Preparation method and application of long-acting recombinant canine insulin fusion protein

Abstract

The present invention belongs to the technical field of biological genetic engineering, and specifically relates to a long-acting recombinant canine insulin fusion protein and its preparation method and application. The fusion protein formed by connecting proinsulin and 7p has high biological activity, and the recombinant canine long-acting insulin is co-expressed in an Escherichia coli expression system using a molecular chaperone, and a high-purity soluble target protein is obtained through denaturation, renaturation and purification. The target protein is mainly located in the supernatant, avoiding the formation of inclusion bodies, simplifying the purification process, and optimizing the induced expression conditions, so that the proportion of soluble protein is significantly increased, and the protein is mainly located in the supernatant. The high expression amount and high purity fusion protein obtained by final purification have good biological activity, which solves the problems of difficulty in obtaining animal insulin, short half-life in vivo and easy to cause immune response. The long-acting recombinant canine insulin expressed by the present invention provides a more suitable treatment plan for canine diabetes treatment, and has industrialization prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and specifically relates to a long-acting recombinant canine insulin fusion protein and a preparation method and application thereof. Background Art

[0002] Insulin is a double-chain (α, β) polypeptide hormone secreted by pancreatic β cells. It is the only known protein hormone in the body that can regulate blood glucose levels. When insulin is deficient and / or ineffective, it can cause blood glucose levels to rise, leading to diabetes. Chronic subcutaneous insulin injection therapy is a routine regimen for treating diabetes in dogs and cats. However, due to the short half-life of insulin, two to five injections a day are required to control blood glucose levels, causing severe pain and discomfort to sick pets. At the same time, the economic and time costs of frequent injections for owners usually lead to a lack of compliance with treatment regimens and insufficient dosage, resulting in poor long-term health outcomes, and even causing a large proportion of owners to choose to euthanize their pets.

[0003] Insulin can be extracted and purified from the pancreas of animals (pigs or cows), but due to the structural differences between different biological insulins, isolated animal insulins such as Vetsulin TM(Intervet) (porcine insulin zinc suspension) is prone to cause immune reactions such as rash, hypotension and even shock, and its clinical application is limited; with the development of molecular biology technology, the development of genetically engineered recombinant insulin has provided a new technical reference for the treatment of diabetes. There are currently two main ideas for extending the half-life of insulin: 1) by increasing the molecular weight of insulin protein to reduce its clearance in the kidney, such as Hanmi Pharmaceuticals by conjugating immunoglobulin Fc with insulin analogs to increase the molecular weight of insulin protein to achieve the purpose of extending the half-life; 2) increasing the inactive reserves of insulin in the blood or body, relying on the slow release of insulin in the blood or body to supplement the pharmaceutical activity of the degraded part of insulin. For example, the glargine insulin developed by Sanofi Aventis replaces the aspartic acid at the 21st position of the A chain of human insulin with glycine, and adds two arginines at the carboxyl end of the B chain, so that insulin can form a more stable hexamer in the blood. The isoelectric point of pH 6.7 makes it easy for insulin to form micro-precipitates in the neutral environment of subcutaneous tissue, which prolongs its absorption, decomposition and action time. The detemir insulin developed by Novo Nordisk modifies insulin with myristic acid side chains to accelerate the formation of hexamer and reduce its absorption rate. At the same time, it allows insulin to reversibly bind to albumin at the administration site, further slowing down its diffusion rate to target tissues and blood circulation, thereby exerting its long-term hypoglycemic effect. Degludec insulin also modifies insulin with 16-carbon fatty diacids to form multiple hexamers. After entering the capillaries, the degludec insulin molecule reversibly binds to albumin in the blood through its fatty acid side chains, prolonging the action time. Existing studies on the recombinant expression of proinsulin in Escherichia coli show that the expressed proteins are all in the form of inclusion bodies, and protein purification requires a renaturation process.

[0004] Therefore, the development of a long-acting insulin for dogs and cats for this disease is an effective and less burdensome treatment option. Summary of the invention

[0005] In view of the problems of low animal insulin production, short half-life and immunogenicity, as well as the current situation of lack of recombinant insulin for pets, the purpose of the present invention is to provide a long-acting recombinant canine insulin fusion protein and its preparation method and application.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides a long-acting recombinant canine insulin fusion protein, the protein structure of which is fused and expressed by canine proinsulin and a targeting polypeptide, and a tag peptide is added to the C-terminus of the protein;

[0008] The canine proinsulin includes canine proinsulin (NCBI accession number: NP_001123565.1), and also includes a protein formed by connecting the insulin A chain and the insulin B chain obtained by removing the canine proinsulin C peptide using a connecting peptide;

[0009] The connecting peptides include Linker1, Linker2, and Linker3 (also called L1, L2, and L3);

[0010] The targeting polypeptide includes 7peptide (HAIYPRH, 7P);

[0011] Furthermore, the long-acting recombinant canine insulin fusion protein includes cProINS-7P-H, cProINSB-L1-cProINSA-7P-H (cINSGL1-7P-H), cProINSB-L2-cProI NSA-7P-H (cINSGL2-7P-H), and cProINSB-L3-cProINSA-7P-H (cINS GL3-7P-H), and their amino acid sequences are shown in SEQ ID NO.1-4, respectively, and their optimized nucleic acid sequences are shown in SEQ ID NO.5-8, respectively.

[0012] The present invention also provides a method for preparing a long-acting recombinant canine insulin fusion protein, comprising the following steps:

[0013] 1) After codon optimization of canine proinsulin, targeting polypeptide and tag peptide, full gene synthesis is performed to obtain a recombinant canine insulin fusion protein nucleic acid sequence;

[0014] 2) cloning the above recombinant canine insulin fusion protein nucleic acid sequence into an expression vector to obtain an expression plasmid;

[0015] 3) using a molecular chaperone and the above expression plasmid to co-transform into competent cells to obtain a recombinant prokaryotic strain;

[0016] 4) Inducing expression of the recombinant prokaryotic strain and purifying the expression sample to obtain the long-acting recombinant canine insulin fusion protein.

[0017] Furthermore, the canine proinsulin in step 1) includes canine proinsulin (NCBI accession number NP_001123565.1, amino acid sequence as shown in SEQ ID NO.9, nucleic acid sequence after codon optimization as shown in SEQ ID NO.10), and also includes a protein formed by connecting the insulin A chain and insulin B chain obtained by removing the canine proinsulin C peptide using a connecting peptide;

[0018] The connecting peptides include Linker1, Linker2, and Linker3 (also called L1, L2, and L3), whose amino acid sequences are shown in SEQ ID NOs.11-13, respectively, and whose nucleic acid sequences after codon optimization are shown in SEQ ID NOs.14-16, respectively;

[0019] The targeting polypeptide includes 7peptide (7P), whose amino acid sequence is shown in SEQ ID NO.17, and whose nucleic acid sequence after codon optimization is shown in SEQ ID NO.18;

[0020] The tag peptide is histidine, and its amino acid sequence is shown in SEQ ID NO.19, and its nucleic acid sequence after codon optimization is shown in SEQ ID NO.20;

[0021] The codon optimization was performed using the E. coli system.

[0022] Furthermore, the expression vector in step 2) includes but is not limited to pET-3a, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET- 33b, pET-34b, pET-35b, pET-37b, pET-39b, pET-40b, pET-41a, pET-42a, pET-43.1a, pET-44a, pET-45b, pET-47b, pE One of T-48b, pET-49b, pET-50b, pET-51b, pET-52b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pColaDuet-1.

[0023] Furthermore, the molecular chaperone in step 3) includes but is not limited to one of pKJE7, pGro7, pTf16, pG-KJE8, and pG-Tf2;

[0024] The competent cells include but are not limited to one of BL21, cBL21, BL21(DE3), BL21codonplus(DE3), BL21 Star(DE3), B834(DE3), Tuner(DE3), Rosetta(DE3), Rosetta2(DE3), ArcticExpress(DE3), Origami(DE3), Origami 2(DE3), OrigamiB(DE3), SHuffle T7, ER2566, OverExpress C43(DE3), BLR(DE3), HMS174(DE3), GT115, MG1655, and NovaBlu e(DE3).

[0025] Furthermore, the inducing agent used in step 4) for inducing expression is IPTG, and its concentration is 0.02-8mM, and the temperature is 15-37°C; preferably 0.2mM, 25°C.

[0026] The present invention also provides an application of the long-acting recombinant canine insulin fusion protein in preparing a pet blood sugar balancing drug.

[0027] The beneficial effects of the present invention are as follows:

[0028] The long-acting recombinant canine insulin fusion protein of the present invention has a protein structure with high expression level and high stability. Through the preparation method of the present invention, the fusion protein formed by connecting proinsulin and 7p has high biological activity, and the recombinant canine long-acting insulin is co-expressed in an Escherichia coli expression system by using a molecular chaperone, so that a high-purity soluble target protein is obtained through denaturation, renaturation and purification. The target protein is mainly located in the supernatant, the formation of inclusion bodies is avoided, the purification process is simplified, and the induced expression conditions are optimized, so that the proportion of soluble protein is significantly increased, the protein is mainly located in the supernatant, and the fusion protein with high expression level and high purity obtained by final purification has good biological activity, which solves the problems of difficulty in obtaining animal insulin, short half-life in vivo and easy to cause immune response. The long-acting recombinant canine insulin expressed by the present invention provides a more suitable treatment plan for canine diabetes treatment and has industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the structure of the fusion protein constructed in Example 1;

[0030] Figure 2 This is a diagram showing the PCR identification results of the bacterial solution of the recombinant prokaryotic strain of Example 1;

[0031] Figure 3 This is the SDS-PAGE result of the recombinant prokaryotic strain expression sample of Example 1;

[0032] Figure 4 This is the SDS-PAGE result of the induced expression sample of the recombinant E. coli with co-transfected molecular chaperone pTf16;

[0033] Figure 5 This is the SDS-PAGE result of the sample induced expression of the co-transfected molecular chaperone pG-Tf2 recombinant E. coli;

[0034] Figure 6 The graph shows the hypoglycemic effect of each group of samples on the type 1 diabetes model in mice. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0036] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0037] Example 1

[0038] Preparation of a long-acting recombinant canine insulin fusion protein

[0039] 1) Full gene synthesis and construction of expression vector:

[0040] The peptide 7peptide (7P) was fused with canine proinsulin (cProI NS), and a histidine tag was added to the C-terminus of the protein to form cProINS-7P-H.

[0041] In order to further improve the protein stability and biological activity, different linkers (L1-L3) were used to connect the canine insulin B chain and the canine insulin A chain based on the removal of the C-peptide from canine proinsulin to form cProINSB-L1-cProINSA-7P-H, cProINSB-L2-cProINSA-7P-H, cProINSB-L3-cProINSA-7P-H (cINSGL1-7P-H, cINSGL2-7P-H, cINSGL3-7P-H);

[0042] After E. coli codon optimization, each protein sequence was sent to Nanjing GenScript Biotechnology Co., Ltd. for full gene synthesis. The schematic diagram of the synthesized protein structure is shown in the figure below. Figure 1As shown, and cloned into the Escherichia coli expression vector pET30a(+), obtaining the recombinant plasmids cProINS-7P-H-pET30a(+), cINSGL1-7P-H-pET30a(+), cINSGL2-7P-H-pET30a(+), and cINSGL3-7P-H-pET30a(+);

[0043] The expression vector can also be pET-3a, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-31b, pET-32a, pET-33b, pET-34b, One of pET-35b, pET-37b, pET-39b, pET-40b, pET-41a, pET-42a, pET-43.1a, pET-44a, pET-45b, pET-47b, pET-48b, pET-49b, pET-50b, pET-51b, pET-52b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pColaDuet-1, has the same effect;

[0044] 2) Co-transformation expression

[0045] The molecular chaperones pTf16 and pG-Tf2 were co-transformed with cProINS-7P-H-pET30a(+), cINSGL1-7P-H-pET30a(+), cINSGL2-7P-H-pET30a(+), and cINSGL3-7P-H-pET30a(+) to express competent ArcticExpress(DE3). At the same time, a control group without co-transformation of molecular chaperones pTf16 and pG-Tf2 was set up, namely cProINS-7P-H-pET30a(+), cINSGL1-7P-H-pET30a(+), cINSGL2-7P-H-pET30a(+), and cINSGL3-7P-H-pET30a(+) were transformed with competent ArcticExpress(DE3);

[0046] The competent cell can also be one of BL21, cBL21, BL21(DE3), BL21codonplus(DE3), BL21Star(DE3), B834(DE3), Tuner(DE3), Rosetta(DE3), Rosetta2(DE3), Origami(DE3), Origami 2(DE3), OrigamiB(DE3), SHuffle T7, ER2566, OverExpress C43(DE3), BLR(DE3), HMS174(DE3), GT115, MG1655, and NovaBlue(DE3);

[0047] The molecular chaperone may also be one of pKJE7, pGro7, pTf16, pG-KJE8, and pG-Tf2;

[0048] 2.1 Take out the expression competent cells (Arctic Express (DE3)) from the -80℃ refrigerator, melt them in an ice bath, add 1ul of the constructed plasmid and 50ul of the competent cells to a new sterile 1.5mL EP tube at a ratio of 1:10, gently pipette to mix, and place on ice for 30min;

[0049] 2.2 Then heat stress at 42℃ for 90s, and then ice bath for 5min;

[0050] 2.3 Add 200 μL of antibiotic-free LB liquid medium to the centrifuge tube and culture at 37°C with gentle shaking for 60 min;

[0051] 2.4 Spread the resuspended bacterial solution evenly on the LB solid medium containing 50 μg / mL Kana, place it in a 37°C incubator and culture it on its front for about 15 minutes. After the liquid is completely absorbed, invert the plate and culture it for 12-16 hours;

[0052] The recombinant prokaryotic strains were obtained through the above, and PCR identification was performed. The identification system and procedures are shown in Table 1 and Table 2:

[0053] Table 1 PCR identification system

[0054]

[0055] Table 2 PCR identification procedures for recombinant bacterial solution

[0056]

[0057] The results obtained by the above PCR identification are as follows Figure 2 As shown, the corresponding recombinant positive strains were screened in each group, and the size of the PCR amplification band was consistent with the expectation. Figure 2Middle M: ​​DNAMarker 5000; 1-2: cProINS-7P-H-pET30a(+) group bacterial liquid PCR identification; 3: cProINS-7P-H-pET30a(+)-pTf16 group bacterial liquid PCR identification; 4: cProINS-7P-H-pET30a(+)-pG-Tf2 group bacterial liquid PCR identification; 5: cINSGL1-7P-H-pET30a(+) group bacterial liquid PCR identification; 6: cINSGL1-7P-H-pET30a(+)-pTf16 group bacterial liquid PCR identification; 7-8: cINSGL1-7P-H-pET30a(+)-pG-Tf2 group bacterial liquid PCR identification; 9-10: cIN PCR identification of bacterial liquid of SGL2-7P-H-pET30a(+) group; 11: PCR identification of bacterial liquid of cINSGL2-7P-H-pET30a(+)-pTf16 group; 12: PCR identification of bacterial liquid of cINSGL2-7P-H-pET30a(+)-pG-Tf2 group; 13-14: PCR identification of bacterial liquid of cINSGL3-7P-H-pET30a(+) group; 15: PCR identification of bacterial liquid of cINSGL3-7P-H-pET30a(+)-pTf16 group; 16: PCR identification of bacterial liquid of cINSGL3-7P-H-pET30a(+)-pG-Tf2 group.

[0058] 3) Inducible expression

[0059] 3.1 Use a sterilized pipette tip to carefully pick up a single colony with Kana resistance growing on the LB plate, and place it in 10 mL of LB liquid medium containing 50 μg / mL Kana for activation culture. Pick two parallel groups from each plate and culture at 37°C, 200 rpm, and shake for 6 h until OD600 = 2.0;

[0060] 3.2 Inoculate the above bacterial solution into 100 mL of LB liquid medium containing 50 μg / mL Kana at a ratio of 1%, and culture at 37°C, 200 rpm, and shake for 3 h until OD600 = 0.6-0.8;

[0061] 3.3 Add 0.2 mM IPTG to the above bacterial solution and induce overnight shaking culture at 25°C and 200 r / min;

[0062] 3.4 Take the overnight expression bacterial solution for ultrasonic disruption (ultrasonication conditions: 5s ultrasonication, 5s interval, 10min, 30% power), centrifuge the disrupted sample (12,000rpm, 4°C, centrifuge for 10min) to collect the supernatant and precipitate;

[0063] The supernatant and precipitate were taken for SDS-PAGE to check the soluble expression of the protein. The results of SDS-PAGE analysis of the recombinant E. coli induced expression sample without co-transformation of molecular chaperones were as follows: Figure 3 As shown (in the figure, M: Protein Marker (14.4-97.4kDa); 1-3: total bacterial solution, supernatant and precipitate of cProINS-7P-H-pET30a(+) group recombinant bacteria induced expression; 4-6: total bacterial solution, supernatant and precipitate of cINSGL1-7P-H-pET30a(+) group recombinant bacteria induced expression; 7-9: total bacterial solution, supernatant and precipitate of cINSGL2-7P-H-pET30a(+) group recombinant bacteria induced expression; 10-12: total bacterial solution, supernatant and precipitate of cINSGL3-7P-H-pET30a(+) group recombinant bacteria induced expression; N: ArcticExpress(DE3) negative control).

[0064] After the recombinant Escherichia coli without co-transfection of molecular chaperone pTf16 was induced by 0.2mM IPTG, the target proteins induced to express in the cProINS-7P-H group, cINSGL1-7P-H group, cINSGL2-7P-H group, and cINSGL3-7P-H group were inclusion bodies, and most of them were located in the precipitate after fragmentation.

[0065] The SDS-PAGE of the co-transfected molecular chaperone pTf16 recombinant Escherichia coli induced expression samples was as follows Figure 4 As shown (M: ProteinMarker (14.4-97.4kDa); 1-3: total bacterial solution, supernatant and precipitate of cProINS-7P-H-pET30a(+)-pTf16 group recombinant bacteria induced expression; 4-6: total bacterial solution, supernatant and precipitate of cINSGL1-7P-H-pET30a(+)-pTf16 group recombinant bacteria induced expression; 7-9: total bacterial solution, supernatant and precipitate of cINSGL2-7P-H-pET30a(+)-pTf16 group recombinant bacteria induced expression; 10-12: total bacterial solution, supernatant and precipitate of cINSGL3-7P-H-pET30a(+)-pTf16 group recombinant bacteria induced expression; N: ArcticExpress (DE3) negative control;)

[0066] The SDS-PAGE of the co-transfected molecular chaperone pG-Tf2 recombinant E. coli induced expression samples was as follows Figure 5As shown (M: ProteinMarker (3.3-31.0 kDa); 1-3: total bacterial solution, supernatant and precipitate of cProINS-7P-H-pET30a(+)-pG-Tf2 group recombinant bacteria induced expression; 4-6: total bacterial solution, supernatant and precipitate of cINSGL1-7P-H-pET30a(+)-pG-Tf2 group recombinant bacteria induced expression; 7-9: total bacterial solution, supernatant and precipitate of cINSGL2-7P-H-pET30a(+)-pG-Tf2 group recombinant bacteria induced expression; 10-12: total bacterial solution, supernatant and precipitate of cINSGL3-7P-H-pET30a(+)-pG-Tf2 group recombinant bacteria induced expression; N: ArcticExpress (DE3) negative control;)

[0067] The molecular chaperone pTf16 was used to co-transform the competent ArcticExpress (DE3) with cProINS-7P-H-pET30a(+), cINSGL1-7P-H-pET30a(+), cINSGL2-7P-H-pET30a(+), and cINSGL3-7P-H-pET30a(+). After induction with 0.2 mM IPTG, most of the target proteins induced and expressed in the cINSGL1-7P-H group, cINSGL2-7P-H group, and cINSGL3-7P-H group were soluble proteins, and most of them were located in the supernatant after fragmentation. However, most of the target proteins induced and expressed by cProINS-7P-H were inclusion bodies, and most of them were located in the precipitate after fragmentation. It can be seen that the co-transformation expression with the molecular chaperone pTf16 can achieve the soluble expression and high-yield expression of cINSGL1-7P-H, cINSGL2-7P-H, and cINSGL3-7P-H fusion proteins. When the molecular chaperone pG-Tf2 was used for co-expression, the total expression of the target protein and the content of the target protein in the supernatant were not as good as those of the pTf16 group ( Figure 4 , Figure 5 ), so the molecular chaperone pTf16 is preferred for the expression of insulin fusion protein.

[0068] 4) Purification

[0069] The supernatant samples of the preferred group of molecular chaperone pTf16 were subjected to nickel column affinity chromatography for protein adsorption and elution purification, and the concentration was determined after ultrafiltration and concentration, and the imidazole was removed by dialysis. The concentrations of the purified samples of each group are shown in Table 3. The samples of each group were subjected to subsequent enzyme digestion.

[0070] Table 3 Concentration of each group of samples after purification

[0071] Group cProINSG-7P-H cINSGL1-7P-H cINSGL2-7P-H cINSGL3-7P-H concentration 0.25mg / mL 1.48mg / mL 1.42mg / mL 1.45mg / mL

[0072] 5) Enzyme digestion

[0073] Prepare trypsin solution with ultrapure water to a concentration of 1 mg / mL; measure the pure insulin fusion protein solution; add trypsin solution, adding 5 ul enzyme solution for every 10 mL sample; add a magnetic stirrer and stir the reaction at room temperature for 24 hours; after the enzyme cleavage is completed, add 6 M hydrochloric acid to adjust the pH to 3.5 to terminate the reaction.

[0074] After enzyme digestion, the samples were further purified for subsequent activity determination.

[0075] Test example

[0076] The fusion protein expressed in Example 1 was used for activity detection:

[0077] The chemical method of inducing type 1 diabetes model is achieved by injecting chemicals to destroy pancreatic β cells. Streptozotocin (STZ) is a pancreatic β cell toxic agent that can specifically destroy β cells by inducing the breakage of pancreatic β cell DNA, which can lead to insulin-deficient diabetes. The degree of damage to β cells by STZ is dose-dependent and is widely used in the establishment of type 1 diabetes models.

[0078] Thirty-five 6-month-old male C57BL / 6J mice were selected and divided into cProINSG-7P-H group, cINSGL1-7P-H group, cINSGL2-7P-H group, cINSGL3-7P-H group, model control group, insulin (INS) control group, and blank control group. Except for the blank control group, the diabetic model needs to be established. Fasting for 6 hours before the start of the experiment, STZ (150 mg / kg) dissolved in sodium citrate buffer (100mM, pH 4.5) was intraperitoneally injected according to the weight of the mice, and the blank control group was injected with an equal amount of sodium citrate buffer. After 5 days, fasting for 2 hours to detect the changes in blood glucose in mice, and diabetic mice with blood glucose higher than 300 mg / dL were considered to have successfully established models and could be used for subsequent experiments. After the model was successfully established, the cProINSG-7P-H group, cINSGL1-7P-H group, cINSGL2-7P-H group, cINSGL3-7P-H group, and insulin (INS) control group were subcutaneously injected at 20 μg / kg, and the model control group and blank control group were injected with an equal volume of normal saline.

[0079] The blood sugar levels before and after drug injection were measured to evaluate the hypoglycemic effect of each group of samples. Figure 6As shown, it can be seen that the insulin (INS) control group reached the blood sugar lowering peak 2 hours after injection, and then the blood sugar rose rapidly, while the cProINSG-7P-H group, cINSGL1-7P-H group, cINSGL2-7P-H group, and cINSGL3-7P-H group of the present invention were able to achieve rapid blood sugar lowering for a longer period of time, lasting nearly 24 hours, and the blood sugar level remained stable.

Claims

1. A long-acting recombinant canine insulin fusion protein, characterized in that: The structure of the fusion protein is composed of canine proinsulin, a targeting polypeptide and a tag peptide from the N-terminus to the C-terminus, wherein the amino acid sequence of the canine proinsulin is selected from SEQ ID NO.9, 1-63 of SEQ ID NO.2, 1-59 of SEQ ID NO.3 or 1-59 of SEQ ID NO.4, and the amino acid sequence of the targeting polypeptide is shown in SEQ ID NO.

17.

2. The long-acting recombinant canine insulin fusion protein according to claim 1, characterized in that: The long-acting recombinant canine insulin fusion proteins are cProINS-7P-H, cProINSB-L1-cProINSA-7P-H, cProINSB-L2-cProINSA-7P-H, and cProINSB-L3-cProINSA-7P-H, and their amino acid sequences are shown in SEQ ID NOs. 1-4, respectively, and their optimized nucleic acid sequences are shown in SEQ ID NOs. 5-8, respectively.

3. A method for preparing the long-acting recombinant canine insulin fusion protein according to claim 1 or 2, characterized in that: The steps include: 1) After codon optimization, canine proinsulin, targeting polypeptide and tag peptide were subjected to full gene synthesis to obtain the nucleic acid sequence of recombinant canine insulin fusion protein; 2) cloning the above recombinant canine insulin fusion protein nucleic acid sequence into an expression vector to obtain an expression plasmid; The expression vector includes but is not limited to E. coli expression vectors pET-3a, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET- 33b, pET-34b, pET-35b, pET-37b, pET-39b, pET-40b, pET-41a, pET-42a, pET-43.1a, pET-44a, pET-45b, pET-47b, pE One of T-48b, pET-49b, pET-50b, pET-51b, pET-52b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pColaDuet-1; 3) Using molecular chaperones and the above expression plasmids to co-transform into competent cells to obtain recombinant prokaryotic strains; 4) Induce expression and purify the recombinant prokaryotic strain to obtain the long-acting recombinant canine insulin fusion protein.

4. The method for preparing the long-acting recombinant canine insulin fusion protein according to claim 3, characterized in that: Step 3) The molecular chaperone includes but is not limited to one of pKJE7, pGro7, pTf16, pG-KJE8, and pG-Tf2.

5. The method for preparing the long-acting recombinant canine insulin fusion protein according to claim 3, characterized in that: Step 3) The competent cells include but are not limited to one of BL21, cBL21, BL21 (DE3), BL21codonplus (DE3), BL21 Star (DE3), B834 (DE3), Tuner (DE3), Rosetta (DE3), Rosetta2 (DE3), Arctic Express (DE3), Origami (DE3), Origami 2 (DE3), OrigamiB (DE3), SHuffle T7, ER2566, OverExpress C43 (DE3), BLR (DE3), HMS174 (DE3), GT115, MG1655, and NovaBlue (DE3).

6. The method for preparing the long-acting recombinant canine insulin fusion protein according to claim 3, characterized in that: Step 4) The inducing agent used for the induced expression is IPTG, and its concentration is 0.02-8 mM, and the temperature is 15-37°C.

7. Use of the long-acting recombinant canine insulin fusion protein according to claim 1 or 2 in the preparation of a pet blood sugar balancing drug.

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