CN114958883B

CN114958883B - Recombinant engineering bacterium for expressing GLP-1 analogue and construction method thereof

Info

Publication number: CN114958883B
Application number: CN202210693488.5A
Authority: CN
Inventors: 曹海燕; 林兆生; 朱志伟; 王娟; 王含
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: 2022-04-12
Filing date: 2022-04-12
Publication date: 2023-01-31
Anticipated expiration: 2042-04-12
Also published as: CN114716533B; CN114716533A; CN114958883A

Abstract

The invention relates to an acylated long-acting GLP-1 derivative, wherein the GLP-1 (7-37) analogue is subjected to fatty acid acylation modification to obtain the long-acting GLP-1 derivative, the invention also relates to a recombinant engineering bacterium for expressing the GLP-1 (7-37) analogue and a construction method, the 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

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

The application is a divisional application of Chinese patent application with the application date of 2022, 12.04.12. 202210381817.2 and the name of 'an acylated long-acting GLP-1 derivative'.

Technical Field

The invention relates to the field of polypeptide technology and derivatives thereof, in particular to recombinant engineering bacteria for expressing GLP-1 analogues and a construction method 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 2 diabetes, with the majority of patients being type 2 diabetes patients (statistically, about 90%). Type 2 diabetes (T2 DM), formerly known as non-insulin dependent diabetes mellitus (NIDDM) or adult-onset diabetes (adult-onset diabetes), is characterised by hyperglycemia, relative insulin deficiency, insulin resistance, etc. At present, clinically used medicaments for treating type 2 diabetes 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, almost no risk of hypoglycemia, and a weight-loss effect and cardiovascular protection, and are becoming the main therapeutic drugs and research hotspots for type 2 diabetes.

GLP-1 (glucagon-like peptide 1) is a secretive insulinotropic hormone which is secreted into blood by intestinal tract cells after food stimulation and can stimulate insulin secretion, the insulin secretion capacity caused by the GLP-1 is about 50-70% of the total insulin secretion, and the function of stimulating insulin secretion has the characteristic of glucose concentration dependence, and has the functions of promoting insulin secretion, inhibiting glucagon release, stimulating insulin beta cell proliferation, inducing insulin beta cell regeneration, preventing insulin beta cell apoptosis, improving insulin sensitivity, increasing glucose utilization and the like. GLP-1 derivatives are a class of secretin drugs, and belong to GLP-1 receptor agonists; 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 peptide 1 and receptor agonist research progress [ J ] Tianjin medicine, 2012, 40 (2): 181-184.); thus, GLP-1 and its analogs and derivatives play an important role in the development and progression of type 1 and 2 diabetes.

The results of studies conducted by Nauck M et al on 10 patients with type 2 diabetes with poorly controlled blood glucose, administered to the patients GLP-1 or placebo, respectively, showed that after GLP-1 infusion, the patients had significantly increased insulin and C-peptide levels, significantly decreased glucagon levels, and fasting blood glucose levels became normal after 4 hours; after the blood sugar level is normal, the insulin level of the patient can not rise any more and the blood sugar level can be maintained stable and can not further drop although GLP-1 is continuously infused, which indicates that GLP-1 is released into the blood under the stimulation of nutrient substances (particularly carbohydrate), the insulin secretion promotion effect of GLP-1 is glucose concentration dependent, and the GLP-1 can play the role of reducing the blood sugar when the blood sugar rises, inhibiting the secretion of glucagon, increasing satiety and reducing hunger so as to achieve the effect of reducing the blood sugar (normal-insulin-dependent) diabetes by exogenous glucose-like peptide 1 (7-36 amide) in type 2 (non-insulin-dependent) diabetes polypeptides ([ J ]. Diabolosis, nauck M.,1993,36)). Lancet et al have shown that GLP-1 produces weight loss via a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and ingestion, and delay of gastric content emptying. 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 1 on glucose control, insulin sensitivity, and β -cell function in type 2 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 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 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 drugs has been proved by a plurality of clinical trial studies to effectively control blood sugar, reduce the weight of patients, reduce systolic blood pressure and improve the function of islet beta cells by the aid of the rope Ma Lutai.

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. Thus, there is still a need to provide new GLP-1 derivatives with excellent glucose and weight reduction potential.

Disclosure of Invention

In order to solve the technical problems, the invention provides a GLP-1 (7-37) polypeptide analogue and a long-acting derivative thereof. The long-acting GLP-1 (7-37) derivative provided by the invention has excellent weight loss and blood sugar reduction capabilities, the weight loss effect and the blood sugar reduction 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 [ Val ] ⁸ ]GLP-1 (7-37) refers to GLP-1 (7-37) analogs in which the histidine naturally occurring at position 8 has been substituted with valine Val.

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-epsilon ²⁶ - ((4S) -4- (hexadecanoylamino) -carboxy-butyryl) [ Arg ³⁴ Lys ²⁶ ]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:

YX ₈ EGTFTSDVSSYLEX ₂₂ QAAX ₂₆ EFIX ₃₀ WLVX ₃₄ X ₃₅ X ₃₆ X ₃₇ ；

wherein:

X ₈ selected from Aib, V, I, T, L, G or S;

X ₂₂ selected from G or E;

X ₂₆ is selected from K or R;

X ₃₀ selected from A, K, E or R;

X ₃₄ is selected from R or G;

X ₃₅ is selected from R or G;

X ₃₆ is selected from R or G;

X ₃₇ is selected from R or G;

and X ₂₆ And X ₃₀ Only one of which is K.

Preferably, said X ₈ Selected from V, X ₂₂ Selected from E, X ₂₆ Selected from R, X ₃₀ Selected from K, X ₃₄ Selected from R, X ₃₅ Is selected from G.

More preferably, (1) said X ₃₆ Selected from R, X ₃₇ Is selected from G; or，(2)X ₃₆ Selected from G, X ₃₇ Is selected from R.

When the GLP-1 (7-37) analogue is [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]When GLP-1 (7-37) is used, the amino acid sequence is shown in SEQ ID NO. 1.

When the GLP-1 (7-37) analogue is [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Gly ³⁶ Arg ³⁷ ]When GLP-1 (7-37) is used, the amino acid sequence is shown in SEQ ID NO. 2.

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

As a preferable embodiment of the present invention, the fatty acid side chain structure 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 amino acid residue via a linker, preferably to the epsilon amino group of amino acid K.

As a preferred embodiment of the present invention, said fatty acid side chain is linked to the epsilon amino group of amino acid K 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, preferably n is 1.

The above-mentioned preferred linker moiety (when n is 1) can be represented by γ -Glu-OEG-OEG; wherein OEG is "2- [2- (2-aminoethoxy) ethoxy group]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, preferably said fatty acid side chain is 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][Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]GLP-1 (7-37) (abbreviated as HS-Y1), [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]The amino acid sequence of GLP-1 (7-37) is shown in SEQ ID NO. 1; or

N-ε ³⁰ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4 (S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Gly ³⁶ Arg ³⁷ ]GLP-1 (7-37) (abbreviated as HS-Y2), [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Gly ³⁶ Arg ³⁷ ]The amino acid sequence of GLP-1 (7-37) is shown in SEQ ID NO. 2.

In general, histidine H at position 7 in the amino acid sequence of GLP-1 (7-37) is believed to be critical for maintaining GLP-1 activity, and thus there were no changes to this site in numerous GLP-1 (7-37) -based studies. However, the inventor of the application unexpectedly finds that the site is mutated into tyrosine Tyr in research, so that the activity of GLP-1 is retained, and the activity is enhanced in aspects of weight loss and blood sugar reduction. Perhaps under the influence of changes in the amino acid sequence at other positions, changes at that position may instead synergistically potentiate its GLP-1 activity. Therefore, based on the discovery, the invention obtains a group of drugs which are expected to be more effective in reducing blood sugar and weight.

On the other hand, the invention provides a recombinant engineering bacterium for highly expressing the GLP-1 (7-37) analogue, wherein the recombinant 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 (7-37) analogue, and the method comprises the following steps: (1) Sequentially and serially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 (7-37) analogue coding gene sequence to prepare a gene expression fragment of the GLP-1 (7-37) analogue; (2) Inserting the gene expression fragment into a prokaryotic expression plasmid to obtain an expression plasmid of the GLP-1 (7-37) analogue; (3) And transferring the expression plasmid into escherichia coli to prepare the recombinant engineering bacteria for expressing the GLP-1 (7-37) 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 digestion sequence is DDDDK.

In vitro binding activity indicates:

(1) Compared with the rope Ma Lutai, the acylated long-acting GLP-1 derivative provided by the invention has GLP-1R binding affinity superior to that of the rope Ma Lutai. The blood sugar reduction experiment in a diabetes animal model also shows that the acylated long-acting GLP-1 derivative has the blood sugar reduction effect similar to or obviously superior to that of the rope Ma Lutai. The selection of the mutation site of the invention is proved to bring significant beneficial hypoglycemic activity to GLP-1.

(2) 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.

Furthermore, the present invention provides a pharmaceutical composition comprising said acylated long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In another aspect, the invention provides the use of the acylated 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 acylated 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 equivalent or excellent blood sugar reducing capability in a diabetic patient, and the 7 th amino acid is modified into Y, so that the GLP-1 derivative has excellent enzymolysis resistance, excellent stability and half-life potential;

(2) Compared with the rope Ma Lutai, the long-acting GLP-1 derivative provided by the invention has more excellent and remarkable weight-reducing capacity and more excellent weight-reducing 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 mouse models;

each group of data corresponding to each time point in the figure is a blank control group, a model control group, a cable Ma Lu peptide group, an HS-Y1 group and an HS-Y2 group from left to right in sequence;

figure 2 is a bar graph of the effect of long acting GLP-1 derivatives on the rate of body weight change in mouse models.

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 acylated 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 engineered bacterium capable of efficiently expressing the derivatives of the present invention is constructed by the following methods (taking HS-Y1 as an example):

(1) Construction of the code [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]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 coding gene segment shown as SEQ ID NO. 3; the fragment is inserted into a prokaryotic expression plasmid pET-30a (+) through NdeI and XhoI sites and is sequenced and verified to obtain an expression plasmid which is called pET-30a (+) - [ Tyr + ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]-GLP-1(7-37)。

(2) Construction of expression [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]Recombinant engineering bacterium of-GLP-1 (7-37)

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 transformed competent cells were pipetted and applied to a plate containing a kanamycin-resistant LB agar medium, the cells were spread out uniformly, the plate was placed at 37 ℃ until the liquid was absorbed, the plate was inverted, cultured overnight at 37 ℃, 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, cultured overnight at 37 ℃, 500. Mu.L of overnight culture broth was added to 1.5mL of a sterile centrifuge tube, and 500. Mu.L of 50% sterile glycerol was added and mixed uniformly to obtain glycerol cryopreserved cells, which were stored at-80 ℃.

(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 first-level seed culture solution cultured overnight, inoculating into 200mL of 2YT culture medium according to the proportion of 1:5, simultaneously adding 200 μ L kanamycin, uniformly mixing, placing in a constant-temperature oscillator, culturing at 37 ℃ and 200rpm for 3h, and culturing with OD600 more than 3.0 to obtain 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.

Centrifuging the collected bacteria liquid for 30min at 8000g to obtain thallus cytoplasm, obtaining about not less than 290g of bacteria/L fermentation liquid, and measuring the expression amount of target protein of the centrifugally collected bacteria, wherein the expression amount is not less than 10g/L.

(4) Recombinant [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg 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, 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, inc.) which is balanced by equilibrium liquid 3 (10 mM ammonium acetate and 20% acetonitrile), eluting by the equilibrium liquid 3, then eluting by a gradient of 0-100% eluent (10 mM ammonium acetate and 80% acetonitrile), analyzing by RP-HPLC, and obtaining the GLP-1 intermediate product with the purity higher than 70% by the purification process.

The tag sequence was cleaved using EK enzyme: adding 20mM PB buffer solution with pH7.4 into the intermediate product to dilute the intermediate product three times, adding EK enzyme according to EK enzyme: intermediate product =1 15 at the temperature of 20 ℃, uniformly mixing, carrying out enzyme digestion overnight, and analyzing the enzyme cleavage rate by RP-HPLC to be approximately 80%.

[Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]-fine purification 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) and analyzed for 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 20mM to allow acid precipitation overnight, RP-HPLC detecting yield above 90%, centrifuging at 13000g at 4 deg.C for 30min, collecting precipitate, storing at-20 deg.C to obtain [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]-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: tyr obtained in step (4) ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg 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 molar ratio of polypeptide to octadecanedioic acid mono-tert-butyl ester-glutamic acid (1-tert-butyl ester) -AEEA-AEEA-OSU-1:4, dissolving the fatty acid powder in acetonitrile, mixing a polypeptide sample with the fatty acid solution, standing the mixed solution at 4 ℃ for one hour, adding water into the sample to dilute the mixtureReleasing 5 times, adjusting pH to 4.8 with 1M citric acid (or 10% acetic acid) to terminate reaction, standing at 4 deg.C for acid precipitation for 10min, centrifuging at 4 deg.C for 30min with 13000g after acid precipitation, and storing at-80 deg.C.

Deprotection and purification of fatty acid: adding TFA to the obtained precipitate to a final concentration of about 10mg/mL of polypeptide, shaking to dissolve the precipitate, standing at room temperature for deprotection for 30min, and dropping 4M NaOH to adjust pH to 7.5-8.5 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 by acid precipitation, acid precipitating at 5363 deg.C 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][Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ ]GLP-1 (7-37) (abbreviated as HS-Y1).

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 [ Tyr ⁷ Val ⁸ Glu ²² Arg ²⁶ Lys ³⁰ Arg ³⁴ Gly ³⁶ Arg ³⁷ ]-a gene fragment of GLP-1 (7-37) having the sequence shown in SEQ ID No.4; the remaining steps were carried out as in this example to prepare HS-Y2.

Example 2: in vitro cell affinity activity assay

(1) Different long-acting GLP-1 derivative injection prepared

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-Y1) and the experimental group 2 (HS-Y2) 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 digesting 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.

Detecting the in vitro activity of the derivative of the GLP-1 analogue by using a Fire-Lumi luciferase detection kit: preparing a determination culture solution, diluting a sample to 320nM step by using a DMEM blank medium, wherein the single dilution multiple does not exceed 10 times, and then performing 5-time serial dilution in a 96-well plate to obtain 8 gradients, wherein each dilution is performed by 2 duplicate wells.

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 for 6h under conditions. The sample plate was removed from the incubator and allowed to stand at room temperature. Adding 100 mu L 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 1:

TABLE 1

Sample (I)	HS-Y1	HS-Y2	Rope Ma Lutai
				EC50(nM)	0.336	0.1746	0.6611

As can be seen from the results in the table, the HS-Y1 and HS-Y2 of the present invention have lower EC50 values than that of cable Ma Lutai, and HS-Y1 and HS-Y2 are about 1/2 and 1/4 of cable Ma Lutai, respectively, indicating that they have about 2-fold and 4-fold better binding affinity to human insulin receptor than cable Ma Lutai, respectively.

Example 3: study of hypoglycemic Effect in mice

(1) Experimental materials:

a. experimental pharmaceutical formulation:

respectively preparing injections of different GLP-1 derivatives, wherein the specific formula is 1.133mg/mL Na ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivatives;

b. animal experiments:

selecting 50 healthy SPF male KM mice with the age of 6-8 weeks, weighing 18-20g, and dividing into a blank control group, a model control group, a positive control group (a cable Ma Lu peptide group), an example 1 group and an example 2 group, wherein:

blank control group: comprises 10 mice, and is prepared by intragastric administration of pure water and subcutaneous injection of a blank solvent into abdomen;

model control group: comprises 10 mice, glucose (4 g/kg) is perfused, and the abdomen is injected with a blank subcutaneously;

positive control group: comprises 10 mice, glucose (4 g/kg) is used for gastric lavage, and the abdomen is injected with medicine subcutaneously;

example 1 group: comprises 10 mice, glucose (4 g/kg) is perfused into stomach, and the abdomen is injected with medicine subcutaneously;

example 2 group: the mice contained 10 mice, were gavaged with glucose (4 g/kg), and were injected with the drug subcutaneously in the abdomen.

(2) The experimental method comprises the following steps:

a. the administration method comprises the following steps:

the mice of each experimental group identified above were dosed according to the specific dosing schedule shown in table 2:

TABLE 2

b. Blood sugar value detection:

fasting for 16h before blood glucose assay, blood glucose for 0h after administration and before oral glucose, blood glucose for 0.5h, 1h, 2h after oral glucose, followed by oral glucose for 23h and blood glucose for 1h after oral glucose, results are shown in table 3 and fig. 1:

TABLE 3

Group of	0h	0.5h	1h	2h	24h
						Blank control group	4.51±0.55	7.01±1.24**	6.01±1.14**	4.73±0.69**	10.83±1.13*
Model control group	4.49±0.54	18.76±3.44	15.98±3.15	9.21±1.12	12.26±1.84
						Positive control group	4.32±0.45	10.94±2.88**	6.21±1.39**	4.32±0.79**	8.95±1.94**
EXAMPLE 1 group	4.4±0.74	8.4±4.03**	4.91±1.5**	3.69±0.49**	8.42±1.43**
						EXAMPLE 2 group	4.23±0.54	10.24±4.53**	5.66±1.67**	4.52±0.75**	9.07±2.46**

Note: "" means, p < 0.05 relative to model control; ". Indicates, p < 0.01 relative to model control.

FIG. 1 is a graph showing the trend of blood glucose change of experimental mice of this example, wherein each time point is a blank control group, a model control group, a positive control group (Soy Ma Lutai), an example 1 group (HS-Y1) and an example 2 group (HS-Y2) from left to right. As can be seen from figure 1, the HS-Y1 and HS-Y2 of the invention are superior to the cable Ma Lutai in controlling the blood sugar of mice in the whole experiment, and have better blood sugar control capability.

Example 4: study of weight loss Effect in mice

(1) Experimental Material

a. Experimental formulation:

GLP-1 derivative injection (HS-Y1 and HS-Y2) is prepared, and the specific formula is 1.133mg/mL Na ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivative 0.045mg/mL.

b. Animal experiments:

selecting healthy SPF male KM mice with the age of 6-8 weeks, weighing 18-20g, and dividing into a normal diet group (ND group) and a high fat diet group (HFD group), wherein the ND group is fed with common maintenance diet, the HFD group is fed with 60% high fat diet, and the feeding period is 12-18 weeks;

detecting the weight of an HFD group, screening unmolded mice, randomly grouping the remaining mice according to the weight, and dividing each group into 5 mice, namely a model control group (blank solvent), a positive control group (cable Ma Lutai, 0.45 mg/kg), an experimental group 1 (HS-Y1, 0.45 mg/kg), an experimental group 2 (HS-Y1, 0.75 mg/kg), an experimental group 3 (HS-Y2, 0.45 mg/kg) and an experimental group 4 (HS-Y2, 0.75 mg/kg);

(2) Experimental methods

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 4:

TABLE 4

b. Detection indexes are as follows:

weight: detecting the body weight at each administration;

(3) Results of the experiment

The test results of this example are shown in table 5, which corresponds to the bar chart shown in fig. 2:

TABLE 5

As can be seen from Table 5 and FIG. 2, HS-Y1 and HS-Y2 of the present invention, although the H at position 7 in the amino acid sequence was changed to Y, unexpectedly maintained good GLP-1 activity, had good weight loss ability, and the effect was superior to that of cable Ma Lutai.

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.

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Claims

1. A gene segment for coding a fusion polypeptide containing GLP-1 (7-37) analogues has a sequence shown in SEQ ID NO.3 or SEQ ID NO. 4.

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 engineering bacterium is constructed according to 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 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 building method according to claim 8, 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.