WO2023231969A1 - 一种含有糜蛋白酶抑制肽的胰岛素杂交肽及其应用 - Google Patents

一种含有糜蛋白酶抑制肽的胰岛素杂交肽及其应用 Download PDF

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WO2023231969A1
WO2023231969A1 PCT/CN2023/096851 CN2023096851W WO2023231969A1 WO 2023231969 A1 WO2023231969 A1 WO 2023231969A1 CN 2023096851 W CN2023096851 W CN 2023096851W WO 2023231969 A1 WO2023231969 A1 WO 2023231969A1
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seq
chain
insulin
peptide
amino acid
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PCT/CN2023/096851
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English (en)
French (fr)
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王伟
申竹芳
刘忞之
周思含
李彩娜
申欣
曹慧
雷蕾
杨燕
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中国医学科学院药物研究所
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Publication of WO2023231969A1 publication Critical patent/WO2023231969A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention belongs to the field of protein engineering and relates to polypeptide molecules capable of inhibiting chymotrypsin (chymotrypsin).
  • the invention also relates to hybrid peptides formed by chymotrypsin inhibitory peptides and insulin molecules or their analogues and their polyethylene glycol, acyl group modified or amidated analogues or pharmaceutically acceptable salts thereof.
  • the insulin hybrid peptide still maintains the inhibitory activity of chymotrypsin, thereby improving the stability and efficacy of its in vivo administration, resisting the degradation of small intestinal metabolic enzymes, promoting its direct absorption into the blood circulation through the small intestine, and realizing the hybrid peptide of insulin or its analogues. Oral administration.
  • Insulin is a blood sugar level-controlling hormone secreted by the pancreas to transport excess glucose from the blood to cells, providing a source of energy and maintaining normal blood sugar levels.
  • patients with diabetes are unable to maintain normal insulin function due to insulin deficiency, insulin resistance, and loss of beta cell function. Therefore, diabetic patients cannot utilize glucose in the blood as a source of energy, but instead exhibit symptoms of hyperglycemia with high glucose levels and excrete glucose in the urine, leading to various complications. Therefore, those diabetic patients with abnormal insulin secretion (type I diabetes) or insulin resistance (type II diabetes) basically need insulin treatment, and by injecting insulin, they can maintain normal blood sugar levels.
  • insulin Since insulin has an extremely short half-life in the body, like other protein and peptide hormones, it cannot show sustained therapeutic effects, so there is a problem that insulin must be continuously and repeatedly administered to exert its effect. Frequent administration of insulin can cause severe pain and discomfort to patients. Therefore, there is a need to improve insulin use in terms of patient compliance, safety, and convenience.
  • peptide drugs including insulin are difficult to be administered orally.
  • insulin bioavailability [1,2,3], which mainly include absorption enhancers such as sodium caprate and 8 -Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SANC) [4], protease inhibitor (aprotinin), soybean trypsin inhibitor (soybean trypsin inhibitor) and leupeptin (leupeptin)) [5], nanocarriers (such as polymers, lipids and inorganic nanoparticles), and microneedles and microcapsule protein drug delivery devices (such as RaniPill TM Capsule) [6].
  • absorption enhancers such as sodium caprate and 8 -Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SANC) [4]
  • protease inhibitor aprotinin
  • soybean trypsin inhibitor soybean trypsin inhibitor
  • leupeptin leupeptin
  • nanocarriers such as polymers, lipids and inorganic nanop
  • Oral insulin administration can use enteric preparation technology to overcome the degradation of gastric acid and pepsin, but the small intestine still contains a variety of protein metabolism enzymes secreted by the pancreas, the most important of which are trypsin and chymotrypsin. Only by overcoming the degradation of small intestinal proteases can the residence time of insulin molecules in the small intestinal environment be prolonged, thereby promoting the absorption of small intestinal epithelium. Although the addition of protease inhibitors and absorption enhancers promotes the intestinal absorption of insulin [8,9], it is still difficult to overcome the protease degradation of insulin after it enters the blood circulation.
  • the insulin molecule is composed of two polypeptide chains, the A chain and the B chain, containing 21 and 30 amino acids respectively, connected to each other by two disulfide bonds. Structurally, it is relatively stable against trypsin degradation [10].
  • the main protease that degrades insulin in the small intestinal microenvironment is chymotrypsin.
  • the existing technology uses D-amino acid substitution, PEG modification, fatty acylation, etc., which still does not solve the problem of oral administration of insulin.
  • the present invention isolates and identifies a series of inhibitory peptides that inhibit chymotrypsin, and then forms hybrid peptides with insulin or its analogs to solve the stability problem of insulin molecules degraded by proteases.
  • the invention provides a hybrid peptide formed by insulin or an analog thereof and a chymotrypsin inhibitor peptide, which are connected through a connecting peptide; wherein the insulin or an analog thereof contains a B-chain and an A-chain and two They are connected through two pairs of disulfide bonds; among them, the chymotrypsin inhibitor peptide is connected to the B-chain or A-chain of insulin or its analogues through the connecting peptide.
  • the present invention provides hybrid peptides formed by connecting five chymotrypsin inhibitory peptides to the B-chain or A-chain of insulin or its analogues.
  • chymotrypsin inhibitory peptides are screened and identified through in vitro enzymology methods, which are selected from polypeptides with the following amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
  • SEQ ID NO:4 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.
  • insulin molecules or analogs thereof of synthetic hybrid peptides are provided. These insulins or analogs thereof include A-chain and B-chain; wherein the A-chain can be selected from polypeptides having the following amino acid sequence: SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33; the B-chain can be selected from polypeptides with the following amino acid sequences: SEQ ID NO: 34, SEQ ID NO :35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40.
  • A-chain can be selected from polypeptides having the following amino acid sequence: SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33
  • the B-chain can be selected from polypeptides with the following amino acid sequences: SEQ ID NO: 34, SEQ ID NO :35, SEQ ID NO:36, SEQ ID NO
  • Another aspect of the present invention provides a connecting peptide connecting chymotrypsin inhibitory peptide and insulin or its analog A-chain or B-chain.
  • the connecting peptide contains three amino acid residues: Gly, Ser and Pro, and its length is Peptides or deletions of one or more amino acid residues.
  • the invention provides a hybrid peptide of insulin or its analogues, which contains a structural domain from the N-terminal to the C-terminal direction: (N-terminal)-chymotrypsin inhibitory peptide-insulin B-chain---insulin A -chain-(C-terminus), in which the C-terminus of the chymotrypsin inhibitor peptide is directly fused to the N-terminus of the B-chain of insulin or its analogues through a connecting peptide;
  • the "(N-terminal)-chymotrypsin inhibitor "Peptide-insulin B-chain” is a polypeptide formed by the fusion of chymotrypsin inhibitor peptide and insulin B-chain, selected from polypeptides with the following amino acid sequences: SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48
  • the invention provides a hybrid peptide of insulin or its analogues, which requires the maturation of its precursor protein.
  • the precursor protein has a domain in the direction from N-terminus to C-terminus: (N-terminus)-chymotrypsin-insulin B-chain-C-polypeptide-insulin A-chain-(C-terminus), where chymotrypsin
  • the C-terminus of the protease inhibitor peptide is directly fused to the N-terminus of the B-chain of insulin or its analogues through a connecting peptide;
  • the "(N-terminal)-chymotrypsin inhibitor peptide-insulin B-chain” is a chymotrypsin inhibitor
  • the polypeptide formed by the fusion of the peptide and the insulin B-chain is selected from polypeptides having the following amino acid sequences: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO
  • the precursor protein needs to be processed in three steps: protease Kex2 enzyme cleavage, tobacco mosaic virus protease TEV enzyme cleavage and nickel ion-induced chemical shearing.
  • the processing methods can be combined, and can be processed step by step; for In order to obtain a hybrid peptide of insulin or its analogues that does not contain two "RR" at the C-terminus of the B-chain, carboxypeptidase B can be used; in order to obtain a hybrid peptide of insulin or its analogues with an amidation-modified C-terminus , treatment with peptidylglycine ⁇ -amidating monooxygenase can be used.
  • the precursor protein of an insulin or analog hybrid peptide may be selected from the group consisting of polypeptides having the following amino acid sequences: SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173 and SEQ ID NO:174.
  • hybrid peptide can be further treated with carboxypeptidase B; it can be further treated with peptidylglycine ⁇ -amidating monooxygenase to obtain a hybrid peptide with an amidation-modified C-terminus.
  • hybrid peptide formed by fusion of the chymotrypsin inhibitory peptides CH57 (SEQ ID NO: 4) and CH10 (SEQ ID NO: 19) with the N-terminus of the B-chain of human insulin or its analogues is as follows:
  • nucleotide coding sequences of the above-mentioned hybrid peptide precursor proteins can be selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184.
  • the invention provides another hybrid peptide, which contains a domain from the N-terminal to the C-terminal direction: (N-terminal)-insulin B-chain-chymotrypsin inhibitory peptide---insulin A-chain-(C -terminal), wherein the N-terminus of the chymotrypsin inhibitor peptide is directly fused to the C-terminus of the B-chain of insulin or its analogues through a connecting peptide;
  • the "(N-terminal)-insulin B-chain-chymotrypsin inhibitor "Peptide” is a polypeptide formed by the fusion of insulin B-chain and chymotrypsin inhibitory peptide, selected from polypeptides with the following amino acid sequences: SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58; the A-chain is selected from poly
  • the invention provides a hybrid peptide of insulin or its analogues, which requires the maturation of its precursor protein.
  • the precursor protein has a domain in the direction from N-terminus to C-terminus: (N-terminus)-insulin B-chain-chymotrypsin inhibitor-C-polypeptide-insulin A-chain-(C-terminus), where chymotrypsin
  • the N-terminus of the protease inhibitory peptide is directly fused to the C-terminus of the B-chain of insulin or its analogues through a connecting peptide;
  • the "(N-terminal)-insulin B-chain-chymotrypsin inhibitory peptide" is insulin B-
  • the polypeptide formed by the fusion of chain and chymotrypsin inhibitory peptide is selected from polypeptides with the following amino acid sequences: SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO
  • the precursor protein needs to pass through the protease Kex2
  • the three-step treatment of enzyme digestion, tobacco mosaic virus protease TEV enzyme digestion and nickel ion-induced chemical shearing can be combined, and can be processed step by step; in order to obtain the C-terminal end of the B-chain that does not contain 2 "RR" hybrid peptides of insulin or its analogs can be treated with carboxypeptidase B; in order to obtain amidation modification of the C-terminus of the hybrid peptide of insulin or its analogs, peptidylglycine ⁇ -amidation monomer can be used Oxygenase treatment of hybrid peptides.
  • the precursor protein of an insulin or analog hybrid peptide may be selected from the group consisting of polypeptides having the following amino acid sequences: SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195 and SEQ ID NO :196.
  • protease Kex2 tobacco mosaic virus protease TEV and nickel ion-induced chemical shearing; in order to obtain the hybridization of insulin or its analogues that does not contain two "RR" at the C-terminus of the B-chain
  • the peptide can be treated with carboxypeptidase B; it can be treated with peptidylglycine alpha-amidating monooxygenase to obtain a hybrid peptide whose C-terminus is amidated.
  • hybrid peptide formed by fusion of the chymotrypsin inhibitory peptides CH57 (SEQ ID NO: 4) and CH10 (SEQ ID NO: 19) with the C-terminus of the B-chain of human insulin or its analogues is as follows:
  • nucleotide coding sequences of the above-mentioned hybrid peptide precursor proteins can be selected from SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207 and SEQ ID NO:208.
  • the invention provides a hybrid peptide, which contains a structural domain from the N-terminal to the C-terminal direction: (N-terminal)-insulin B-chain---chymotrypsin inhibitor-insulin A-chain-(C- end), wherein the C-terminus of the chymotrypsin inhibitor peptide is directly fused to the N-terminus of the A-chain of insulin or its analogues through a connecting peptide, and the B-chain of insulin or its analogues is selected from a polypeptide with the following amino acid sequence: SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:40; the "chymotrypsin inhibitor-insulin A-chain-(C-terminal)" is The polypeptide formed by the fusion of chymotrypsin inhibitor peptide and insulin A-chain is selected from polypeptides with the following amino acid sequences: SEQ ID NO: 59, SEQ ID NO:
  • the invention provides a hybrid peptide of insulin or its analogues, which requires the maturation of its precursor protein.
  • the precursor protein has a domain in the direction from N-terminus to C-terminus: (N-terminus)-insulin B-chain-C-polypeptide-chymotrypsin-insulin A-chain-(C-terminus), where chymotrypsin
  • the C-terminal end of the protease inhibitor peptide is directly fused to the N-terminal end of the A-chain of insulin or its analogues through a connecting peptide;
  • the B-chain of insulin or its analogues is selected from a polypeptide having the following amino acid sequence: SEQ ID NO: 34,
  • the "chymotrypsin inhibitor-insulin A-chain-(C-terminal)" described in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, and SEQ ID NO:40 is chymotrypsin inhibitor and insulin
  • Peptide SEQ ID NO: 70. SEQ ID NO:71.
  • the precursor protein needs to undergo three steps of protease Kex2 enzyme cleavage, tobacco mosaic virus protease TEV enzyme cleavage and nickel ion-induced chemical shearing.
  • the processing methods can be combined, and can be processed step by step; in order to obtain B- Hybrid peptides of insulin or its analogues that do not contain two "RR"s at the C-terminus of the chain can continue to be treated with carboxypeptidase B; in order to obtain hybrid peptides of insulin or its analogues with an amidation-modified C-terminus, you can continue Hybrid peptides were treated with peptidylglycine alpha-amidating monooxygenase.
  • the precursor protein of an insulin or analog hybrid peptide may be selected from the group consisting of polypeptides having the following amino acid sequences: SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217 and SEQ ID NO:218.
  • hybrid peptide can be treated with carboxypeptidase B; it can be treated with peptidylglycine ⁇ -amidation monooxygenase to obtain a hybrid peptide with an amidation-modified C-terminus.
  • hybrid peptide formed by fusion of the chymotrypsin inhibitory peptides CH57 (SEQ ID NO: 4) and CH10 (SEQ ID NO: 19) with the N-terminus of the A-chain of human insulin or its analogues is as follows:
  • nucleotide coding sequences of the above-mentioned precursor proteins can be selected from SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227 and SEQ ID NO:228.
  • the invention provides another hybrid peptide, which contains a domain from the N-terminal to the C-terminal direction: (N-terminal)-insulin B-chain---insulin A-chain-chymotrypsin inhibitory peptide-(C -terminus), wherein the N-terminus of the chymotrypsin inhibitor peptide is directly fused to the C-terminus of the A-chain of insulin or its analogues through a connecting peptide; the B-chain of insulin or its analogues is selected from polypeptides with the following amino acid sequences: SEQ ID NO:34, SEQ ID NO: 35.
  • polypeptide formed by the fusion is selected from polypeptides having the following amino acid sequences: SEQ ID NO: 63, SEQ ID NO: 64.
  • the present invention provides a hybrid peptide of insulin or its analogues, which needs to be generated through the maturation processing of its precursor protein.
  • the precursor protein has a domain in the direction from N-terminus to C-terminus: (N-terminus)-insulin B-chain-C-polypeptide-insulin A-chain-chymotrypsin inhibitor-(C-terminus), where chymotrypsin
  • the N-terminus of the protease inhibitor peptide is directly fused to the C-terminus of the A-chain of insulin or its analogues through a connecting peptide;
  • the B-chain of insulin or its analogues is selected from a polypeptide with the following amino acid sequence: SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:40;
  • the "insulin A-chain-chymotrypsin inhibitory peptide-(C-terminal)" is insulin A-chain and The poly
  • the precursor protein is processed through three steps of protease Kex2 enzyme cleavage, tobacco mosaic virus protease TEV enzyme cleavage and nickel ion-induced chemical shearing.
  • the processing methods can be combined, and can be processed step by step; in order to obtain the B-chain Hybrid peptides of insulin or its analogs that do not contain two "RR"s at the C-terminus can continue to be treated with carboxypeptidase B; in order to obtain hybrid peptides of insulin or its analogs with an amidation-modified C-terminus, you can continue to use Peptidylglycine ⁇ -amidation monooxygenase processes hybrid peptides.
  • the precursor protein of an insulin or analog hybrid peptide may be selected from the group consisting of polypeptides having the following amino acid sequences: SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237 and SEQ ID NO:238.
  • hybrid peptide can be treated with carboxypeptidase B; it can be treated with peptidylglycine ⁇ -amidation monooxygenase to obtain a hybrid peptide with an amidation-modified C-terminus.
  • hybrid peptide formed by fusion of the chymotrypsin inhibitory peptides CH57 (SEQ ID NO: 4) and CH10 (SEQ ID NO: 19) with the C-terminus of the A-chain of human insulin or its analogues is as follows:
  • nucleotide coding sequences of the above-mentioned precursor proteins can be selected from SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247 and SEQ ID NO:248.
  • the present invention provides yet another hybrid peptide, which contains a structural domain from the N-terminal to the C-terminal direction: (N-terminal)-insulin B-chain---insulin A-chain-chymotrypsin inhibitory peptide-A- chain-(C-terminal), in which chyme
  • the albumen inhibitory peptide directly forms a fusion peptide with the A-chain of insulin or its analog;
  • the B-chain of insulin or its analog is selected from polypeptides with the following amino acid sequences: SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO :36, SEQ ID NO:39, SEQ ID NO:40;
  • the "insulin A-chain-chymotrypsin inhibitor-A-chain-(C-terminus)" is an intermediate fusion chymotrypsin inhibitor peptide of the insulin A-chain
  • the polypeptide formed is selected from the group consisting of polypeptides having the following amino acid sequences: SEQ ID NO:65,
  • the present invention provides a hybrid peptide of insulin or its analogues, which needs to be generated through the maturation processing of its precursor protein.
  • the precursor protein has a domain in the direction from N-terminus to C-terminus: (N-terminus)-insulin B-chain-C-polypeptide-insulin A-chain-chymotrypsin inhibitor-A-chain-(C-terminus ), wherein the chymotrypsin inhibitor peptide directly forms a fusion peptide with the A-chain of insulin or its analog;
  • the insulin or analog B-chain is selected from polypeptides with the following amino acid sequences: SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:40;
  • the "insulin A-chain-chymotrypsin inhibitory peptide-A-chain-(C-terminal)" is the intermediate fusion chymotrypsin of the insulin A-chain.
  • the polypeptide formed by the protease inhibitor peptide is selected from the polypeptides with the following amino acid sequences: SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68; the C-polypeptide is selected from the polypeptides with the following amino acid sequences. :SEQ ID NO:70 ⁇ SEQ ID NO:71.
  • the precursor protein is processed through three steps of protease Kex2 enzyme cleavage, tobacco mosaic virus protease TEV enzyme cleavage and nickel ion-induced chemical shearing.
  • the processing methods can be combined, and can be processed step by step; in order to obtain the B-chain Hybrid peptides of insulin or its analogs whose C-terminus does not contain two "RR"s can continue to be treated with carboxypeptidase B; in order to obtain hybrid peptides of insulin or its analogs whose C-terminus is amidated, you can use Peptidylglycine ⁇ -amidation monooxygenase processes hybrid peptides.
  • the precursor protein of an insulin or analog hybrid peptide may be selected from the group consisting of polypeptides having the following amino acid sequences: SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257 and SEQ ID NO:258.
  • hybrid peptide can be treated with carboxypeptidase B; it can be treated with peptidylglycine ⁇ -amidation monooxygenase to obtain a hybrid peptide with an amidation-modified C-terminus.
  • hybrid peptides of chymotrypsin inhibitory peptides CH57 (SEQ ID NO: 4) and CH10 (SEQ ID NO: 19) directly form fusion peptides with the A-chain of human insulin or its analogues are as follows:
  • nucleotide coding sequences of the above-mentioned precursor proteins can be selected from SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267 and SEQ ID NO:268.
  • the present invention provides a method for heterologous biological preparation of hybrid peptides containing chymotrypsin inhibitor peptide and insulin or its analogues.
  • the biological preparation process is to first synthesize its precursor protein, and then undergo a series of Processing steps such as protease digestion and chemically induced shearing, and finally preparation by chromatographic purification.
  • the N-terminus of its precursor protein contains the tobacco mosaic virus protease (TEV) cleavage site (ENLYFQ) shown in SEQ ID NO:69, between the B-chain and A-chain of the insulin molecule or its analogues.
  • TSV tobacco mosaic virus protease
  • the C-polypeptide between is selected from the polypeptides of the following sequences: SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73.
  • the precursor protein of the heterologous fusion expression hybrid peptide in the present invention contains two proteases Kex2 and two TEV cleavage sites, in which Kex2 cleaves the signal peptide and insulin or other proteins that guide the secretion and expression of the precursor protein.
  • TEV cuts and processes the precursor protein of the hybrid peptide to form a mature hybrid peptide ; Then undergo chemical shearing induced by nickel ions, and/or treatment with carboxypeptidase B and peptidylglycine ⁇ -amidation monooxygenase or without treatment.
  • the present invention provides a use of a hybrid peptide formed by insulin or an analog thereof and a chymotrypsin inhibitor peptide in the treatment of type I diabetes and type II diabetes.
  • the present invention provides a method of administration of hybrid peptides by subcutaneous injection and oral administration in the treatment of type I diabetes and type II diabetes.
  • the present invention can improve the stability of insulin molecules by fusing chymotrypsin inhibitor peptide with insulin or its analogues, promote its oral administration, improve patients' medication compliance and reduce side effects, and has beneficial effects economic value.
  • Figure 1 Determination of the Michaelis-Menten constant K m of chymotrypsin. Using Prism software, plot the concentration of the substrate AAPFpNA against the initial velocity V 0 to obtain the value of the Michaelis-Menten constant K m of the substrate AAPFpNA hydrolyzed by chymotrypsin. The experiment was repeated three times, and the calculated values were expressed as “mean ⁇ standard deviation”.
  • Figure 2 Determination of the inhibitory activity of chymotrypsin inhibitor peptides. By adding different concentrations of chymotrypsin inhibitor peptides (CH5, CH11, CH18 and CH25), their inhibitory effects on chymotrypsin were examined, and their ability to produce 50% inhibitory effect was determined. concentration (IC 50 value). Three replicate wells were set up in the experiment, and the calculated values were expressed as “mean ⁇ standard deviation”.
  • Figure 3 Determination of the inhibitory activity of chymotrypsin inhibitor peptides. By adding different concentrations of chymotrypsin inhibitor peptides (CH33, CH34 and CH35), their inhibitory effects on chymotrypsin were examined, and the concentration at which they could produce 50% inhibitory effect was determined ( IC 50 value). Three replicate wells were set up in the experiment, and the calculated values were expressed as “mean ⁇ standard deviation”.
  • Figure 4 Determination of the inhibitory activity of chymotrypsin inhibitor peptides. By adding different concentrations of chymotrypsin inhibitor peptides (CH26 and CH51), their inhibitory effects on chymotrypsin were examined, and the concentration at which they could produce 50% inhibitory effect (IC 50 value). Three replicate wells were set up in the experiment, and the calculated values were expressed as “mean ⁇ standard deviation”.
  • Figure 5 Determination of the inhibitory activity of chymotrypsin inhibitor peptides. By adding different concentrations of chymotrypsin inhibitor peptides (CH10, CH54, CH55 and CH57), their inhibitory effects on chymotrypsin were examined, and their ability to produce 50% inhibitory effect was determined. concentration (IC 50 value). Three replicate wells were set up in the experiment, and the calculated values were expressed as “mean ⁇ standard deviation”.
  • Figure 6 Determination of the inhibitory activity of chymotrypsin inhibitor peptides. By adding different concentrations of chymotrypsin inhibitor peptides (CH10, CH60, CH61 and CH62), their inhibitory effects on chymotrypsin were examined, and their ability to produce 50% inhibitory effect was determined. concentration (IC 50 value). Three replicate wells were set up in the experiment, and the calculated values were expressed as “mean ⁇ standard deviation”.
  • Figure 7 Determination of the inhibitory activity of chymotrypsin inhibitor peptides. By adding different concentrations of chymotrypsin inhibitor peptides (CH65, CH69, CH70 and CH71), their inhibitory effects on chymotrypsin were examined, and their ability to produce 50% inhibitory enzyme was determined. The concentration of the effect (IC 50 value). Three replicate wells were set up in the experiment, and the calculated values were expressed as “mean ⁇ standard deviation”.
  • FIG. 8 Schematic diagram of the methanolic yeast expression vector pDeut-Aox1-ScKex2, which contains two promoters AOX1 and DAS2 with different methanol-induced expression strengths.
  • the relatively weak promoter DAS2 drives the expression of Saccharomyces cerevisiae protease Kex2.
  • Figure 9 Gene editing Cas9/gRNA expression vector targeting the AOX1 gene of methanolic yeast.
  • Figure 10 Precursor protein expression and maturation cleavage process of insulin or its analog hybrid peptide.
  • the ⁇ MF signal peptide guides the precursor protein to target to the endoplasmic reticulum.
  • the precursor protein is transported by the endoplasmic reticulum after the signal peptide is removed by cleavage.
  • the trans-Golgi apparatus is cleaved by protease Kex2 to remove the precursor region of ⁇ MF and the "RR" post-peptide bond at the C-terminus of the B-chain; then it is packaged and secreted out of the cell; then it is cleaved by protease TEV and carboxypeptidase B ( Carboxy peptidase B) and nickel ion induced cleavage to obtain insulin analog hybrid peptide.
  • Natural protease inhibitors have been used as additives in experimental studies to protect insulin molecules from protease degradation.
  • an inhibitor selected from the soybean trypsin inhibitor BBI family contains two protease-inhibiting active loops (Loop), inhibiting human trypsin and chymotrypsin; this protease inhibitor has been used in PCT
  • loop protease-inhibiting active loops
  • Sunflower Trypsin Inhibitor-1 (SFTI-1) is a head-to-tail cyclic peptide isolated from sunflower seeds containing only 14 amino acid residues
  • PCT Patent publication number WO2020023386 also describes its use as a protease inhibitor, an oral pharmaceutical component for the treatment of diabetes.
  • SFTI-1 forms a rigid structure containing 2 short ⁇ -sheets, an intramolecular disulfide bond, and head-to-tail cyclization.
  • SFTI-1 has a stable cyclic structure, it is difficult to directly couple with therapeutic peptides and can only be used as an additive.
  • SFTI-1 only has antitrypsin activity and does not have antichymotrypsin activity.
  • SFTI-1 cannot act as a chymotrypsin inhibitor to protect the degradation of insulin molecules by chymotrypsin.
  • the P1 position of serpins determines the specificity of different serine proteases.
  • the P1 position of chymotrypsin is Tyr and Phe.
  • Only a few documents have reported that peptide inhibitors derived from the BBI family peptide skeleton have Anti-chymotrypsin activity [11-14], but the inhibitory activity is weak; while the active peptides disclosed in the literature that inhibit chymotrypsin only function as chymotrypsin inhibitors.
  • Book The invention is based on the core molecular skeleton that inhibits trypsin SFTI-1. It changes the protease specificity of the inhibitory peptide molecular skeleton by replacing the P1 site, and then replaces different recognition sites and evaluates the inhibitory activity.
  • polypeptide molecular skeleton for inhibiting chymotrypsin was obtained: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17.
  • SEQ ID NO:18 SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.
  • the above-mentioned chymotrypsin inhibitory peptide provided by the present invention directly forms a fusion hybrid peptide with a biologically active peptide, which changes the fact that as an additive to inhibit chymotrypsin, it only protects the active polypeptide from the degradation of part of the chymotrypsin at the release site, and can be accompanied by Active peptides such as insulin enter the blood circulation and are always protected against degradation by chymotrypsin.
  • the insulin molecule itself has good resistance to trypsin.
  • the most important metabolic enzyme is chymotrypsin, which accounts for 9% of the total protein secreted by the pancreas [15].
  • chymotrypsin inhibitory peptide is used to directly form a hybrid peptide with insulin or its analog molecules.
  • the formed hybrid peptide protects it from degradation by proteases from the microenvironment of the small intestine to entering the blood circulation and exerting its medicinal effect. .
  • polypeptide synthesis The polypeptides of the invention can be prepared by various methods.
  • polypeptides can be synthesized by commonly used solid phase synthesis methods, such as methods involving t-BOC or FMOC protection of the ⁇ -amino group, which are well known in the art.
  • solid phase synthesis methods such as methods involving t-BOC or FMOC protection of the ⁇ -amino group, which are well known in the art.
  • amino acids are added sequentially into a growing chain of amino acids.
  • Solid-phase synthesis methods are particularly suitable for synthesizing peptides or relatively short peptides in large-scale production.
  • Determination of enzyme inhibitory activity Determine the inhibition constant of the synthesized active polypeptide protease inhibitor (molecular skeleton).
  • the inhibitory activity of porcine ⁇ -chymotrypsin was determined by competitive binding using the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPFpNA).
  • the experimental determination of the inhibitory activity of porcine ⁇ -chymotrypsin was carried out in 20mM CaC12, 50mM Tris-HC1 buffer (pH 7.8). Peptide concentration was determined using optical density (OD) at 280 nm.
  • the Michaelis-Menten constant (Km) for enzyme hydrolysis of a substrate is calculated from the initial rate of substrate hydrolysis at 405 nm.
  • the absorbance value of the substrate was measured at 405 nm after complete hydrolysis. All data were processed using nonlinear regression.
  • the chymotrypsin inhibitor obtained by the present invention can be widely used with insulin analogs to form anti-chymotrypsin inhibitors.
  • the degraded hybrid peptide improves its stability and biological activity in vivo and in vitro.
  • insulin or its analogs are not limited to the insulin or its analogs selected as examples disclosed in the present invention, and may be analogs that replace one or several amino acid residues in its B-chain, for example, such as SEQ ID NO:74(FVQQHLCGSHLVEALYLVCGERGFFYTPKT), SEQ ID NO:75(FVNQHLCGSHLVEALYLVCGERGFFYTPKQ), SEQ ID NO:76(FVNQHLCGSHLVEALYLVCGERGFFYTPKE), SEQ ID NO:77(FVQQHLCGSHLVEALYLVCGERGFFYTPKE), SEQ ID NO:78(FVN QHLCGSHLVEALYLVCGERGFFYTDPT), SEQ ID NO:79
  • the hybrid peptide in the present invention uses eukaryotic cells to perform heterologous secretion expression of its precursor protein, in which the protease Kex2 in the expression host cell cleaves the precursor protein to promote its secretion expression; and then uses the tobacco mosaic virus to secrete the precursor protein.
  • Protease TEV enzyme has broad substrate properties [16] and cleaves the N-terminal fusion peptide (ENLYFQ, SEQ ID NO: 69) of the B-chain and A-chain of the secreted precursor protein (insulin analog).
  • G ⁇ SRHW nickel ion-induced cleavage peptide
  • carboxypeptidase B to treat or not treat, and you can obtain hybrid peptides that do not contain or contain two basic amino acids "RR" at the C-terminal; the C-terminal of the B-chain or A-chain contains
  • nickel ions induce cleavage of the peptide (G ⁇ SRHW), after nickel ion-mediated shearing, the C- The C-terminus of the mature B-chain or A-chain is fused with a glycine (Gly) residue and can be treated with recombinant amidase to form an amidation protective modification.
  • C-polypeptide Also known as C-peptide (Peptide), natural C-peptide is a single-chain polypeptide composed of 31 amino acids, connecting the B-chain and A-chain in the proinsulin molecule.
  • the polypeptide described in the present invention as connecting the B-chain and A-chain in the precursor protein of insulin or its analog hybrid peptide may have the same or different amino acid structure as the natural C-peptide.
  • polypeptide sequences disclosed herein are shown from left to right, with the left end of the sequence being the N-terminus of the polypeptide and the right end of the sequence being the C-terminus of the polypeptide.
  • hybrid peptide used in the present invention refers to two peptide segments connected by natural peptide bonds, and can be prepared by solid-phase synthesis or biological expression.
  • linker peptide as used herein broadly refers to a glycine, serine or proline-rich peptide segment that facilitates the formation of a turn structure, capable of linking two polypeptides together and forming a chemical structure.
  • the concept also applies to C-polypeptides whose function is to connect the B-chain and the A-chain to form a precursor protein.
  • proteases refers to polypeptide molecules that inhibit the function of proteases.
  • proteases include chymotrypsin found in the mammalian gastrointestinal tract and proteases with similar chymotrypsin-like activity in the blood circulation.
  • the fluorenylmethoxycarbonyl (Fmoc) solid-phase chemical synthesis method is used to synthesize them one by one from the C-terminus to the N-terminus; after the synthesis of the linear peptide with amino acid side chain protection is completed, it is cleaved from the resin.
  • Fmoc fluorenylmethoxycarbonyl
  • Resin Fmoc-L-Ala-Wang resin, Fmoc-N-(2,2,4,6,7-pentamethylchroman-5 -Sulfonyl)-L-arginine-Wang resin (Fmoc-Arg(Pbf)-Wang resin), Fmoc-N-trityl-L-asparagine-Wang resin (Fmoc-Asn(Trt )-Wang resin), Fmoc-O-tert-butyl-L-aspartic acid-Wang resin (Fmoc-Asp(OtBu)-Wang resin), Fmoc-L-glycine-Wang resin (Fmoc-Gly- Wang resin), Fmoc-L-phenylalanine-Wang resin (Fmoc-Phe-Wang resin), Fmoc-L-proline-Wang resin (Fmoc-Pro-Wang resin), Fmoc-O-tert Butyl-
  • SEQ ID NO:2 selects Fmoc-Asp(OtBu)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1. First, add amino acid raw materials corresponding to the polypeptide sequence in sequence, and synthesize with protective groups. peptide segment, that is
  • SEQ ID NO:3 selects Fmoc-Pro-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1. First, add amino acid raw materials corresponding to the polypeptide sequence in sequence, and synthesize peptide segments with protective groups.
  • SEQ ID NO:4 selects Fmoc-Phe-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO:1.
  • Remove Fmoc then add lysis solution to remove the resin and amino acid side chain protecting groups, and oxidize to form disulfide bonds.
  • SEQ ID NO: 5 selects Fmoc-Phe-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO: 1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups. , namely Fmoc-Arg(Pbf)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin, remove Fmoc , then add lysis solution to remove the resin and amino acid side chain protecting groups, and oxidize to form disulfide bonds. Finally, separate and purify to obtain the target peptide SEQ ID NO: 5 (CH58), with an actual measured molecular weight of 1297.0Da ([M+2H] 2+ 649.5).
  • SEQ ID NO:6 selects Fmoc-Asn(Trt)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • the peptide fragment of the protecting group is Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn-Pro -Asn(Trt)-Wang resin, remove Fmoc, then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds, and finally separate and purify to obtain the target peptide SEQ ID NO: 6 (CH59),
  • SEQ ID NO:7 selects Fmoc-Asn(Trt)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1. First, add amino acid raw materials corresponding to the polypeptide sequence in sequence, and synthesize with protective groups.
  • SEQ ID NO:8 selects Fmoc-Pro-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO:1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups. , namely Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Pro -Wang resin, remove Fmoc, then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, separate and purify to obtain the target peptide SEQ ID NO: 8 (CH61). The actual measured molecular weight is 1363.2 Da([M+2H] 2+ 682.6).
  • SEQ ID NO:9 selects Fmoc-Asn(Trt)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • SEQ ID NO:10 selects Fmoc-Asn(Trt)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • SEQ ID NO:11 selects Fmoc-Phe-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence in sequence, and synthesize peptides with protective groups. , namely Fmoc-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin, remove Fmoc , then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, the target peptide SEQ ID NO: 11 (CH64) is separated and purified. The actual measured molecular weight is 1269.2Da ([M+2H] 2+ 635.6).
  • SEQ ID NO:12 selects Fmoc-Asp(OtBu)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1. First, add amino acid raw materials corresponding to the polypeptide sequence in sequence, and synthesize with protective groups.
  • SEQ ID NO: 13 selects Fmoc-Pro-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO: 1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups. , namely Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Pro- Wang resin, remove Fmoc, then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, separate and purify the target peptide SEQ ID NO: 13 (CH66), with an actual measured molecular weight of 1523.2Da. ([M+2H] 2+ 762.6).
  • SEQ ID NO:14 selects Fmoc-Phe-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO:1. First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups.
  • SEQ ID NO: 15 selects Fmoc-Phe-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO: 1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups. , namely Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Abu-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe- Wang resin, remove Fmoc, then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, separate and purify the target peptide SEQ ID NO: 15 (CH68), with an actual measured molecular weight of 1439.2Da. ([M+2H] 2+ 720.6).
  • SEQ ID NO:16 selects Fmoc-Phe-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO:1. First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups.
  • SEQ ID NO: 17 selects Fmoc-Phe-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO: 1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups. , namely Fmoc-Cys(Trt)-Glu(OtBu)-Arg(Pbf)-Ala-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe -Wang resin, remove Fmoc, then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, separate and purify to obtain the target peptide SEQ ID NO: 17 (CH70), the actual measured molecular weight is 1497.2 Da([M+2H] 2+ 749.6).
  • SEQ ID NO: 18 selects Fmoc-Phe-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO: 1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups. , namely Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin, take off Fmoc, and then add lysis solution to remove the resin and amino acid side chain protecting groups, and oxidize to form disulfide bonds. Finally, the target peptide SEQ ID NO: 18 (CH71) is separated and purified. The actual measured molecular weight is 1409.2Da([M+2H ] 2+ 705.6).
  • SEQ ID NO: 19 selects Fmoc-Gly-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO: 1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptide segments with protective groups. , namely Fmoc-Phe-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Gly-Wang resin, remove Fmoc, and then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, the target peptide SEQ ID NO: 19 (CH10) is separated and purified. The actual measured molecular weight is 1375.55Da ( [M+Na] + 1398.55).
  • SEQ ID NO:20 selects Fmoc-Gly-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO:1.
  • First, add the amino acid raw materials corresponding to the polypeptide sequence and synthesize the peptide segment with a protecting group. , namely Fmoc-Phe-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Gly-Wang resin, off Remove Fmoc, then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, separate and purify to obtain the target peptide SEQ ID NO: 20 (CH5). The actual measured molecular weight is 1360.02Da([M+ K+H]2+ 700.01).
  • SEQ ID NO: 21 selects Fmoc-Gly-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO: 1.
  • SEQ ID NO:22 is synthesized according to the method described in SEQ ID NO:1. First, amino acid raw materials corresponding to the polypeptide sequence are added sequentially to synthesize a peptide segment with a protecting group, namely Fmoc-Ala-Cys(Trt)-Thr.
  • SEQ ID NO:23 selects Fmoc-Asn(Trt)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1. First, add amino acid raw materials corresponding to the polypeptide sequence in sequence, and synthesize with protective groups.
  • the peptide segment is Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Pro -Asn(Trt)-Wang resin, remove Fmoc, then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds, and finally separate and purify to obtain the target peptide SEQ ID NO: 23 (CH25),
  • SEQ ID NO:24 selects Fmoc-Asn(Trt)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • the peptide segment is Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Wang resin, remove Fmoc, and then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds.
  • the target peptide SEQ ID NO: 24 (CH26) is separated and purified.
  • SEQ ID NO:25 selects Fmoc-Tyr(tBu)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • SEQ ID NO:26 selects Fmoc-Ala-Wang resin as the starting material and synthesizes it according to the method described in SEQ ID NO:1.
  • First, add amino acid raw materials corresponding to the polypeptide sequence and synthesize peptides with protective groups. , namely Fmoc-Phe-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Ala-Wang resin, remove Fmoc, and then add lysis solution to remove the resin and amino acid side chain protective groups, and oxidize to form disulfide bonds. Finally, the target peptide SEQ ID NO: 26 (CH34) is separated and purified. The actual measured molecular weight is 1390.80Da ( [M+2H] 2+ 696.40).
  • SEQ ID NO:27 selects Fmoc-Arg(Pbf)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • SEQ ID NO:28 selects Fmoc-Tyr(tBu)-Wang resin as the starting material, and synthesizes it according to the method described in SEQ ID NO:1.
  • Example 2 Design of polypeptide molecules that inhibit chymotrypsin and evaluation of their inhibitory activity
  • the final concentration of chymotrypsin is approximately 30 nM, and the final concentration of AAPFpNA is approximately 0.5 mM.
  • BBI Bowman-Birk family protease inhibitors
  • the present invention combines the specificity of the P1 site of chymotrypsin (specifically recognizes Tyr and Phe) and the conservation of P1' and P3', and designs and synthesizes a series of chymotrypsin cyclized with a core structure of "CTYSIPPQC".
  • Polypeptides (Table 1), in vitro inhibitory activity assay yielded two polypeptides CH10 and CH5 with high inhibitory activity, with IC 50 values of 0.06 ⁇ M and 0.19 ⁇ M respectively (Table 2). The results confirmed that the P1 site is effective against chymotrypsin.
  • the inhibitory activity has a greater impact; then the P4 and P7' amino acid residues were replaced on the basis of CH10 to obtain CH11, CH35 and CH51, whose inhibitory activity was reduced by nearly 7 times, indicating that the P4 and P7' amino acid residues can enhance Intermolecular force with the active site of chymotrypsin (Table 2); by comparing the inhibitory activity differences of CH33, CH34 and CH10, it can be seen that P8'glycine with strong polarity is more conducive to enhancing the molecular interaction between it and the protease Interaction (Table 2); extension of the P5 site (CH25, CH26) and amino acid substitution (CH18) of the P4' site did not significantly change the inhibitory activity (Table 2, Figures 2, 3, 4).
  • SFTI-1 Compared with other BBI family protease inhibitor molecules, SFTI-1 (GRCT K SIPPICFPD) has The special structure contains only one pair of disulfide bonds and has a head-to-tail cyclized structure. According to the specificity of the P1 site of chymotrypsin, the Lys of the P1 site was directly mutated to Tyr or Phe, and the peptides CH54 and CH55 were designed and synthesized (Table 1). Both of them have good inhibitory activity against chymotrypsin, and their IC 50 0.03 ⁇ M and 0.02 ⁇ M respectively ( Figure 5). In order to further simplify their structures, CH56, CH57 and CH58 were synthesized (Table 1).
  • Polypeptides CH59-CH62 with amino acid substitutions at P4 and P7' positions were simultaneously synthesized (Table 1). Their inhibitory activity was slightly weaker than that of CH57.
  • the IC 50 for inhibiting chymotrypsin was 0.18 ⁇ M, 0.09 ⁇ M, 0.12 ⁇ M, and 0.15 ⁇ M respectively (Table 2 , Figure 6); a series of disulfide bond expanded peptides CH65-CH71 were also synthesized on the basis of CH57 (Table 1), among which CH65 and CH68-CH71 both showed good inhibitory activity against chymotrypsin, and their IC 50 were respectively It is 0.48 ⁇ M, 0.45 ⁇ M, 0.30 ⁇ M, 0.25 ⁇ M, 0.41 ⁇ M (Table 2, Figure 7).
  • CH10 has almost no enzyme inhibitory activity at a concentration of 0.0001 ⁇ M, but there are two duplicate wells due to a large sampling error, so the values of these two duplicate wells are discarded.
  • CH10 has almost no enzyme inhibitory activity at a concentration of 0.0001 ⁇ M, but there are two duplicate wells due to a large sampling error, so the values of these two duplicate wells are discarded.
  • the most widely used vector is Invitrogen's pPIC9K expression plasmid.
  • This expression vector has unstable expression of resistance selection markers and only one multiple cloning site (multiple cloning site). MCS), low homologous integration efficiency and other problems, in order to improve the integrated expression of heterologous target genes, the plasmid expression vector is modified.
  • the construction process is as follows:
  • Kex2 (ScKex2, SEQ ID NO: 101), using restriction endonucleases EcoR I and Not I, is subcloned into the expression cassette controlled by the DAS2 promoter, which can achieve high intracellular expression of ScKex2; after ligation ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the plasmid expression vector pDeut-Aox1-ScKex2 ( Figure 8).
  • Example 4 Construction of gene editing expression vector pGAD-Cas9-gAOX1 targeting methanol yeast alcohol oxidase 1 (AOX1)
  • the present invention uses gene editing technology to perform directional integration and expression of the target gene through a gRNA-mediated dual plasmid transformation system.
  • Plasmid pGADT7 was used as the backbone to construct a gRNA expression vector targeting the AOX1 gene. The construction process was as follows:
  • Example 5 Construction of precursor protein expression vector containing hybrid peptide of insulin or analogues
  • Methanolic yeast cells are eukaryotic monocytes with strong secretory expression ability and are easy to ferment and culture.
  • the present invention uses the signal peptide and precursor region of Saccharomyces cerevisiae ⁇ MF to guide the secretion expression of the hybrid peptide precursor protein.
  • the hybrid peptide precursor protein produced by translation in the endoplasmic reticulum (ER) is cleaved by a signal enzyme to remove the signal peptide of ⁇ MF, and then transported to the trans-Golgi apparatus, and then ⁇ MF is cleaved by yeast ScKex2, which is highly expressed and localized in the trans-Golgi apparatus.
  • the precursor region and the C-polypeptide (C-Peptide) or linker peptide (Linker) between the B-chain and A-chain in the hybrid peptide of insulin or its analogues are then packaged and secreted extracellularly in the reverse Golgi apparatus; After purification by affinity chromatography, tobacco mosaic virus protease TEV is used to cut and remove the fused peptide and its connecting peptide, and finally the target protein is purified.
  • Figure 10 briefly outlines the process from hybrid peptide precursor protein to mature hybrid peptide. .
  • the detailed construction process of the hybrid peptide precursor protein coding frame is as follows:
  • Precursor protein expression vector of human insulin or its analogues artificially synthesizes precursor protein encoding genes encoding different B-chains of insulin or its analogues and having the same A-chain (SEQ ID NO: 29) (Table 3 ), then use the synthetic primers Ins-BamH1/Ins-Spe1 (SEQ ID NO: 118 and 119) (Table 4) to directly amplify the DNA coding frame, and use the restriction endonucleases BamH I and Spe I to subclone into the expression plasmid pDeut-Aox1-ScKex2, after concatenation ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pAOX1-ScKex2-Ins-1, pAOX1-ScKex2-Ins-2, pAOX1-ScKex2-Ins-3 and pAOX1-ScKex2- Ins-4.
  • the correct positive cloning plasmid was linearized using restriction endonuclease Pme I, and electrotransformed into methanol yeast cell GS115 together with the gRNA expression plasmid pGAD-Cas9-gAOX1.
  • the above 2 PCR-amplified DNAs Fragment perform SOE-PCR filling reaction, then use the product of PCR filling reaction as template, use primer Ins-BamH1-2/Ins-Spe1 (Ins-Spe1-1) to perform PCR amplification of chymotrypsin inhibitory peptide and insulin or Its analog B-chain N-terminal fusion coding frame (ORF), and finally connected ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pIns1-B-CH57N, pIns2-B-CH57N, pIns3-B-CH57N, pIns4-B-CH57N, pIns1-B- CH10N, pIns2-B-CH10N, pIns3-B-CH10N, pIns4-B-CH10N.
  • the PCR amplified DNA fragment is subjected to the corresponding SOE-PCR filling reaction, and then the product of the PCR filling reaction is used as a template, and the primer Ins -BamH1/Ins-Spe1 (or Ins-Spe1-1) is used to PCR amplify the C-terminal fusion coding frame (ORF) of the B-chain of insulin or its analogue chymotrypsin inhibitor peptide, and finally connect ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pIns1-B-CH57C, pIns2-B-CH57C, pIns3-B-CH57C, pIns4-B-CH57C, pIns1-B- CH10C, pIns2-B-CH10C, pIns3-B-CH10C, pIns3-B-CH10
  • the DNA fragment amplified by PCR Carry out the corresponding SOE-PCR filling reaction, then use the product of the PCR filling reaction as a template, and then use the primers Ins-BamH1/Ins-Spe1 to perform PCR amplification of insulin or its analog B-chain C-terminal fusion chymotrypsin inhibition Peptide fusion coding frame (ORF), in which C-peptide (C-Peptide) is replaced by a more flexible linking peptide (Linker sequence is GGSGGSG), and finally connected ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pIns1-B-CH57CT, pIns2-B-CH57CT, pIns3-B-CH57CT, pIns4-B-CH57CT, pIns1-B- CH10CT, pIns2-B-CH10CT, pIns3-B-CH10CT, pIns4-
  • the above four encoding insulin or analogues respectively correspond to the connecting peptide and A. - chain precursor protein coding region, perform the corresponding SOE-PCR filling reaction on the PCR amplified DNA fragment, and then use the product of the PCR filling reaction as a template, using the primer Ins-BamH1/Ins-Spe1 (or Ins -Spe1-1) perform PCR to amplify the fusion coding frame (ORF) of the A-terminus of the A-chain of insulin or its analogue fused to the chymotrypsin inhibitor peptide, and finally connect ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pIns1-A-CH57N, pIns2-A-CH57N, pIns3-A-CH57N, pIns4-A-CH57N, pIns1-A- CH10N, pIns2-A-CH10N, p
  • Peptide and A-chain DNA fragment use primers Ins-A-CH57C2/TFLD1-R1 or Ins-A-CH10C2/TFLD1-R1 (Table 4) to perform PCR amplification of the above four insulin or analogue corresponding connecting peptides and
  • the PCR-amplified DNA fragment was subjected to the corresponding SOE-PCR Filling reaction, then using the product of the PCR filling reaction as a template, using the primers Ins-BamH1/Ins-Spe1 to perform PCR amplification of the C-terminal fusion coding frame (ORF) of the A-chain of insulin or its analogue fused to the chymotrypsin inhibitor peptide ), and finally connect ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pIns1-A-CH57C, pIns2-A-CH57C, pIns3-A
  • primers AOX1-P1/A-CH-LR1 (Table 4) were used to perform PCR amplification of DNA fragments encoding the signal peptide, precursor region of ⁇ MF and the B-chain of insulin or its analogues, and primer A- CH-LR2/TFLD1-R1 (Table 4) was used to PCR amplify the above eight DNA fragments encoding the connecting peptide and A-chain precursor protein of insulin or analogues respectively.
  • the PCR amplified DNA fragments were subjected to the corresponding SOE. -PCR filling reaction, then use the product of the PCR filling reaction as a template, and use the primers Ins-BamH1/Ins-Spe1 to perform PCR amplification of the coding frame of the C-terminal fusion chymotrypsin inhibitor peptide of insulin or its analog A-chain ( ORF), in which the connecting peptide between the B-chain and A-chain of insulin or its analog is replaced with SEQ ID NO:70 (RREAEDGSGGSGENLYFQ); the last connection ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pIns1-A-CH57CT, pIns2-A-CH57CT, pIns3-A-CH57CT, pIns4-A-CH57CT, pIns1-A- CH10CT, pIns2-A-CH10CT, pIns3
  • the fusion coding frame (ORF) of the chymotrypsin inhibitor peptide is inserted into the middle of the A-chain of the analog, and finally connected ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vectors pIns1-A-CH57M, pIns2-A-CH57M, pIns3-A-CH57M, pIns4-A-CH57M, pIns1-A- CH10M, pIns2-A-CH10M, pIns3-A-CH10M, pIns4-A-CH10M.
  • Example 6 DNA transformation of methanolic yeast, screening of positive transformants and electrotransformation method for induced expression of target protein:
  • yeast genome extraction According to the yeast genome extraction kit, operate as follows:
  • Positive clones were screened using YPD solid medium containing 100 ⁇ g ⁇ mL-1G418 resistance.
  • the genome of the recombinant strain was randomly selected from 6 positive clones, and the primer AOX1_P1/TFLD1-R1 was used for the first round of PCR amplification, and then the primer AOX1_P2/Ins-Spe1 was used for the second round of PCR verification.
  • the target gene positive clone amplified the band. At about 1kb, the DNA fragment is purified and recovered, and then sequenced for verification.
  • the present invention selects the cold shock-induced expression system pCold-MBP (Takara Company), heterologous expression through Shuffle T7 host strain to increase protein solubility and promote correct protein folding.
  • pCold-MBP Cold-Nhe1/Cold-Nde1
  • Table 5 PCR amplify the DNA fragment on the plasmid pCold-MBP
  • the endonucleases Nhe I and Nde I replace the corresponding vector structural fragments on the vector and eliminate the His-Tag tag at the N-terminus of the MBP tag that promotes soluble expression, thus obtaining plasmid pCold-MBP-T.
  • the primers TEV-BamH1/TEV-Sal1 (SEQ ID NO:156 and 157) (Table 5) were used to directly amplify the synthetic tobacco mosaic virus protease TEV coding frame (SEQ ID NO:158), and restriction endonucleation was used to The enzymes BamH I and Sal I are directionally inserted into the plasmid pCold-MBP-T, and after ligation ( II One Step Cloning Kit), transformation, positive clone screening and sequencing verification to obtain the expression plasmid vector pCold-TEV, which can achieve soluble expression of TEV and self-cleavage of the fusion tag MBP.
  • PAM peptidylglycine alpha-amidating monooxygenase
  • pReceiver-M02 (GeneCopoeia) containing the PAM gene (GenBank: NM_000919) as a template
  • primers pReceiver-M03F/PAM-1 (SEQ ID NO: 159 and 161), PAM-2/pReceiver-M03R (SEQ ID NO: 160 and 162) (Table 5) were used to PCR amplify DNA fragments containing the PHM domain encoding amino acid residues 39-350 and the PAL domain encoding amino acids 494-835, and then the two DNA fragments were PCR filling reaction, and then using the PCR filling reaction product as a template, using the primers PAM-BamH1/PAM-Sal1 (SEQ ID NO: 163 and 164) (Table 5) to perform PCR amplification of the truncated PAM mutant, where A connecting peptide segment (GSGGSG) was introduced between these two domains.
  • proteases were purified through nickel ion affinity chromatography to obtain the proteases, and then an enzyme digestion reaction system containing insulin or its analog hybrid peptide was established: 50mM Hepes (pH 7.0), 1 ⁇ M CuSO 4 , 50 ⁇ g/ml catalase (catalase), 1mM ascorbic acid (ascorbate), add precursor protein substrate, protease TVE and PAM, directly perform enzyme cleavage reaction of precursor protein at 30°C, add after the reaction 1/10 volume of 0.1M EDTA and 50% TFA to inactivate the enzyme digestion reaction.
  • reaction buffer 1.0mM NiCl 2 , 0.1M CHES, 0.1M acetone oxime, 0.1M NaCl, pH 8.2
  • G ⁇ SRHW nickel ion-induced shear fusion tag
  • Example 8 Stability analysis of hybrid peptides of insulin or its analogs against chymotrypsin hydrolysis
  • Control experiment Take three sterile EP tubes, and add 1.5 ⁇ L, 1mM insulin or its analog hybrid peptide, 23.5 ⁇ L, 50mM Tris and 20mM CaCl 2 (pH 7.8) buffer and 3.75 ⁇ L, 10% TFA, centrifuge at 8000rpm for 30s and mix.
  • the enzymatic hydrolysis process of human insulin, insulin analogs and their hybrid peptides by chymotrypsin is as follows: Take three sterile EP tubes, add 9 ⁇ L, 1mM human insulin, insulin analogs and their hybrid peptides and 138 ⁇ L, 20mM into each EP tube CaC1 2 , 50mM Tris-HC1 buffer (pH 7.8). At the same time, prepare a certain volume of 0.05 ⁇ g/ ⁇ L chymotrypsin enzyme solution in another sterile EP tube. Then place the four EP tubes containing peptides and enzymes at 37°C for preheating for 5 minutes, and add 3 ⁇ L to each EP tube containing peptides. Chymotrypsin enzyme solution and mix well.
  • the final concentration of human insulin, insulin analogs and their hybrid peptides is 60 ⁇ M, and the final concentration of chymotrypsin is 1.0 ng/ ⁇ L.
  • Each time point is repeated three times.
  • RP-HPLC is used to detect the peak area of the polypeptide at each time point.
  • the ratio of the remaining peak area of the sample at detection time T (h) to the peak area of the prototype polypeptide at 0h is calculated as the remaining percentage (%) of the polypeptide. .
  • Table 6 Stability analysis of human insulin and its hybrid peptides (Ins3-A-CH10M, and Ins3-A-CH57M) against chymotrypsin
  • Example 9 In vivo activity of insulin or its analogues and insulin hybrid peptide via subcutaneous injection and duodenal administration
  • Insulin analogs such as insulin lispro LisPro showed good hypoglycemic activity compared with the positive control; insulin hybrid peptides also containing chymotrypsin inhibitory peptide (Ins3-B-CH57N, Ins3-B-CH57C, Ins3- A-CH10M, Ins3-A-CH57M) also showed good hypoglycemic activity (Table 7); indicating that the structural chymotrypsin inhibitory peptide does not affect its interaction with the insulin receptor, whether at the N-terminus of its B-chain, the C-terminus of its B-chain or in the middle of its A-chain.
  • the N-terminus of the A-chain and the C-terminus of the A-chain prepared by biological expression are weakly fused with chymotrypsin inhibitors (Ins3-A-CH57N and Ins3-A-CH57C). The reason may be that they are not formed correctly. Disulfide bond structure, may be a false negative result.
  • Table 7 Hypoglycemic activity of human insulin and its analogues administered subcutaneously * ,p ⁇ 0.05; ** ,p ⁇ 0.01; *** ,p ⁇ 0.001.
  • Table 7 Hypoglycemic activity of human insulin and its analogues administered subcutaneously (continued) * ,p ⁇ 0.05; ** ,p ⁇ 0.01; *** ,p ⁇ 0.001.
  • Ins3-A-CH57M can significantly reduce the blood glucose levels of mice for 40 minutes, with blood glucose levels reduced by 12.1%, 11.9% and 15.3% respectively (Table 8). It shows that the chymotrypsin inhibitor peptide has a good protective effect on insulin when it is at the C-terminus of the B-chain and in the middle of the A-chain, improves their stability in the body, and promotes the absorption of the insulin hybrid peptide into the blood through the small intestinal epithelium. cycle.
  • Table 8 Hypoglycemic activity of human insulin and its analogues after duodenal administration * ,p ⁇ 0.05; ** ,p ⁇ 0.01; *** ,p ⁇ 0.001.
  • Table 8 Hypoglycemic activity of human insulin and its analogues after duodenal administration (continued) * ,p ⁇ 0.05; ** ,p ⁇ 0.01; *** ,p ⁇ 0.001.

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Abstract

本发明属于蛋白质工程领域，涉及一种小肠糜蛋白酶(chymotrpsin)抑制肽与胰岛素或其类似物形成的杂交肽；杂合的胰岛素类似物能抵御糜蛋白酶的降解进而提高其稳定性，不仅可提高其直接注射给药的疗效，还可以穿透小肠上皮直接吸收进入血循环，实现胰岛素杂交肽类药物的口服给药。本发明还包括制备含有糜蛋白酶抑制肽和胰岛素或其类似物的杂交肽方法。

Description

一种含有糜蛋白酶抑制肽的胰岛素杂交肽及其应用 技术领域
本发明属于蛋白质工程领域，涉及具有抑制糜蛋白酶(胰凝乳蛋白酶)的多肽分子，本发明还涉及糜蛋白酶抑制肽与胰岛素分子或其类似物形成的杂交肽及其被聚乙二醇化、酰基化或酰胺化修饰的类似物或其药学上可接受的盐。所述胰岛素杂交肽依然保持抑制糜蛋白酶抑制活性，从而提高其体内给药的稳定性和疗效，可抵御小肠代谢酶降解，促进其直接经小肠吸收进入血循环，实现胰岛素或其类似物的杂交肽口服给药。
背景技术
胰岛素是一种由胰腺分泌的血糖水平控制激素，用于将血液中多余的葡萄糖输送到细胞，从而提供能量来源并维持正常的血糖水平。然而，由于胰岛素缺乏、胰岛素抵抗和β细胞功能丧失，糖尿病患者无法维持正常的胰岛素功能。因此，糖尿病患者不能利用血液中的葡萄糖作为能量来源，而是表现出高葡萄糖水平的高血糖症状，并在尿液中排出葡萄糖，从而导致各种并发症。因此，那些胰岛素分泌异常(I型糖尿病)或胰岛素抵抗(II型糖尿病)的糖尿病患者基本上需要胰岛素治疗，通过注射胰岛素，他们可以保持血糖水平正常。
由于胰岛素在体内的半衰期极短，与其他蛋白质和肽激素一样，它无法显示出持续的治疗效果，因此存在一个问题，即必须连续反复施用胰岛素才能发挥其作用。频繁施用胰岛素会给患者带来严重的疼痛和不适。因此需要从患者依从性、安全性和方便性等方面改进胰岛素的使用。
由于胰岛素的分子量高，易被胃酸、胃蛋白酶和肠道代谢酶(gastrointestinal tract,GIT)水解，以及小肠粘膜上皮的物理障碍，导致包括胰岛素在内的多肽药物难以口服给药。为了克服这些障碍，已经进行了许多不同的研究，在提高胰岛素生物利用度方面取得了积极的研究结果[1,2,3]，其中主要包括吸收促进剂如癸酸钠(sodium caprate)和8-(2-羟基苯甲酰胺基)辛酸钠(sodium N-[8-(2-hydroxybenzoyl)amino]caprylate，SANC)[4]、蛋白酶抑制剂(抑肽酶(aprotinin),大豆胰蛋白酶抑制剂(soybean trypsin inhibitor)和亮肽素 (leupeptin))[5]、纳米载体(如聚合物、脂质和无机纳米颗粒)以及微针和微囊类蛋白药物递送装置(如RaniPillTM Capsule)[6]。这些技术也已进行了临床实验研究，但口服胰岛素的生物利用度似乎也仅在1-2％[6,7]。口服胰岛素临床实验没有进入更深入的研究，其主要原因可能与成本效益有关。
口服胰岛素给药可以采用肠溶制剂技术克服胃酸和胃蛋白酶的降解，但在小肠内依然含有多种胰腺分泌的蛋白代谢酶，其中最主要的是胰蛋白酶和糜蛋白酶。只有克服小肠蛋白酶的降解，才能延长胰岛素分子在小肠内环境中驻留时间，进而促进小肠上皮的吸收。尽管蛋白酶抑制剂和吸收促进剂的添加促进了胰岛素的小肠吸收[8，9]，但依然难以克服胰岛素进入血循环后的蛋白酶降解。
胰岛素分子由两条多肽链组成，即A链和B链，分别包含21和30个氨基酸，通过两个二硫键相互连接。结构上对胰蛋白酶的降解相对稳定[10]，在小肠微环境中降解胰岛素的蛋白酶主要是糜蛋白酶。为提高胰岛素分子抵御蛋白酶降解的稳定性，现有技术是采用D-型氨基酸的取代、PEG修饰和脂肪酰化等，依然没有解决胰岛素口服给药的难题。针对现有技术的不足，本发明分离鉴定一系列抑制糜蛋白酶的抑制肽，然后与胰岛素或其类似物形成杂交肽，解决胰岛素分子被蛋白酶降解的稳定性问题。
发明内容
本发明提供一种含有胰岛素或其类似物和糜蛋白酶抑制肽所形成的杂交肽，二者之间通过连接肽连接；其中胰岛素或其类似物，含有一条B-链和一条A-链且二者通过两对二硫键连接；其中糜蛋白酶抑制肽通过连接肽与胰岛素或其类似物的B-链或A-链连接。如图10所示，本发明提供了5种糜蛋白酶抑制肽与胰岛素或其类似物的B-链或A-链连接形成的杂交肽。
本发明在一个具体的实施方案中，通过体外酶学的方法筛选鉴定了糜蛋白酶抑制肽，其选自具有以下氨基酸序列的多肽：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:28。
在本发明的一方面提供了合成杂交肽的胰岛素分子或其类似物，这些胰岛素或其类似物包括A-链和B-链；其中A-链可选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33；B-链可选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40。
本发明另一方面提供了一种连接糜蛋白酶抑制肽和胰岛素或其类似物A-链或B-链之间的连接肽，连接肽含有Gly、Ser和Pro三种氨基酸残基，其长度为一个或多个氨基酸残基的肽段或缺失。
本发明提供了一种胰岛素或其类似物的杂交肽，其含有从N-末端到C-末端方向的结构域：(N-末端)-糜蛋白酶抑制肽-胰岛素B-链---胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物B-链的N-末端融合；所述的“(N-末端)-糜蛋白酶抑制肽-胰岛素B-链”为糜蛋白酶抑制肽和胰岛素B-链融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49和SEQ ID NO:50；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33。
在一个实施方案中，本发明提供的一种胰岛素或其类似物的杂交肽，需要经过其前体蛋白的成熟加工生成。前体蛋白具有从N-末端到C-末端方向的结构域：(N-末端)-糜蛋白酶抑制肽-胰岛素B-链-C-多肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物的B-链N-末端融合；所述的“(N-末端)-糜蛋白酶抑制肽-胰岛素B-链”为糜蛋白酶抑制肽和胰岛素B-链融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49和SEQ ID NO:50；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:70、SEQ ID NO:71。其中，前体蛋白需要经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，其处理方法可以组合的方式，可以是分步依次处理；为 了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物杂交肽，可以使用羧肽酶B处理；为了获得C-末端被酰胺化修饰的胰岛素或其类似物的杂交肽，可以使用肽基甘氨酸α-酰胺化单加氧酶的处理。
在一个具体的实施方案中，一种胰岛素或其类似物杂交肽的前体蛋白可选自具有以下氨基酸序列的多肽：SEQ ID NO:165、SEQ ID NO:166、SEQ ID NO:167、SEQ ID NO:168、SEQ ID NO:169、SEQ ID NO:170、SEQ ID NO:171、SEQ ID NO:172、SEQ ID NO:173和SEQ ID NO:174。这些前体蛋白需要经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切的处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可再使用羧肽酶B继续处理；可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理，得到C-末端被酰胺化修饰的杂交肽。举例，糜蛋白酶抑制肽CH57(SEQ ID NO:4)和CH10(SEQ ID NO:19)与人胰岛素或其类似物B-链的N-末端融合形成的杂交肽如下：

上述这些杂交肽前体蛋白的核苷酸编码序列可选自SEQ ID NO:175、SEQ ID NO:176、SEQ ID NO:177、SEQ ID NO:178、SEQ ID NO:179、SEQ ID NO:180、SEQ ID NO:181、SEQ ID NO:182、SEQ ID NO:183和SEQ ID NO:184。
本发明提供了另一种杂交肽，其含有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-糜蛋白酶抑制肽---胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物B-链的C-末端融合；所述的“(N-末端)-胰岛素B-链-糜蛋白酶抑制肽”为胰岛素B-链和糜蛋白酶抑制肽融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33。
在一个实施方案中，本发明提供的一种胰岛素或其类似物的杂交肽，需要经过其前体蛋白的成熟加工生成。前体蛋白具有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-糜蛋白酶抑制肽-C-多肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物的B-链C-末端融合；所述的“(N-末端)-胰岛素B-链-糜蛋白酶抑制肽”为胰岛素B-链和糜蛋白酶抑制肽融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:72和SEQ ID NO:73。其中，前体蛋白需要经过蛋白酶Kex2 酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，其处理方法可以组合的方式，可以是分步依次处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可以使用羧肽酶B处理；为了获得胰岛素或其类似物的杂交肽的C-末端被酰胺化修饰，可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理杂交肽。
在一个具体的实施方案中，一种胰岛素或其类似物杂交肽的前体蛋白可选自具有以下氨基酸序列的多肽：SEQ ID NO:185、SEQ ID NO:186、SEQ ID NO:187、SEQ ID NO:188、SEQ ID NO:189、SEQ ID NO:190、SEQ ID NO:191、SEQ ID NO:192、SEQ ID NO:193、SEQ ID NO:194、SEQ ID NO:195和SEQ ID NO:196。这些前体蛋白需要经过蛋白酶Kex2、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切的处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可再继续使用羧肽酶B处理；可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理，得到C-末端被酰胺化修饰的杂交肽。举例，糜蛋白酶抑制肽CH57(SEQ ID NO:4)和CH10(SEQ ID NO:19)与人胰岛素或其类似物B-链C-末端融合形成的杂交肽如下：

上述这些杂交肽前体蛋白的核苷酸编码序列可选自SEQ ID NO:197、SEQ ID NO:198、SEQ ID NO:199、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:202、SEQ ID NO:203、SEQ ID NO:204、SEQ ID NO:205、SEQ ID NO:206、SEQ ID NO:207和SEQ ID NO:208。
本发明提供了一种杂交肽，其含有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链---糜蛋白酶抑制肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物A-链的N-末端融合,胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“糜蛋白酶抑制肽-胰岛素A-链-(C-末端)”为糜蛋白酶抑制肽和胰岛素A-链融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62。
在一个实施方案中，本发明提供的一种胰岛素或其类似物的杂交肽，需要经过其前体蛋白的成熟加工生成。前体蛋白具有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-C-多肽-糜蛋白酶抑制肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物A-链的N-末端融合；胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40所述的“糜蛋白酶抑制肽-胰岛素A-链-(C-末端)”为糜蛋白酶抑制肽和胰岛素A-链融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO: 70、SEQ ID NO:71。其中，前体蛋白需要经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，其处理方法可以组合的方式，可以是分步依次处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可以继续使用羧肽酶B处理；为了获得C-末端被酰胺化修饰的胰岛素或其类似物杂交肽，可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理杂交肽。
在一个具体的实施方案中，一种胰岛素或其类似物杂交肽的前体蛋白可选自具有以下氨基酸序列的多肽：SEQ ID NO:209、SEQ ID NO:210、SEQ ID NO:211、SEQ ID NO:212、SEQ ID NO:213、SEQ ID NO:214、SEQ ID NO:215、SEQ ID NO:216、SEQ ID NO:217和SEQ ID NO:218。这些前体蛋白需要经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切的处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可再继续使用羧肽酶B处理；可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理，得到C-末端被酰胺化修饰的杂交肽。举例，糜蛋白酶抑制肽CH57(SEQ ID NO:4)和CH10(SEQ ID NO:19)与人胰岛素或其类似物A-链N-末端融合形成的杂交肽如下：

上述这些前体蛋白的核苷酸编码序列可选自SEQ ID NO:219、SEQ ID NO:220、SEQ ID NO:221、SEQ ID NO:222、SEQ ID NO:223、SEQ ID NO:224、SEQ ID NO:225、SEQ ID NO:226、SEQ ID NO:227和SEQ ID NO:228。
本发明提供了另一种杂交肽，其含有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链---胰岛素A-链-糜蛋白酶抑制肽-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物A-链的C-末端融合；胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO: 35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-(C-末端)”为胰岛素A-链和糜蛋白酶抑制肽融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:63、SEQ ID NO:64。
在另一个实施方案中，本发明提供的一种胰岛素或其类似物的杂交肽，需要经过其前体蛋白的成熟加工生成。前体蛋白具有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-C-多肽-胰岛素A-链-糜蛋白酶抑制肽-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物A-链的C-末端融合；胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-(C-末端)”为胰岛素A-链和糜蛋白酶抑制肽融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:63、SEQ ID NO:64；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:70、SEQ ID NO:71。其中，前体蛋白经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，其处理方法可以组合的方式，可以是分步依次处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可以继续使用羧肽酶B处理；为了获得C-末端被酰胺化修饰的胰岛素或其类似物杂交肽，可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理杂交肽。
在一个具体的实施方案中，一种胰岛素或其类似物杂交肽的前体蛋白可选自具有以下氨基酸序列的多肽：SEQ ID NO:229、SEQ ID NO:230、SEQ ID NO:231、SEQ ID NO:232、SEQ ID NO:233、SEQ ID NO:234、SEQ ID NO:235、SEQ ID NO:236、SEQ ID NO:237和SEQ ID NO:238。这些前体蛋白需要经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切的处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可再继续使用羧肽酶B处理；可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理，得到C-末端被酰胺化修饰的杂交肽。举例，糜蛋白酶抑制肽CH57(SEQ ID NO:4)和CH10(SEQ ID NO:19)与人胰岛素或其类似物A-链C-末端融合形成的杂交肽如下：
上述这些前体蛋白的核苷酸编码序列可选自SEQ ID NO:239、SEQ ID NO:240、SEQ ID NO:241、SEQ ID NO:242、SEQ ID NO:243、SEQ ID NO:244、SEQ ID NO:245、SEQ ID NO:246、SEQ ID NO:247和SEQ ID NO:248。
本发明提供了又一种杂交肽，其含有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链---胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)，其中糜蛋 白酶抑制肽直接与胰岛素或其类似物的A-链形成融合肽；胰岛素或类似物B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)”为胰岛素A-链中间融合糜蛋白酶抑制肽形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68。
在另一个实施方案中，本发明提供的一种胰岛素或其类似物的杂交肽，需要经过其前体蛋白的成熟加工生成。前体蛋白具有从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-C-多肽-胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)，其中糜蛋白酶抑制肽直接与胰岛素或其类似物的A-链形成融合肽；胰岛素或类似物B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)”为胰岛素A-链中间融合糜蛋白酶抑制肽形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:70、SEQ ID NO:71。其中，前体蛋白经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，其处理方法可以组合的方式，可以是分步依次处理；为了获得B-链的C-末端不含有2个“RR”的胰岛素或其类似物杂交肽，可以继续使用羧肽酶B处理；为了获得C-末端被酰胺化修饰的胰岛素或其类似物的杂交肽，可以使用肽基甘氨酸α-酰胺化单加氧酶处理杂交肽。
在一个具体的实施方案中，一种胰岛素或其类似物杂交肽的前体蛋白可选自具有以下氨基酸序列的多肽：SEQ ID NO:249、SEQ ID NO:250、SEQ ID NO:251、SEQ ID NO:252、SEQ ID NO:253、SEQ ID NO:254、SEQ ID NO:255、SEQ ID NO:256、SEQ ID NO:257和SEQ ID NO:258。这些前体蛋白需要经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切的处理；为了获得B-链C-末端不含有2个“RR”的胰岛素或其类似物的杂交肽，可再继续使用羧肽酶B处理；可以继续使用肽基甘氨酸α-酰胺化单加氧酶处理，得到C-末端被酰胺化修饰的杂交肽。举例，糜蛋白酶抑制肽CH57(SEQ ID NO:4)和CH10(SEQ ID NO:19)直接与人胰岛素或其类似物A-链形成融合肽的杂交肽如下：

上述这些前体蛋白的核苷酸编码序列可选自SEQ ID NO:259、SEQ ID NO:260、SEQ ID NO:261、SEQ ID NO:262、SEQ ID NO:263、SEQ ID NO:264、SEQ ID NO:265、SEQ ID NO:266、SEQ ID NO:267和SEQ ID NO:268。
本发明在一个实施方案中，提供了一个异源生物制备含有糜蛋白酶抑制肽和胰岛素或其类似物所形成杂交肽的方法，其生物制备过程是首先合成其前体蛋白，然后经过一系列的蛋白酶酶切和化学诱导剪切等处理步骤，最后经色谱纯化制备。其前体蛋白的N-端含有如SEQ ID NO:69所示的烟草花叶病毒蛋白酶(TEV)剪切位点(ENLYFQ)，在胰岛素分子或其类似物的B-链和A-链之间的C-多肽选自如下序列的多肽：SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73。如图10所示，本发明中异源融合表达杂交肽的前体蛋白含有2个蛋白酶Kex2和2个TEV剪切位点，其中Kex2剪切引导前体蛋白分泌表达的信号肽和胰岛素或其类似物B-链与连接肽之间C-末端的“RR”肽键(“RR”双碱性氨基酸残基后的肽键)；TEV剪切加工杂交肽的前体蛋白形成成熟的杂交肽；再经镍离子诱导化学剪切，和/或羧肽酶B和肽基甘氨酸α-酰胺化单加氧酶的处理或不处理。
本发明在一个实施方案中提供了一种含有胰岛素或其类似物和糜蛋白酶抑制肽所形成的杂交肽在治疗I型糖尿病和II型糖尿病中的应用。
本发明在另一个实施方案中提供了杂交肽在治疗I型糖尿病和II型糖尿病时可通过皮下注射和口服给药的给药方式。
有益技术效果：本发明可通过糜蛋白酶抑制肽与胰岛素或其类似物融合的方式提高胰岛素分子的稳定性，促进其口服给药得以实现，可以提高患者用药的依从性和减少副作用，具有有益的经济学价值。
为了更容易理解和将本发明投入实践中，现将参考附图仅以示例的方式描述其一个或多个优选实施例。
附图说明
本发明的各种特征在权利要求书中具有特殊性。参考以下详细描述，将获得对本发明的特征和优点的更好理解，在所述说明性实施例中利用本发明的原理，所述附图包括：
图1.糜蛋白酶的米氏常数Km的测定.利用Prism软件、以底物AAPFpNA的浓度对初速度V0作图，即得到糜蛋白酶水解底物AAPFpNA的米氏常数Km值。实验设置三个重复，计算值以“平均值±标准差”表示。
图2.糜蛋白酶抑制肽抑制活性的测定 通过加入不同浓度的糜蛋白酶抑制肽(CH5、CH11、CH18和CH25)，考察它们对糜蛋白酶的抑制作用，并测定它们能够产生50％抑酶作用的浓度(IC50值)。实验设置三个复孔，计算值以“平均值±标准差”表示。
图3.糜蛋白酶抑制肽抑制活性的测定 通过加入不同浓度的糜蛋白酶抑制肽(CH33、CH34和CH35)，考察它们对糜蛋白酶的抑制作用，并测定它们能够产生50％抑酶作用的浓度(IC50值)。实验设置三个复孔，计算值以“平均值±标准差”表示。
图4.糜蛋白酶抑制肽抑制活性的测定 通过加入不同浓度的糜蛋白酶抑制肽(CH26和CH51)，考察它们对糜蛋白酶的抑制作用，并测定它们能够产生50％抑酶作用的浓度(IC50值)。实验设置三个复孔，计算值以“平均值±标准差”表示。
图5.糜蛋白酶抑制肽抑制活性的测定 通过加入不同浓度的糜蛋白酶抑制肽(CH10、CH54、CH55和CH57)，考察它们对糜蛋白酶的抑制作用，并测定它们能够产生50％抑酶作用的浓度(IC50值)。实验设置三个复孔，计算值以“平均值±标准差”表示。
图6.糜蛋白酶抑制肽抑制活性的测定 通过加入不同浓度的糜蛋白酶抑制肽(CH10、CH60、CH61和CH62)，考察它们对糜蛋白酶的抑制作用，并测定它们能够产生50％抑酶作用的浓度(IC50值)。实验设置三个复孔，计算值以“平均值±标准差”表示。
图7.糜蛋白酶抑制肽抑制活性的测定 通过加入不同浓度的糜蛋白酶抑制肽(CH65、CH69、CH70和CH71)，考察它们对糜蛋白酶的抑制作用，并测定它们能够产生50％抑酶 作用的浓度(IC50值)。实验设置三个复孔，计算值以“平均值±标准差”表示。
图8.甲醇酵母表达载体pDeut-Aox1-ScKex2的示意图含有2个甲醇诱导表达强弱不同的启动子AOX1和DAS2，表达相对较弱的启动子DAS2启动酿酒酵母蛋白酶Kex2的表达。
图9.靶向甲醇酵母AOX1基因的基因编辑Cas9/gRNA表达载体。
图10.胰岛素或其类似物杂交肽的前体蛋白表达和成熟剪切过程αMF信号肽引导前体蛋白靶向内质网，在内质网由信号肽剪切去除信号肽后前体蛋白转运质反面高尔基体，由蛋白酶Kex2剪切去除αMF的前体区和B-链C-末端的“RR”后肽键；进而包装分泌到细胞外；再经过蛋白酶TEV剪切、羧肽酶B(Carboxy peptidase B)和镍离子诱导剪切，获得胰岛素类似物杂交肽。
具体实施方式
天然的蛋白酶抑制剂作为添加剂已用于保护胰岛素分子免于蛋白酶降解的实验研究。在这些多肽类蛋白酶抑制剂中，一个选自大豆胰蛋白酶抑制剂BBI家族的抑制剂含有2个抑制蛋白酶的活性环(Loop)，抑制人胰蛋白酶和糜蛋白酶；这种蛋白酶抑制剂已在PCT专利WO2014191545、WO2019239405和WO2017161184中有公开描述。
对比BBI多肽抑制剂，向日葵胰蛋白酶抑制剂-1(Sunflower Trypsin Inhibitor-1,SFTI-1)是一个从向日葵种子中分离的仅含有14个氨基酸残基的头-尾环化的环肽，PCT专利公开号WO2020023386也描述了其可作为蛋白酶抑制剂，即一种口服药物组分用于糖尿病的治疗。SFTI-1形成一个刚性结构，包含2个短的β-折叠、一个分子内二硫键和头-尾环化。SFTI-1尽管具有结构稳定的环状结构，但很难与治疗性多肽进行直接偶联，仅可作为添加剂应用。但是SFTI-1仅具有抗胰蛋白酶活性，不具有抗糜蛋白酶活性,SFTI-1不能作为糜蛋白酶抑制剂保护糜蛋白酶对胰岛素分子的降解。
丝氨酸蛋白酶抑制肽的P1位点决定不同丝氨酸蛋白酶的专一性，糜蛋白酶(Chymotrypsin)的P1位点为Tyr、Phe，仅有少数几篇文献报道了基于BBI家族多肽骨架衍生的多肽抑制剂具有抗糜蛋白酶活性[11-14]，但抑制活性较弱；而文献中披露的具有抑制糜蛋白酶的活性肽仅作为糜蛋白酶抑制剂的功能应用。本 发明依据抑制胰蛋白酶SFTI-1的核心分子骨架为基础，通过替换P1位点改变抑制肽分子骨架的蛋白酶专一性，再进行不同识别位点的替换和抑制活性评价，经过一系列的优化实验，获得了抑制糜蛋白酶的多肽分子骨架为：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:28。本发明提供的上述糜蛋白酶的抑制肽直接与生物活性肽形成融合的杂交肽，改变了作为抑制糜蛋白酶的添加剂仅在释放位点保护了活性多肽免受部分糜蛋白酶的降解，而且能伴随着活性多肽如胰岛素进入血循环，一直保护其抗糜蛋白酶的降解作用。
胰岛素分子本身对胰蛋白酶具有较好的抗性，在小肠代谢酶中最主要的代谢酶是糜蛋白酶，占胰腺分泌的总蛋白的9％[15]。在本发明中利用糜蛋白酶抑制肽直接与胰岛素或其类似物分子形成杂交肽，所形成的杂交肽从小肠的微环境到进入血循环直至发挥药效的整个过程中一直保护其免受蛋白酶的降解。
多肽合成：本发明的多肽可以通过各种方法制备。例如，多肽可通过常用的固相合成方法合成，例如涉及本领域公知的α-氨基的t-BOC或FMOC保护的方法。在这里，氨基酸按顺序添加到一个不断增长的氨基酸链中。固相合成方法特别适合于大规模生产中合成多肽或相对较短的多肽。
酶抑制活性的测定：测定合成的活性多肽类蛋白酶抑制剂(分子骨架)的抑制常数。使用显色底物N-琥珀酰-Ala-Ala-Pro-Phe-p-硝基苯胺(AAPFpNA)，通过竞争性结合来测定猪α-糜蛋白酶的抑制活性。对猪α-糜蛋白酶的抑制活性相关实验测定在20mM CaC12、50mM Tris-HC1缓冲液(pH 7.8)中进行。用280nm处的光密度(OD)测定多肽浓度。酶水解底物的米氏常数(Km)由405nm处的底物水解初始速率计算得出。在完全水解后的405nm处测定底物的吸光度值。所有数据均采用非线性回归处理。
胰岛素类似物：
本发明所获得的糜蛋白酶抑制剂可广泛地与胰岛素类似物形成抗糜蛋白酶 降解的杂交肽，提高其稳定性和体内外的生物活性。其中，胰岛素或其类似物并不仅限于本发明中公开的作为示例而选择的胰岛素或其类似物，可以是其B-链中替换一个或几个氨基酸残基的类似物，举例，如SEQ ID NO:74(FVQQHLCGSHLVEALYLVCGERGFFYTPKT)、SEQ ID NO:75(FVNQHLCGSHLVEALYLVCGERGFFYTPKQ)、SEQ ID NO:76(FVNQHLCGSHLVEALYLVCGERGFFYTPKE)、SEQ ID NO:77(FVQQHLCGSHLVEALYLVCGERGFFYTPKE)、SEQ ID NO:78(FVNQHLCGSHLVEALYLVCGERGFFYTDPT)、SEQ ID NO:79(FVNQHLCGSHLVQALYLVCGERGFFYTPKT)、SEQ ID NO:80(FVNQHLCGSHLVEALALVCGERGFFYTPKT)、SEQ ID NO:81(FVNQHLCGSHLVEALYLVCGEEGFFYTPKT)、SEQ ID NO:82(FVNQHLCGSHLVEALYLVCGEEGFFYTLPT)、SEQ ID NO:83(FVNQHLCGSHLVEALYLVCGERGFFYTVPT)、SEQ ID NO:84(FVNQHLCGSHLVEALYLVCGERGFFYTAPT)；或者A-链中替换一个或几个氨基酸残基的类似物，举例，如SEQ ID NO:85(GIVEQCCTSICSLEQLENYCN)、SEQ ID NO:86(GIVEQCCTSICSLYQLENYCS)、SEQ ID NO:87(GIVEQCCHSICSLYQLENYCN)、SEQ ID NO:88(GIVEQCCWSICSLYQLENYCN)、SEQ ID NO:89(GIVEQCCMSICSLYQLENYCN)。
杂交肽前体蛋白的蛋白酶剪切与成熟：
本发明中的杂交肽，利用真核细胞进行其前体蛋白的异源分泌表达，其中在表达宿主细胞内蛋白酶Kex2进行前体蛋白的剪切促进其分泌表达；然后再利用烟草花叶病毒的蛋白酶TEV酶具有宽泛的底物特性[16]，对分泌表达的前体蛋白(胰岛素类似物)B-链和A-链的N-端融合肽段(ENLYFQ，SEQ ID NO:69)进行剪切修饰，释放正确的B-链或A-链；同时在前体蛋白(胰岛素类似物)B-链和A-链的C-端引入镍离子诱导剪切的肽段(G↓SRHW)(SEQ ID NO:72、SEQ ID NO:73)[17]。此外，还可以再使用羧肽酶B处理或不处理，可分别获得C-末端不含有或含有两个碱性氨基酸“RR”的杂合肽；B-链或A-链的C-末端含有镍离子诱导剪切肽段(G↓SRHW)时，经镍离子介导剪切后会在其C- 端融合一个甘氨酸(Gly)残基，可以使用重组酰胺化酶进行处理，使得成熟的B-链或A-链的C-末端形成一个酰胺化的保护性修饰。
C-多肽：也称为C-肽(Peptide)，天然的C-肽是有31个氨基酸组成的单链多肽，在胰岛素原分子中连接B-链和A-链。本发明描述为连接胰岛素或其类似物杂交肽的前体蛋白中B-链和A-链之间的多肽，可以与天然的C-肽氨基酸结构相同或不同。
定义：
除非本文特别定义，否则本文中使用的所有术语具有与本发明领域的普通技术人员所理解的相同的含义。提供以下定义是为了提供在描述本发明的说明书和权利要求中使用的术语的清晰度。
单数形式“一个/一种(a)”、“一个/一种(an)”和“所述(the)”包括复数，除非上下文另有明确指示。
技术人员清楚的是，本文公开的多肽序列从左至右进行显示，其中序列的左端为多肽的N-端，序列的右端为多肽的C-端。
本发明使用的术语“杂交肽”指由天然的肽键连接的两个肽段，且可以通过固相合成或生物表达制备。
本文使用的术语“连接肽”广义上是指促进转折结构形成的一个富含甘氨酸、丝氨酸或脯氨酸的肽段，能够将两个多肽连接在一起并形成一个化学结构。在本发明中概念上也适用于C-多肽，其功能是连接B-链和A-链形成前体蛋白。
本文所用的术语“蛋白酶抑制剂”是指抑制蛋白酶功能的多肽分子。在本发明中蛋白酶抑制剂抑制哺乳动物胃肠道中发现的糜蛋白酶和血循环中类似糜蛋白酶样活性的蛋白酶。
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引用以下实施例来说明本发明的实施方案并且仅为了更好的理解本发明，但不应被解释为限制本发明的范围或精神。
实施例
实施例1多肽固相合成
按照每条多肽氨基酸残基的序列，采用芴甲氧羰基(Fmoc)固相化学合成方法从C-端到N-端逐一合成；当氨基酸侧链保护的线性肽合成完成后，从树脂上切割线性肽，去除线性肽中氨基酸残基的保护基，再进行分子内巯基的氧化环化形成二硫键，最后利用高压液相色谱反相C18柱色谱纯化获得目标多肽。
一、原料
(1)树脂：Fmoc-L-丙氨酸-王氏树脂(Fmoc-Ala-Wang resin)、Fmoc-N-(2,2,4,6,7-五甲基苯并二氢呋喃-5-磺酰基)-L-精氨酸-王氏树脂(Fmoc-Arg(Pbf)-Wang resin)、Fmoc-N-三苯甲基-L-天冬酰胺-王氏树脂(Fmoc-Asn(Trt)-Wang resin)、Fmoc-O-叔丁基-L-天冬氨酸-王氏树脂(Fmoc-Asp(OtBu)-Wang resin)、Fmoc-L-甘氨酸-王氏树脂(Fmoc-Gly-Wang resin)、Fmoc-L-苯丙氨酸-王氏树脂(Fmoc-Phe-Wang resin)、Fmoc-L-脯氨酸-王氏树脂(Fmoc-Pro-Wang resin)、Fmoc-O-叔丁基-L-酪氨酸-王氏树脂(Fmoc-Tyr(tBu)-Wang resin)。
(3)试剂：哌啶、DMF(N,N-二甲基甲酰胺)、DCM(二氯甲烷)、4-Picoline(4-甲基吡啶)、DIEA(二异丙基乙胺)、HATU(2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯)、HOBT(1-羟基苯并三唑)、TBTU(O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸)、DIC(二异丙基碳二亚胺)，TFA(三氟乙酸)、EDT(1，2乙二硫醇)、TIPS(三异丙基硅烷)、TA(苯甲硫醚)、苯酚、乙醚、DMSO(二甲亚砜)、纯水。
二、合成方法
SEQ ID NO:1(Gly-Arg-Cys-Thr-Phe-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro-Asp)
(1)称取Fmoc-Asp(OtBu)-Wang resin，放到玻璃反应柱加DCM溶胀30min，减压抽掉DCM。
(2)用DMF洗涤树脂3遍，加入哌啶/DMF(1:4,v/v)溶液反应20min除去保护基Fmoc，减压抽掉溶液，用DMF洗涤6遍。
(3)分别称取第二个氨基酸Fmoc-Pro-OH、TBTU加入到树脂中，DMF溶解并加入DIEA，反应30min，取树脂做Kaiser Test检验反应，观察到溶液亮黄、树脂黄时，说明反应完全，减压抽掉溶剂。
(4)重复步骤(2)和(3)，最终得到带有保护基团的肽段，即Fmoc-Gly-Arg(Pbf)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Pro-Asp(OtBu)-Wang resin，脱去Fmoc，然后用DMF、DCM和甲醇各洗三遍，抽干树脂。
(5)加入裂解液(TFA、EDT、TA、苯酚、纯水按一定比例混合)去除树脂和氨基酸侧链保护基，砂芯过滤，向滤液加入乙醚析出，离心，洗涤固体3次，抽干。
(6)用H2O/乙腈(9:1,v/v)溶解，体积放大到100mL，加入稀氨水调至碱性(pH≈8)，取小样测试巯基活性，黄色说明巯基存在，加入双氧水2-3滴，反应5-10min，再次检测，溶液呈现透明，说明氧化完全(90％以上)，加冰醋酸调至酸性(pH≈6)，其化学结构由质谱表征，结果正确后利用高压液相色谱反相C18柱色谱纯化获得目标多肽。
(7)SEQ ID NO:1(CH54)的实测分子量为1550.4Da([M+2H]2+＝776.2)。
SEQ ID NO:2(Gly-Arg-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro-Asp)
SEQ ID NO:2选取Fmoc-Asp(OtBu)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即
Fmoc-Gly-Arg(Pbf)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Pro-Asp(OtBu)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链 保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:2(CH55)，其实测分子量为1566.4Da([M+2H]2+＝784.2)。
SEQ ID NO:3(Gly-Arg-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro)
SEQ ID NO:3选取Fmoc-Pro-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Gly-Arg(Pbf)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Pro-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:3(CH56)，其实测分子量为1452.2Da([M+2H]2+＝727.1)。
SEQ ID NO:4(Gly-Arg-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:4选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Gly-Arg(Pbf)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:4(CH57)，其实测分子量为1354.2Da([M+2H]2+＝678.1)。
SEQ ID NO:5(Arg-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:5选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Arg(Pbf)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:5(CH58)，其实测分子量为1297.0Da([M+2H]2+＝649.5)。
SEQ ID NO:6(Gly-Thr-Cys-Thr-Phe-Ser-Ile-Pro-Pro-Ile-Cys-Asn-Pro-Asn)
SEQ ID NO:6选取Fmoc-Asn(Trt)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保 护基团的肽段，即Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn-Pro-Asn(Trt)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:6(CH59)，其实测分子量为1461.0Da([M+2H]2+＝731.5)。
SEQ ID NO:7(Gly-Thr-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Asn-Pro-Asn)
SEQ ID NO:7选取Fmoc-Asn(Trt)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Pro-Asn(Trt)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:7(CH60)，其实测分子量为1477.2Da([M+2H]2+＝739.6)。
SEQ ID NO:8(Gly-Thr-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Asn-Pro)
SEQ ID NO:8选取Fmoc-Pro-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Pro-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:8(CH61)，其实测分子量为1363.2Da([M+2H]2+＝682.6)。
SEQ ID NO:9(Gly-Thr-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Asn)
SEQ ID NO:9选取Fmoc-Asn(Trt)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:9(CH62)，其实测 分子量为1266.0Da([M+2H]2+＝634.0)。
SEQ ID NO:10(Thr-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Asn)
SEQ ID NO:10选取Fmoc-Asn(Trt)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Thr(tBu)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:10(CH63)，其实测分子量为1209.3Da([M-H]-＝1208.3)。
SEQ ID NO:11(Lys-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:11选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:11(CH64)，其实测分子量为1269.2Da([M+2H]2+＝635.6)。
SEQ ID NO:12
(Cys-Gly-Arg-Ala-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro-Asp)
SEQ ID NO:12选取Fmoc-Asp(OtBu)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Pro-Asp(OtBu)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:12(CH65)，其实测分子量为1637.2Da([M+2H]2+＝819.6)。
SEQ ID NO:13(Cys-Gly-Arg-Ala-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro)
SEQ ID NO:13选取Fmoc-Pro-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Pro-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:13(CH66)，其实测分子量为1523.2Da([M+2H]2+＝762.6)。
SEQ ID NO:14(Cys-Gly-Arg-Ala-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:14选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:14(CH67)，其实测分子量为1425.2Da([M+2H]2+＝713.6)。
SEQ ID NO:15(Cys-Gly-Arg-Ala-Abu-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:15选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Abu-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:15(CH68)，其实测分子量为1439.2Da([M+2H]2+＝720.6)。
SEQ ID NO:16(Cys-Lys-Arg-Ala-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:16选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Cys(Trt)-Lys(Boc)-Arg(Pbf)-Ala-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-C ys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:16(CH69)，其实测分子量为1496.2Da([M+2H]2+＝749.1)。
SEQ ID NO:17(Cys-Glu-Arg-Ala-Thr-Tyr-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:17选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Cys(Trt)-Glu(OtBu)-Arg(Pbf)-Ala-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:17(CH70)，其实测分子量为1497.2Da([M+2H]2+＝749.6)。
SEQ ID NO:18(Cys-Gly-Arg-Ala-Thr-Phe-Ser-Ile-Pro-Pro-Ile-Cys-Phe)
SEQ ID NO:18选取Fmoc-Phe-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Cys(Trt)-Gly-Arg(Pbf)-Ala-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:18(CH71)，其实测分子量为1409.2Da([M+2H]2+＝705.6)。
SEQ ID NO:19(Phe-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Gln-Cys-Tyr-Gly)
SEQ ID NO:19选取Fmoc-Gly-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Phe-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Gly-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:19(CH10)，其实测分子量为1375.55Da([M+Na]+＝1398.55)。
SEQ ID NO:20(Phe-Cys-Thr-Phe-Ser-Ile-Pro-Pro-Gln-Cys-Tyr-Gly)
SEQ ID NO:20选取Fmoc-Gly-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Phe-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Gly-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:20(CH5)，其实测分子量为1360.02Da([M+K+H]2+＝700.01)。
SEQ ID NO:21
(Ser-Cys-Thr-Phe-Ser-Ile-Pro-Pro-Gln-Cys-Tyr-Gly)
SEQ ID NO:21选取Fmoc-Gly-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Ser(tBu)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Gly-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:21(CH11)，其实测分子量为1300.55Da([M+H]+)。
SEQ ID NO:22
(Ala-Cys-Thr-Tyr-Ser-Ile-Pro-Ala-Lys-Cys-Phe)
SEQ ID NO:22按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Ala-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Ala-Lys(Boc)-Cys(Trt)-Phe-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:22(CH18)，其实测分子量为1200.80Da([M+2H]2+＝601.40)。
SEQ ID NO:23
(Gly-Thr-Cys-Thr-Phe-Ser-Ile-Pro-Pro-Ile-Cys-Asn-Pro-Asn)
SEQ ID NO:23选取Fmoc-Asn(Trt)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Pro-Asn(Trt)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:23(CH25)，其实测分子量为1461.00Da([M+2H]2+＝731.50)。
SEQ ID NO:24
(Gly-Thr-Cys-Thr-Phe-Ser-Ile-Pro-Pro-Ile-Cys-Asn)
SEQ ID NO:24选取Fmoc-Asn(Trt)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Gly-Thr(tBu)-Cys(Trt)-Thr(tBu)-Phe-Ser(tBu)-Ile-Pro-Pro-Ile-Cys(Trt)-Asn(Trt)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:24(CH26)，其实测分子量为1249.50Da([M+Na]+＝1272.50)。
SEQ ID NO:25
(Phe-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Gln-Cys-Tyr)
SEQ ID NO:25选取Fmoc-Tyr(tBu)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Phe-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:25(CH33)，其实测分子量为1318.80Da([M+2H]2+＝660.40)。
SEQ ID NO:26
(Phe-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Gln-Cys-Tyr-Ala)
SEQ ID NO:26选取Fmoc-Ala-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Phe-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Ala-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:26(CH34)，其实测分子量为1390.80Da([M+2H]2+＝696.40)。
SEQ ID NO:27
(Phe-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Gln-Cys-Arg)
SEQ ID NO:27选取Fmoc-Arg(Pbf)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Phe-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Arg(Pbf)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:27(CH35)，其实测分子量为1312.20Da([M+2H]2+＝657.10)。
SEQ ID NO:28
(Pro-Cys-Thr-Tyr-Ser-Ile-Pro-Pro-Gln-Cys-Tyr)
SEQ ID NO:28选取Fmoc-Tyr(tBu)-Wang resin为起始原料，按照SEQ ID NO:1所述的方法进行合成，先依次添加与多肽序列相应的氨基酸原料、合成带有保护基团的肽段，即Fmoc-Pro-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Pro-Pro-Gln(Trt)-Cys(Trt)-Tyr(tBu)-Wang resin，脱去Fmoc，再加入裂解液去除树脂和氨基酸侧链保护基，并氧化形成二硫键，最终分离、纯化得到目的肽段SEQ ID NO:28(CH51)，其实测分子量为1268.80Da([M+2H]2+＝635.40)。
实施例2抑制糜蛋白酶的多肽分子的设计及抑制活性评价
米氏常数Km值的测定:
(1)往96孔板中加入198μL、20mM CaC12、50mM Tris-HC1缓冲液(pH 7.8)，37℃预热15min。再加入2μL不同浓度的底物pNA(DMSO配置)，500rpm混匀1min，置于37℃孵育20min，测定OD405nm的吸光度值。200μL反应体系中，pNA的终浓度分别为0、0.025、0.05、0.075、0.1、0.125、0.15、0.25、和0.3mM。每个浓度做三个复孔，以pNA浓度对OD405nm值作图，求得标准曲线。
(2)往96孔板中加入190μL、20mM CaC12、50mM Tris-HC1缓冲液(pH 7.8)及8μL、0.75μM的糜蛋白酶，37℃预热5min。再加入2μL不同浓度的底物AAPFpNA(DMSO配置)，500rpm混匀1min，置于37℃反应20min，测定OD405 nm的吸光度值。200μL反应体系中，AAPFpNA的终浓度分别为0、0.125、0.25、0.285、0.33、0.4和0.5mM。每个浓度做三个复孔，以时间对OD405nm值作图，求得相应的曲线。以曲线的斜率除以标准曲线的斜率和酶浓度，得到初速度V0(mM/(min*mM protein))。利用Prism软件、以底物AAPFpNA的浓度对初速度V0作图，即得到糜蛋白酶水解AAPFpNA的米氏常数Km值。
抑制常数Ki值的测定:
(1)将不同浓度的糜蛋白酶抑制肽(CHs)骨架、20mM CaC12、50mM Tris-HC1缓冲液(pH 7.8)加入预冷的96孔板中，总体积190μL，37℃预热5min(500rpm离心1min，静置4min)。加入8μL、750nM的糜蛋白酶，37℃孵育10min(500rpm离心1min，静置9min)。加入2μL、50mM的底物AAPFpNA，500rpm混匀1min，置于37℃反应90min，测定OD405nm的吸光度值。每个浓度做三个复孔，空白对照只加缓冲液和底物，作为最小吸收值(Min OD405nm)；阴性对照只加缓冲液、酶和底物，作为最大吸收值(Max OD405nm)。
(2)200μL反应体系中，糜蛋白酶的终浓度约30nM，AAPFpNA的终浓度约0.5mM。
(3)数据统计
酶的剩余活性(％)＝(1-(Max OD405nm-Sample OD405nm)/(Max OD405nm-Min OD405nm))*100
以底物浓度对酶的剩余活性作图，求得CHs骨架抑制糜蛋白酶的半数抑制浓度(IC50)，再代入公式Ki＝IC50/(1+S/Km)(S、IC50和Km分别是底物浓度、半数抑制浓度和米氏常数)即可求得CHs骨架抑制糜蛋白酶的抑制常数Ki。
结果：
利用不同的AAPFpNA浓度对一定浓度的糜蛋白酶催化水解产生pNA测定OD405nm的吸光度值，参照标准曲线，利用Prism软件、以底物AAPFpNA的浓度对初速度V0作图，即得到糜蛋白酶水解AAPFpNA的米氏常数Km值为0.38mM(R2＝0.9988)(图1)。
天然Bowman-Birk家族的蛋白酶抑制剂(Bowman-Birk inhibitor，BBI)大多含有两个独立的抑制位点，分别抑制胰蛋白酶和糜蛋白酶；大豆BBI是由71个氨基酸残基组成，由7个二硫键交联，其中一个抑制胰蛋白酶的位点位于Lys16-Ser17，另一个抑制糜蛋白酶的位点位于Leu 43-Ser44，它与胰蛋白酶或胰凝乳蛋白酶形成1:1复合物，并与这两种酶形成三元复合物。这种天然结构的BBI抑制蛋白酶活性较弱，有文献报道[McBride JD,Freeman N,Domingo GJ,Leatherbarrow RJ.Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library.J Mol Biol,1996,259:819-827.]可通过简化BBI结构可获得单一抑制活性的由二硫键连接的环肽。本发明结合糜蛋白酶P1位点的特异性(特异性地识别Tyr和Phe)以及P1'和P3'的保守性，设计合成了由二硫键环化且具有核心结构为“CTYSIPPQC”的一系列多肽(表1)，体外的抑制活性测定获得2个具有较高抑制活性的多肽CH10和CH5，其IC50值分别为0.06μM和0.19μM(表2)，结果证实P1位点对糜蛋白酶的抑制活性影响较大；然后在CH10的基础上对其P4和P7'氨基酸残基进行替换获得了CH11、CH35和CH51,其抑制活性降低了近7倍，说明P4和P7'氨基酸残基可增强与糜蛋白酶的活性位点的分子间作用力(表2)；通过对比CH33、CH34和CH10的抑制活性差异，可见具有较强极性的P8'甘氨酸更有利于增强其与蛋白酶之间的分子间作用(表2)；P5位点的延长(CH25、CH26)和P4'位点的氨基酸替换(CH18)对抑制活性没有大的改变(表2，图2,3,4)。
SFTI-1(GRCTKSIPPICFPD)相对其它BBI家族的蛋白酶抑制剂分子，其结 构特殊仅含有一对二硫键，具有首-尾环化的结构。根据糜蛋白酶P1位点的特异性，直接突变其P1位点的Lys为Tyr或Phe，设计合成了多肽CH54和CH55(表1)，二者均具有很好的抑制糜蛋白酶活性，其IC50分别为0.03μM和0.02μM(图5)。为进一步简化其结构合成了CH56、CH57和CH58(表1)，三者的抑制可以看出P8'位的Pro会产生空间位阻，降低其抑制作用，导致其IC50(0.62μM)相对于CH55(IC50＝0.02μM)降低30多倍(表2)；同时对比CH57和CH58的抑制活性，说明P5位的Gly有利于增强抑制肽与糜蛋白酶活性中心区的相互作用，P5位点的氨基酸残基对其抑制活性影响较大；结果也表明CH58是与糜蛋白酶活性中心相互作用的核心骨架(表2)。同时合成P4和P7'位点氨基酸取代的多肽CH59-CH62(表1)，其抑制活性较CH57稍弱，抑制糜蛋白酶的IC50分别为0.18μM、0.09μM、0.12μM、0.15μM(表2，图6)；还在CH57的基础上合成一系列二硫键扩环的多肽CH65-CH71(表1),其中CH65、CH68-CH71均显示出较好的抑制糜蛋白酶活性，其IC50分别为0.48μM、0.45μM、0.30μM、0.25μM、0.41μM(表2，图7)。
表1.糜蛋白酶的抑制肽的分子结构及其活性


a：表中的抗糜蛋白酶骨架的分子内两个半胱氨酸之间形成了二硫键；Xaa＝
L-α-Aminobutyric acid,Abu。
表2.糜蛋白酶的抑制肽的活性测定

*：CH10在0.0001μM浓度几乎没有抑酶活性，但有两个复孔由于加样误差很大，故舍弃这两个复孔的值。
表2.糜蛋白酶的抑制肽的活性测定


*：CH10在0.0001μM浓度几乎没有抑酶活性，但有两个复孔由于加样误差很大，故舍弃这两个复孔的值。
表2.糜蛋白酶的抑制肽的活性测定(续表)
表2.糜蛋白酶的抑制肽的活性测定(续表)
表2.糜蛋白酶的抑制肽的活性测定(续表)
表2.糜蛋白酶的抑制肽的活性测定(续表)
实施例3甲醇酵母表达载体pDeut-Aox1-ScKex2构建
在利用甲醇酵母为宿主进行异源蛋白的分泌表达时，使用最广泛的载体是Invitrogen公司pPIC9K表达质粒，该表达载体存在抗性筛选标记表达不稳定、仅一个多克隆位点(multiple cloning site，MCS)、同源整合效率低等问题，为提高异源目的基因的整合表达进行质粒表达载体的改造，构建过程如下：
⑴人工合成含有酿酒酵母细胞色素C异构酶1(Cytochrome c isoform 1，CYC1)的启动子和终止子并融合大肠杆菌Kan编码基因的融合表达框(SEQ ID NO:90),然后使用引物kan-1/kan-2(SEQ ID NO:91和92)扩增融合基因片段，利用限制性内切酶BsrG I和Nde I定向亚克隆到原始的pPIC9K质粒表达载体，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到质粒pScCyc1-Kan。
⑵人工合成一个含有酿酒酵母αMF(mating factor alpha 1)的信号肽和分泌前体区、甲醇酵母FLD1的终止子和DAS2启动子的融合基因片段(SEQ ID NO:93),然后使用引物A4-B1-/A4-B-2(SEQ ID NO:94和95)扩增整个融合基因片段，利用限制性内切酶BamH I插入到质粒pScCYC-Kan中，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到质粒pA4-DAS2-Kan。
⑶利用甲醇酵母基因组DNA为模板，使用引物AOX1-3F1/AOX1-3F2(SEQ ID NO:96和97)扩增AOX1基因的3'-末端的同源DNA片段(SEQ ID NO:98)，再使用限制性内切酶Sap I位点插入AOX1基因的3'-末端的同源DNA片段，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到质粒表达载体pDeut-Aox1vector。
⑷为了促进胰岛素或其类似物前体蛋白的分泌表达，需要增强蛋白酶Kex2的表达，使用引物ScKEX2-E1/ScKEX2-N2(SEQ ID NO:99和100)直接扩增编码框优化的酿酒酵母蛋白酶Kex2(ScKex2,SEQ ID NO:101)，使用限制性内切酶EcoR I和Not I亚克隆到DAS2启动子控制的表达框，可实现ScKex2胞内高表达；经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到质粒表达载体pDeut-Aox1-ScKex2(图8)。
实施例4.靶向甲醇酵母乙醇氧化酶1(AOX1)的基因编辑表达载体pGAD-Cas9-gAOX1的构建
甲醇同源整合的效率很低，为提高其定向整个到甲醇酵母AOX1位点的效率，本发明利用基因编辑技术由gRNA介导的双质粒转化体系进行目的基因的定向整合表达。利用质粒pGADT7为骨架进行靶向AOX1基因的gRNA表达载体的构建，构建过程如下：
⑴首先人工合成含有靶向AOX1启动子区的gRNA表达框(gAOX1)和甲醇酵母复制子元件PARS2(SEQ ID NO:102)，然后使用引物gRNA-Nhe1/PARS2-Not1(SEQ ID NO:103和104)直接扩增融合基因片段，使用限制性内切酶Nhe I和Not I位点亚克隆亚克隆到质粒pGADT7，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验 证即得到质粒载体pGADT7-gAOX1。
⑵以含有Cas9编码框的质粒pX260(AddGene)为模板，使用引物X260-Nde1/X260-Xho1(SEQ ID NO:105和106)直接扩增Cas9编码框，利用限制性内切酶Nde I和Xho I亚克隆到质粒pGADT7-gAOX1，经过连接II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到质粒载体pCas9-gAOX1。
⑶利用甲醇酵母基因组DNA为模板使用引物GAP_X260-1/GAP_X260-2(SEQ ID NO:107和108)扩增甲醇酵母三磷酸甘油醛脱氢酶基因启动子(GAD)(SEQ ID NO:109)DNA片段，然后使用限制性内切酶Cla I和Nde I位点亚克隆到质粒pCas9-gAOX1，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到质粒载体pGAD-Cas9-gAOX1(图9)。
实施例5.含有胰岛素或类似物的杂交肽的前体蛋白表达载体的构建
甲醇酵母细胞是分泌表达能力很强的真核单核细胞，易于发酵培养。本发明使用酿酒酵母αMF的信号肽和前体区引导杂交肽前体蛋白的分泌表达。首先，在内质网(ER)翻译产生的杂交肽前体蛋白由信号酶剪切去除αMF的信号肽，然后转运至反面高尔基体，再由高表达并定位反面高尔基体的酵母ScKex2剪切αMF的前体区和胰岛素或其类似物杂交肽中B-链和A-链之间的C-多肽(C-Peptide)或连接肽(Linker)，进而在反面高尔基体被包装分泌到胞外；经亲和层析纯化后再利用烟草花叶病毒蛋白酶TEV剪切去除融合的肽段及其连接肽，最后纯化获得目的蛋白，图10简要概述了由杂交肽前体蛋白到成熟杂交肽的过程。详细的杂交肽前体蛋白编码框的构建过程如下：
⑴人胰岛素或其类似物的前体蛋白表达载体：人工合成编码胰岛素或其类似物的不同B-链，同时具有相同A-链(SEQ ID NO:29)的前体蛋白编码基因(表3)，然后利用合成的引物Ins-BamH1/Ins-Spe1(SEQ ID NO:118和119)(表4)直接扩增DNA编码框，利用限制性内切酶BamH I和Spe I亚克隆到表达质粒pDeut-Aox1-ScKex2，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pAOX1-ScKex2-Ins-1、pAOX1-ScKex2-Ins-2、pAOX1-ScKex2-Ins-3和pAOX1-ScKex2-Ins-4。提纯正 确的阳性克隆质粒，利用限制性内切酶Pme I线性化，与gRNA表达质粒pGAD-Cas9-gAOX1一起电转化甲醇酵母细胞GS115。
表3人胰岛素或其类似物前体蛋白编码基因
⑵人胰岛素或其类似物B-链N-末端融合糜蛋白酶抑制肽的前体蛋白表达载体
使用已构建的含有胰岛素或其类似物的前体蛋白表达质粒pAOX1-ScKex2-Ins-1、pAOX1-ScKex2-Ins-2、pAOX1-ScKex2-Ins-3和pAOX1-ScKex2-Ins-4为模板，分别使用引物AOX1-P1/CH57-B-N1或AOX1-P1/CH10-B-N1(表4)进行PCR扩增编码αMF的信号肽和前体区的DNA片段，同利用引物CH57-B-N2/TFLD1-R1或CH10-B-N2/TFLD1-R1(表4)进行PCR扩增编码胰岛素或类似物前体蛋白的DNA片段，上述2个PCR扩增的DNA 片段，进行SOE-PCR补平反应，再利用PCR补平反应的产物为模板，利用引物Ins-BamH1-2/Ins-Spe1(Ins-Spe1-1)进行PCR扩增糜蛋白酶抑制肽与胰岛素或其类似物B-链N-末端融合的编码框(ORF)，最后连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pIns1-B-CH57N、pIns2-B-CH57N、pIns3-B-CH57N、pIns4-B-CH57N、pIns1-B-CH10N、pIns2-B-CH10N、pIns3-B-CH10N、pIns4-B-CH10N。
⑶人胰岛素或其类似物B-链C-末端融合糜蛋白酶抑制肽的前体蛋白表达载体
使用已构建的含有胰岛素或其类似物的前体蛋白表达质粒pAOX1-ScKex2-Ins-1、pAOX1-ScKex2-Ins-2、pAOX1-ScKex2-Ins-3和pAOX1-ScKex2-Ins-4为模板，分别使用引物AOX1-P1/Ins1-B-CH57C1或AOX1-P1/Ins1-B-CH10C1、AOX1-P1/Ins2-B-CH57C1或AOX1-P1/Ins2-B-CH10C1、AOX1-P1/Ins3-B-CH57C1或AOX1-P1/Ins3-B-CH10C1和AOX1-P1/Ins3-B-CH57C1或AOX1-P1/Ins3-B-CH10C1(表4)分别进行PCR扩增编码αMF的信号肽、前体区和胰岛素或其类似物B-链编码的DNA片段，同时利用引物Ins1-B-CH57C2/TFLD1-R1或Ins1-B-CH10C2/TFLD1-R1(表4)进行PCR扩增上述4个分别编码胰岛素或类似物对应连接肽和A-链的前体蛋白的DNA片段，把PCR扩增的DNA片段，进行对应的SOE-PCR补平反应，再利用PCR补平反应的产物为模板，利用引物Ins-BamH1/Ins-Spe1(或Ins-Spe1-1)进行PCR扩增胰岛素或其类似物B-链C-末端融合糜蛋白酶抑制肽的融合编码框(ORF)，最后连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pIns1-B-CH57C、pIns2-B-CH57C、pIns3-B-CH57C、pIns4-B-CH57C、pIns1-B-CH10C、pIns2-B-CH10C、pIns3-B-CH10C、pIns4-B-CH10C。
再以质粒pIns1-B-CH57C、pIns2-B-CH57C、pIns3-B-CH57C、pIns4-B-CH57C、pIns1-B-CH10C、pIns2-B-CH10C、pIns3-B-CH10C、pIns4-B-CH10C为模板，分别使用AOX1-P1/CH57-G-R1或AOX1-P1/CH10-G-R1(表4)进行PCR扩增编码αMF和胰岛素或其类似物B-链C-末端融合糜蛋白酶抑制肽的整个编码框DNA片段，同时使用引物CH-G-F1/TFLD1-R1(表4)进行PCR扩增上述8个分别胰岛素或类似物对应连接肽和A-链的前体蛋白编码区，把PCR扩增的DNA片段， 进行对应的SOE-PCR补平反应，再利用PCR补平反应的产物为模板，再利用引物Ins-BamH1/Ins-Spe1进行PCR扩增胰岛素或其类似物B-链C-末端融合糜蛋白酶抑制肽的融合编码框(ORF)，其中C-肽(C-Peptide)被替换为柔性更好的连接肽(Linker序列为GGSGGSG)，最后连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pIns1-B-CH57CT、pIns2-B-CH57CT、pIns3-B-CH57CT、pIns4-B-CH57CT、pIns1-B-CH10CT、pIns2-B-CH10CT、pIns3-B-CH10CT、pIns4-B-CH10CT。
⑷人胰岛素或其类似物A-链N-末端融合糜蛋白酶抑制肽的前体蛋白表达载体
使用已构建的含有胰岛素或其类似物的前体蛋白表达质粒pAOX1-ScKex2-Ins-1、pAOX1-ScKex2-Ins-2、pAOX1-ScKex2-Ins-3和pAOX1-ScKex2-Ins-4为模板，分别使用引物AOX1-P1/Ins-A-CH57N1和AOX1-P1/Ins-A-CH10N1(表4)进行PCR扩增编码αMF的信号肽、前体区和胰岛素或其类似物B-链及其连接肽的DNA片段，同利用引物Ins-A-CH57N2/TFLD1-R1或Ins-A-CH10N2/TFLD1-R1(表4)进行PCR扩增上述4个分别编码胰岛素或类似物对应连接肽和A-链的前体蛋白编码区，把PCR扩增的DNA片段，进行对应的SOE-PCR补平反应，再利用PCR补平反应的产物为模板，利用引物Ins-BamH1/Ins-Spe1(或Ins-Spe1-1)进行PCR扩增胰岛素或其类似物A-链A-末端融合糜蛋白酶抑制肽的融合编码框(ORF)，最后连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pIns1-A-CH57N、pIns2-A-CH57N、pIns3-A-CH57N、pIns4-A-CH57N、pIns1-A-CH10N、pIns2-A-CH10N、pIns3-A-CH10N、pIns4-A-CH10N。
⑸人胰岛素或其类似物A-链C-末端融合糜蛋白酶抑制肽的前体蛋白表达载体
使用已构建的含有胰岛素或其类似物的前体蛋白表达质粒pAOX1-ScKex2-Ins-1、pAOX1-ScKex2-Ins-2、pAOX1-ScKex2-Ins-3和pAOX1-ScKex2-Ins-4为模板，分别使用引物AOX1-P1/Ins-A-CH57C1和AOX1-P1/Ins-A-CH10C1(表4)进行PCR扩增编码αMF的信号肽、前体区和胰岛素或其类似物B-链、连接肽和A-链的DNA片段，同利用引物Ins-A-CH57C2/TFLD1-R1或Ins-A-CH10C2/TFLD1-R1(表4)进行PCR扩增上述4个胰岛素或类似物对应连接肽和A-链的前体蛋白编码区，把PCR扩增的DNA片段，进行对应的SOE-PCR 补平反应，再利用PCR补平反应的产物为模板，利用引物Ins-BamH1/Ins-Spe1进行PCR扩增胰岛素或其类似物A-链C-末端融合糜蛋白酶抑制肽的融合编码框(ORF)，最后连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pIns1-A-CH57C、pIns2-A-CH57C、pIns3-A-CH57C、pIns4-A-CH57C、pIns1-A-CH10C、pIns2-A-CH10C、pIns3-A-CH10C、pIns4-A-CH10C。
再使用质粒pIns1-A-CH57C、pIns2-A-CH57C、pIns3-A-CH57C、pIns4-A-CH57C、pIns1-A-CH10C、pIns2-A-CH10C、pIns3-A-CH10C、pIns4-A-CH10C为模板，分别使用引物AOX1-P1/A-CH-LR1(表4)进行PCR扩增编码αMF的信号肽、前体区和胰岛素或其类似物B-链的DNA片段，同时利用引物A-CH-LR2/TFLD1-R1(表4)进行PCR扩增上述8个分别编码胰岛素或类似物对应连接肽和A-链前体蛋白的DNA片段，把PCR扩增的DNA片段，进行对应的SOE-PCR补平反应，再利用PCR补平反应的产物为模板，利用引物Ins-BamH1/Ins-Spe1进行PCR扩增胰岛素或其类似物A-链C-末端融合糜蛋白酶抑制肽的编码框(ORF)，其中胰岛素或其类似物的B-链和A-链之间的连接肽替换为SEQ ID NO:70(RREAEDGSGGSGENLYFQ)；最后连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pIns1-A-CH57CT、pIns2-A-CH57CT、pIns3-A-CH57CT、pIns4-A-CH57CT、pIns1-A-CH10CT、pIns2-A-CH10CT、pIns3-A-CH10CT、pIns4-A-CH10CT。
⑹人胰岛素或其类似物A-链中间融合糜蛋白酶抑制肽的前体蛋白表达载体
使用已构建的含有胰岛素或其类似物的前体蛋白表达质粒pAOX1-ScKex2-Ins-1、pAOX1-ScKex2-Ins-2、pAOX1-ScKex2-Ins-3和pAOX1-ScKex2-Ins-4为模板，分别使用引物AOX1-P1/Ins-A-CH57M1和AOX1-P1/Ins-A-CH10M1(表4)进行PCR扩增编码αMF的信号肽、前体区和胰岛素或其类似物B-链、连接肽和部分A-链的DNA片段，同时利用引物Ins-A-CH57M2/TFLD1-R1或Ins-A-CH10M2/TFLD1-R1(表4)进行PCR扩增上述4个胰岛素或类似物对应连接肽和部分A-链的前体蛋白编码区，把PCR扩增的DNA片段，进行对应的SOE-PCR补平反应，再利用PCR补平反应的产物为模板，利用引物Ins-BamH1/Ins-Spe1(或Ins-Spe1-1)进行PCR扩增胰岛素或其 类似物A-链中间插入糜蛋白酶抑制肽的融合编码框(ORF)，最后连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pIns1-A-CH57M、pIns2-A-CH57M、pIns3-A-CH57M、pIns4-A-CH57M、pIns1-A-CH10M、pIns2-A-CH10M、pIns3-A-CH10M、pIns4-A-CH10M。
表4用于构建人胰岛素或其类似物杂交肽前体蛋白表达载体的引物


实施例6.甲醇酵母的DNA转化、阳性转化子的筛选和目的蛋白的诱导表达电转化方法：
(1)挑取新活化的毕赤酵母GS115平板上的单菌落，于5～10mL YPD液体培养基中，30℃、220rpm培养8～10小时。按照1.2～1.5％(V/V)的比例转接到新鲜的YPD培养基中，相同条件培养过夜。
(2)待菌液OD600nm值为1.3～1.5时，每4～6mL菌液收集到一个1.5mL离心管中，8000rpm离心5分钟。
(3)依次用预冷的1mL无菌水洗菌体2次，用1mL 1M无菌山梨醇溶液洗菌体2次，每次洗毕后于8000rpm离心5分钟。
(4)每4～6mL离心后的菌体用80μL 1M预冷的无菌山梨醇溶液重悬菌体，即为处理过的一管感受态细胞。
(5)将一管GS115感受态细胞与2～3μg(体积不超过10μL)目的DNA片段混匀，加入到预冷的无菌电转杯中，冰上静置5分钟。擦净电转杯外壁的水滴，在电转仪上选择毕赤酵母选项即“Pic”，电击后记下相应的参数，并立即加入1mL1M预冷的无菌山梨醇YPD溶液，冰上静置1小时。于30℃静置复壮4～6小时。利用移液器轻轻混匀后，每200μL涂布于一个YPD相应抗性平板上。置于30℃培养箱培养2～4天。
酵母基因组提取：按酵母基因组提取试剂盒操作如下：
(1)取1-5mL酵母培养物(不超过5×107酵母细胞)，12000rpm离心1分钟，尽量吸除上清。
(2)酵母细胞壁的破除：向菌体中加入600μL蜗牛酶，充分混匀，并在摇床上220rpm，30℃处理2小时。4000rpm离心10分钟，弃上清，收集沉淀。
(3)加入200μL缓冲液GA重悬，充分混匀。
(4)加入20μL Proteinase K溶液，混匀。
(5)加入220μL缓冲液GB，充分颠倒混匀，70℃放置10分钟，溶液应变清亮，短暂离心去除管壁水珠。
(6)加入220μL无水乙醇，充分颠倒混匀，短暂离心去除管壁水珠。
(7)将上一步所得溶液和絮状沉淀都加入一个吸附柱CB3中(吸附柱放入收集管中)，12000rpm离心30s，倒掉废液，放回CB3。
(8)向吸附柱CB3中加入500μL缓冲液GD，12000rpm离心30s，倒掉废液，放回CB3。
(9)向吸附柱CB3中加入600μL漂洗液PW，12000rpm离心30s，倒掉废液，放回CB3。重复一次
(10)将吸附柱CB3放入收集管中置于12000rpm离心2分钟，室温放置10分钟，以除去吸附柱中残留的乙醇。
(11)将吸附柱CB3置于一个干净的离心管中，向吸附膜的中央加入50μL ddH2O，37℃温育15min，12000rpm离心2分钟将质粒溶液收集到离心管中。
阳性克隆的PCR鉴定：
用含有100μg·mL-1G418抗性的YPD固体培养基筛选阳性克隆。重组菌株随机选取6株阳性克隆提取基因组，使用引物AOX1_P1/TFLD1-R1进行第一轮PCR扩增，然后再使用引物AOX1_P2/Ins-Spe1进行第二轮PCR验证，目的基因阳性克隆扩增条带在1kb左右，纯化回收DNA片段，再测序验证。
目的蛋白表达的快速筛选：
挑取经PCR鉴定单克隆接种于4mL BMGY培养基中，28℃、240RPM培养约16-20h，OD600＝8-10。按OD600＝1将其转入4mL BMMY，28℃、240RPM继续培养72-96h，每天添加1-1.5％无水甲醇。按照伯乐微孔过滤仪(Bio-DotApparatus)(Bio-rad)的操作进行斑点印记杂交(Dot-Blot)，选取杂交信号强的克隆进行摇瓶发酵培养。
目的蛋白的表达纯化：
选取斑点印记筛选获得目的蛋白表达水平高的阳性克隆，利用分装有250mL BMMY培养基的1L摇瓶进行诱导发酵培养；发酵培养72-96h后，离心收集上清；然后利用镍离子亲和层析进行胞外分泌蛋白的纯化。利用咪唑梯度洗脱即获得目的蛋白。
实施例7.蛋白酶TEV和PAM的表达、纯化和酶切反应
构建TEV酶表达载体：本发明选择冷休克诱导表达系统pCold-MBP(Takara 公司)，通过Shuffle T7宿主菌进行异源表达，以增加蛋白的可溶性，促进蛋白正确折叠。为方便利用His-Tag纯化目标蛋白，先使用引物Cold-Nhe1/Cold-Nde1(SEQ ID NO:154和155)(表5)进行PCR扩增质粒pCold-MBP上的DNA片段，再利用限制性内切酶Nhe I和Nde I置换载体上对应的载体结构片段，消除促进可溶性表达的MBP标签N-末端的His-Tag标签，即得到质粒pCold-MBP-T。然后利用引物TEV-BamH1/TEV-Sal1(SEQ ID NO:156和157)(表5)直接扩增人工合成的烟草花叶病毒蛋白酶TEV编码框(SEQ ID NO:158)，利用限制性内切酶BamH I和Sal I定向插入到质粒pCold-MBP-T，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pCold-TEV，可实现TEV的可溶性表达和融合标签MBP的自剪切。
构建肽基甘氨酸α-酰胺化单加氧酶(peptidylglycine alpha-amidating monooxygenase,PAM)表达载体：PAM含有肽基甘氨酸α-酰胺化单加氧酶PHM(peptidylglycine alpha-hydroxylating monooxygenase)和肽酰氨基乙醛酸裂解酶PAL(peptidylamidoglycolate lyase)结构域2个催化结构域。利用含有PAM基因(GenBank:NM_000919)质粒pReceiver-M02(GeneCopoeia)为模板，使用引物pReceiver-M03F/PAM-1(SEQ ID NO:159和161)、PAM-2/pReceiver-M03R(SEQ ID NO:160和162)(表5)分别进行PCR扩增含有编码第39-350个氨基酸残基的PHM结构域和第494-835个氨基酸的PAL结构域的DNA片段，然后把这2个DNA片段进行PCR补平反应，再以PCR补平反应产物为模板，利用引物PAM-BamH1/PAM-Sal1(SEQ ID NO:163和164)(表5)进行PCR扩增截短的PAM突变体，其中在这2个结构域之间引入了连接肽段(GSGGSG)。利用限制性内切酶定向插入到质粒pCold-MBP-T，经过连接(II One Step Cloning Kit试剂盒)、转化、阳性克隆筛选和测序验证即得到表达质粒载体pCold-PAM，可通过TEV酶剪切去除其N-末端的融合标签MBP。
上述这2个蛋白酶通过镍离子亲和层析纯化获得蛋白酶，然后建立含有胰岛素或其类似物杂交肽的酶切反应体系：50mM Hepes(pH 7.0),1μM CuSO4,50μg/ml过氧化氢酶(catalase),1mM抗坏血酸(ascorbate)，加入前体蛋白底物、蛋白酶TVE和PAM，在30℃直接进行前体蛋白的酶切反应，反应结束后加入 1/10体积0.1M EDTA和50％TFA灭活酶切反应。最后使用超滤浓缩替换为镍离子诱导剪切融合标签(G↓SRHW)的反应缓冲液(1.0mM NiCl2,0.1M CHES,0.1M acetone oxime,0.1M NaCl,pH 8.2)，在22℃反应16h，然后直接利用RP-HPLC进行色谱纯化，即获得本发明提供的含有胰岛素或其类似物的杂交肽。
表5用于构建蛋白酶TEV和PAM表达载体的引物
实施例8.胰岛素或其类似物的杂交肽抗糜蛋白酶酶解的稳定性分析
对照实验：取三个无菌的EP管，每个EP管中加入1.5μL、1mM胰岛素或其类似物的杂交肽，23.5μL、50mM Tris和20mM CaCl2(pH 7.8)缓冲液及3.75μL、10％TFA，8000rpm离心30s混匀。
人胰岛素、胰岛素类似物及其杂交肽对糜蛋白酶酶解过程如下：取三个无菌的EP管，每个EP管中加入9μL、1mM人胰岛素、胰岛素类似物及其杂交肽及138μL、20mM CaC12、50mM Tris-HC1缓冲液(pH 7.8)。同时，在另一个无菌的EP管中配置一定体积的0.05μg/μL的糜蛋白酶酶液。然后将含有多肽和酶的四个EP管同时置于37℃预热5min，往每份含多肽的EP管中分别加入3μL 糜蛋白酶酶液并混匀。开始计时，于反应的0.5、1.0、2.0、3.0和4.0h分别取出25μL反应液，加入3.75μL、10％TFA终止反应，8000rpm离心30s混匀。
上述实验样品在25μL反应体系中，人胰岛素、胰岛素类似物及其杂交肽的终浓度是60μM，糜蛋白酶的终浓度是1.0ng/μL。每个时间点有三次重复，利用RP-HPLC检测各时间点多肽的峰面积，计算检测时间T(h)样品的剩余峰面积和0h原型多肽的峰面积之比为多肽的剩余百分比(％)。
结果:人胰岛素经糜蛋白酶酶解4h后仅剩余约10.3％，其对糜蛋白酶酶解的稳定性较低，而引入含有糜蛋白酶抑制骨架CH57或CH10的胰岛素类似物杂交肽呈现出非常好的抗胰蛋白酶酶解的稳定性，经酶解4h剩余量超过近80％。
表6:人胰岛素及其杂交肽(Ins3-A-CH10M、和Ins3-A-CH57M)对糜蛋白酶的稳定性分析
实施例9.胰岛素或其类似物和胰岛素杂交肽经皮下注射和十二指肠给药的体内活性
胰岛素类似物和胰岛素杂交肽经皮下注射给药的体内降血糖活性：正常ICR小鼠，皮下注射给予优泌林(0.4U/kg)及胰岛素类似物或相应体积的生理盐水(0.1mL/10g B.W.，n＝6-9)，分别于给药后40min和90min尾尖采血，以葡萄糖氧化酶法测定血糖水平，计算各时刻血糖值。计算值以“平均值±标准误”表示，p<0.05被认为具有统计学差异。
结果：胰岛素类似物如赖脯胰岛素LisPro与阳性对照相比，显示出很好的降血糖活性；同样含有糜蛋白酶抑制肽的胰岛素杂交肽(Ins3-B-CH57N、Ins3-B-CH57C、Ins3-A-CH10M、Ins3-A-CH57M)也显示出很好的降血糖活性(表 7)；说明无论是在其B-链的N-末端、B-链的C-末端还是A-链的中间均未显示结构上糜蛋白酶抑制肽影响其与胰岛素受体之间的相互作用。但是生物表达制备的A-链的N-端和A-链的C-端融合糜蛋白酶抑制肽活性较弱(Ins3-A-CH57N和Ins3-A-CH57C)，其原因可能是没有形成正确的二硫键结构，可能是假阴性结果。
表7：人胰岛素及其类似物皮下给药的降血糖活性

*,p<0.05；**,p<0.01；***,p<0.001.
表7：人胰岛素及其类似物皮下给药的降血糖活性(续)

*,p<0.05；**,p<0.01；***,p<0.001.
胰岛素类似物和胰岛素杂交肽经十二指肠给药的体内降血糖活性：
正常ICR小鼠，经吸入乙醚麻醉后，手术取出十二指肠，注入胰岛素类似物或相应体积的生理盐水(0.1mL/10g B.W.，n＝6-10)，然后缝合伤口，分别于给药后40min和90min尾尖采血，以葡萄糖氧化酶法测定血糖水平，计算各时刻血糖值。计算值以“平均值±标准误”表示，p<0.05被认为具有统计学差异。
结果：与正常对照组相比，只有胰岛素杂交肽Ins3-B-CH57C、Ins3-A-CH10M和 Ins3-A-CH57M十二指肠给药后能够显著降低小鼠40min的血糖值，血糖值分别降低12.1％、11.9％和15.3％(表8)。说明糜蛋白酶抑制肽在B-链的C-末端和A-链的中间时对胰岛素均有很好的保护作用，提高了它们的体内稳定性，且促进了胰岛素杂交肽经小肠上皮吸收进入血液循环。
表8：人胰岛素及其类似物十二指肠给药的降血糖活性

*,p<0.05；**,p<0.01；***,p<0.001.
表8：人胰岛素及其类似物十二指肠给药的降血糖活性(续)

*,p<0.05；**,p<0.01；***,p<0.001.

Claims (22)

  1. 一种由胰岛素或其类似物和糜蛋白酶抑制肽所形成的杂交肽，二者之间通过连接肽连接；其中胰岛素或其类似物，含有一条B-链和一条A-链且二者通过两对二硫键连接；其中糜蛋白酶抑制肽通过连接肽与胰岛素或其类似物的B-链或A-链连接。
  2. 根据权利要求1所述的杂交肽，其特征在于，其中所述的糜蛋白酶抑制肽选自具有以下氨基酸序列的多肽：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:28。
  3. 根据权利要求1所述的杂交肽，其特征在于，所述的胰岛素或其类似物包括A-链和B-链；其中A-链可选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33；B-链可选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40。
  4. 根据权利要求1所述的杂交肽，其特征在于，其中所述的胰岛素或其类似物与糜蛋白酶抑制肽之间的连接肽是含有Gly、Ser和Pro氨基酸残基且长度为一个或多个氨基酸残基的肽段或缺失。
  5. 根据权利要求1-3所述的杂交肽，其特征在于，其从N-末端到C-末端方向的结构域：(N-末端)-糜蛋白酶抑制肽-胰岛素B-链---胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物的B-链N-末端融合；所述的“(N-末端)-糜蛋白酶抑制肽-胰岛素B-链”为糜蛋白酶抑制肽和胰岛素B-链融合形成的多肽可选自具有以下氨基酸序列的多肽：SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49和SEQ ID NO:50；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33。
  6. 根据权利要求5所述的杂交肽，其特征在于，所述的杂交肽前体蛋白从N-末端到C-末端方向的结构域：(N-末端)-糜蛋白酶抑制肽-胰岛素B-链-C-多肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物的B-链N-末端融合；所述的“(N-末端)-糜蛋白酶抑制肽-胰岛素B-链”为糜蛋白酶抑制肽和胰岛素B-链融合形成的多肽可选自具有以下氨基酸序列的多肽：SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49和SEQ ID NO:50；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:70、SEQ ID NO:71；其中，前体蛋白经过蛋白酶Kex2酶切、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，和/或羧肽酶B和肽基甘氨酸α-酰胺化单加氧酶的处理或不处理。
  7. 根据权利要求6所述的杂交肽，其特征在于，所述的杂交肽前体蛋白选自具有以下氨基酸序列的多肽：SEQ ID NO:165、SEQ ID NO:166、SEQ ID NO:167、SEQ ID NO:168、SEQ ID NO:169、SEQ ID NO:170、SEQ ID NO:171、SEQ ID NO:172、SEQ ID NO:173和SEQ ID NO:174；
  8. 根据权利要求1-3所述的杂交肽，其特征在于，其从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-糜蛋白酶抑制肽---胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物的B-链C-末端融合；所述的“(N-末端)-胰岛素B-链-糜蛋白酶抑制肽”为胰岛素B-链和糜蛋白酶抑制肽融合形成的多肽可选自具有以下氨基酸序列的多肽：SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33。
  9. 根据权利要求8所述的杂交肽，其特征在于，所述的杂交肽前体蛋白从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-糜蛋白酶抑制肽-C-多肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物的B-链C-末端融合；所述的“(N-末端)-胰岛素B-链-糜蛋白酶抑制肽”为胰岛素B-链和糜蛋白酶抑制肽融合形成的多肽可选自具有以下氨基酸 序列的多肽：SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；A-链选自具有以下氨基酸序列的多肽：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:72和SEQ ID NO:73；其中，前体蛋白经过蛋白酶Kex2、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，和/或羧肽酶B和肽基甘氨酸α-酰胺化单加氧酶的处理或不处理。
  10. 根据权利要求9所述的杂交肽，其特征在于，所述的杂交肽前体蛋白选自具有以下氨基酸序列的多肽：SEQ ID NO:185、SEQ ID NO:186、SEQ ID NO:187、SEQ ID NO:188、SEQ ID NO:189、SEQ ID NO:190、SEQ ID NO:191、SEQ ID NO:192、SEQ ID NO:193、SEQ ID NO:194、SEQ ID NO:195和SEQ ID NO:196。
  11. 根据权利要求1-3所述的杂交肽，其特征在于，其从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链---糜蛋白酶抑制肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物的A-链N-末端融合，胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“糜蛋白酶抑制肽-胰岛素A-链-(C-末端)”为糜蛋白酶抑制肽和胰岛素A-链融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62。
  12. 根据权利要求11所述的杂交肽，其特征在于，所述的杂交肽前体蛋白从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-C-多肽-糜蛋白酶抑制肽-胰岛素A-链-(C-末端)，其中糜蛋白酶抑制肽的C-末端直接通过连接肽与胰岛素或其类似物的A-链N-末端融合，胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“糜蛋白酶抑制肽-胰岛素A-链-(C-末端)”为糜蛋白酶抑制肽和胰岛素A-链融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:70、SEQ ID NO:71；其中，前体蛋白经过 蛋白酶Kex2、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，和/或羧肽酶B和肽基甘氨酸α-酰胺化单加氧酶的处理或不处理。
  13. 根据权利要求12所述的杂交肽，其特征在于，所述的杂交肽前体蛋白选自具有以下氨基酸序列的多肽：SEQ ID NO:209、SEQ ID NO:210、SEQ ID NO:211、SEQ ID NO:212、SEQ ID NO:213、SEQ ID NO:214、SEQ ID NO:215、SEQ ID NO:216、SEQ ID NO:217和SEQ ID NO:218。
  14. 根据权利要求1-3所述的杂交肽，其特征在于，其从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链---胰岛素A-链-糜蛋白酶抑制肽-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物的A-链C-末端融合；胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-(C-末端)”为胰岛素A-链和糜蛋白酶抑制肽融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:63、SEQ ID NO:64。
  15. 根据权利要求14所述的杂交肽，其特征在于，所述的杂交肽前体蛋白从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-C-多肽-胰岛素A-链-糜蛋白酶抑制肽-(C-末端)，其中糜蛋白酶抑制肽的N-末端直接通过连接肽与胰岛素或其类似物的A-链C-末端融合；胰岛素或其类似物的B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-(C-末端)”为胰岛素A-链和糜蛋白酶抑制肽融合形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:63、SEQ ID NO:64；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:70、SEQ ID NO:71；其中，前体蛋白经过蛋白酶Kex2、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，和/或羧肽酶B和肽基甘氨酸α-酰胺化单加氧酶的处理或不处理。
  16. 根据权利要求15所述的杂交肽，其特征在于，所述的杂交肽前体蛋白选自具有以下氨基酸序列的多肽：SEQ ID NO:229、SEQ ID NO:230、SEQ ID NO:231、SEQ ID NO:232、SEQ ID NO:233、SEQ ID NO:234、SEQ ID NO:235、SEQ ID NO:236、SEQ ID NO:237和SEQ ID NO:238。
  17. 根据权利要求1-3所述的杂交肽，其特征在于，其从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链---胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)，其中糜蛋白酶抑制肽直接与胰岛素或其类似物的A-链形成融合肽；胰岛素或类似物B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)”为胰岛素A-链中间融合糜蛋白酶抑制肽形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68。
  18. 根据权利要求17所述的杂交肽，其特征在于，所述的杂交肽前体蛋白从N-末端到C-末端方向的结构域：(N-末端)-胰岛素B-链-C-多肽-胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)，其中糜蛋白酶抑制肽直接与胰岛素或其类似物的A-链形成融合肽；胰岛素或类似物B-链选自具有以下氨基酸序列的多肽：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40；所述的“胰岛素A-链-糜蛋白酶抑制肽-A-链-(C-末端)”为胰岛素A-链中间融合糜蛋白酶抑制肽形成的多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68；C-多肽选自具有以下氨基酸序列的多肽：SEQ ID NO:70、SEQ ID NO:71；其中，前体蛋白经过蛋白酶Kex2、烟草花叶病毒蛋白酶TEV酶切和镍离子诱导化学剪切三步处理，和/或羧肽酶B和肽基甘氨酸α-酰胺化单加氧酶的处理或不处理。
  19. 根据权利要求18所述的杂交肽，其特征在于，所述的杂交肽前体蛋白选自具有以下氨基酸序列的多肽：SEQ ID NO:249、SEQ ID NO:250、SEQ ID NO:251、SEQ ID NO:252、SEQ ID NO:253、SEQ ID NO:254、SEQ ID NO:255、SEQ ID NO:256、SEQ ID NO:257和SEQ ID NO:258。
  20. 一种制备权利要求1-19中含有糜蛋白酶抑制肽和胰岛素或其类似物所形成杂交肽的方法，其特征在于，其前体蛋白的N-端含有如SEQ ID NO:69所示的烟草花叶病毒蛋白酶(TEV)剪切位点，在胰岛素分子或其类似物的B-链和A-链之间的连接肽选自如下序列的多肽：SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73；其中，前体蛋白经过蛋白酶Kex2、烟草花叶病毒蛋白酶 TEV酶切和镍离子诱导化学剪切三步处理，和/或羧肽酶B和肽基甘氨酸α-酰胺化单加氧酶的处理或不处理。
  21. 权利要求1-19中的杂交肽在制备治疗I型糖尿病或II型糖尿病药物中的应用。
  22. 根据权利要求21的应用，其特征在于，所述的杂交肽在治疗I型糖尿病和II型糖尿病时可施用皮下注射和口服给药。
PCT/CN2023/096851 2022-05-29 2023-05-29 一种含有糜蛋白酶抑制肽的胰岛素杂交肽及其应用 WO2023231969A1 (zh)

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