CN117986380A - Insulin conjugate and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to an insulin conjugate and application thereof. The amino acid structure of the insulin conjugate of the invention is: GLP-1 (7-37) analog- (GQAP) ₃ -insulin analog. The conjugate provided by the invention can be used for treating insulin-dependent diabetes mellitus, has excellent treatment effect, can play roles in reducing appetite and weight, and solves the side effect that the traditional use of insulin and derivatives thereof can cause weight gain of patients.

Description

Insulin conjugate and application thereof

The present application claims full priority from patent application number 202211379884.7 filed on 11/4/2022. The entire contents of this application are incorporated herein by reference in their entirety.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an insulin conjugate and application thereof.

Background

Diabetes is a group of metabolic disorders of carbohydrates, proteins, fats, etc. caused by absolute or relative hyposecretion of insulin and/or dysfunction of insulin utilization, and is mainly marked by hyperglycemia, and can be caused by various factors such as heredity and environment. Diabetes is one of the three major fatal diseases in humans, with mortality rates inferior to cardiovascular and cerebrovascular diseases and cancers.

Insulin is the only hormone in the body that reduces blood glucose, and at the same time promotes glycogen, fat, protein synthesis, exogenous insulin and insulin derivatives are mainly used to treat diabetes. Insulin consists of A, B peptide chains, 11 Human Insulin (Insulin Human) A chains have 21 amino acids, 15 Human Insulin (Insulin Human) B chains have 30 amino acids, and total 51 amino acids; wherein the sulfhydryl groups in the four cysteines A7 (Cys) -B7 (Cys) and A20 (Cys) -B19 (Cys) form two disulfide bonds, so that A, B two chains are connected, and a disulfide bond exists between A6 (Cys) and A11 (Cys) in the A chain. Insulin is secreted by islet beta cells within the pancreas by stimulation with endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, and the like. The biological action of insulin at the cellular level is initiated by binding to specific receptors on the target cell membrane; insulin receptors are specific sites on the membrane of the target cell where insulin acts, and can only bind to insulin or proinsulin containing insulin molecules, with a high degree of specificity.

Glucagon-like peptide 1 (glp-1, glucogen-likepeptide-1) is a glucagon secreted by intestinal L cells and has the effects of promoting insulin secretion, inhibiting glucagon release, stimulating islet beta cell proliferation, inducing islet beta cell regeneration, preventing islet beta cell apoptosis, improving insulin sensitivity, increasing glucose utilization, and the like. GLP-1 and its analogs and derivatives therefore play an important role in the treatment of the occurrence and progression of type I and II diabetes. Moreover, GLP-1 and analogues thereof have the advantages of effectively reducing blood sugar, reducing weight, regulating blood pressure and blood fat, benefiting cardiovascular and having no risk of hypoglycemia.

Currently, there are few studies and reports on insulin and GLP-1 related polypeptide conjugates, and the present invention is specifically proposed in view of this.

Disclosure of Invention

In order to solve the technical problems, the invention provides a conjugate of insulin and GLP-1 (7-37) -related polypeptide drugs, which is formed by connecting insulin analogues and GLP-1 (7-37) analogues through specific connecting peptides. The conjugate of the invention carries out conjugation modification on the two, introduces the target activity of the latter on the basis of insulin with complete hypoglycemic effect, can play the roles of reducing appetite and reducing weight, and solves the side effect that the traditional use of insulin and derivatives thereof can cause weight increase of patients.

The insulin derivative in the conjugate of the present invention may be natural insulin or an insulin analogue, more preferably an insulin analogue having a long-acting effect.

As used herein, the term "insulin analogue" refers to a peptide having at least 80% amino acid sequence homology with natural insulin, some groups on the amino acid residues of which may be chemically substituted, deleted or modified, and which derivative has the function of regulating blood glucose levels in vivo.

The term "GLP-1 (7-37) analog" refers to an analog obtained by adding, deleting or mutating one to more amino acids based on human natural GLP-1 (7-37).

The term "insulin conjugate" refers to a polypeptide formed by the covalent linkage of insulin to other polypeptides, including Linker.

The term "insulin" refers to a hormone that is a 51 amino acid residue polypeptide (5808 daltons), which plays an important role in many critical cellular processes. The mature form of human insulin consists of 51 amino acids arranged into a chain of 5808Da total molecular weight (GlyAl-AsnA) and a B chain (PheB 1-ThrB 30). The molecule is stabilized by two interchain chains (A20-B19, A7-B7) and one interchain disulfide bond (A6-A11). Insulin of the present invention includes natural, synthetically provided, or genetically engineered (e.g., recombinant) sources, and in various embodiments of the present invention, insulin may be human insulin.

The term "inclusion-promoting sequence" refers to a polypeptide sequence that is linked prior to a protein or polypeptide of interest for promoting expression or formation of inclusion bodies.

The term "peptide" refers to a molecule comprising amino acid sequences linked by peptide bonds, whether in length, post-translational modification or function.

The first aspect of the present invention provides an insulin conjugate or a pharmaceutically acceptable salt thereof, the amino acid structure of the insulin conjugate is as follows:

GLP-1 (7-37) analog- (GQAP) ₃ -insulin analog;

That is, the GLP-1 (7-37) analog, the linker peptide having the amino acid sequence of (GQAP) ₃ and the insulin analog are sequentially linked by a covalent bond, (GQAP) ₃ is linked to the N-terminus of the insulin B chain and the C-terminus of the GLP-1 (7-37) analog, respectively.

The insulin analogue consists of an insulin A chain and an insulin B chain, wherein the amino acid sequence of the insulin A chain is shown as SEQ ID NO. 2, and the amino acid sequence of the insulin B chain is shown as SEQ ID NO. 3; insulin B chain and insulin A chain are linked by intermolecular disulfide bonds.

Further preferably, the epsilon-amino group of lysine of insulin B chain is connected with a fatty acid side chain so as to prolong the action time and achieve the long-acting effect.

As a preferred embodiment of the present invention, the amino acid sequence of the GLP-1 (7-37) analogue is shown as SEQ ID NO. 1.

As a further preferred embodiment of the present invention, the fatty acid side chain may be COOH (CH ₂)_14～20 CO-. Gamma. -Glu-AEEA-AEEA-;

Preferably COOH (CH ₂)_18～20 CO-. Gamma. -Glu-AEEA-AEEA-;

Further preferably, COOH (CH ₂)₁₈ CO-. Gamma. -Glu-AEEA-AEEA) is used.

Wherein AEEA refers to 2- [2- (2-aminoethoxy) ethoxy ] acetyl.

The upper part of the amino acid of the insulin of the present invention is represented by the codes A7, B7, A20 and B19, wherein A and B represent the insulin A chain and insulin B chain, respectively, and wherein the numbers represent the positions of the amino acids corresponding to the natural human insulin A chain and the natural human insulin B chain. If A7 represents the amino acid corresponding to the 7 th position of the A chain of natural human insulin in the insulin sequence; b7 represents the amino acid corresponding to position 7 of the B chain of natural human insulin in the insulin sequence. For natural human insulin, A7, B7, A20 and B19 are all cysteine (Cys), and two disulfide bonds are formed between A7 (Cys) -B7 (Cys) and A20 (Cys) -B19 (Cys) by utilizing the sulfhydryl groups of the cysteine respectively, so that A, B two chains are connected, and in addition, a disulfide bond exists between A6 (Cys) and A11 (Cys) in the A chain; the insulin analogue moieties in the derivatives of the invention are also linked using the disulfide bonds described above.

In a second aspect, the present invention also provides a soluble pharmaceutical composition comprising an insulin conjugate or a pharmaceutically acceptable salt thereof. The pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise preservative and osmotic pressure regulator; the preservative may be phenol and/or m-cresol and the osmotic pressure regulator is glycerol. Preferably, the auxiliary materials comprise phenol, m-cresol and glycerol. More preferably, the auxiliary materials include phenol and glycerol. Preferably, the pH of the soluble pharmaceutical composition of the invention is=7 to 8.5, preferably 7.5 to 8.5, or 7.8 to 8.3.

Further, the invention provides pharmaceutical compositions comprising more than 2 zinc atoms per 6 molecules of insulin conjugate; preferably 2 to 12 zinc atoms per 6 molecules of insulin conjugate, or 4 to 12 zinc atoms per 6 molecules of insulin conjugate, or 5 to 12 zinc atoms per 6 molecules of insulin conjugate, or 8 to 10 zinc atoms per 6 molecules of insulin conjugate.

The invention also provides a preparation method of the insulin conjugate, which can adopt a chemical synthesis method or a fermentation expression method for preparing recombinant genetically engineered bacteria.

In particular, the third aspect of the present invention provides a fusion protein, which can be obtained by fermenting and expressing recombinant genetically engineered bacteria, for further obtaining the insulin conjugate of the first aspect of the present invention. Specifically, the fusion protein is formed by sequentially connecting at least an inclusion body promoting sequence, an enzyme cleavage site fragment and an insulin conjugate precursor; the amino acid sequence of the inclusion promoting sequence is shown as SEQ ID NO. 4, and the amino acid sequence of the enzyme digestion site fragment is shown as SEQ ID NO. 5.

The amino acid structure of the insulin conjugate precursor is shown below:

GLP-1 (7-37) analog- (GQAP) ₃ -insulin analog precursor;

The insulin analogue precursor consists of an insulin A chain, an insulin B chain and a linker structure, wherein the amino acid sequence of the insulin A chain is shown as SEQ ID NO. 2, and the amino acid sequence of the insulin B chain is shown as SEQ ID NO. 3. The insulin B chain is connected with the insulin A chain through a linker, and the amino acid sequence of the linker is shown as SEQ ID NO. 6; wherein, (GQAP) ₃ is linked to the N-terminus of the insulin B chain and the C-terminus of the GLP-1 (7-37) analog, respectively.

That is, the insulin conjugate precursor is formed by sequentially connecting GLP-1 (7-37) analogues, (GQAP) ₃, an insulin B chain, a linker and an insulin A chain.

As a preferred embodiment of the present invention, the amino acid sequence of the GLP-1 (7-37) analogue is shown as SEQ ID NO. 1.

As a specific embodiment of the present invention, the amino acid sequence of the fusion protein is selected from SEQ ID NO.7.

In a fourth aspect, the invention provides a polynucleotide fragment for use in the fusion protein described above, the nucleotide sequence of the polynucleotide fragment being selected from SEQ ID NO.8.

In a fifth aspect the invention provides an expression vector comprising a polynucleotide fragment according to the fifth aspect of the invention. The expression vector can be selected from recombinant pET-28a (+), pET-30a (+) or pET-32a (+) plasmid, and preferably recombinant pET-30a (+) expression vector.

The sixth aspect of the present invention provides a recombinant escherichia coli engineering bacterium, which comprises the expression vector, specifically, escherichia coli is selected from BL21 (DE 3), and the construction method can be adopted:

(1) The synthesized polynucleotide fragment is specifically shown as SEQ ID NO. 8;

(2) Cloning the polynucleotide fragment into a plasmid pET-30a (+) to construct an expression vector;

(3) And (3) transforming the expression vector into escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli engineering bacteria.

In a seventh aspect, the present invention provides a method for preparing an insulin conjugate, comprising at least the steps of:

(1) Culturing the recombinant escherichia coli engineering bacteria to express fusion proteins;

(2) The fusion protein is denatured, renatured and digested to obtain insulin conjugate precursor;

(3) Attaching a fatty acid side chain to the insulin conjugate precursor to obtain an insulin conjugate;

The fatty acid side chain is COOH (CH ₂)_14～20 CO-gamma-Glu-AEEA-AEEA-, linked to the epsilon-amino group of the lysine of the insulin B chain;

Preferably, the fatty acid side chain is COOH (CH ₂)_16～18 CO-gamma-Glu-AEEA-AEEA-;

more preferably COOH (CH ₂)₁₈ CO-. Gamma. -Glu-AEEA-AEEA-.

Specifically, step (1) in the preparation method of the insulin conjugate comprises:

s11, activating strains to obtain an activated seed culture solution;

s12, fermentation culture: inoculating the activated seed culture solution to a fermentation medium;

S13, induction expression: when the OD 600 value is 128-132, IPTG is added to induce expression, and the final concentration of the IPTG is 0.9-1.1 mmol.

According to the research on the expression quantity of the target protein, the induction OD600 value is more preferably 130.4, the IPTG is added to induce expression, and the final concentration of the IPTG is preferably 1.0mmol.

The eighth aspect of the present invention proposes the use of the above-mentioned insulin conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament for the treatment of metabolic diseases. In particular, metabolic disorders include, but are not limited to: diabetes (type I diabetes, type II diabetes), overweight and obesity, steatohepatitis (NASH, ASH), cardiovascular disease, fatty liver, cirrhosis, nonalcoholic fatty liver disease, metabolic syndrome, and various diabetic complications.

Compared with the prior art, the technical scheme provided by the invention has the following advantages:

the conjugate provided by the invention can be used for treating insulin-dependent diabetes mellitus, has excellent treatment effect, can play roles in reducing appetite and weight, and solves the side effect that the traditional use of insulin and derivatives thereof can cause weight gain of patients.

Drawings

FIG. 1 shows the result of SDS-PAGE analysis after induced expression of recombinant engineering bacteria;

FIG. 2 is a graph of the growth production of recombinant engineering bacteria;

FIG. 3 is a graph showing the results of a hypoglycemic experiment in a hyperglycemic animal model;

Fig. 4 shows the results of weight loss experiments in experimental animals.

Detailed Description

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

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 otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.

Example 1:

this example is intended to illustrate the preparation of insulin conjugates.

(1) Expression of conjugate proteins

The inclusion-promoting body sequence (SEQ ID NO: 4), the enzyme digestion sequence (SEQ ID NO: 5) and the insulin conjugate precursor are connected in series to be used as fusion proteins, and the amino acid structure of the insulin conjugate precursor is formed by sequentially connecting GLP-1 (7-37) analogues, (GQAP) ₃, an insulin B chain, a linker and an insulin A chain; the amino acid sequence of the fusion protein is SEQ ID NO. 7.

The coding gene sequence of the fusion protein is constructed, and a chemical synthesis mode is used for obtaining a gene fragment, and the nucleotide sequence is shown as SEQ ID NO.8.

The above fragment was inserted into prokaryotic expression plasmid pET-30a (+) through NdeI and XhoI sites and sequenced to verify that the resulting expression plasmid for transformation assay was designated pET-30a (+) -HSP008-126.

(2) Construction of recombinant engineering bacteria expressing GLP-1 analogues of the application:

2 mu L of plasmid containing target genes is added into 50 mu L of BL21 competent cells (TransGenBiotech), and the mixture is placed on ice for 30min; heat shock at 42 ℃ for 90sec, and rapidly placing in ice for 5min; to the centrifuge tube, 900. Mu.L of sterile LB liquid medium (without antibiotics) was added, and after mixing, the mixture was incubated at 37℃for 1 hour at 200rpm to resuscitate the bacteria. 100. Mu.L of the mixture was pipetted onto LB agar medium plates containing kanamycin resistance. The plates were inverted and incubated overnight at 37 ℃. The next day, monoclonal colonies were picked using an inoculating loop, inoculated in 8mL of sterile LB liquid medium (containing antibiotics), shake-cultured at 37℃at 220rpm until OD600 was about 3-4. Seed solution deposited as the corresponding GLP-1 analog.

(3) Inducible expression of recombinant engineering bacteria expressing GLP-1 analogues of the application:

The single clone on the transformation plate is picked up and inoculated in a test tube containing 10mL LB culture solution of 50 mug/mL kanamycin, and is shaken at 37 ℃ and 220rpm for overnight; the next day, the strain is inoculated into 10mL LB culture solution of 50 mug/mL kanamycin according to the inoculation amount of 1 percent, and the strain is shaken at 220rpm at 37 ℃ until the OD600 of the strain is 0.6-0.8; adding IPTG to the rest culture to a final concentration of 0.5mM, shaking at 37 ℃ and 220rpm, and inducing the expression of the fusion protein; SDS-PAGE detection analysis was performed. The experimental results are shown in FIG. 1.

(4) Fermenting and expressing recombinant engineering bacteria:

50 mu L of the preserved seed liquid bacterial liquid is added into 50mL of 2xYT (low-salt LB) liquid culture medium containing 50 mu g/mL of kanamycin, the mixture is uniformly mixed and placed in a constant temperature oscillator, the mixture is cultured at 37 ℃ and 200rpm overnight, 40mL of the mixture is taken and connected into 200mL of 2xYT culture medium containing 50 mu g/mL of kanamycin, the mixture is uniformly mixed and placed in the constant temperature oscillator, the culture is carried out at 37 ℃ and 200rpm, the OD600 is more than 3.0, and the secondary seed culture liquid is obtained. Taking 60mL of secondary seeds according to the weight ratio of 1:10 (600 mL) is inoculated into a fermentation medium (600 mL) and cultivated in a 2L fermentation tank, when the OD600 value of the detected culture bacterial liquid reaches about 130, IPTG is started to be inoculated, the final concentration is 1mmol/L, the culture is finished after the induction for 24h, the culture is finished, centrifugation is carried out at 8000rpm for 30min, the bacterial cells are collected, the bacterial cell yield is about 250g/L fermentation liquid, and the centrifugally collected bacterial cells are delivered to an analysis department for measuring the target protein expression quantity, wherein the expression quantity is not lower than 10g/L. Specifically, the fermentation data for one batch are shown in table 1:

TABLE 1

Batch number	HSP008-126-20221206-1
		Primary seed OD600 (16 h)	6.44
Second grade seed OD600 (3 h)	3.01
		Inoculation volume (mL)	60.00
Initial volume (L)	0.70
		Initial OD600	NA
PH control	6.80
		Induction temperature	32.00
Induction of OD600	130.40
		IPTG final concentration (mM)	1.00
Bacterial harvesting OD600	247.20
		Bacterial liquid volume (L)	1.50
Total wet weight of thallus (g)	381.16
		Cell mass(g/L)	254.11
Weight of bacterial liquid (g)	1494.08
		Concentration (mg/mL)	1.047
Expression level g/g (in% of the cells)	4.38
		Expression level g/L	11.92

As is clear from Table 1, when the OD600 of induction was about 130, the expression level of the target protein was high. The microbial growth profile is shown in figure 2.

(5) Preparation of insulin conjugate precursors

100G of cells were weighed, resuspended in 500mL of an aqueous solution (pH=8.0) containing 50mM Tris-HCl and 50mM NaCl, sonicated for 30min with a sonicator to disrupt the cells, and the resulting homogenate was centrifuged at 13000g at 4℃for 30min, after centrifugation was completed, the precipitate was collected, dissolved with an aqueous solution (pH=8.0) containing 8M urea, 40mM cysteine and 50mM Tris-HCl, and diluted 10-fold with water to give a sample solution before cleavage.

Tag sequence excision using EK enzyme: dilute hydrochloric acid is added into the intermediate product, the pH is adjusted to 7.4, and EK enzyme (1U/. Mu.L) is added into the intermediate product according to the sample solution before enzyme digestion=1:15 (volume ratio) and is subjected to enzyme digestion overnight after being uniformly mixed.

Purification after cleavage: the digested sample was concentrated using Capto S (commercially available from cytiva) equilibrated with 50mM citric acid (pH=3.0), and after rinsing with 50mM citric acid solution, eluted with a gradient of 0 to 100% eluent (aqueous solution containing 50mM citric acid, 1M sodium chloride, pH=3.0) to give the crude product.

Precision before modification: the solution was concentrated by using UniHybrid to 200 C8 (available from Sony micro technology Co., ltd.) equilibrated with equilibration solution 3 (aqueous solution containing 0.1% TFA, 20% acetonitrile), and after rinsing equilibration solution 3, the solution was eluted with a gradient of 0 to 100% eluent (aqueous solution containing 0.1% TFA, 80% acetonitrile) to give a PR-UPLC purity of about 90% or higher.

(6) Preparation of insulin conjugates

Fatty acid modification: adding water into the insulin conjugate precursor to prepare 0.5-10 mg/mL solution, adding 1M sodium hydroxide to adjust the pH to 11.0-11.5, shaking uniformly to completely dissolve the protein, and quantifying the polypeptide concentration by UV; the molar ratio of polypeptide to octadecanedioic acid mono-tert-butyl-glutamic acid (1-tert-butyl) -AEEA-AEEA-OSU-is 1:4 weighing fatty acid powder, dissolving in acetonitrile, mixing polypeptide sample with fatty acid solution, standing the mixed solution at 4deg.C for one hour, diluting the sample with water for 5 times, adjusting pH to 4.8 with 1M citric acid (or 10% acetic acid) to terminate reaction, standing at 4deg.C for 10min, centrifuging at 13000g at 4deg.C for 30min, and storing the precipitate at-80deg.C.

Deprotection and purification of fatty acids: adding TFA to the obtained precipitate with the final concentration of the polypeptide of about 10mg/mL, oscillating to dissolve the precipitate, standing at room temperature for deprotection for 30min, and then dripping 4M NaOH to adjust the pH to 7.5-8.5 to terminate the reaction.

Concentrating the reaction solution after the reaction is terminated by using a protein purification chromatography system (Saikovia SDL 100) at a flow rate of 3mL/min, pumping UniHybrid-200 C8 (purchased from Souzhou Nami micro-technology Co., ltd.) which is balanced by using a balance solution 3 (containing 0.1% TFA and 20% acetonitrile) in advance, eluting the balance solution 3, then carrying out gradient elution by using 0-100% eluent (containing 0.1% TFA and 80% acetonitrile), and collecting an eluting peak, wherein the purity of the eluting peak is about 90% through RP-UPLC detection; diluting the elution peak with water for 3 times, regulating pH to 4.80,4 ℃ by acid precipitation, carrying out acid precipitation for 30min, adding PB buffer solution (pH 7.0) into the precipitate after centrifugation, re-dissolving, and freezing at-80 ℃ to obtain the insulin conjugate HS-008-126-C20.

Example 2:

this example is a graph illustrating the hypoglycemic effect of the insulin conjugate HS-008-126-C20 prepared in example 1 on a hyperglycemic animal model.

(1) Experimental animals:

6-8 weeks old/male C57BL/6J mice, 18 mice, and the weight is 18-20 g;

(2) The experimental method comprises the following steps:

and (3) molding: mice are fed with high-fat feed for 4-6 weeks after being fed adaptively for two weeks, then the mice are fasted for 16 hours and then are injected with STZ (80 mpk) in an intraperitoneal mode to induce a hyperglycemia model, blood sugar is detected 7 days after induction, and random blood sugar values are above 16.8mmol/L, so that modeling is successful.

Grouping and administration: model mice were grouped according to blood glucose and body weight and dosed according to the contents of table 2, with positive controls dosed substantially equimolar to the experimental group;

TABLE 2

(3) Results statistics

FIG. 3 shows the results of the blood glucose test, and FIG. 3 shows that the HS-008-126-C20 and Icodec week insulin+soma mixed solution of the invention has basically the same blood glucose reducing activity within 0-48 hours, and the blood glucose reducing performance within 48-72 hours is superior to that of the positive control group, and is consistent with the results of the in vitro activity test.

Example 3: weight loss efficacy experiment

This example is a graph showing the weight loss effect of the insulin conjugate HS-008-126-C20 prepared in example 1 on normal experimental animals.

(1) Experimental animals:

The experimental animals are BKS-LeprREM/Gpt mice, the number of the experimental animals is 18, the week age is 6, and the experimental animals are male; (2) experimental method:

and (3) molding: BKS-LeprREM/Gpt mice were selected and randomly grouped according to body weight, each group containing 6 mice, and divided into a blank control group, a positive control group (Icodec) and an experimental group (HS-008-126-C20); the positive control group and the experimental group were administered equimolar.

The administration mode is as follows: abdominal subcutaneous injections were given at a frequency of once every 2 days for 5 consecutive administrations, see in particular Table 4:

TABLE 4 Table 4

(3) Results statistics

The results of the weight-loss experiments are shown in fig. 4, and as can be seen from fig. 4, the weight-loss effect of the insulin conjugate of the invention is remarkable and is far higher than Icodec under the condition of equimolar administration. Therefore, the insulin conjugate of the invention not only has excellent insulin hypoglycemic activity, but also can remarkably inhibit weight gain.

The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the 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 and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (15)

1. An insulin conjugate, or a pharmaceutically acceptable salt thereof, having the amino acid structure shown below:

GLP-1 (7-37) analog- (GQAP) ₃ -insulin analog;

The insulin analogue consists of an insulin A chain and an insulin B chain, wherein the amino acid sequence of the insulin A chain is shown as SEQ ID NO. 2, the amino acid sequence of the insulin B chain is shown as SEQ ID NO. 3, and the insulin B chain and the insulin A chain are connected through intermolecular disulfide bonds;

The (GQAP) ₃ is linked to the N-terminus of the insulin B chain and the C-terminus of the GLP-1 (7-37) analog, respectively;

the epsilon-amino group of the lysine of the insulin B chain is connected with a fatty acid side chain, and the fatty acid side chain is COOH (CH ₂)_14～20 CO-gamma-Glu-AEEA-AEEA-.

2. The insulin conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the amino acid sequence of the GLP-1 (7-37) analogue is as shown in SEQ ID No. 1.

3. A soluble pharmaceutical composition comprising the insulin conjugate of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, wherein the soluble pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant comprising at least one of a preservative and an osmolality regulator;

Preferably, the preservative is selected from phenol and/or m-cresol and the osmolality regulator is selected from glycerol.

4. The soluble pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable salt is a 2-12 zinc atoms per 6 molecule insulin conjugate;

Preferably 4 to 12 zinc atoms per 6 molecules of insulin conjugate, more preferably 8 to 10 zinc atoms per 6 molecules of insulin conjugate.

5. The fusion protein is characterized by being formed by sequentially connecting at least an inclusion body promoting sequence, an enzyme cleavage site fragment and an insulin conjugate precursor; the amino acid sequence of the inclusion body promoting sequence is shown as SEQ ID NO. 4, and the amino acid sequence of the enzyme digestion site fragment is shown as SEQ ID NO. 5; the amino acid structure of the insulin conjugate precursor is shown below:

GLP-1 (7-37) analog- (GQAP) ₃ -insulin analog precursor;

The insulin analogue precursor consists of an insulin A chain, an insulin B chain and a linker; the amino acid sequence of the insulin A chain is shown as SEQ ID NO. 2, and the amino acid sequence of the insulin B chain is shown as SEQ ID NO. 3; the insulin B chain is connected with the insulin A chain through a linker, and the amino acid sequence of the linker is shown as SEQ ID NO. 6;

The (GQAP) ₃ is linked to the N-terminus of the insulin B chain and the C-terminus of the GLP-1 (7-37) analog, respectively.

6. The fusion protein of claim 5, wherein the GLP-1 (7-37) analog has the amino acid sequence shown in SEQ ID NO. 1.

7. The fusion protein of claim 5, wherein the amino acid sequence of the fusion protein is selected from the group consisting of SEQ ID No.7.

8. A polynucleotide fragment for encoding the fusion protein of claim 6 or 7, wherein the nucleotide sequence of the polynucleotide fragment is selected from the group consisting of SEQ ID No.8.

9. An expression vector comprising the polynucleotide fragment of claim 8.

10. The expression vector of claim 9, wherein the expression vector is a recombinant pET-28a (+), a recombinant pET-30a (+) expression vector, a recombinant pET-32a (+) expression vector.

11. A recombinant escherichia coli engineering bacterium, characterized in that the recombinant escherichia coli engineering bacterium comprises the expression vector as set forth in claim 9 or 10, and the escherichia coli is selected from BL21 (DE 3).

12. The recombinant escherichia coli engineering bacterium according to claim 11, wherein the construction method of the recombinant escherichia coli engineering bacterium at least comprises the following steps:

(1) Synthesizing the polynucleotide fragment of claim 8;

(2) Cloning the polynucleotide fragment into a plasmid pET-30a (+) to construct an expression vector;

(3) And (3) transforming the expression vector into escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli engineering bacteria.

13. A method of preparing an insulin conjugate comprising at least the steps of:

(1) Culturing the recombinant escherichia coli engineering bacterium of claim 11 or 12, and expressing the fusion protein;

(2) The fusion protein is denatured, renatured and digested to obtain an insulin conjugate precursor;

(3) Attaching a fatty acid side chain to the insulin conjugate precursor to obtain the insulin conjugate;

The fatty acid side chain is COOH (CH ₂)_14～20 CO-gamma-Glu-AEEA-AEEA-, linked to the epsilon-amino group of the lysine of the insulin B chain.

14. The method of claim 13, wherein step (1) comprises:

s11, activating strains to obtain an activated seed culture solution;

S12, fermentation culture: inoculating the activated seed culture solution to a fermentation medium;

S13, induction expression: when the OD 600 value is 128-132, IPTG is added to induce expression, and the final concentration of the IPTG is 0.9-1.1 mmol.

15. Use of an insulin conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 4, for the manufacture of a medicament for the treatment of metabolic disorders.