WO2021136521A1 - Polypeptide and use thereof - Google Patents

Polypeptide and use thereof Download PDF

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Publication number
WO2021136521A1
WO2021136521A1 PCT/CN2020/142229 CN2020142229W WO2021136521A1 WO 2021136521 A1 WO2021136521 A1 WO 2021136521A1 CN 2020142229 W CN2020142229 W CN 2020142229W WO 2021136521 A1 WO2021136521 A1 WO 2021136521A1
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Prior art keywords
protein
befa
amino acid
polypeptide
terminus
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PCT/CN2020/142229
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French (fr)
Chinese (zh)
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李利佳
古想娣
吴都
郭林峰
李晓平
胡育龙
李宇晟
李玉
许玲华
李静
陈小锋
李文佳
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东莞市东阳光生物药研发有限公司
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Publication of WO2021136521A1 publication Critical patent/WO2021136521A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/04Anorexiants; Antiobesity agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/20Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/24Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/50Fusion polypeptide containing protease site

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to polypeptides and their applications. More specifically, the present invention relates to polypeptides, methods for improving BefA protein stability or preparing BefA protein mutants, fusion proteins, nucleic acids, recombinant cells and drugs The composition and its application in the field of metabolic diseases such as diabetes.
  • the lack of insulin secreting cells has always been considered as one of the causes of type I diabetes.
  • Type I diabetes the immune system mistakenly attacks and destroys beta cells.
  • researchers have found that the lack of functional ⁇ cells is also an important cause of type II diabetes, and the function of pancreatic islets in patients with type II diabetes gradually declines in the late stage. Therefore, the development of drugs that can increase the number of healthy ⁇ -cells is very important for solving the late stage of type I diabetes or type II diabetes, and is the main focus of diabetes research.
  • diabetic patients are often accompanied by metabolic syndromes such as obesity, fatty liver, and liver function impairment. Therefore, it is very necessary to develop drugs with more clinical benefits, such as weight loss and liver function protection drugs.
  • the current hypoglycemic biologic drugs on the market mainly include: insulin and its analogs, GLP-1 and GLP-1 analogs, such as liraglutide and dulaglutide.
  • the mechanism of action of insulin and its analogues is a direct supplementation of insulin (or analogues) for type I diabetes to achieve the effect of insulin lowering blood sugar, but there is a risk of hypoglycemia, and it does not reduce the weight of type 2 diabetes patients and protect liver function
  • GLP-1 and its analogues are receptor agonists of GLP-1, which exert a hypoglycemic effect by stimulating the receptor of GLP-1.
  • GLP-1 The mode of action of GLP-1 is to increase the secretion of insulin, inhibit the secretion of glucagon, delay gastric emptying, and reduce food intake.
  • these drugs have a short half-life and have adverse gastrointestinal reactions such as nausea and vomiting.
  • the above drugs can reduce blood sugar, they cannot completely reverse the damaged pancreatic islets. Patients often need long-term medication, which increases medical expenditure and reduces the patient's medication experience. Long-term medication may also lead to drug tolerance.
  • BefA ⁇ -cell proliferation factor A
  • BefA protein expressed by prokaryotic cells in vitro has the activity of promoting ⁇ cell proliferation, but BefA is easily degraded, and it is difficult to obtain high-purity and complete molecular weight BefA protein, which greatly limits the in vivo and in vitro drug efficacy research and clinical application of BefA protein. application.
  • the research and development personnel of the present application were surprised and consciously surprised to find that the N-terminus of BefA protein was modified, including adding amino acids at the N-terminus, or making the N-terminus amino acids methylated, formylated, or carbamoyl.
  • the present invention proposes a polypeptide.
  • the N-terminal has a modification, and the modification includes an increase in the N-terminal amino acid, methylation, formylation, carbamylation, At least one of succinylation, cyclization, propionylation, hexadecanoylation, tetradecylation, and acetylation.
  • the polypeptide according to the embodiment of the present invention maintains the activity of the BefA protein to promote ⁇ cell proliferation, and has higher stability than the polypeptide having the sequence (1), (2) or (3).
  • the above-mentioned polypeptide may further include at least one of the following additional technical features:
  • the increase of the N-terminal amino acid includes the addition of one or more amino acids at the N-terminal, and the added amino acid includes at least one of glycine, alanine, isoleucine, methionine, and valine.
  • Acid serine, threonine, glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine.
  • the increase of the N-terminal amino acid is an increase of an amino acid at the N-terminal, and the amino acid is glycine, alanine, isoleucine, methionine, valine, serine, and threonine.
  • Acid glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO:7.
  • polypeptide having the amino acid sequence shown in SEQ ID NO: 7 according to the embodiment of the present invention has high stability, is not easy to be degraded, and the purity after purification can reach 99%.
  • the present invention proposes a method for improving the stability of BefA protein or preparing BefA protein mutants.
  • the N-terminus of the amino acid sequence of the BefA protein is modified, and the modification includes an increase in the N-terminus amino acid, methylation, formylation, carbamylation, succinylation, cyclization, At least one of propionylation, hexadecanoylation, tetradecylation and acetylation.
  • the method according to the embodiment of the present invention greatly improves the stability of the BefA protein, and the prepared BefA protein mutant not only has high stability, but also maintains the activity of the BefA protein to significantly promote the proliferation of ⁇ cells.
  • the above method may further include at least one of the following additional technical features:
  • the BefA protein has an amino acid sequence shown in (1) SEQ ID NO: 1 to 6; or (2) compared with (1), it has at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 99% identical; or (3) an amino acid sequence having one or more amino acid substitutions, deletions and/or additions compared to (1).
  • adding one or more amino acids to the N-terminus of the BefA protein amino acid sequence can be achieved by gene cloning, so as to obtain BefA protein mutants.
  • those skilled in the art can pre-synthesize a nucleic acid capable of expressing a BefA protein mutant with one or more amino acids added to the N-terminus, and then introduce the nucleic acid into the recipient cell for expression, and obtain one or more amino acids added to the N-terminus.
  • BefA protein mutants can be achieved by gene cloning, so as to obtain BefA protein mutants.
  • those skilled in the art can pre-synthesize a nucleic acid capable of expressing a BefA protein mutant with one or more amino acids added to the N-terminus, and then introduce the nucleic acid into the recipient cell for expression, and obtain one or more amino acids added to the N-terminus. BefA protein mutants.
  • the BefA protein mutant can be further connected to a chaperone protein through a connecting peptide, and the connecting peptide is suitable for being cleaved by a protease, so as to form a free BefA protein mutant again.
  • the BefA protein mutant is further connected to the chaperone protein through the connecting peptide, which can significantly increase the yield of the BefA protein mutant, but afterwards, the chaperone protein and the connecting peptide need to be completely excised under the action of a suitable protease to form a free one again. BefA protein mutant.
  • adding one or more amino acids to the N-terminus of the BefA protein amino acid sequence can be achieved by gene cloning and restriction enzyme digestion, so as to obtain BefA protein mutants.
  • those skilled in the art can pre-synthesize nucleic acid capable of expressing the first BefA protein mutant with some amino acids at the N-terminus, and then introduce the nucleic acid into recipient cells for expression, to obtain the first protein mutant with some amino acids at the N-terminus.
  • BefA protein mutant; the first BefA protein mutant is further connected to the chaperone protein through a connecting peptide.
  • the chaperone protein and the connecting peptide need to be set at the N-terminus of the first BefA protein mutant, and the connecting peptide is suitable for protease Cleavage, and then under the action of suitable protease cleavage, the chaperone protein and part of the connecting peptide are excised, so as to retain some amino acids at the N-terminus of the first BefA protein mutant.
  • the remaining part of the connecting peptide and The first part of the amino acids added by gene cloning together constitute the added amino acids at the N-terminus of BefA protein.
  • adding one or more amino acids to the N-terminus of the amino acid sequence of the BefA protein can also be achieved by introducing restriction enzymes.
  • restriction enzymes For example, those skilled in the art can directly construct a fusion protein of a chaperone protein and the BefA protein, a connecting peptide is arranged between the chaperone protein and the BefA protein, and the N-terminal of the connecting peptide is connected to the C of the chaperone protein.
  • the C-terminus of the connecting peptide is connected to the N-terminus of the BefA protein.
  • the connecting peptide is suitable for being cleaved by a protease to form a free target protein.
  • the BefA protein has one or more amino acids added, and the free target protein is the BefA protein mutant. That is to say, the amino acids added to the N-terminus of the free target protein are at least part of the amino acids on the connecting peptide. In other words, a new amino acid was introduced at the N-terminus of the BefA protein by introducing a connecting peptide with a restriction site.
  • At least one amino acid is successfully connected to the N-terminus of BefA protein.
  • the chaperone protein includes at least one selected from maltose binding protein, CBP, GST, SUMO, Trx, and NusA.
  • the chaperone protein contains or does not contain a His tag and a Flag tag.
  • the protease is cysteine protease, enterokinase, thrombin, factor Xa protease, human rhinovirus 3C protease, SUMO enzyme or KEX-2 of tobacco etch virus.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO: 8.
  • the present invention proposes a fusion protein.
  • the fusion protein includes a chaperone protein, a connecting peptide, and a BefA protein, the N-terminus of the connecting peptide is connected to the C-terminus of the chaperone protein, and the C-terminus of the connecting peptide is connected to the BefA protein.
  • the connecting peptide is suitable for being cleaved by a protease to form a free BefA protein mutant.
  • the N-terminus of the free BefA protein mutant has one or more amino acids added.
  • the fusion protein according to the embodiment of the present invention has a high expression level, and under the action of a suitable digestive enzyme, a BefA protein mutant with at least one amino acid added to the N-terminal can be successfully obtained.
  • the fusion protein may further include at least one of the following additional technical features:
  • the chaperone protein includes at least one selected from maltose binding protein, CBP, GST, SUMO, Trx, and NusA.
  • the chaperone protein contains or does not contain a His tag and a Flag tag.
  • the protease is cysteine protease, enterokinase, thrombin, factor Xa protease, human rhinovirus 3C protease, SUMO enzyme or KEX-2 of tobacco etch virus.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO: 8.
  • the present invention provides a nucleic acid.
  • the nucleic acid encodes the aforementioned polypeptide or the aforementioned fusion protein.
  • the nucleic acid may further include at least one of the following additional technical features:
  • the nucleic acid has the nucleotide sequence shown in SEQ ID NO: 9 or the nucleotide sequence shown in SEQ ID NO: 10.
  • the nucleic acid with the nucleotide sequence shown in SEQ ID NO: 9 encodes a polypeptide with the amino acid sequence shown in SEQ ID NO: 7, and the nucleic acid encoding with the nucleotide sequence shown in SEQ ID NO: 10 has SEQ ID NO : A fusion protein of the amino acid sequence shown in 8.
  • the present invention proposes a recombinant cell.
  • the recombinant cell carries the aforementioned nucleic acid.
  • the recombinant cell according to the embodiment of the present invention can express the aforementioned polypeptide or fusion protein.
  • the aforementioned recombinant cell may further include at least one of the following additional technical features:
  • the recombinant cell is Escherichia coli, yeast or mammalian cell.
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition includes the aforementioned polypeptide.
  • the pharmaceutical composition according to the embodiment of the present invention can be used for the treatment or prevention of diabetes.
  • the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features:
  • the pharmaceutical composition further includes a pharmaceutically acceptable auxiliary agent.
  • the pharmaceutical composition can be prepared into a specific pharmaceutical preparation and administered to a patient in a specific manner.
  • the present invention proposes the use of the aforementioned polypeptide or the aforementioned pharmaceutical composition in the preparation of a medicine, which is used to promote the proliferation of ⁇ cells.
  • the present invention proposes the use of the aforementioned polypeptide or the aforementioned pharmaceutical composition in the preparation of medicines for treating or preventing diabetes or protecting liver function.
  • the BefA protein in the present invention refers to Beta Cell Expansion Factor A (Beta Cell Expansion Factor A, BefA).
  • Figure 1 is a schematic diagram of a recombinant expression plasmid pET28a-BefA according to an embodiment of the present invention
  • Figure 2 is a schematic diagram of PCR identification of pET28a-BefA positive clones according to an embodiment of the present invention
  • Figure 3 is a schematic diagram of a recombinant expression plasmid pET28a-MBP-BefA according to an embodiment of the present invention
  • Figure 4 is a schematic diagram of PCR identification of pET28a-MBP-BefA positive clones according to an embodiment of the present invention
  • Figure 5A is an SDS-PAGE profile of a BefA nickel column affinity chromatography sample according to an embodiment of the present invention
  • Fig. 5B is a mass spectrometry test result of a BefA nickel column affinity chromatography sample according to an embodiment of the present invention
  • Fig. 5C is an SDS-PAGE chart of a BefA ion exchange chromatography sample according to an embodiment of the present invention.
  • Fig. 6A is a schematic diagram of SDS-PAGE of BefA mutant according to an embodiment of the present invention.
  • Fig. 6B is a mass spectrometric detection result of BefA mutant according to an embodiment of the present invention.
  • Fig. 7 is a schematic diagram of BefA protein promoting the proliferation of INS-1 cells according to an embodiment of the present invention.
  • Fig. 8 is a schematic diagram of BefA mutant promoting the proliferation of INS-1 cells according to an embodiment of the present invention.
  • Fig. 9 is a curve of random blood glucose change in ob/ob mice after injection of BefA protein according to an embodiment of the present invention.
  • Fig. 10 is a curve of weight change of ob/ob mice after injection of BefA protein according to an embodiment of the present invention.
  • FIG. 11 is a schematic diagram of the absolute weight of ob/ob mouse liver after injection of BefA protein according to an embodiment of the present invention.
  • Figure 12 is a schematic diagram of aspartate aminotransferase (AST) activity in ob/ob mice after injection of BefA protein according to an embodiment of the present invention
  • FIG. 13 is a schematic diagram of alanine aminotransferase (ALT) activity in ob/ob mice after injection of BefA protein according to an embodiment of the present invention
  • FIG. 14 is a schematic diagram of a random blood glucose change curve in db/db mice after injection of BefA protein mutant according to an embodiment of the present invention.
  • Fig. 15 is a curve of body weight change of db/db mice after injection of BefA protein mutant according to an embodiment of the present invention.
  • 16 is a schematic diagram of the absolute weight of the liver of db/db mice after injection of BefA protein mutants according to an embodiment of the present invention.
  • FIG 17 is a schematic diagram of alanine aminotransferase (ALT) activity in db/db mice after injection of BefA protein mutants according to an embodiment of the present invention
  • Figure 18 is a schematic diagram of the aspartate aminotransferase (AST) activity of db/db mice after injection of BefA protein mutants according to an embodiment of the present invention.
  • the "peptide bond” in this application refers to the chemical bond between each amino acid when the amino acids are connected to form a protein, which is called a peptide bond.
  • a peptide The bond is a chemical bond (-NH-CO-) formed by connecting the carboxyl group (-COOH) of one amino acid and the amino group (-NH 2) of another amino acid when two amino acids are connected.
  • the end of a polypeptide that ends with a carboxyl group is called the C-terminus, and the end that ends with an amino group is called the N-terminus.
  • connection or "connection” between the polypeptide and the polypeptide described in this application can be either a direct connection or an indirect connection.
  • connection can be either a direct connection or an indirect connection.
  • the polypeptide and the polypeptide can be connected by short peptides or amino acids.
  • the invention overcomes the above-mentioned problems of easy degradation of BefA protein and small application research range, and provides a method for preparing high-purity BefA protein. That is, by constructing a BefA fusion protein and designing a protease cleavage site in the fusion protein, it has a gene sequence shaped like an ABC structure, where A is the chaperone MBP (maltose binding protein) protein gene, and B is the encoding containing TEV (tobacco etch virus). The nucleotide sequence of the cysteine protease enzyme cleavage site and the connecting peptide, C is the BefA protein gene.
  • A is the chaperone MBP (maltose binding protein) protein gene
  • B is the encoding containing TEV (tobacco etch virus).
  • TEV tobacco etch virus
  • the fusion protein is purified and cleaved with TEV protease to obtain a BefA mutant with a glycine (G) at the N-terminal.
  • the purified protein has a purity of more than 95% and is tested to have biological activity.
  • BefA protein and BefA mutant can promote the proliferation of INS-1 (rat insulinoma cells) to a certain extent in vitro.
  • BefA protein can improve the liver function of ob/ob mice, and BefA mutant can improve db/db Liver function of mice.
  • the MBP chaperone gene and encoding contain TEV (Cysteine Protease of Tobacco Etch Virus) restriction site and connecting peptide (425aa in total).
  • TEV Cysteine Protease of Tobacco Etch Virus
  • the nucleic acid sequence is optimized according to the optimization principle of Escherichia coli (BL21(DE3)), Obtain the coding gene MBP, and finally give it to the gene synthesis company for full gene synthesis (pET28a-MBP).
  • the upstream primer F1 GCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAAGATCCGTTTTCTGG, containing the XbaI restriction site; the downstream primer R1: CCGCTCGAGTT AATGGTGATGATGATGATGAC, containing the XhoI Hi restriction tag site and 6 ⁇ His restriction site.
  • Add other reaction solution for PCR and amplify fragment A (844bp) containing BefA. Fragment A was digested with XhoI and XbaI to obtain fragment B (792bp).
  • Fragment B was ligated with the vector fragment (5236bp) recovered from pET28a plasmid by XhoI and XbaI digestion to obtain plasmid pET28a-BefA, as shown in Figure 1.
  • the plasmid pET28a-BefA was transformed into E. coli BL21 (DE3) by chemical transformation method.
  • the clones were screened for resistance on the Kana antibiotic plate, and the selected clones were amplified by PCR using primers F1 and R1 as templates to obtain a DNA fragment consistent with the theoretical size (792bp), as shown in Figure 2.
  • the sequencing results showed that the cloned gene fragment was consistent with the theory.
  • the upstream primer F2 CATGCCATGGAAATAAAAACAGGTGCACG, containing the NcoI restriction site; the downstream primer R2: GCTTTCACTTTTGCCCAATCTCCCTGAAAATACAGGTTTT.
  • upstream primer F3 AAAACCTGTATTTTCAGGGAGATTGGGCAAAAGTGAAAGC, downstream primer R3: CCGCTCGAGTTAACGGGTCAGATCTTTAT, containing XhoI restriction site.
  • Inoculate the glycerol tube of positive strains obtained by screening and identification into 5mL LB liquid medium containing 50 ⁇ g/mL kanamycin at 0.2% inoculum, and activate overnight at 37°C at 250rpm; inoculate with 2% inoculum to contain 50 ⁇ g/mL In a 2000mL Erlenmeyer flask of Kanamycin LB liquid medium, the volume of 20%, 37°C250rpm culture to OD 600 0.6 ⁇ 0.8, add IPTG to a final concentration of 1mM, 37°C250rpm to induce expression for 4 ⁇ 6h; The fermentation broth was centrifuged (8000rpm 10min), and the bacteria were collected.
  • the cells are resuspended in purified water, the cells are crushed (ultrasonic or homogenized), and the crushed liquid is centrifuged at low temperature and high speed (12000 rpm, 30 min, 4° C.), and the supernatant is obtained for purification.
  • the BefA protein uses nickel column affinity chromatography and ion exchange chromatography, and the BefA protein has been degraded, and no high purity BefA protein can be obtained.
  • HisTrap FF column uses 5 column volumes of 20mM ⁇ 50mM Tris, pH7.0 ⁇ 8.5, 100mM ⁇ 200mM NaCl buffer to wash the column, and transfer the supernatant of the broken solution to the column, use 20mM ⁇ 50mM Tris, pH7.0 ⁇ 8.5, 100mM ⁇ 200mM NaCl, 30mM imidazole buffer to wash away unbound impurities, and then use 20mM ⁇ 50mM Tris, pH7.0 ⁇ 8.5, 0 ⁇ 200mM NaCl, 50mM ⁇ 500mM imidazole buffer to elute the target protein.
  • SP550C (Borgron) uses 5 column volumes of 20mM-50mM acetic acid-sodium acetate, pH3.0-5.0 buffer to wash the column, and the cell is resuspended in 20mM-50mM acetic acid-sodium acetate, pH3.0-5.0 buffer After crushing, the supernatant of the crushed solution was applied to the column, and the unbound impurities were washed away with 20mM-50mM acetic acid-sodium acetate, pH3.0-5.0, 100mM-500mM NaCl buffer, imidazole buffer, and 20mM-50mM acetic acid-sodium acetate , PH3.0 ⁇ 5.0, 500mM ⁇ 1M NaCl buffer to elute the target protein.
  • the sample obtained by ion exchange chromatography was tested by SDS-PAGE, and the result is shown in Figure 5C.
  • HisTrap FF column uses 5 column volumes of 20mM-50mM Tris, pH 7.0-8.5, 0-200mM NaCl buffer to wash the column, and transfer the MBP-BefA crushing liquid supernatant to the column, use 20mM-50mM Tris, pH7 .0 ⁇ 8.5, 0 ⁇ 200mM NaCl, 10 ⁇ 100mM imidazole buffer to wash away unbound impurities, and then use 20mM ⁇ 50mM Tris, pH7.0 ⁇ 8.5, 0 ⁇ 200mM NaCl, 200mM ⁇ 500mM imidazole buffer to remove the target protein MBP-BefA eluted.
  • the MBP-BefA protein was dialyzed and changed to 20mM-50mM Tris, pH7.4-8.5 buffer.
  • 1mg recombinant TEV protease can completely cleave 100mg MBP-BefA fusion protein at 4°C ⁇ 30°C, 20mM ⁇ 50mM Tris, pH7.0 ⁇ 8.5, 0 ⁇ 0.5mM EDTA, 0 ⁇ 1M DTT for 4 ⁇ 15h.
  • HisTrap FF column uses 5 column volumes of 20 mM-50 mM Tris, pH 7.0-8.5 buffer to wash the column, apply MBP-BefA digestion sample to the column, and collect the penetrating solution for ion exchange chromatography.
  • Q Sepharose FF (Borgron) uses 5 column volumes of 20mM-50mM Tris, pH7.0-8.5 buffer to wash the column, put the affinity chromatography two penetrating solution on the column, and collect the penetrating solution to obtain BefA mutant .
  • the results of SDS-PAGE detection and MS analysis of the purified sample are shown in Figures 6A and 6B and Table 2.
  • the purified sample (about 25KD) has only one band, indicating that the purity of the protein obtained is very high; according to Figure 6B, it can be seen that the molecular weight of the purified sample is 25724, which is similar to the BefA mutation. The theoretical molecular weight is consistent, indicating that the purified sample is a BefA mutant; according to Table 2, it can be seen that the purified BefA mutant has a purity of about 99%.
  • the purified BefA protein mutant has less degradation and higher protein purity. It shows that the BefA protein mutant has better stability.
  • INS-1 rat islet cell tumor cells
  • INS-1 rat islet cell tumor cells
  • INS-1 cells were plated at 3*10 4 to 6*10 4 cells/mL at 100 ⁇ L/well to a 96-well plate, and cultured at 37°C, 5% CO 2 for 48 hours. Change the medium to sugar-free 1640+0.1% BSA, starve for 24h. The starvation medium was discarded, and the BefA protein (or BefA modification of BefA mutant BefA modification) diluted to 10 -9 M to 10 -7 M was added with 1640+1% FBS medium to stimulate, 100 ⁇ L/well. Incubate at 37°C with 5% CO 2 for 72 hours.
  • Ob/ob mice were divided into 2 groups, 8 animals in each group, and 30 mg/kg BefA protein and PBS buffer were injected subcutaneously once a day for 4 weeks. Oral sugar was administered on the 7th day of administration. Tolerance OGTT test, that is, animals are fasted for 16h, and glucose 2g/kg is given by gavage at the same time of D7 administration. The blood glucose level of animals is measured before glucose administration and 15min, 30min, 1h and 2h after glucose administration. During the experiment, the change of animal body weight was monitored. On the day of the end of the experiment, blood was collected from the animal to prepare plasma to detect the plasma ALT/AST level, and the animal was dissected and its liver was weighed.
  • FIG. 9-13 The effect of BefA protein on the glucose tolerance, body weight, liver weight, liver index ALT and AST of ob/ob mice is shown in Figure 9-13.
  • the results in Figure 9 show that the test results show that BefA protein can significantly reduce the blood glucose level after glucose administration and improve glucose tolerance;
  • the results of Figures 10-11 show that BefA protein has a significant effect on reducing the body weight of ob/ob mice on different days. And it also has a significant reduction effect on the absolute weight of the liver;
  • the results of Figures 12-13 show that the BefA protein significantly reduces the liver function indexes ALT and AST of ob/ob mice.
  • Spontaneously diabetic db/db mice were divided into 2 groups, 8 animals in each group, were injected subcutaneously with 30 mg/kg of BefA mutant and the control group were injected with PBS buffer subcutaneously for 4 weeks, of which the administration was tested on the first day of the experiment Random blood glucose before and 1, 2, 4, 6, 8 and 24h after administration. During the experiment, the change of animal body weight was monitored. On the day of the end of the experiment, blood was collected from the animal to prepare plasma to detect the plasma ALT/AST level. See Figure 14-18.
  • the results in Figure 14 show that the BefA mutant can significantly reduce the animal's random blood glucose levels 4-6h after the administration on the first day of administration; the results in Figures 15-16 show that the BefA mutant has a certain reduction in the body weight of db/db mice Trend, has a significant reduction effect on the absolute weight of the liver; Figure 17-18 results show: BefA mutants have a significant reduction in the liver function indexes ALT and AST of db/db mice.
  • Both BefA protein and BefA mutants can significantly promote the proliferation of INS-1 cells, significantly reduce blood sugar or improve glucose tolerance; BefA protein and BefA mutants can reduce body weight or have a tendency to reduce weight; BefA protein and BefA mutants are both It can lower the absolute liver of animals and lower the level of liver ALT/AST. Both BefA protein and BefA mutants can lower blood sugar, reduce body weight and have liver function protection functions, that is, BefA mutants retain the pharmacological activity of BefA protein.

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Abstract

Disclosed is a polypeptide. The polypeptide has a modification at the N-terminus compared with polypeptides having the following amino acid sequences: (1) a polypeptide having the amino acid sequences represented by SEQ ID NOs: 1 to 6; or (2) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to (1); or (3) a polypeptide with substitution, deletion and/or addition of one or more amino acids compared with (1), wherein the modification comprises at least one modification selected from addition, methylation, formylation, carbamylation, succinylation, cyclization, lysine propionylation, hexadecanoylation, myristylation, and acetylation of N-terminal amino acids. The polypeptide not only maintains the activity of BefA proteins to promote the proliferation of beta-cells, but also has the characteristics of higher stability and difficult degradation. Furthermore, the polypeptide also has the potential to lower glucose, promote weight loss, and protect the liver.

Description

多肽及其应用Peptides and their applications
优先权信息Priority information
本发明请求于2020年1月2日向中国国家知识产权局提交的、专利申请号CN202010003240.2、申请名称为“多肽及其应用”的中国专利申请的优先权,并且其全部内容通过引用并入本发明。The present invention requests the priority of the Chinese patent application filed with the State Intellectual Property Office of China on January 2, 2020, with the patent application number CN202010003240.2 and the application name "polypeptide and its application", and the entire content of which is incorporated by reference this invention.
技术领域Technical field
本发明涉及生物医药领域,具体地,本发明涉及多肽及其应用,更具体地,本发明涉及多肽、提高BefA蛋白稳定性或制备BefA蛋白突变体的方法、融合蛋白、核酸、重组细胞和药物组合物及其在糖尿病等代谢疾病领域的应用。The present invention relates to the field of biomedicine. Specifically, the present invention relates to polypeptides and their applications. More specifically, the present invention relates to polypeptides, methods for improving BefA protein stability or preparing BefA protein mutants, fusion proteins, nucleic acids, recombinant cells and drugs The composition and its application in the field of metabolic diseases such as diabetes.
背景技术Background technique
胰岛素分泌细胞的缺失一直被认为是I型糖尿病的病因之一。在I型糖尿病中,免疫系统错误地攻击和破坏β细胞。近年来,研究者发现,缺乏具有功能的β细胞也是II型糖尿病的一个重要诱因,II型糖尿病病人晚期胰岛功能逐渐下降。因此,开发能够增加健康β细胞数量的药物对于解决I型糖尿病或II型糖尿病晚期是非常重要的,是糖尿病研究的主要重点。同时,糖尿病病人常伴随着肥胖、脂肪肝、肝功能受损等代谢综合征,所以,开发临床获益更多的药物,如减重,保护肝功等的药物,也是非常必要的。目前市场上的降糖生物药主要包括:胰岛素及其类似物、GLP-1和GLP-1类似物,如利拉鲁肽,杜拉糖肽。胰岛素及其类似物作用机制是一个针对I型糖尿病的直接补充胰岛素(或类似物)作用,达到胰岛素降糖的作用,但是存在低血糖风险,且其没有降低2型糖尿病患者体重,保护肝功能的作用。GLP-1及其类似物,是GLP-1的受体激动剂,通过激动GLP-1的受体而发挥降糖作用。GLP-1的作用方式是增加胰岛素的分泌,抑制高血糖素的分泌、延缓胃排空,减少进食量。但该类药物半衰期短,有恶心、呕吐等消化道不良反应。以上药物,虽能起到降糖作用,但是无法完全逆转受损的胰岛,患者往往需要长期给药,增加了医药支出和降低了患者用药体验,长期服药也有可能导致药物耐受现象。The lack of insulin secreting cells has always been considered as one of the causes of type I diabetes. In Type I diabetes, the immune system mistakenly attacks and destroys beta cells. In recent years, researchers have found that the lack of functional β cells is also an important cause of type II diabetes, and the function of pancreatic islets in patients with type II diabetes gradually declines in the late stage. Therefore, the development of drugs that can increase the number of healthy β-cells is very important for solving the late stage of type I diabetes or type II diabetes, and is the main focus of diabetes research. At the same time, diabetic patients are often accompanied by metabolic syndromes such as obesity, fatty liver, and liver function impairment. Therefore, it is very necessary to develop drugs with more clinical benefits, such as weight loss and liver function protection drugs. The current hypoglycemic biologic drugs on the market mainly include: insulin and its analogs, GLP-1 and GLP-1 analogs, such as liraglutide and dulaglutide. The mechanism of action of insulin and its analogues is a direct supplementation of insulin (or analogues) for type I diabetes to achieve the effect of insulin lowering blood sugar, but there is a risk of hypoglycemia, and it does not reduce the weight of type 2 diabetes patients and protect liver function The role of. GLP-1 and its analogues are receptor agonists of GLP-1, which exert a hypoglycemic effect by stimulating the receptor of GLP-1. The mode of action of GLP-1 is to increase the secretion of insulin, inhibit the secretion of glucagon, delay gastric emptying, and reduce food intake. However, these drugs have a short half-life and have adverse gastrointestinal reactions such as nausea and vomiting. Although the above drugs can reduce blood sugar, they cannot completely reverse the damaged pancreatic islets. Patients often need long-term medication, which increases medical expenditure and reduces the patient's medication experience. Long-term medication may also lead to drug tolerance.
为了改善现有降糖药物的治疗效果,减少副作用,开发出具有改善或者逆转受损胰岛组织的药物,从根本上逆转糖尿病的发展,显得尤为关键,治疗糖尿病的药物仍需要进一步开发。In order to improve the therapeutic effect of the existing hypoglycemic drugs and reduce the side effects, the development of drugs capable of improving or reversing the damaged islet tissue, and fundamentally reversing the development of diabetes, appears to be particularly critical. Drugs for the treatment of diabetes still need to be further developed.
Jennifer Hampton Hill等在研究斑马鱼早期幼虫发育过程中,发现胰腺β细胞群的正常扩增需要肠道微生物群,而特定的细菌可以恢复β细胞数量到正常水平。经过实验筛查发现这 些细菌共享一类以前未被描述的蛋白质,命名为BefA(β细胞增殖因子A)。BefA蛋白可以促进斑马鱼幼虫发育过程中β细胞增殖的活性,故对BefA蛋白进行研究。Jennifer Hampton Hill et al. studied the development of early zebrafish larvae and found that the normal expansion of pancreatic β-cell population requires intestinal microbiota, and specific bacteria can restore the number of β-cells to a normal level. Experimental screening revealed that these bacteria share a previously undescribed protein named BefA (β-cell proliferation factor A). BefA protein can promote the activity of β-cell proliferation during the development of zebrafish larvae, so BefA protein was studied.
发明内容Summary of the invention
本申请是基于发明人对以下事实和问题的发现和认识作出的:This application is based on the inventor's discovery and understanding of the following facts and problems:
现有技术中,体外原核细胞表达的BefA蛋白具有促进β细胞增殖的活性,但BefA容易降解,难以得到高纯度的完整分子量的BefA蛋白,因此大大限制了BefA蛋白的体内外药效研究以及临床应用。本申请的研发人员在BefA蛋白药物的开发过程中意外而惊喜地发现,对BefA蛋白的N端进行修饰,包括在N端增加氨基酸,或使N端氨基酸甲基化、甲酰化、氨甲酰化、琥珀酰化、环化、丙酰化、十六烷酰化、十四烷化和乙酰化,可大幅提高BefA蛋白的稳定性,纯化后BefA蛋白纯度可达95%以上,且获得的该BefA蛋白突变体具有促进β细胞增殖的活性。In the prior art, BefA protein expressed by prokaryotic cells in vitro has the activity of promoting β cell proliferation, but BefA is easily degraded, and it is difficult to obtain high-purity and complete molecular weight BefA protein, which greatly limits the in vivo and in vitro drug efficacy research and clinical application of BefA protein. application. During the development process of BefA protein drugs, the research and development personnel of the present application were surprised and pleasantly surprised to find that the N-terminus of BefA protein was modified, including adding amino acids at the N-terminus, or making the N-terminus amino acids methylated, formylated, or carbamoyl. Acylation, succinylation, cyclization, propionylation, hexadecanoylation, tetradecylation and acetylation can greatly improve the stability of BefA protein. After purification, the purity of BefA protein can reach more than 95% and obtain The BefA protein mutant of has the activity of promoting the proliferation of β cells.
为此,在本发明的第一方面,本发明提出了一种多肽。根据本发明的实施例,所述与具有下列氨基酸序列的多肽相比,其N端具有修饰,所述修饰包括选自N端氨基酸的增加、甲基化、甲酰化、氨甲酰化、琥珀酰化、环化、丙酰化、十六烷酰化、十四烷化和乙酰化的至少之一。For this reason, in the first aspect of the present invention, the present invention proposes a polypeptide. According to an embodiment of the present invention, compared with the polypeptide having the following amino acid sequence, the N-terminal has a modification, and the modification includes an increase in the N-terminal amino acid, methylation, formylation, carbamylation, At least one of succinylation, cyclization, propionylation, hexadecanoylation, tetradecylation, and acetylation.
(1)具有SEQ ID NO:1-6所示氨基酸序列的多肽;或(1) A polypeptide having the amino acid sequence shown in SEQ ID NO: 1-6; or
(2)与(1)相比具有至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少99%同一性的多肽;或(2) A polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity compared with (1); or
(3)与(1)相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽。(3) Compared with (1), a polypeptide having one or more amino acid substitutions, deletions and/or additions.
Figure PCTCN2020142229-appb-000001
Figure PCTCN2020142229-appb-000001
Figure PCTCN2020142229-appb-000002
Figure PCTCN2020142229-appb-000002
根据本发明实施例的多肽保持了BefA蛋白促进β细胞增殖的活性,相比于具有(1)、(2)或(3)序列的多肽,具有更高的稳定性。The polypeptide according to the embodiment of the present invention maintains the activity of the BefA protein to promote β cell proliferation, and has higher stability than the polypeptide having the sequence (1), (2) or (3).
根据本发明的实施例,上述多肽还可以进一步包括如下附加技术特征至少之一:According to an embodiment of the present invention, the above-mentioned polypeptide may further include at least one of the following additional technical features:
根据本发明的实施例,所述N端氨基酸的增加包括N端增加一个或一个以上的氨基酸,且增加的氨基酸至少有一个甘氨酸、丙氨酸、异亮氨酸、甲硫氨酸、缬氨酸、丝氨酸、苏氨酸、谷氨酸、组氨酸或脯氨酸;优选甘氨酸、丙氨酸、甲硫氨酸、缬氨酸或异亮氨酸;更优选甘氨酸。According to an embodiment of the present invention, the increase of the N-terminal amino acid includes the addition of one or more amino acids at the N-terminal, and the added amino acid includes at least one of glycine, alanine, isoleucine, methionine, and valine. Acid, serine, threonine, glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine.
根据本发明的实施例,所述的N端氨基酸的增加为N端增加一个氨基酸,所述氨基酸为甘氨酸、丙氨酸、异亮氨酸、甲硫氨酸、缬氨酸、丝氨酸、苏氨酸、谷氨酸、组氨酸或脯氨酸;优选甘氨酸、丙氨酸、甲硫氨酸、缬氨酸或异亮氨酸;更优选甘氨酸。According to an embodiment of the present invention, the increase of the N-terminal amino acid is an increase of an amino acid at the N-terminal, and the amino acid is glycine, alanine, isoleucine, methionine, valine, serine, and threonine. Acid, glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine.
根据本发明的实施例,所述多肽具有SEQ ID NO:7所示的氨基酸序列。According to an embodiment of the present invention, the polypeptide has the amino acid sequence shown in SEQ ID NO:7.
Figure PCTCN2020142229-appb-000003
Figure PCTCN2020142229-appb-000003
根据本发明实施例的上述具有SEQ ID NO:7所示的氨基酸序列的多肽,稳定性高、不易 降解,纯化后的纯度可达99%。The above-mentioned polypeptide having the amino acid sequence shown in SEQ ID NO: 7 according to the embodiment of the present invention has high stability, is not easy to be degraded, and the purity after purification can reach 99%.
在本发明的第二方面,本发明提出了一种提高BefA蛋白稳定性或制备BefA蛋白突变体的方法。根据本发明的实施例,对BefA蛋白氨基酸序列的N端进行修饰,所述修饰包括选自N端氨基酸的增加、甲基化、甲酰化,氨甲酰化、琥珀酰化、环化、丙酰化、十六烷酰化、十四烷化和乙酰化的至少之一。根据本发明实施例的方法大幅提高了BefA蛋白的稳定性,且制备得到的BefA蛋白突变体不仅稳定性高,又保持了BefA蛋白显著促进β细胞增殖的活性。In the second aspect of the present invention, the present invention proposes a method for improving the stability of BefA protein or preparing BefA protein mutants. According to an embodiment of the present invention, the N-terminus of the amino acid sequence of the BefA protein is modified, and the modification includes an increase in the N-terminus amino acid, methylation, formylation, carbamylation, succinylation, cyclization, At least one of propionylation, hexadecanoylation, tetradecylation and acetylation. The method according to the embodiment of the present invention greatly improves the stability of the BefA protein, and the prepared BefA protein mutant not only has high stability, but also maintains the activity of the BefA protein to significantly promote the proliferation of β cells.
根据本发明的实施例,上述方法还可以进一步包括如下附加技术特征至少之一:According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的实施例,所述BefA蛋白具有(1)SEQ ID NO:1~6所示氨基酸序列;或(2)与(1)相比具有至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少99%同一性的;或(3)与(1)相比具有一个或者多个氨基酸的取代、缺失和/或添加的氨基酸序列。According to an embodiment of the present invention, the BefA protein has an amino acid sequence shown in (1) SEQ ID NO: 1 to 6; or (2) compared with (1), it has at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 99% identical; or (3) an amino acid sequence having one or more amino acid substitutions, deletions and/or additions compared to (1).
根据本发明的实施例,使BefA蛋白氨基酸序列的N端增加一个或一个以上的氨基酸可通过基因克隆的方式实现,以便获得BefA蛋白突变体。例如,本领域技术人员可通过预先合成能够表达N端增加一个或一个以上的氨基酸的BefA蛋白突变体的核酸,进而将该核酸导入受体细胞中进行表达,获得N端增加一个或一个以上氨基酸的BefA蛋白突变体。根据本发明再一实施例,所述BefA蛋白突变体还可进一步通过连接肽与伴侣蛋白连接,所述连接肽适于被蛋白酶切割,以便再次形成游离的BefA蛋白突变体。BefA蛋白突变体进一步通过连接肽与伴侣蛋白连接,可显著提高了BefA蛋白突变体的产量,但之后还需要再在适宜蛋白酶的作用下,将伴侣蛋白和连接肽完全切除,以再次形成游离的BefA蛋白突变体。According to an embodiment of the present invention, adding one or more amino acids to the N-terminus of the BefA protein amino acid sequence can be achieved by gene cloning, so as to obtain BefA protein mutants. For example, those skilled in the art can pre-synthesize a nucleic acid capable of expressing a BefA protein mutant with one or more amino acids added to the N-terminus, and then introduce the nucleic acid into the recipient cell for expression, and obtain one or more amino acids added to the N-terminus. BefA protein mutants. According to another embodiment of the present invention, the BefA protein mutant can be further connected to a chaperone protein through a connecting peptide, and the connecting peptide is suitable for being cleaved by a protease, so as to form a free BefA protein mutant again. The BefA protein mutant is further connected to the chaperone protein through the connecting peptide, which can significantly increase the yield of the BefA protein mutant, but afterwards, the chaperone protein and the connecting peptide need to be completely excised under the action of a suitable protease to form a free one again. BefA protein mutant.
根据本发明的实施例,使BefA蛋白氨基酸序列的N端增加一个或一个以上的氨基酸可通过基因克隆和酶切引入的方式实现,以便获得BefA蛋白突变体。例如,本领域技术人员可通过预先合成能够表达N端增加了部分氨基酸的第一BefA蛋白突变体的核酸,进而将该核酸导入受体细胞中进行表达,获得N端增加了部分氨基酸的第一BefA蛋白突变体;将该第一BefA蛋白突变体进一步通过连接肽与伴侣蛋白连接,此时伴侣蛋白和连接肽需要设置于第一BefA蛋白突变体的N端,所述连接肽适于被蛋白酶切割,进而在适宜蛋白酶的酶切作用下,将伴侣蛋白和部分连接肽切除,以便在第一BefA蛋白突变体的N端再多保留部分氨基酸,此时,酶切后剩余的部分连接肽以及最先通过基因克隆方式增加的部分氨基酸共同构成了BefA蛋白的N端增加的氨基酸。According to an embodiment of the present invention, adding one or more amino acids to the N-terminus of the BefA protein amino acid sequence can be achieved by gene cloning and restriction enzyme digestion, so as to obtain BefA protein mutants. For example, those skilled in the art can pre-synthesize nucleic acid capable of expressing the first BefA protein mutant with some amino acids at the N-terminus, and then introduce the nucleic acid into recipient cells for expression, to obtain the first protein mutant with some amino acids at the N-terminus. BefA protein mutant; the first BefA protein mutant is further connected to the chaperone protein through a connecting peptide. At this time, the chaperone protein and the connecting peptide need to be set at the N-terminus of the first BefA protein mutant, and the connecting peptide is suitable for protease Cleavage, and then under the action of suitable protease cleavage, the chaperone protein and part of the connecting peptide are excised, so as to retain some amino acids at the N-terminus of the first BefA protein mutant. At this time, the remaining part of the connecting peptide and The first part of the amino acids added by gene cloning together constitute the added amino acids at the N-terminus of BefA protein.
根据本发明的实施例,使BefA蛋白氨基酸序列的N端增加一个或一个以上的氨基酸也可通过酶切引入的方式实现。例如,本领域技术人员可直接构建伴侣蛋白与所述BefA蛋白的融合蛋白,所述伴侣蛋白与所述BefA蛋白之间设置有连接肽,所述连接肽的N端与所述伴侣蛋白的C端连接,所述连接肽的C端与所述BefA蛋白的N端连接,所述连接肽适于被 蛋白酶切割,以便形成游离的目标蛋白,所述游离的目标蛋白的N端相比于所述BefA蛋白,增加了一个或多个氨基酸,该游离的目标蛋白即为BefA蛋白突变体。也就是说游离的目标蛋白的N端所增加的氨基酸为连接肽上的至少部分氨基酸。换句话说,通过引入了具有酶切位点的连接肽的方式在BefA蛋白的N端引入了新的氨基酸。According to an embodiment of the present invention, adding one or more amino acids to the N-terminus of the amino acid sequence of the BefA protein can also be achieved by introducing restriction enzymes. For example, those skilled in the art can directly construct a fusion protein of a chaperone protein and the BefA protein, a connecting peptide is arranged between the chaperone protein and the BefA protein, and the N-terminal of the connecting peptide is connected to the C of the chaperone protein. The C-terminus of the connecting peptide is connected to the N-terminus of the BefA protein. The connecting peptide is suitable for being cleaved by a protease to form a free target protein. The BefA protein has one or more amino acids added, and the free target protein is the BefA protein mutant. That is to say, the amino acids added to the N-terminus of the free target protein are at least part of the amino acids on the connecting peptide. In other words, a new amino acid was introduced at the N-terminus of the BefA protein by introducing a connecting peptide with a restriction site.
根据本发明实施例的上述使BefA蛋白氨基酸序列的N端增加一个或一个以上的氨基酸的方法,成功实现了在BefA蛋白的N端连接至少一个氨基酸。According to the above-mentioned method of adding one or more amino acids to the N-terminus of the BefA protein amino acid sequence according to the embodiment of the present invention, at least one amino acid is successfully connected to the N-terminus of BefA protein.
根据本发明的实施例,所述伴侣蛋白包括选自麦芽糖结合蛋白、CBP、GST、SUMO、Trx、NusA的至少之一,任选地,所述伴侣蛋白含有或不含有His标签、Flag标签。According to an embodiment of the present invention, the chaperone protein includes at least one selected from maltose binding protein, CBP, GST, SUMO, Trx, and NusA. Optionally, the chaperone protein contains or does not contain a His tag and a Flag tag.
根据本发明的实施例,所述蛋白酶为烟草蚀纹病毒的半胱氨酸蛋白酶、肠激酶、凝血酶、Xa因子蛋白酶、人鼻病毒3C蛋白酶、SUMO酶或KEX-2。According to an embodiment of the present invention, the protease is cysteine protease, enterokinase, thrombin, factor Xa protease, human rhinovirus 3C protease, SUMO enzyme or KEX-2 of tobacco etch virus.
根据本发明的实施例,所述融合蛋白具有SEQ ID NO:8所示的氨基酸序列。According to an embodiment of the present invention, the fusion protein has the amino acid sequence shown in SEQ ID NO: 8.
Figure PCTCN2020142229-appb-000004
Figure PCTCN2020142229-appb-000004
在本发明的第三方面,本发明提出了一种融合蛋白。根据本发明的实施例,所述融合蛋白包括伴侣蛋白、连接肽以及BefA蛋白,所述连接肽的N端与所述伴侣蛋白的C端相连,所述连接肽的C端与所述BefA蛋白的N端相连,所述连接肽适于被蛋白酶切割,以便形成游离的BefA蛋白突变体,所述游离的BefA蛋白突变体的N端相比于BefA蛋白,增加了一个或多个氨基酸。根据本发明实施例的融合蛋白表达量高,且在适宜消化酶的作用下,可成功获得N端至少增加一个氨基酸的BefA蛋白突变体。In the third aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein includes a chaperone protein, a connecting peptide, and a BefA protein, the N-terminus of the connecting peptide is connected to the C-terminus of the chaperone protein, and the C-terminus of the connecting peptide is connected to the BefA protein. The connecting peptide is suitable for being cleaved by a protease to form a free BefA protein mutant. Compared with the BefA protein, the N-terminus of the free BefA protein mutant has one or more amino acids added. The fusion protein according to the embodiment of the present invention has a high expression level, and under the action of a suitable digestive enzyme, a BefA protein mutant with at least one amino acid added to the N-terminal can be successfully obtained.
根据本发明的实施例,所述融合蛋白还可以进一步包括如下附加技术特征至少之一:According to an embodiment of the present invention, the fusion protein may further include at least one of the following additional technical features:
根据本发明的实施例,所述伴侣蛋白包括选自麦芽糖结合蛋白、CBP、GST、SUMO、Trx、NusA的至少之一,任选地,所述伴侣蛋白含有或不含有His标签、Flag标签。According to an embodiment of the present invention, the chaperone protein includes at least one selected from maltose binding protein, CBP, GST, SUMO, Trx, and NusA. Optionally, the chaperone protein contains or does not contain a His tag and a Flag tag.
根据本发明的实施例,所述蛋白酶为烟草蚀纹病毒的半胱氨酸蛋白酶、肠激酶、凝血酶、 Xa因子蛋白酶、人鼻病毒3C蛋白酶、SUMO酶或KEX-2。According to an embodiment of the present invention, the protease is cysteine protease, enterokinase, thrombin, factor Xa protease, human rhinovirus 3C protease, SUMO enzyme or KEX-2 of tobacco etch virus.
根据本发明的实施例,所述融合蛋白具有SEQ ID NO:8所示的氨基酸序列。According to an embodiment of the present invention, the fusion protein has the amino acid sequence shown in SEQ ID NO: 8.
在本发明的第四方面,本发明提出了一种核酸。根据本发明的实施例,所述核酸编码前面所述的多肽或前面所述的融合蛋白。In the fourth aspect of the present invention, the present invention provides a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the aforementioned polypeptide or the aforementioned fusion protein.
根据本发明的实施例,所述核酸还可以进一步包括如下附加技术特征至少之一:According to an embodiment of the present invention, the nucleic acid may further include at least one of the following additional technical features:
根据本发明的实施例,所述核酸具有SEQ ID NO:9所示的核苷酸序列或SEQ ID NO:10所示的核苷酸序列。According to an embodiment of the present invention, the nucleic acid has the nucleotide sequence shown in SEQ ID NO: 9 or the nucleotide sequence shown in SEQ ID NO: 10.
Figure PCTCN2020142229-appb-000005
Figure PCTCN2020142229-appb-000005
Figure PCTCN2020142229-appb-000006
Figure PCTCN2020142229-appb-000006
具有SEQ ID NO:9所示的核苷酸序列的核酸编码具有SEQ ID NO:7所示的氨基酸序列的多肽,具有SEQ ID NO:10所示的核苷酸序列的核酸编码具有SEQ ID NO:8所示的氨基酸序列的融合蛋白。The nucleic acid with the nucleotide sequence shown in SEQ ID NO: 9 encodes a polypeptide with the amino acid sequence shown in SEQ ID NO: 7, and the nucleic acid encoding with the nucleotide sequence shown in SEQ ID NO: 10 has SEQ ID NO : A fusion protein of the amino acid sequence shown in 8.
在本发明的第五方面,本发明提出了一种重组细胞。根据本发明的实施例,所述重组细胞携带前面所述的核酸。根据本发明实施例的重组细胞可表达前面所述的多肽或融合蛋白。In the fifth aspect of the present invention, the present invention proposes a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the aforementioned nucleic acid. The recombinant cell according to the embodiment of the present invention can express the aforementioned polypeptide or fusion protein.
根据本发明的实施例,上述重组细胞还可以进一步包括如下附加技术特征至少之一:According to an embodiment of the present invention, the aforementioned recombinant cell may further include at least one of the following additional technical features:
根据本发明的实施例,所述重组细胞为大肠杆菌、酵母菌或哺乳动物细胞。According to an embodiment of the present invention, the recombinant cell is Escherichia coli, yeast or mammalian cell.
在本发明的第六方面,本发明提出了一种药物组合物。根据本发明的实施例,所述药物组合物包括前面所述的多肽。根据本发明实施例的药物组合物可用于糖尿病的治疗或预防。In the sixth aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the aforementioned polypeptide. The pharmaceutical composition according to the embodiment of the present invention can be used for the treatment or prevention of diabetes.
根据本发明的实施例,上述药物组合物还可以进一步包括如下附加技术特征至少之一:According to an embodiment of the present invention, the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features:
根据本发明的实施例,所述药物组合物进一步包括药学上可接受的辅剂。进而所述药物组合物可制备成特定的药物制剂,通过特定的方式给与患者。According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable auxiliary agent. Furthermore, the pharmaceutical composition can be prepared into a specific pharmaceutical preparation and administered to a patient in a specific manner.
在本发明的第七方面,本发明提出了前面所述的多肽或前面所述的药物组合物在制备药物中的用途,所述药物用于促进β细胞增殖。In the seventh aspect of the present invention, the present invention proposes the use of the aforementioned polypeptide or the aforementioned pharmaceutical composition in the preparation of a medicine, which is used to promote the proliferation of β cells.
在本发明的第八方面,本发明提出了前面所述的多肽或前面所述的药物组合物在制备药物中的用途,所述药物用于治疗或预防糖尿病或保护肝功。In the eighth aspect of the present invention, the present invention proposes the use of the aforementioned polypeptide or the aforementioned pharmaceutical composition in the preparation of medicines for treating or preventing diabetes or protecting liver function.
本发明所述的BefA蛋白是指β细胞增殖因子A(Beta Cell Expansion Factor A,BefA)。The BefA protein in the present invention refers to Beta Cell Expansion Factor A (Beta Cell Expansion Factor A, BefA).
附图说明Description of the drawings
图1是根据本发明实施例的重组表达质粒pET28a-BefA的示意图;Figure 1 is a schematic diagram of a recombinant expression plasmid pET28a-BefA according to an embodiment of the present invention;
图2是根据本发明实施例的pET28a-BefA阳性克隆PCR鉴定示意图;Figure 2 is a schematic diagram of PCR identification of pET28a-BefA positive clones according to an embodiment of the present invention;
图3是根据本发明实施例的重组表达质粒pET28a-MBP-BefA的示意图;Figure 3 is a schematic diagram of a recombinant expression plasmid pET28a-MBP-BefA according to an embodiment of the present invention;
图4是根据本发明实施例的pET28a-MBP-BefA阳性克隆PCR鉴定的示意图;Figure 4 is a schematic diagram of PCR identification of pET28a-MBP-BefA positive clones according to an embodiment of the present invention;
图5A是根据本发明实施例的BefA镍柱亲和层析样品SDS-PAGE图谱;Figure 5A is an SDS-PAGE profile of a BefA nickel column affinity chromatography sample according to an embodiment of the present invention;
图5B是根据本发明实施例的BefA镍柱亲和层析样品质谱检测结果;Fig. 5B is a mass spectrometry test result of a BefA nickel column affinity chromatography sample according to an embodiment of the present invention;
图5C是根据本发明实施例的BefA离子交换层析样品SDS-PAGE图谱;Fig. 5C is an SDS-PAGE chart of a BefA ion exchange chromatography sample according to an embodiment of the present invention;
图6A是根据本发明实施例的BefA突变体SDS-PAGE的示意图;Fig. 6A is a schematic diagram of SDS-PAGE of BefA mutant according to an embodiment of the present invention;
图6B是根据本发明实施例的BefA突变体质谱检测结果;Fig. 6B is a mass spectrometric detection result of BefA mutant according to an embodiment of the present invention;
图7是根据本发明实施例的BefA蛋白促INS-1细胞增殖的示意图;Fig. 7 is a schematic diagram of BefA protein promoting the proliferation of INS-1 cells according to an embodiment of the present invention;
图8是根据本发明实施例的BefA突变体促INS-1细胞增殖的示意图;Fig. 8 is a schematic diagram of BefA mutant promoting the proliferation of INS-1 cells according to an embodiment of the present invention;
图9是根据本发明实施例的注射BefA蛋白后ob/ob小鼠随机血糖变化曲线;Fig. 9 is a curve of random blood glucose change in ob/ob mice after injection of BefA protein according to an embodiment of the present invention;
图10是根据本发明实施例的注射BefA蛋白后ob/ob小鼠体重变化曲线;Fig. 10 is a curve of weight change of ob/ob mice after injection of BefA protein according to an embodiment of the present invention;
图11是根据本发明实施例的注射BefA蛋白后ob/ob小鼠肝绝对重量的示意图;11 is a schematic diagram of the absolute weight of ob/ob mouse liver after injection of BefA protein according to an embodiment of the present invention;
图12是根据本发明实施例的注射BefA蛋白后ob/ob小鼠谷草转氨酶(AST)活性的示意图;Figure 12 is a schematic diagram of aspartate aminotransferase (AST) activity in ob/ob mice after injection of BefA protein according to an embodiment of the present invention;
图13是根据本发明实施例的注射BefA蛋白后ob/ob小鼠谷丙转氨酶(ALT)活性的示意图;Figure 13 is a schematic diagram of alanine aminotransferase (ALT) activity in ob/ob mice after injection of BefA protein according to an embodiment of the present invention;
图14是根据本发明实施例的注射BefA蛋白突变体后db/db小鼠随机血糖变化曲线的示意图;14 is a schematic diagram of a random blood glucose change curve in db/db mice after injection of BefA protein mutant according to an embodiment of the present invention;
图15是根据本发明实施例的注射BefA蛋白突变体后db/db小鼠体重变化曲线;Fig. 15 is a curve of body weight change of db/db mice after injection of BefA protein mutant according to an embodiment of the present invention;
图16是根据本发明实施例的注射BefA蛋白突变体后db/db小鼠肝绝对重量的示意图;16 is a schematic diagram of the absolute weight of the liver of db/db mice after injection of BefA protein mutants according to an embodiment of the present invention;
图17是根据本发明实施例的注射BefA蛋白突变体后db/db小鼠谷丙转氨酶(ALT)活性的示意图;Figure 17 is a schematic diagram of alanine aminotransferase (ALT) activity in db/db mice after injection of BefA protein mutants according to an embodiment of the present invention;
图18是根据本发明实施例的注射BefA蛋白突变体后db/db小鼠谷草转氨酶(AST)活性的示意图。Figure 18 is a schematic diagram of the aspartate aminotransferase (AST) activity of db/db mice after injection of BefA protein mutants according to an embodiment of the present invention.
具体实施方式Detailed ways
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention, but should not be construed as limiting the present invention.
需要说明的是,如无特别说明,本申请所述的“肽键”是指在氨基酸相互连接形成蛋白质时,各个氨基酸之间相互连接的那个化学键,就称为肽键,换句话说,肽键是在两个氨基酸连接时,由一个氨基酸的羧基(-COOH)与另一个氨基酸的氨基(-NH 2)相互连接所形成的化学键(-NH-CO-)。多肽中以羧基结尾的端叫C端,以氨基结尾的端叫N端。 It should be noted that, unless otherwise specified, the "peptide bond" in this application refers to the chemical bond between each amino acid when the amino acids are connected to form a protein, which is called a peptide bond. In other words, a peptide The bond is a chemical bond (-NH-CO-) formed by connecting the carboxyl group (-COOH) of one amino acid and the amino group (-NH 2) of another amino acid when two amino acids are connected. The end of a polypeptide that ends with a carboxyl group is called the C-terminus, and the end that ends with an amino group is called the N-terminus.
本申请所述的多肽与多肽的“相连”或“连接”,既可以是直接相连,也可是间接相连。例如,当采用间接相连时,多肽与多肽之间可通过短肽或氨基酸相连。The "connection" or "connection" between the polypeptide and the polypeptide described in this application can be either a direct connection or an indirect connection. For example, when an indirect connection is used, the polypeptide and the polypeptide can be connected by short peptides or amino acids.
根据文献(Jennifer Hampton Hill,A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development,elifesciences,Dec.2016)的氨基酸序列,重组工程菌经诱导表达纯化得到的BefA蛋白出现严重的降解。According to the amino acid sequence of the literature (Jennifer Hampton Hill, A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development, elifesciences, Dec. 2016), the BefA protein obtained by the recombinant engineering bacteria after inducing expression and purification is severely degraded.
本发明克服了上述BefA蛋白易降解以及应用研究范围小等问题,提供了一种高纯度BefA蛋白的制备方法。即通过构建BefA融合蛋白,并在融合蛋白内设计蛋白酶切位点,具有形如A-B-C结构的基因序列,其中A为伴侣MBP(麦芽糖结合蛋白)蛋白基因,B为编码包含TEV(烟草蚀纹病毒的半胱氨酸蛋白酶)酶切位点和连接肽的核苷酸序列,C为BefA蛋白基因。重组工程菌经诱导表达后,融合蛋白纯化后经TEV蛋白酶酶切,可以获得N端多一个甘氨酸(G)的BefA突变体,纯化后蛋白纯度达95%以上,经检测具备生物活性。The invention overcomes the above-mentioned problems of easy degradation of BefA protein and small application research range, and provides a method for preparing high-purity BefA protein. That is, by constructing a BefA fusion protein and designing a protease cleavage site in the fusion protein, it has a gene sequence shaped like an ABC structure, where A is the chaperone MBP (maltose binding protein) protein gene, and B is the encoding containing TEV (tobacco etch virus). The nucleotide sequence of the cysteine protease enzyme cleavage site and the connecting peptide, C is the BefA protein gene. After the recombinant engineered bacteria is induced to express, the fusion protein is purified and cleaved with TEV protease to obtain a BefA mutant with a glycine (G) at the N-terminal. The purified protein has a purity of more than 95% and is tested to have biological activity.
BefA蛋白以及BefA突变体在体外均具有一定程度地促进INS-1(大鼠胰岛素瘤细胞)细胞增殖的作用,BefA蛋白能改善ob/ob小鼠的肝功能,且BefA突变体能改善db/db小鼠的肝功能。Both BefA protein and BefA mutant can promote the proliferation of INS-1 (rat insulinoma cells) to a certain extent in vitro. BefA protein can improve the liver function of ob/ob mice, and BefA mutant can improve db/db Liver function of mice.
实施例1 重组表达质粒pET28a-BefA,pET28a-MBP-B的构建Example 1 Construction of recombinant expression plasmid pET28a-BefA, pET28a-MBP-B
1、β细胞增殖因子A(BefA),MBP伴侣蛋白的全基因合成1. Full gene synthesis of β cell proliferation factor A (BefA), MBP chaperone protein
根据文献(Jennifer Hampton Hill,A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development,elifesciences,Dec.2016)公布的人肠道微生物Enterococcus gallinaru菌株分泌的BefA氨基酸序列(共258aa),按照大肠杆菌(BL21(DE3))的优化原则,对核酸序列进行优化。同时对序列中的酶切位点进行严格限定,序列3'端添加终止密码子(TAA),获得编码基因BefA,最终交给基因合成公司进行全基因合成(pUC57-BefA)。According to the literature (Jennifer Hampton Hill, A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development, elifesciences, Dec. 2016), the amino acid sequence of BefA secreted by the enterococcus gallinaru strain of human intestines (258aa in total), according to E. coli (BL21) (DE3)) optimize the nucleic acid sequence. At the same time, the restriction site in the sequence is strictly limited. A stop codon (TAA) is added to the 3'end of the sequence to obtain the coding gene BefA, which is finally handed over to the gene synthesis company for full gene synthesis (pUC57-BefA).
MBP伴侣基因与编码包含TEV(烟草蚀纹病毒的半胱氨酸蛋白酶)酶切位点和连接肽(共425aa),按照大肠杆菌(BL21(DE3))的优化原则,对核酸序列进行优化,获得编码基因MBP,最终交给基因合成公司进行全基因合成(pET28a-MBP)。The MBP chaperone gene and encoding contain TEV (Cysteine Protease of Tobacco Etch Virus) restriction site and connecting peptide (425aa in total). The nucleic acid sequence is optimized according to the optimization principle of Escherichia coli (BL21(DE3)), Obtain the coding gene MBP, and finally give it to the gene synthesis company for full gene synthesis (pET28a-MBP).
2、重组pET28a-BefA表达菌株构建2. Construction of recombinant pET28a-BefA expression strain
以合成的质粒pUC57-BefA为模板,通过上游引物F1:GCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAAGATCCGTTTTCTGG,含有XbaI酶切位点;下游引物R1:CCGCTCGAGTT AATGGTGATGATGATGATGAC,含有XhoI酶切位点和6×His标签。加入其它反应液进行PCR,扩增得到含有BefA的片段A(844bp)。片段A经XhoI和XbaI双酶切获得片段B(792bp),片段B与pET28a质粒经XhoI和XbaI双酶切回收的载体片段(5236bp)进行连接,得到质粒pET28a-BefA,见图1。质粒pET28a-BefA通过化学转化法转化至大肠杆菌BL21(DE3)。在卡那抗生素平板上对克隆进行抗性筛选,挑选的克隆,通过引物F1和R1为模板进行PCR扩增,得到与理论大小(792bp)相符的DNA片段,见图2。利用BefA基因片段的两端设计引物进行测序,测序结果显示克隆基因片段与理论一致。Using the synthetic plasmid pUC57-BefA as a template, the upstream primer F1: GCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAAGATCCGTTTTCTGG, containing the XbaI restriction site; the downstream primer R1: CCGCTCGAGTT AATGGTGATGATGATGATGAC, containing the XhoI Hi restriction tag site and 6×His restriction site. Add other reaction solution for PCR, and amplify fragment A (844bp) containing BefA. Fragment A was digested with XhoI and XbaI to obtain fragment B (792bp). Fragment B was ligated with the vector fragment (5236bp) recovered from pET28a plasmid by XhoI and XbaI digestion to obtain plasmid pET28a-BefA, as shown in Figure 1. The plasmid pET28a-BefA was transformed into E. coli BL21 (DE3) by chemical transformation method. The clones were screened for resistance on the Kana antibiotic plate, and the selected clones were amplified by PCR using primers F1 and R1 as templates to obtain a DNA fragment consistent with the theoretical size (792bp), as shown in Figure 2. Using the two ends of the BefA gene fragment to design primers for sequencing, the sequencing results showed that the cloned gene fragment was consistent with the theory.
3、重组pET28a-MBP-BefA表达菌株构建3. Construction of recombinant pET28a-MBP-BefA expression strain
以合成的质粒pET28a-MBP为模板,通过上游引物F2:CATGCCATGGAAATAAAAACAGGTGCACG,含有NcoI酶切位点;下游引物R2:GCTTTCACTTTTGCCCAATCTCCCTGAAAATACAGGTTTT。加入其它反应液进行PCR,扩增得到含有MBP的片段B(1301bp),以合成质粒pUC57-BefA为模板,通过上游引物F3:AAAACCTGTATTTTCAGGGAGATTGGGCAAAAGTGAAAGC,下游引物R3:CCGCTCGAGTTAACGGGTCAGATCTTTAT,含有XhoI酶切位点。加入其它反应液进行PCR,扩增得到含有BefA的片段C(743bp)。以PCR扩增获得的片段B和片段C为模板,通过上游引物F2,下游引物R3,加入其它反应液进行Overlap PCR,扩增得到含有MBP-BefA的片段(约2000bp)。片段MBP-BefA与pET28a质粒经NcoI和XhoI双酶切回收的载体片段(5231bp)进行连接,得到质粒pET28a-MBP-BefA,见图3。质粒pET28a-MBP-BefA通过化学转化法转化至大肠杆菌BL21(DE3),在卡那抗生素平板上对克隆进行抗性筛选,挑选的克隆,通过引物F2和R3为模板进行PCR扩增,得到与理论大小相符的DNA片段,见图4。利用MBP-BefA基因片段的两端设计引物进行测序,测序结果显示克隆基因片段与理论一致。Using the synthetic plasmid pET28a-MBP as a template, the upstream primer F2: CATGCCATGGAAATAAAAACAGGTGCACG, containing the NcoI restriction site; the downstream primer R2: GCTTTCACTTTTGCCCAATCTCCCTGAAAATACAGGTTTT. Add other reaction solution for PCR, and amplify fragment B (1301bp) containing MBP, using synthetic plasmid pUC57-BefA as template, using upstream primer F3: AAAACCTGTATTTTCAGGGAGATTGGGCAAAAGTGAAAGC, downstream primer R3: CCGCTCGAGTTAACGGGTCAGATCTTTAT, containing XhoI restriction site. Add other reaction solution for PCR, and amplify fragment C (743bp) containing BefA. Using the fragment B and fragment C obtained by PCR amplification as templates, the upstream primer F2, the downstream primer R3 are added to other reaction solutions to perform Overlap PCR, and a fragment (about 2000 bp) containing MBP-BefA is amplified. Fragment MBP-BefA is ligated with the vector fragment (5231bp) recovered by double digestion with NcoI and XhoI from pET28a plasmid to obtain plasmid pET28a-MBP-BefA, as shown in Figure 3. The plasmid pET28a-MBP-BefA was transformed into E. coli BL21(DE3) by chemical transformation method, and the clones were screened for resistance on the Kana antibiotic plate. The selected clones were amplified by PCR with primers F2 and R3 as templates, and The DNA fragments of the theoretical size are shown in Figure 4. The two ends of the MBP-BefA gene fragment were used to design primers for sequencing. The sequencing results showed that the cloned gene fragment was consistent with the theory.
实施例2 工程细胞表达生产BefA/BefA突变体Example 2 Expression and production of BefA/BefA mutants by engineered cells
1、摇瓶发酵BefA/MBP-BefA蛋白1. Shake bottle fermentation of BefA/MBP-BefA protein
以筛选与鉴定得到的阳性菌株甘油管,按0.2%接种量接种至5mL含50μg/mL卡那霉素LB液体培养基中,37℃250rpm活化过夜;按2%接种量接种至含50μg/mL卡那霉素LB液体培养基的2000mL三角瓶中,装液量20%,37℃250rpm培养至OD 600=0.6~0.8时,加入IPTG至终浓度为1mM,37℃250rpm诱导表达4~6h;发酵液经过离心(8000rpm 10min),收集菌体。菌体用纯化水重悬后,破碎菌体(超声波破碎或匀浆破碎),破碎液经过低温高速离心(12000rpm30min 4℃),获得上清液进行纯化。 Inoculate the glycerol tube of positive strains obtained by screening and identification into 5mL LB liquid medium containing 50μg/mL kanamycin at 0.2% inoculum, and activate overnight at 37°C at 250rpm; inoculate with 2% inoculum to contain 50μg/mL In a 2000mL Erlenmeyer flask of Kanamycin LB liquid medium, the volume of 20%, 37℃250rpm culture to OD 600 =0.6~0.8, add IPTG to a final concentration of 1mM, 37℃250rpm to induce expression for 4~6h; The fermentation broth was centrifuged (8000rpm 10min), and the bacteria were collected. After the cells are resuspended in purified water, the cells are crushed (ultrasonic or homogenized), and the crushed liquid is centrifuged at low temperature and high speed (12000 rpm, 30 min, 4° C.), and the supernatant is obtained for purification.
2、纯化BefA蛋白2. Purification of BefA protein
BefA蛋白使用镍柱亲和层析和离子交换层析,BefA蛋白均发生了降解,均未能得到纯 度较高的BefA蛋白。The BefA protein uses nickel column affinity chromatography and ion exchange chromatography, and the BefA protein has been degraded, and no high purity BefA protein can be obtained.
方法一:BefA破碎液上清镍柱亲和层析具体纯化步骤如下:Method 1: The specific purification steps of BefA crushing liquid supernatant nickel column affinity chromatography are as follows:
HisTrap FF column(GE)使用5个柱体积的20mM~50mM Tris,pH7.0~8.5,100mM~200mM NaCl缓冲液冲洗柱子,将破碎液上清上柱,使用20mM~50mM Tris,pH7.0~8.5,100mM~200mM NaCl,30mM咪唑缓冲液洗去未结合的杂质,接着用20mM~50mM Tris,pH7.0~8.5,0~200mM NaCl,50mM~500mM咪唑缓冲液将目的蛋白洗脱下来。HisTrap FF column (GE) uses 5 column volumes of 20mM~50mM Tris, pH7.0~8.5, 100mM~200mM NaCl buffer to wash the column, and transfer the supernatant of the broken solution to the column, use 20mM~50mM Tris, pH7.0~ 8.5, 100mM~200mM NaCl, 30mM imidazole buffer to wash away unbound impurities, and then use 20mM~50mM Tris, pH7.0~8.5, 0~200mM NaCl, 50mM~500mM imidazole buffer to elute the target protein.
镍柱亲和层析得到的样品进行SDS-PAGE检测和MS分析,其结果如图5A和图5B所示以及表1所示。根据图5A SDS-PAGE结果可以看出,纯化得到的样品除主条带(26KD)外,还有一些杂带;根据图5B可以看出主峰分子量为26489,与BefA蛋白理论分子量一致,说明纯化得到的样品主要成分是BefA蛋白。根据表1可以看出,纯化样品中BefA蛋白约占66%,其余大部分为BefA蛋白降解产物。说明使用镍柱亲和层析未能得到高纯度的BefA蛋白。The samples obtained by affinity chromatography on the nickel column were subjected to SDS-PAGE detection and MS analysis. The results are shown in Figure 5A and Figure 5B and Table 1 below. According to the results of SDS-PAGE in Figure 5A, it can be seen that in addition to the main band (26KD), the purified sample has some miscellaneous bands. According to Figure 5B, it can be seen that the molecular weight of the main peak is 26489, which is consistent with the theoretical molecular weight of BefA protein, indicating the purification. The main component of the obtained sample is BefA protein. According to Table 1, it can be seen that the BefA protein in the purified sample accounts for about 66%, and most of the rest are BefA protein degradation products. It shows that the use of nickel column affinity chromatography failed to obtain high-purity BefA protein.
表1:Table 1:
Figure PCTCN2020142229-appb-000007
Figure PCTCN2020142229-appb-000007
方法二:BefA破碎液上清阳离子交换层析具体纯化步骤如下:Method 2: The specific purification steps of BefA crushed liquid supernatant cation exchange chromatography are as follows:
SP550C(博格隆)使用5个柱体积的20mM~50mM乙酸-乙酸钠,pH3.0~5.0缓冲液冲洗柱子,菌体使用20mM~50mM乙酸-乙酸钠,pH3.0~5.0缓冲液重悬后破碎,将破碎液上清上柱,使用20mM~50mM乙酸-乙酸钠,pH3.0~5.0,100mM~500mM NaCl缓冲液咪唑缓冲液洗去未结合的杂质,用20mM~50mM乙酸-乙酸钠,pH3.0~5.0,500mM~1M NaCl缓冲液将目的蛋白洗脱下来。离子交换层析得到的样品进行SDS-PAGE检测,其结果如图5C所示。SP550C (Borgron) uses 5 column volumes of 20mM-50mM acetic acid-sodium acetate, pH3.0-5.0 buffer to wash the column, and the cell is resuspended in 20mM-50mM acetic acid-sodium acetate, pH3.0-5.0 buffer After crushing, the supernatant of the crushed solution was applied to the column, and the unbound impurities were washed away with 20mM-50mM acetic acid-sodium acetate, pH3.0-5.0, 100mM-500mM NaCl buffer, imidazole buffer, and 20mM-50mM acetic acid-sodium acetate , PH3.0~5.0, 500mM~1M NaCl buffer to elute the target protein. The sample obtained by ion exchange chromatography was tested by SDS-PAGE, and the result is shown in Figure 5C.
根据图5C可以看出,离子交换层析得到的样品,除主要条带(约25KD),还存在较多的杂带,说明纯化得到的BefA蛋白纯度较低,使用离子交换层析未能得到高纯度的BefA蛋白。 说明BefA蛋白稳定性较差。According to Figure 5C, it can be seen that in addition to the main band (about 25KD), there are more miscellaneous bands in the sample obtained by ion exchange chromatography, indicating that the purity of the purified BefA protein is low, which cannot be obtained by ion exchange chromatography. High purity BefA protein. It shows that BefA protein has poor stability.
3、纯化MBP-BefA蛋白突变体3. Purification of MBP-BefA protein mutants
MBP-BefA破碎液上清经过亲和层析一-酶切-亲和层析二-离子交换层析,得到BefA突变体,其结果见图6。具体纯化步骤如下:The supernatant of the MBP-BefA crushed liquid was subjected to affinity chromatography-enzyme digestion-affinity chromatography-two-ion exchange chromatography to obtain BefA mutants. The results are shown in Figure 6. The specific purification steps are as follows:
(1)亲和层析一(1) Affinity chromatography one
HisTrap FF column(GE)使用5个柱体积的20mM~50mM Tris,pH7.0~8.5,0~200mM NaCl缓冲液冲洗柱子,将MBP-BefA破碎液上清上柱,使用20mM~50mM Tris,pH7.0~8.5,0~200mM NaCl,10~100mM咪唑缓冲液洗去未结合的杂质,接着用20mM~50mM Tris,pH7.0~8.5,0~200mM NaCl,200mM~500mM咪唑缓冲液将目的蛋白MBP-BefA洗脱下来。将MBP-BefA蛋白透析换液至20mM~50mM Tris,pH7.4~8.5缓冲液。HisTrap FF column (GE) uses 5 column volumes of 20mM-50mM Tris, pH 7.0-8.5, 0-200mM NaCl buffer to wash the column, and transfer the MBP-BefA crushing liquid supernatant to the column, use 20mM-50mM Tris, pH7 .0~8.5, 0~200mM NaCl, 10~100mM imidazole buffer to wash away unbound impurities, and then use 20mM~50mM Tris, pH7.0~8.5, 0~200mM NaCl, 200mM~500mM imidazole buffer to remove the target protein MBP-BefA eluted. The MBP-BefA protein was dialyzed and changed to 20mM-50mM Tris, pH7.4-8.5 buffer.
(2)TEV酶酶切(2) TEV enzyme digestion
1mg重组TEV蛋白酶在4℃~30℃,20mM~50mM Tris,pH7.0~8.5,0~0.5mM EDTA,0~1M DTT条件下4~15h,可以完全切割100mg MBP-BefA融合蛋白。1mg recombinant TEV protease can completely cleave 100mg MBP-BefA fusion protein at 4℃~30℃, 20mM~50mM Tris, pH7.0~8.5, 0~0.5mM EDTA, 0~1M DTT for 4~15h.
(3)镍柱亲和层析二(3) Nickel column affinity chromatography 2
HisTrap FF column(GE)使用5个柱体积的20mM~50mM Tris,pH7.0~8.5缓冲液冲洗柱子,将MBP-BefA酶切样品上柱,收集穿透液进行离子交换层析。HisTrap FF column (GE) uses 5 column volumes of 20 mM-50 mM Tris, pH 7.0-8.5 buffer to wash the column, apply MBP-BefA digestion sample to the column, and collect the penetrating solution for ion exchange chromatography.
(4)离子交换层析(4) Ion exchange chromatography
Q Sepharose FF(博格隆)使用5个柱体积的20mM~50mM Tris,pH7.0~8.5缓冲液冲洗柱子,将亲和层析二穿透液上柱,收集穿透液,得到BefA突变体。纯化得到的样品进行SDS-PAGE检测和MS分析结果如图6A和6B以及表2所示。Q Sepharose FF (Borgron) uses 5 column volumes of 20mM-50mM Tris, pH7.0-8.5 buffer to wash the column, put the affinity chromatography two penetrating solution on the column, and collect the penetrating solution to obtain BefA mutant . The results of SDS-PAGE detection and MS analysis of the purified sample are shown in Figures 6A and 6B and Table 2.
根据图6A SDS-PAGE结果可以看出,纯化得到的样品(约25KD)仅有一条条带,说明得到的蛋白纯度很高;根据图6B可以看出纯化得到的样品分子量为25724,与BefA突变体理论分子量一致,说明纯化得到的样品为BefA突变体;根据表2可以看出,纯化得到的BefA突变体纯度约99%。According to the results of SDS-PAGE in Figure 6A, it can be seen that the purified sample (about 25KD) has only one band, indicating that the purity of the protein obtained is very high; according to Figure 6B, it can be seen that the molecular weight of the purified sample is 25724, which is similar to the BefA mutation. The theoretical molecular weight is consistent, indicating that the purified sample is a BefA mutant; according to Table 2, it can be seen that the purified BefA mutant has a purity of about 99%.
结论:与BefA蛋白相比,纯化得到的BefA蛋白突变体降解较少,蛋白纯度较高。说明BefA蛋白突变体稳定性较好。Conclusion: Compared with BefA protein, the purified BefA protein mutant has less degradation and higher protein purity. It shows that the BefA protein mutant has better stability.
表2:Table 2:
Figure PCTCN2020142229-appb-000008
Figure PCTCN2020142229-appb-000008
Figure PCTCN2020142229-appb-000009
Figure PCTCN2020142229-appb-000009
实施例3 BefA及BefA突变体活性检测Example 3 Activity detection of BefA and BefA mutants
1、INS-1细胞增殖实验1. INS-1 cell proliferation experiment
INS-1(大鼠胰岛细胞瘤细胞)细胞按3*10 4~6*10 4cells/mL,按100μL/well铺板至96孔板,37℃,5%CO 2培养48h。换液至无糖1640+0.1%BSA,饥饿24h。弃去饥饿培养基,加入1640+1%FBS培养基稀释至10 -9M~10 -7M的BefA蛋白(或BefA突变体BefA modification)刺激,100μL/well。37℃,5%CO 2培养72h。加入CCK8试剂,10μL/well,37℃,5%CO 2培养箱培养2h后,检测OD 450。结果见图7-8。BefA蛋白及BefA突变体均能显著促进INS-1细胞的增殖。 INS-1 (rat islet cell tumor cells) cells were plated at 3*10 4 to 6*10 4 cells/mL at 100 μL/well to a 96-well plate, and cultured at 37°C, 5% CO 2 for 48 hours. Change the medium to sugar-free 1640+0.1% BSA, starve for 24h. The starvation medium was discarded, and the BefA protein (or BefA modification of BefA mutant BefA modification) diluted to 10 -9 M to 10 -7 M was added with 1640+1% FBS medium to stimulate, 100 μL/well. Incubate at 37°C with 5% CO 2 for 72 hours. After adding CCK8 reagent, 10μL/well, 37°C, 5% CO 2 incubator for 2 hours, the OD 450 was detected. The results are shown in Figure 7-8. Both BefA protein and BefA mutant can significantly promote the proliferation of INS-1 cells.
2、小鼠药效实验2. Mice efficacy experiment
(1)BefA蛋白ob/ob小鼠药效实验(1) Pharmacodynamic experiment of BefA protein ob/ob mice
ob/ob小鼠分2组,每组8只动物,分别皮下注射30mg/kg的BefA蛋白和PBS缓冲液,每天给药1次,给药4周,其中在给药第7天进行口服糖耐量OGTT测试,即动物禁食16h,D7给药的同时灌胃给予葡萄糖2g/kg,在给予葡萄糖前以及给药葡萄糖后15min、30min、1h及2h检测动物血糖水平。实验过程中监测动物体重变化,在实验结束的当天动物取全血制备血浆检测血浆ALT/AST水平,动物解剖取其肝脏称重。BefA蛋白对ob/ob小鼠的糖耐量、体重、肝重、肝脏指标ALT、AST的影响见图9-13。图9结果显示:试验结果显示,BefA蛋白能够显著降低给糖后的血糖水平,改善糖耐量;图10~11结果显示:BefA蛋白对ob/ob小鼠体重在不同的天具有显著降低效应,且对肝绝对重量也具有明显的降低效应;图12~13结果显示:BefA蛋白对ob/ob小鼠的肝功指标ALT、AST有显著降低。Ob/ob mice were divided into 2 groups, 8 animals in each group, and 30 mg/kg BefA protein and PBS buffer were injected subcutaneously once a day for 4 weeks. Oral sugar was administered on the 7th day of administration. Tolerance OGTT test, that is, animals are fasted for 16h, and glucose 2g/kg is given by gavage at the same time of D7 administration. The blood glucose level of animals is measured before glucose administration and 15min, 30min, 1h and 2h after glucose administration. During the experiment, the change of animal body weight was monitored. On the day of the end of the experiment, blood was collected from the animal to prepare plasma to detect the plasma ALT/AST level, and the animal was dissected and its liver was weighed. The effect of BefA protein on the glucose tolerance, body weight, liver weight, liver index ALT and AST of ob/ob mice is shown in Figure 9-13. The results in Figure 9 show that the test results show that BefA protein can significantly reduce the blood glucose level after glucose administration and improve glucose tolerance; the results of Figures 10-11 show that BefA protein has a significant effect on reducing the body weight of ob/ob mice on different days. And it also has a significant reduction effect on the absolute weight of the liver; the results of Figures 12-13 show that the BefA protein significantly reduces the liver function indexes ALT and AST of ob/ob mice.
(2)BefA突变体db/db小鼠药效实验(2) Pharmacodynamic experiment of BefA mutant db/db mice
自发性糖尿病db/db小鼠分2组,每组8只动物,分别皮下注射30mg/kg的BefA突变体及对照组皮下注射PBS缓冲液,给药4周,其中实验第一天检测给药前及给药后1、2、4、6、8和24h随机血糖。实验过程中监测动物体重变化,在实验结束的当天动物取全血制备血浆检测血浆ALT/AST水平,动物解剖取其肝脏称重。见图14-18。图14结果显示,在给药的第一天BefA突变体在给药后4-6h能够显著降低动物随机血糖水平;图15~16结果显示:BefA突变体对db/db小鼠体重具有一定降低趋势,对肝绝对重量具有明显的降低效应;图17~18结果显示:BefA突变体对db/db小鼠的肝功指标ALT、AST有显著降低。Spontaneously diabetic db/db mice were divided into 2 groups, 8 animals in each group, were injected subcutaneously with 30 mg/kg of BefA mutant and the control group were injected with PBS buffer subcutaneously for 4 weeks, of which the administration was tested on the first day of the experiment Random blood glucose before and 1, 2, 4, 6, 8 and 24h after administration. During the experiment, the change of animal body weight was monitored. On the day of the end of the experiment, blood was collected from the animal to prepare plasma to detect the plasma ALT/AST level. See Figure 14-18. The results in Figure 14 show that the BefA mutant can significantly reduce the animal's random blood glucose levels 4-6h after the administration on the first day of administration; the results in Figures 15-16 show that the BefA mutant has a certain reduction in the body weight of db/db mice Trend, has a significant reduction effect on the absolute weight of the liver; Figure 17-18 results show: BefA mutants have a significant reduction in the liver function indexes ALT and AST of db/db mice.
结论:BefA蛋白及BefA突变体均能显著促进INS-1细胞的增殖,明显降低血糖或改善 糖耐量;BefA蛋白及BefA突变体均能降低体重或具有降低体重趋势;BefA蛋白及BefA突变体均能降低动物绝对肝、降低肝ALT/AST水平。BefA蛋白及BefA突变体均具有降低血糖,降低体重且具有肝功保护功能,即BefA突变体保留了BefA蛋白的药理活性。Conclusion: Both BefA protein and BefA mutants can significantly promote the proliferation of INS-1 cells, significantly reduce blood sugar or improve glucose tolerance; BefA protein and BefA mutants can reduce body weight or have a tendency to reduce weight; BefA protein and BefA mutants are both It can lower the absolute liver of animals and lower the level of liver ALT/AST. Both BefA protein and BefA mutants can lower blood sugar, reduce body weight and have liver function protection functions, that is, BefA mutants retain the pharmacological activity of BefA protein.
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structures, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned embodiments within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims (18)

  1. 一种多肽,其特征在于,所述多肽与具有下列氨基酸序列的多肽相比,其N端具有修饰,所述修饰包括选自N端氨基酸的增加、甲基化、甲酰化、氨甲酰化、琥珀酰化、环化、丙酰化、十六烷酰化、十四烷化和乙酰化的至少之一,A polypeptide characterized in that, compared with a polypeptide having the following amino acid sequence, the N-terminal has a modification, and the modification includes an increase in N-terminal amino acid, methylation, formylation, carbamoyl At least one of succinylation, cyclization, propionylation, hexadecanoylation, tetradecylation and acetylation,
    (1)具有SEQ ID NO:1-6所示氨基酸序列的多肽;或(1) A polypeptide having the amino acid sequence shown in SEQ ID NO: 1-6; or
    (2)与(1)相比具有至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少99%同一性的多肽;或(2) A polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity compared with (1); or
    (3)与(1)相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽。(3) Compared with (1), a polypeptide having one or more amino acid substitutions, deletions and/or additions.
  2. 根据权利要求1所述的多肽,其特征在于,所述N端氨基酸的增加包括N端增加一个或一个以上的氨基酸,且增加的氨基酸至少有一个甘氨酸、丙氨酸、异亮氨酸、甲硫氨酸、缬氨酸、丝氨酸、苏氨酸、谷氨酸、组氨酸或脯氨酸;优选甘氨酸、丙氨酸、甲硫氨酸、缬氨酸或异亮氨酸;更优选甘氨酸。The polypeptide according to claim 1, wherein the increase of the N-terminal amino acid includes the addition of one or more amino acids at the N-terminal, and the added amino acid includes at least one of glycine, alanine, isoleucine, and methyl. Thionine, valine, serine, threonine, glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine .
  3. 根据权利要求1或2所述的多肽,其特征在于,所述的N端氨基酸的增加为N端增加一个氨基酸,所述氨基酸为甘氨酸、丙氨酸、异亮氨酸、甲硫氨酸、缬氨酸、丝氨酸、苏氨酸、谷氨酸、组氨酸或脯氨酸;优选甘氨酸、丙氨酸、甲硫氨酸、缬氨酸或异亮氨酸;更优选甘氨酸。The polypeptide according to claim 1 or 2, wherein the increase in the N-terminal amino acid is an increase in the N-terminal amino acid, and the amino acid is glycine, alanine, isoleucine, methionine, Valine, serine, threonine, glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine.
  4. 根据权利要求1-3中任一项所述的多肽,其特征在于,所述多肽具有SEQ ID NO:7所示的氨基酸序列。The polypeptide according to any one of claims 1-3, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:7.
  5. 一种提高BefA蛋白稳定性或制备BefA蛋白突变体的方法,其特征在于,对BefA蛋白氨基酸序列的N端进行修饰,所述修饰包括选自N端氨基酸的增加、甲基化、甲酰化、氨甲酰化、琥珀酰化、环化、丙酰化、十六烷酰化、十四烷化和乙酰化的至少之一。A method for improving the stability of BefA protein or preparing BefA protein mutants, characterized in that the N-terminus of the amino acid sequence of BefA protein is modified, and the modification includes an increase in N-terminus amino acid, methylation, and formylation. , At least one of carbamylation, succinylation, cyclization, propionylation, hexadecanoylation, tetradecylation and acetylation.
  6. 根据权利要求5所述的方法,其特征在于,所述BefA蛋白具有The method of claim 5, wherein the BefA protein has
    (1)SEQ ID NO:1-6所示氨基酸序列;或(1) SEQ ID NO: the amino acid sequence shown in 1-6; or
    (2)与(1)相比具有至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少99%同一性的序列;或(2) A sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity compared with (1); or
    (3)与(1)相比具有一个或者多个氨基酸的取代、缺失和/或添加的氨基酸序列。(3) Compared with (1), an amino acid sequence having one or more amino acid substitutions, deletions and/or additions.
  7. 根据权利要求5所述的方法,其特征在于,所述N端氨基酸的增加包括N端增加一个或一个以上的氨基酸,且增加的氨基酸至少有一个甘氨酸、丙氨酸、异亮氨酸、甲硫氨酸、缬氨酸、丝氨酸、苏氨酸、谷氨酸、组氨酸或脯氨酸;优选甘氨酸、丙氨酸、甲硫氨酸、缬氨酸或异亮氨酸;更优选甘氨酸。The method according to claim 5, wherein the increase of the N-terminal amino acid comprises the addition of one or more amino acids at the N-terminal, and the added amino acid includes at least one of glycine, alanine, isoleucine, and methyl. Thionine, valine, serine, threonine, glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine .
  8. 根据权利要求7所述的方法,其特征在于,所述的N端氨基酸的增加为N端增加一个氨基酸,所述氨基酸为甘氨酸、丙氨酸、异亮氨酸、甲硫氨酸、缬氨酸、丝氨酸、苏氨酸、谷氨酸、组氨酸或脯氨酸;优选甘氨酸、丙氨酸、甲硫氨酸、缬氨酸或异亮氨酸;更优选甘 氨酸。The method according to claim 7, wherein the increase of the N-terminal amino acid is an increase of an amino acid at the N-terminal, and the amino acid is glycine, alanine, isoleucine, methionine, and valine. Acid, serine, threonine, glutamic acid, histidine or proline; preferably glycine, alanine, methionine, valine or isoleucine; more preferably glycine.
  9. 一种融合蛋白,其特征在于,所述融合蛋白包括伴侣蛋白、连接肽以及BefA蛋白,所述连接肽的N端与所述伴侣蛋白的C端相连,所述连接肽的C端与所述BefA蛋白的N端相连,所述连接肽适于被蛋白酶切割,以便形成游离的BefA蛋白突变体,所述游离的BefA蛋白突变体的N端相比于BefA蛋白,增加了一个或多个氨基酸。A fusion protein, characterized in that the fusion protein comprises a chaperone protein, a connecting peptide and a BefA protein, the N-terminus of the connecting peptide is connected to the C-terminus of the chaperone protein, and the C-terminus of the connecting peptide is connected to the C-terminus of the chaperone protein. The N-terminus of the BefA protein is connected, and the connecting peptide is suitable for being cleaved by a protease to form a free BefA protein mutant. Compared with the BefA protein, the N-terminus of the free BefA protein mutant is increased by one or more amino acids. .
  10. 根据权利要求9所述的融合蛋白,其特征在于,所述伴侣蛋白包括选自麦芽糖结合蛋白、CBP、GST、SUMO、Trx、NusA的至少之一,任选地,所述伴侣蛋白含有或不含有His标签、Flag标签;The fusion protein of claim 9, wherein the chaperone protein comprises at least one selected from the group consisting of maltose binding protein, CBP, GST, SUMO, Trx, and NusA, optionally, the chaperone protein contains or does not Contains His tag and Flag tag;
    任选地,所述蛋白酶为烟草蚀纹病毒的半胱氨酸蛋白酶、肠激酶、凝血酶、Xa因子蛋白酶、人鼻病毒3C蛋白酶、SUMO酶或KEX-2;Optionally, the protease is cysteine protease of tobacco etch virus, enterokinase, thrombin, factor Xa protease, human rhinovirus 3C protease, SUMO enzyme or KEX-2;
    任选地,所述融合蛋白具有SEQ ID NO:8所示的氨基酸序列。Optionally, the fusion protein has the amino acid sequence shown in SEQ ID NO: 8.
  11. 一种核酸,其特征在于,编码权利要求1~4任一项所述的多肽或权利要求9~10任一项所述的融合蛋白。A nucleic acid characterized in that it encodes the polypeptide according to any one of claims 1 to 4 or the fusion protein according to any one of claims 9 to 10.
  12. 根据权利要求11所述的核酸,其特征在于,所述核酸具有SEQ ID NO:9所示的核苷酸序列或SEQ ID NO:10所示的核苷酸序列。The nucleic acid according to claim 11, wherein the nucleic acid has the nucleotide sequence shown in SEQ ID NO: 9 or the nucleotide sequence shown in SEQ ID NO: 10.
  13. 一种重组细胞,其特征在于,携带权利要求11或12所述的核酸。A recombinant cell characterized by carrying the nucleic acid according to claim 11 or 12.
  14. 根据权利要求13所述的重组细胞,其特征在于,所述重组细胞为大肠杆菌、酵母菌或哺乳动物细胞。The recombinant cell according to claim 13, wherein the recombinant cell is Escherichia coli, yeast or mammalian cell.
  15. 一种药物组合物,其特征在于,包括权利要求1~4任一项所述的多肽。A pharmaceutical composition characterized by comprising the polypeptide of any one of claims 1 to 4.
  16. 根据权利要求15所述的药物组合物,其特征在于,进一步包括药学上可接受的辅剂。The pharmaceutical composition according to claim 15, characterized in that it further comprises a pharmaceutically acceptable adjuvant.
  17. 权利要求1~4所述的多肽或权利要求15或16所述的药物组合物在制备药物中的用途,所述药物用于促进β细胞增殖。The use of the polypeptide according to claims 1 to 4 or the pharmaceutical composition according to claim 15 or 16 in the preparation of a medicine, which is used to promote the proliferation of β cells.
  18. 权利要求1~4所述的多肽或权利要求15或16所述的药物组合物在制备药物中的用途,所述药物用于治疗或预防糖尿病、肥胖或保护肝功。The use of the polypeptide according to claims 1 to 4 or the pharmaceutical composition according to claim 15 or 16 in the preparation of medicines for the treatment or prevention of diabetes, obesity or protection of liver function.
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