood sugar and divided into 6 groups, and each group is provided with 5 test animals which are respectively a model control group (solvent), a positive control group (cable Ma Lutai), an experiment group 1 (HS-G1), an experiment group 2 (HS-G2), an experiment group 3 (HS-G3) and an experiment group 4 (HS-G4). 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 same manner as shown in Table 3 in example 3, once every 48 hours, 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 prior to 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 are 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 administration for 9 days;

TABLE 5

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.

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 fig. 2 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. Especially HS-G3 and HS-G4, the weight change rate reaches more than 2 times of that of the rope Ma Lutai, the weight reduction effect is particularly obvious, and even if the HS-G1 and the HS-G2 of the invention are compared, the weight reduction effect is similar to 1.5-2 times.

Example 5: study of weight loss Effect in Normal mice

(1) Experimental Material

a. Experimental formulation:

GLP-1 derivative injection (HS-G3 and HS-G4) is prepared, and the specific formula is 1.133mg/mLNa ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivative 0.045mg/mL.

b. Animal experiments:

selecting 50 healthy SPF male KM mice with the body weight of 18-20g and the ages of 6-8 weeks, and dividing the mice into a Normal Diet (ND) group and an HFD (high fat diet) group, wherein the normal diet group comprises 10 mice and is fed with a common maintenance diet, the HFD group comprises 40 mice and is fed with 60% high fat diet, and the feeding period is 5-8 weeks;

detecting the weight of an HFD group, screening unmolded mice, randomly grouping the remaining mice according to the weight, and dividing the mice into a blank control group (used for judging whether a DIO model succeeds or not and does not participate in a drug test), a positive control group (Somalutide), an experimental group 1, an experimental group 2 and a model control group (blank solvent);

(2) Experimental method

a. The administration mode comprises the following steps:

the medicine is taken once every two days, the administration route is intraperitoneal injection and four times of administration, and the contents of specific administration dosage and the like are shown in a table 7:

TABLE 7

b. Detection indexes are as follows:

weight: detecting body weight at each administration;

(3) Results of the experiment

The test results of this example are shown in Table 8, and the rate of change of body weight after 9 days of administration is shown in FIG. 3;

TABLE 8

Statistical analysis of all data in this experiment was performed using SPSS software, and all values are expressed as mean ± standard deviation (mean ± SD); for normal distribution data, differences between groups were compared using one-way anova; for non-normal distribution data, differences between groups were analyzed using the Kruskal-WallisH test or Mann-WhitneyU test; the correlation analysis adopts a Spearman test; for all analyses, P <0.05 was considered statistically significant.

FIG. 3 is a graph of the change rate of body weight of a normal mouse after administration for 9 days, and it can be seen from the results of FIG. 3 and Table 8 that, for the normal mouse, the long-acting GLP-1 derivatives HS-G3 and HS-G4 provided by the invention have better weight-reducing capability than that of Ma Lutai, and achieve the weight-reducing effect of approximately 2 times and more than 2 times of the former; meanwhile, the long-acting GLP-1 derivative cannot cause excessive lower blood sugar, and has no risk of hypoglycemia. Thus, the long-acting GLP-1 derivatives of the invention are more suitable for the development of weight-reducing indications.

In addition, the results of a group of experiments on HS-G1 and HS-G2 in a normal mouse model show that the two have weight reduction effect equivalent to that of the rope Ma Lutai, and have weight change rates of 8.94% and 6.26% corresponding to the weight change rate of the rope Ma Lutai 8.19.19%.

Example 6: pharmacokinetic Studies

(1) Experimental Material

Experimental mice: SD rats, 50, weighing between 180-200g, male mice of 6-8 weeks of age.

Experimental formulation: the injection of rope Ma Lutai, HS-G3 and HS-G4 has the formula of 1.133mg/mLNa ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivative were 0.015mg/mL, 0.045mg/mL, and 0.075mg/mL, respectively.

(2) Experimental methods

a. Grouping condition: experimental rats were grouped according to table 9:

b. the administration mode comprises the following steps: the administration route is intraperitoneal subcutaneous injection, and the contents of specific administration dosage and the like are shown in a table 9:

TABLE 9

(3) Results of the experiment

Half-lives of different doses were measured for different experiments, and the results are shown in table 10:

watch 10

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 non-normal distribution data, differences between groups were analyzed using the Kruskal-Wallis H test or the Mann-Whitney U test. The correlation analysis was performed using Spearman's test. For all analyses, P <0.05 was considered statistically significant.

From the results of the above experiments, the long-acting GLP-1 derivatives HS-G3 and HS-G4 provided by the invention have more excellent half-lives in rats than cable Ma Lutai, and both have long half-lives of more than about 1.5 times of cable Ma Lutai under different dosages.

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

<110> Beijing-Hui-Heng Biotechnology Ltd

Jilin Huisheng biopharmaceutical Co.,Ltd.

<120> a long-acting GLP-1 derivative

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Claims

1. A gene segment for encoding a fusion polypeptide containing GLP-1 (7-37) analogue, the sequence of which is shown in any one of SEQ ID NO. 7-8.

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-28a (+) expression vector or 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 according to the following method:

(1) Synthesizing the gene fragment of claim 1;

(2) Connecting the coding group to an expression vector pET-30a (+) or pET-28a (+) to construct a recombinant expression vector;

(3) And (3) transforming 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: inserting the gene fragment in the step (1) into an expression vector pET-30a (+) between NdeI and XhoI enzyme cutting sites or pET-28a (+) between NcoI and XhoI enzyme cutting sites to construct a 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 method for constructing the recombinant engineering bacteria of any one of claims 3 to 7, wherein the 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 method is: inserting the gene fragment in the step (1) between NdeI and XhoI enzyme cutting sites of an expression vector pET-30a (+) or between NcoI and XhoI enzyme cutting sites of pET-28a (+) so as to construct a recombinant expression vector.

10. The building method according to claim 8 or 9, wherein the 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.