Novel double-target insulin analogue as well as preparation method and application thereof

Abstract

The invention relates to the technical field of polypeptide drugs, in particular to a novel double-target insulin analogue as well as a preparation method and application thereof. A general formula of a sequence of a precursor of the novel double-target insulin analogue is as follows: a GLP-1 sequence-an Fc sequence-an insulin sequence, the sequence of the GLP-1 is as shown in SEQ ID NO.1, the sequence of the Fc is as shown in SEQ ID NO.2, and the sequence of the insulin is as shown in SEQ ID NO.3. The precursor is subjected to three-enzyme digestion to obtain the double-target insulin analogue, and the double-target insulin analogue is used for preparing medicines for improving cardiovascular diseases, controlling body weight and controlling blood sugar. According to the technical scheme, the technical problems that in the prior art, a preparation process of a polypeptide medicine containing a Glp-1 analogue and an insulin analogue is complex, and the stability and the long-term effect of the medicine are not ideal can be solved. More effective blood sugar control is provided through combination of two mechanisms, common side effects are reduced, and ideal popularization and application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a novel dual-target insulin analog, a preparation method and application thereof. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is a hormone mainly produced by intestinal L cells and belongs to the incretin hormone category. GLP-1 promotes the secretion of insulin by pancreatic β cells in a glucose concentration-dependent manner and reduces the secretion of glucagon by pancreatic α cells, thereby lowering blood sugar. Insulin is a protein hormone secreted by pancreatic β cells in the pancreas in response to endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin is the only hormone in the body that lowers blood sugar and also promotes the synthesis of glycogen, fat, and protein. Exogenous insulin is mainly used to treat diabetes.

[0003] Liraglutide was developed by NovoNordisk. It consists of 31 amino acids and has a relative molecular weight of 3751Da. It can reduce glucose concentration by promoting the secretion of insulin by pancreatic β cells and inhibiting the release of glucagon by pancreatic α cells, thereby achieving the purpose of treating type II diabetes. The structure of liraglutide is 97% similar to that of endogenous GLP-1. It is obtained by replacing the 34th lysine with arginine and adding a 16-carbon palmitoyl fatty acid side chain to the 26th lysine on the basis of the natural GLP-1 molecular structure. The binding rate of liraglutide to albumin is over 98%, and it has a high resistance to DPP-IV degradation and a long half-life. A single injection per day can achieve the effect of 24h blood sugar control.

[0004] Insulin degludec was developed by NovoNordisk and launched in Japan in October 2012 for the treatment of type 1 and type 2 diabetes. Insulin degludec is an ultra-long-acting basal insulin analogue obtained by removing the threonine at position B30 from human insulin and connecting a 16-carbon fatty diacid to the lysine at position B29 through an L-γ-glutamic acid linker. This unique molecular structure allows it to exist in the preparation in a stable, soluble, double hexamer form before injection. The half-life of insulin degludec for type 1 or type 2 diabetes is close to 25 hours, and for patients with type 1 diabetes, its duration is >42 hours.

[0005] The applicant's prior patent CN117771347A provides a pharmaceutical composition preparation of Glp-1 analogs and insulin analogs, as well as its preparation method and application. Since both GLP-1 analogs and insulin analogs have shown ideal hypoglycemic effects, this scheme attempts to prepare the two polypeptide drugs into a mixed preparation. However, the above-mentioned mixed preparation still requires the preparation of two polypeptide drugs separately, the process route is relatively complicated, and the stability, long-term effect and duration of action of each polypeptide drug, especially insulin, need to be further improved. It is urgent to further improve the preparations of Glp-1 analogs and insulin analogs to simplify the operating process and improve the quality of drugs. Summary of the invention

[0006] The present invention aims to provide a novel dual-target insulin analog to solve the technical problems in the prior art of complex preparation process of polypeptide drugs containing Glp-1 analogs and insulin analogs and unsatisfactory drug stability and long-term effect.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A novel dual-target insulin analog, the general formula of the precursor sequence is: GLP-1 sequence-Fc sequence-insulin sequence; the GLP-1 sequence is shown in SEQ ID NO.1, the Fc sequence is shown in SEQ ID NO.2, and the insulin sequence is shown in SEQ ID NO.3.

[0009] Furthermore, a G4S linker sequence is provided between the GLP-1 sequence and the Fc sequence.

[0010] Furthermore, a histidine tag was connected to the N-terminus of the GLP-1 sequence.

[0011] Furthermore, the amino acid sequence of the precursor of the novel dual-target insulin analog is shown in SEQ ID NO.4.

[0012] Furthermore, the nucleotide sequence of the precursor of the novel dual-target insulin analog is shown in SEQ ID NO.5.

[0013] Furthermore, after the precursor is treated with enzymes, a dual-target insulin analog is obtained; the method of the enzyme treatment is: using carboxypeptidase B and Kex2 enzymes to treat the precursor with enzymes to obtain a dual-target insulin analog; its amino acid sequence is shown in SEQ ID NO.7.

[0014] The present technical scheme also provides a method for preparing a novel dual-target insulin analog, which is characterized by: integrating a DNA fragment with a sequence such as that shown in SEQ ID NO.6 into an empty vector to obtain an expression vector; transferring the expression vector into bacteria to obtain an engineered bacterium; inducing the engineered bacterium to express a fusion protein with a sequence such as that shown in SEQ ID NO.4; and obtaining a dual-target insulin analog after extraction, purification and enzyme digestion.

[0015] Furthermore, the method of enzymatic treatment is: at pH 7.5-7.6 and room temperature, the fusion protein shown in SEQ ID NO.4 is treated with carboxypeptidase B and Kex2 enzymes for 16-24 hours; the mass ratio of the sum of the mass of carboxypeptidase B and Kex2 enzymes to the mass of the fusion protein is 1:200, and the mass ratio of carboxypeptidase B and Kex2 enzymes is 1:1.

[0016] This technical solution also provides a novel dual-target insulin analog for use in the preparation of drugs for improving cardiovascular diseases and drugs for controlling body weight.

[0017] This technical solution also provides a novel dual-target insulin analog for use in the preparation of drugs for controlling blood sugar.

[0018] In summary, the principle of this technical solution is:

[0019] Dual-target insulin, also known as dual-acting insulin or bi-acting insulin, is a diabetes treatment designed to mimic the effects of two hormones in the body—insulin and glucagon-like peptide-1 (GLP-1). This drug combines the blood sugar-lowering effects of insulin with the additional benefits of a GLP-1 receptor agonist, providing a more comprehensive diabetes treatment. The following are the main mechanisms of action of dual-target insulin:

[0020] (1) Effects of insulin: Insulin is a hormone secreted by pancreatic β cells. Its main function is to promote cell absorption of blood sugar for energy or conversion into fat storage. By increasing exogenous insulin, dual-target insulin helps lower blood sugar levels, especially during post-meal blood sugar peaks.

[0021] (2) Effects of GLP-1 receptor agonists: GLP-1 is a hormone secreted in the intestine that plays a role in blood sugar control, including enhancing insulin secretion (when blood sugar levels are elevated), reducing glucagon secretion (a hormone that increases blood sugar), delaying gastric emptying, and suppressing appetite. These effects help lower postprandial blood sugar levels and may have a positive impact on weight management.

[0022] By combining these two mechanisms, dual-target insulin aims to provide more effective blood sugar control while reducing the side effects common with traditional insulin therapy, such as weight gain and risk of hypoglycemia. In addition, the addition of a GLP-1 receptor agonist may help improve pancreatic beta cell function and have a positive impact on cardiovascular health.

[0023] The beneficial effects of this technical solution are:

[0024] Dual-target insulin (also called dual-acting insulin or insulin-GLP-1 receptor agonist combination) combines the effects of insulin and GLP-1 receptor agonist, aiming to provide a more comprehensive diabetes treatment effect than traditional insulin. The main advantages of this combination include:

[0025] (1) Improved blood sugar control: Dual-target insulin can more effectively control blood sugar levels by mimicking the effects of insulin and GLP-1 at the same time. The GLP-1 component helps to enhance insulin secretion, reduce glucagon secretion, slow gastric emptying, and may improve the health of pancreatic beta cells. This dual effect can provide more stable blood sugar control and reduce blood sugar fluctuations.

[0026] (2) Reduce the risk of hypoglycemia: Traditional insulin therapy sometimes leads to low blood sugar (hypoglycemia) events, especially when efforts are made to control blood sugar levels very close to the normal range. Dual-target insulin, due to its GLP-1 component, may help reduce the risk of hypoglycemia because the insulin release effect of GLP-1 is glucose-dependent, that is, insulin secretion is enhanced only when blood sugar levels rise.

[0027] (3) Weight management: Many patients who use traditional insulin experience weight gain due to the effects of insulin on promoting the conversion of glucose into fat and reducing fat breakdown. The GLP-1 component of dual-target insulin can help control weight and may even lead to weight loss because GLP-1 can increase satiety, reduce food intake, and may increase energy expenditure.

[0028] (4) Possible cardiovascular benefits: GLP-1 receptor agonists have shown possible beneficial effects on the cardiovascular system, which is particularly important for patients with diabetes because they are generally at higher risk of cardiovascular disease. Although these effects require further study, the use of dual-target insulin may help improve cardiovascular health.

[0029] (5) Convenience: By combining the two drugs into one injection, patients may find it more convenient than taking insulin and GLP-1 receptor agonists separately, which may improve patient compliance and thus improve treatment outcomes.

[0030] Overall, dual-target insulin offers an innovative treatment option that may provide more comprehensive diabetes management than conventional insulin therapy, particularly in terms of improving glycemic control, reducing the risk of hypoglycemia, facilitating weight management, and potentially providing additional cardiovascular benefits. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods and can be completed according to the described recombinant technology (see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); the materials, reagents, etc. used can be obtained from commercial channels.

[0032] Embodiment 1:

[0033] In this study, a fusion protein of insulin and glucagon-like peptide-1 (GLP-1) was designed and recombinantly expressed.

[0034] GLP-1 sequence: HGEGTFTSDVSSYLEEQAAKEFIAWLVK (SEQ ID NO. 1).

[0035] G4S joining sequence: GGGGGGGSGGGGSGGGGS.

[0036] EK restriction site (enterokinase): DDDDK.

[0037] Fc fusion protein sequence:

[0038] AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO. 2).

[0039] Kex2 restriction site (dibasic endopeptidase): KR.

[0040] Insulin sequence:

[0041] FVNQHLCGSHLVEALHLVCGERGFHYTPKT- KR EAEDLQVGQVELGGGPGAGSLQPLALEGSLG KR - GIVEQCCT SICSLEQLENYCN (SEQ ID NO.3, the wavy line represents the insulin B chain, the single underline represents the insulin A chain, the double underline represents the KR sequence recognized by the Kex2 enzyme, and the remaining sequence is the connecting peptide between insulins).

[0042] In summary, the protein sequence of the fusion protein used in this scheme is:

[0043] MHHHHHHLE KR -HGEGGTFTSDVSSYLEEQAAKEFIAWLVK-GGGGGGGSGGGGSGGGGS-DDDDKAESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG-FVNQHLCGSHLVEALHL VCGERGFHYTPKT- KR EAEDLQVGQVELGGGPGAGSLQPLALEGSLG KR -GIVEQCCTSICSLEQLENYCN (SEQ ID NO. 4, double underlines indicate Kex2 cleavage site, wavy line indicates EK cleavage site).

[0044] In SEQ ID NO.3, from N-terminus to C-terminus, it is as follows: 6×His tag, Kex2 enzyme recognition sequence KR, GLP-1 sequence, G4S linker sequence, EK enzyme recognition sequence DDDDK, Fc fusion protein sequence, insulin B chain, Kex2 enzyme recognition sequence KR, linker sequence, Kex2 enzyme recognition sequence KR, insulin A chain. According to experimental studies, the fusion protein of insulin and glucagon-like peptide-1 formed in the above manner has the most ideal effect in terms of protein expression efficiency and the final product obtained.

[0045] The DNA sequence corresponding to the fusion protein of SEQ ID NO.4 is shown in SEQ ID NO.5, which is a codon-optimized sequence expressing the fusion protein of SEQ ID NO.4:

[0046] ATGCATCACCACCATCACCACCTAGAAAAACGCCATGGTGAGGGCACTTTTACCTCCGACGTGTCTAGCTATTTGGAAGAGCAGGCTGCGAAGGAGTTCATCGCCTGGCTGGTCAAGGGCGGCGGTGGCGGAGGCGGCTCCGGTGGTGGCGGCTCTGGTGGCGGCGGCTCTGACGACGACGATAAGGCGGAAAGCAAATATGGTCCGCCGTGCCCACCATGTCCGGCACCGGAAGCGGCTGGTGGTCCGTCAGTATTCCTGTTCCCGCCAAAGCCGAAGGATACCCTGATGATTTCGAGAACACCCGAAGTGACCTGCGTTGTCGTTGATGTTTCGCAAGAGGACCCGGAGGTTCAGTTCAACTGGTATGTGGATGGTGTGGAGGTGCACA(SEQ ID NO.5).

[0047] Nde I (CATATG) was added to the 5' end of the gene, and a stop codon (TGATAA) and HindⅢ (AAGCTT) restriction site were added to the 3' end. Suzhou Junji Company was commissioned to synthesize the gene fragment. After adding the restriction site, the sequence is as follows:

[0048] CATATG TGATAAAAGCTT (SEQ ID NO.6).

[0049] The synthesized fusion protein gene fragment was inserted into the commercially available pET-26b expression vector through the restriction sites of Nde I and Hind III by conventional means in the prior art. The successfully constructed expression plasmid was transformed into the competent Escherichia coli cell BL21 (DE3) by conventional means to obtain an engineered bacterium expressing the fusion protein of SEQ ID NO.4.

[0050] Next, the engineered bacteria were induced to express the fusion protein of SEQ ID NO.4 by conventional means in the prior art, and then the protein was purified, the process including: fermentation, bacterial separation, bacterial disruption, washing (secondary homogenization), water washing, inclusion bodies, inclusion body dissolution, 30Q purification, renaturation, 30Q purification, enzyme digestion, enzyme digestion recovery (protein A) and concentration. The details are as follows:

[0051] (1) Fermentation

[0052] Transfer the strain into 200 mL of fresh LB medium at a volume ratio of 1:200, and culture at 37±1℃, 220-250rpm for 6-10 hours until the bacterial concentration OD600 is ≥2. After microscopic examination shows no contamination from other bacteria, it can be used as seed liquid for fermentation.

[0053] The seed liquid was inoculated into a sterilized fermentation medium (3 g / L yeast powder, 5 g / L peptone, 15.7 g / L glycerol, 5 g / L anhydrous potassium dihydrogen phosphate, 10.3 g / L anhydrous disodium hydrogen phosphate, 0.6 g / L sodium chloride, 1 g / L ammonium chloride, 1 g / L magnesium sulfate heptahydrate) at a volume ratio of 1:10, and the temperature and pH were maintained at 37±1°C and 7.0±0.1, and the dissolved oxygen was controlled to be ≥20% during the culture process until the bacterial concentration OD600 was 20-25;

[0054] After culturing to a bacterial concentration of OD600 = 20-25, add IPTG at a final concentration of 0.5 mmol / L, stop feeding for 0.5 = 1 h and start feeding induction. After 10-12 h of induction, stop induction, measure the volume of the bacterial liquid in the tank, and harvest the bacteria by centrifugation.

[0055] (2) Inclusion body and cell processing

[0056] The bacteria were suspended in water at a mass-to-volume ratio of 1:10 (emulsified in an emulsifier for 3-5 minutes), the suspended bacterial solution was broken, the pressure was 60 MPa, and the homogenization was performed three times, and then centrifuged in a centrifuge bottle to collect the precipitate.

[0057] The centrifuged precipitate after bacterial disruption was washed with 5 nM EDTA at a mass-to-volume ratio of 1:10 for 10 min, and the precipitate was collected by centrifugation (if the precipitate state is poor, a second homogenization is required); the precipitate was then suspended in water at a mass-to-volume ratio of 1:10, and the precipitate was collected by centrifugation to obtain inclusion bodies.

[0058] (3) Dissolution and 30Q purification

[0059] The washed inclusion bodies were dissolved at a mass volume ratio of 1:10, and the composition of the dissolving solution was: 8M urea, 50mM L-cys, 50mM Tris, pH8.0. The dissolved solution was centrifuged to remove the undissolved precipitate, and the dissolving solution was gradient eluted with a 30Q column. The sample was recovered as a single main peak (liquid A: 8M urea, 50mM L-cys, 50mM Tris, pH8.0; solution B: solution A + 0.5M sodium chloride pH8.0), and finally renatured with 20× 50Mm Tris pH8.5 pre-cooled water.

[0060] (4) 30Q purification after renaturation

[0061] After renaturation, the sample contains polymers, which need to be removed to improve the purity of the sample. Therefore, it needs to be purified again with 30Q (Solution A: 20mM PB, pH7.5; Solution B: Solution A + 0.5M sodium chloride pH7.5). After the above steps, a high-purity fusion protein with the sequence of SEQ ID NO.4 is obtained.

[0062] (5) Double enzyme digestion

[0063] The renatured, recovered and re-purified samples were subjected to enzyme digestion. The enzymes used included the sum of the mass of carboxypeptidase B (CPB) and Kex2 enzymes and the mass ratio of the fusion protein of SEQ ID NO.4 at 1:200 (wherein the mass ratio of CPB and Kex2 was 1:1), pH 7.5-7.6; temperature: room temperature 25°C, enzyme digestion time 16-24h. The target protein was separated and obtained using conventional chromatography methods in the prior art. Specifically, a chromatography column protein A (7ml, 20mM PB, pH 7.5; eluent: 50mM glycine pH 3.0) was used, the loading pH was 7.5, and a gradient elution B solution (0-100%) was used. After the above treatment, a sample recovered by double enzyme digestion was obtained.

[0064] In the above process, Kex2 enzyme recognizes KR sequence, removes N-terminal 6×His tag and connecting peptide between insulin. CPB enzyme removes the residual KR sequence at the end of insulin A chain. During the renaturation process, insulin A chain and B chain automatically form the correct conformation connected by disulfide bond. Enzyme cleavage only removes the intermediate connecting peptide and does not affect the structure of disulfide bond connection. After double enzyme cleavage, GLP-1-Fc-insulin is obtained, and the fusion protein GLP-1-Fc-insulin is purified and recovered by conventional means of existing technology. The sequence is shown in SEQ ID NO.7:

[0065] HGEGTFTSDVSSYLEEQAAKEFIAWLVK-GGGGGGGSGGGGSGGGGS-DDDDKAESKYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG-FVNQHLCGSHLVEALHLVCGERGFHYTPK T-GIVEQCCTSICSLEQLENYCN (protein molecular weight: 36137.34).

[0066] (6) Triple enzyme digestion

[0067] The sample recovered by double digestion was further added with EK enzyme for digestion, the mass ratio of enzyme to protein to be digested was 1:200 (EK: fusion protein), pH7.5-7.6; temperature: room temperature 25°C, digestion time 16-24h. The target protein was separated and obtained using conventional chromatography methods in the prior art, specifically, using a chromatography column proteinA (7ml, 20mM PB, pH7.5; eluent: 50mM glycine pH3.0), loading pH7.5, and using gradient elution B solution (0-100%). On the basis of Kex2 and CPB enzyme excision, the N-terminal GLP-1 sequence was digested with EK enzyme to obtain Fc-insulin, the sequence of which is shown in SEQ ID NO.8:

[0068] AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG-FVN QHLCGSHLVEALHLVCGERGFHYTPKT-GIVEQCCTSICSLEQLENYCN (protein molecular weight: 31306.35).

[0069] The efficacy of the obtained GLP-1-Fc-insulin and Fc-insulin was experimentally studied. SPF Kunming mice (weight 30±1g) were used to evaluate the effect of the peptide. After each group of experimental animals was given the corresponding dose, blood was taken from the tail at the corresponding time point to measure the blood sugar level. Refer to the 1211 insulin bioassay method of the four general rules of the 2020 edition of the Chinese Pharmacopoeia. Six mice in each experimental group were subcutaneously administered, and the peptide was appropriately diluted. The administration of each mouse was as follows: GLP-1-Fc-insulin 0.7mg, Fc-insulin 0.45mg (at the above dosage, according to the ratio of the molecular weight of the insulin sequence to the total molecular weight of the sequence, the amount of insulin in GLP-1-Fc-insulin and Fc-insulin is roughly the same). The control group was given normal saline. Blood was taken at the set time point for blood sugar level detection, and the glucose tolerance experiment was to inject 50% glucose 0.1ml / mouse at the set time point, and the blood sugar level was measured 10min later. Among them, the glucose tolerance test is mainly used to evaluate the efficacy of drugs containing GLP-1 analogs. The Fc-insulin group does not contain GLP-1 and no glucose tolerance test was performed. When the blood sugar level in the body is higher than the normal level, the GLP-1 analog plays a role without lowering the normal level of blood sugar. The experimental results are shown in Table 1-3.

[0070] Table 1: Blood glucose test results of the control group (unit: mmol / L)

[0071]

[0072] Table 2: GLP-1-Fc-insulin blood glucose test results (unit: mmol / L)

[0073]

[0074] Table 3: Fc-insulin blood glucose test results (unit: mmol / L)

[0075]

[0076] From the data in the above table, we can see that the duration of action of Fc-insulin is greater than 58h, and the duration of action of GLP-1-Fc-insulin is greater than 58h, indicating that both insulins have a certain long-term effect. Among them, the duration of action refers to: when the blood sugar value of the experimental group is higher than that of the control group, the drug efficacy cannot maintain the compensatory effect of blood sugar in the body, which is the critical value of drug efficacy maintenance; the time from the start of the experiment to the time when the above critical value is reached is the maintenance time. GLP-1-Fc-insulin adds GLP-1 to form a dual-target insulin. Dual-target insulin can more effectively control blood sugar levels by simulating the effects of insulin and GLP-1 at the same time.

[0077] Comparative Example 1

[0078] The corresponding DNA sequence was synthesized and expressed in the manner of Example 1 to obtain CIN-Fc (SEQ ID NO.9):

[0079] MHHHHHHLEKR-FVNQHLCGSHLVEALHLVCGERGFHYTPKT-KREAEDLQVGQVELGGGPGAGSLQPLALEGSL GKR-GIVEQCCTSICSLEQLENYCN-AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLG (protein molecular weight: 36286.96).

[0080] Then, the above polypeptide was double-digested to obtain insulin-Fc (SEQ ID NO.10):

[0081] FVNQHLCGSHLVEALHLVCGERGFHYTPKT-GIVEQCCTSICSLEQLENYCN-AESKYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0082] Different from the Fc-insulin obtained by triple enzyme digestion in Example 1, the Fc segment of this comparative example is connected to the insulin A chain. The Fc segment of the Fc-insulin obtained by triple enzyme digestion in Example 1 is connected to the insulin B chain. Experimental studies were conducted on Fc-insulin and insulin-Fc, referring to the experimental method of Example 1. The experimental results are shown in Tables 4 and 5. The dosage of Fc-insulin and insulin-Fc was 0.4 mg.

[0083] Table 4: Fc-insulin blood glucose test results (unit: mmol / L, obtained by the three-enzyme digestion method of Example 1)

[0084] Animal Labeling 6h 24h 31h 48h 1 6.1 5.3 3.8 10.5 2 4.4 5.3 6.7 7.5 3 3.3 3.2 3.8 3.4 4 7.4 4.4 4.8 13.0 5 5.3 3.6 5.4 10.1 6 8.0 3.8 3.9 6.5 Mean 5.75 4.27 4.73 8.50

[0085] Table 5: Insulin-Fc blood glucose test results (unit: mmol / L, the polypeptide connected to the Fc segment and insulin A chain provided in this comparative example)

[0086] Animal Labeling 6h 24h 31h 1 2.4 3.1 6.9 2 5.6 7.0 6.8 3 6.7 7.6 7.9 4 7.3 8.7 6.6 5 6.4 6.9 7.2 6 4.8 5.6 5.7 Mean 5.53 6.48 6.85

[0087] From the above experimental data, it can be seen that when the Fc segment is connected to the insulin B chain, the glucose-lowering effect of the polypeptide is more ideal. Therefore, in the process of preparing GLP-1-Fc-insulin, the scheme of setting the Fc segment between the insulin B chain and GLP-1 is adopted.

[0088] Comparative Example 2: Efficacy Experiment of Icodec

[0089] The hypoglycemic effect of commercially available insulin ecol was tested with reference to the method of Example 1 (dosage 0.1 mg). The experimental results are shown in Table 6.

[0090] Table 6: Results of insulin blood sugar test (unit: mmol / L)

[0091]

[0092] Comparing Ico insulin and the Fc-insulin of this scheme, the hypoglycemic effect of the drug provided by this scheme is more ideal and longer-lasting. In addition, GLP-1-Fc-insulin has added a dual-target function, and the hypoglycemic effect is better. It should be noted that under the condition of the administration dose of 0.1 mg of Ico insulin in this scheme, the total amount of insulin is roughly the same as the amount of insulin in the 0.7 mg GLP-1-Fc-insulin and 0.45 mg Fc-insulin mentioned above.

[0093] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A novel dual-target insulin analog, characterized in that: The general formula of the sequence of its precursor is: GLP-1 sequence-Fc sequence-insulin sequence; the GLP-1 sequence is shown in SEQ ID NO.1, the Fc sequence is shown in SEQ ID NO.2, and the insulin sequence is shown in SEQ ID NO.

3.

2. A novel dual-target insulin analog according to claim 1, characterized in that: A G4S linker sequence is provided between the GLP-1 sequence and the Fc sequence.

3. A novel dual-target insulin analog according to claim 2, characterized in that: A histidine tag was attached to the N-terminus of the GLP-1 sequence.

4. A novel dual-target insulin analog according to claim 3, characterized in that: The amino acid sequence of its precursor is shown in SEQ ID NO.

4.

5. A novel dual-target insulin analog according to claim 4, characterized in that: The nucleotide sequence of its precursor is shown in SEQ ID NO.

5.

6. A novel dual-target insulin analog according to claim 6, characterized in that: After the precursor is treated with enzymes, a dual-target insulin analog is obtained; the method of the enzyme treatment is: using carboxypeptidase B and Kex2 enzymes to treat the precursor with enzymes to obtain a dual-target insulin analog; its amino acid sequence is shown in SEQ ID NO.

7.

7. A method for preparing a novel dual-target insulin analog according to any one of claims 1 to 6, characterized in that: The DNA fragment with the sequence shown in SEQ ID NO.6 is integrated into an empty vector to obtain an expression vector; the expression vector is transferred into bacteria to obtain an engineered bacterium; the engineered bacterium is induced to express a fusion protein with the sequence shown in SEQ ID NO.4; and after extraction, purification and enzyme digestion, a dual-target insulin analog is obtained.

8. The method for preparing a novel dual-target insulin analog according to claim 7, characterized in that: The method of enzyme digestion treatment is: at pH 7.5-7.6 and room temperature, the fusion protein shown in SEQ ID NO.4 is treated with carboxypeptidase B and Kex2 enzymes for 16-24 hours; the mass ratio of the sum of the mass of carboxypeptidase B and Kex2 enzymes to the mass of the fusion protein is 1:200, and the mass ratio of carboxypeptidase B and Kex2 enzymes is 1:

1.

9. Use of a novel dual-target insulin analogue according to any one of claims 1 to 6 in the preparation of drugs for improving cardiovascular diseases and drugs for controlling body weight.

10. Use of a novel dual-target insulin analogue according to any one of claims 1 to 6 in the preparation of a drug for controlling blood sugar.

Images