Colipase inhibitory peptide and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a colipase inhibitory peptide and application thereof. The colipase inhibitory peptide has colipase inhibitory activity, can significantly reduce fat absorption by inhibiting the activity of colipase, achieves the effects of losing weight and reducing blood fat, is safe and free of toxic and side effects, has good stability and low blood absorptivity, and is beneficial for polypeptide to better inhibit the colipase to play a role in digestive tracts.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a colipase inhibitory peptide and its application. Background Art

[0002] With the change of modern lifestyle, obesity and hyperlipidemia have become global health problems, seriously affecting people's quality of life and lifespan. Traditional lipid-lowering and weight-loss methods include diet control, exercise, and drug treatment, but these methods are often difficult to adhere to in the long term and have limited effects. Currently, although existing weight-loss and lipid-lowering products on the market can alleviate these problems to a certain extent, they have deficiencies such as large side effects and poor patient compliance. Therefore, developing new, efficient, and safe lipid-lowering and weight-loss products has important clinical significance and social value.

[0003] Colipase is a cofactor that acts synergistically with pancreatic lipase and plays a key role in the digestion and absorption of fat. Research shows that colipase can stabilize the active conformation of pancreatic lipase and promote its binding to the lipid hydrolysis interface. During fat digestion, the function of colipase is crucial for maintaining the activity of pancreatic lipase.

[0004] Peptide products have attracted much attention in the field of product research and development due to their significant advantages of high biological activity and low toxicity. In recent years, significant progress has been made in the research and development of peptide products, especially in polypeptide discovery strategies and medicinal chemistry modifications. Summary of the Invention

[0005] Combined with the biological characteristics of colipase, a safe and effective new weight-loss and / or lipid-lowering product is developed by designing and screening polypeptides that can regulate the activity of colipase or its interaction with pancreatic lipase.

[0006] The purpose of the first aspect of the present invention is to provide a polypeptide or its salt.

[0007] The purpose of the second aspect of the present invention is to provide a conjugate.

[0008] The purpose of the third aspect of the present invention is to provide a fusion protein.

[0009] The purpose of the fourth aspect of the present invention is to provide biological materials related to the polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, and / or the fusion protein of the third aspect of the present invention.

[0010] The purpose of the fifth aspect of the present invention is to provide a composition.

[0011] The object of the sixth aspect of the present invention is to provide the use of the polypeptide or its salt according to the first aspect of the present invention, the conjugate according to the second aspect, the fusion protein according to the third aspect, the biological material according to the fourth aspect, and the composition according to the fifth aspect.

[0012] The object of the seventh aspect of the present invention is to provide a product.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The first aspect of the present invention provides a polypeptide or its salt, and the polypeptide comprises the following amino acid sequence: X1X2X3X4X5X6X7X8; wherein,

[0015] X1 is selected from W, P, H, R, L, Y;

[0016] X2 is selected from Q, V, E, T, N, F;

[0017] X3 is selected from H, P, W, Y, D, L;

[0018] X4 is selected from V, Q, N, D, L, E, A;

[0019] X5 is selected from S, P, R, G, F, K, W, T;

[0020] X6 is selected from L, A, P, Q, Y, E, F;

[0021] X7 is selected from P, R, V, S;

[0022] X8 is selected from A, E, K, D, Y, V.

[0023] In some embodiments, in the amino acid sequence X1X2X3X4X5X6X7X8 comprised by the polypeptide, X1 is W or P, X2 is Q, V or E, X3 is H, P or W, X4 is V, Q or N, X5 is S or P, X6 is L or A, X7 is P or R, and X8 is A, E or K;

[0024] In some embodiments, in the amino acid sequence X1X2X3X4X5X6X7X8 comprised by the polypeptide, X1 is W, X2 is Q or E, X3 is H or W, X4 is V or N, X5 is S or P, X6 is L or A, X7 is P or R, and X8 is A or K;

[0025] In some embodiments, the polypeptide comprises the amino acid sequence: X1X2X3X4X5X6X7X8, wherein X1 is W, X2 is E, X3 is W, X4 is N, X5 is P, X6 is A, X7 is R, and X8 is K; or, X1 is W, X2 is Q, X3 is H, X4 is V, X5 is S, X6 is L, X7 is P, and X8 is A; or, X1 is P, X2 is V, X3 is P, X4 is Q, X5 is P, X6 is L, X7 is P, and X8 is E.

[0026] In some embodiments, the present invention provides a polypeptide or a salt thereof, the polypeptide comprising the following amino acid sequence: PX2X3X4X5X6PX8; wherein,

[0027] X2 is selected from Q, V, E, T, F;

[0028] X3 is selected from H, P, W, D, L;

[0029] X4 is selected from V, Q, D, E;

[0030] X5 is selected from S, P, G, F, T;

[0031] X6 is selected from L, A, Q, E, F;

[0032] X8 is selected from E, K, V.

[0033] In some embodiments, the present invention provides a polypeptide or a salt thereof, the polypeptide comprising the following amino acid sequence: X1X2X3VX5X6PX8; wherein,

[0034] X1 is selected from W, L;

[0035] X2 is selected from Q, V, E, N;

[0036] X3 is selected from H, W, Y;

[0037] X5 is selected from S, R, K;

[0038] X6 is selected from L, Q;

[0039] X8 is selected from A, E, K.

[0040] In some embodiments, the present invention provides a polypeptide or a salt thereof, the polypeptide comprising the following amino acid sequence: X1X2WNX5X6X7X8; wherein,

[0041] X1 is selected from W, P, L;

[0042] X2 is selected from E, T, N;

[0043] X5 is selected from P, R;

[0044] X6 is selected from A, P;

[0045] X7 is selected from P, R, V;

[0046] X8 is selected from A, K, Y.

[0047] In some embodiments, the present invention provides a polypeptide or a salt thereof, the polypeptide comprising the following amino acid sequence: X1EX3X4SX6PD; wherein,

[0048] X1 is selected from H, R;

[0049] X3 is selected from P, W;

[0050] X4 is selected from D, L;

[0051] X6 is selected from L, P.

[0052] In some embodiments, the present invention provides a polypeptide or a salt thereof, the polypeptide comprising the following amino acid sequence: YX2X3X4X5LX7K; wherein,

[0053] X2 is selected from T, N;

[0054] X3 is selected from P, L;

[0055] X4 is selected from V, L;

[0056] X5 is selected from P, W;

[0057] X7 is selected from V, S.

[0058] In some embodiments, the present invention provides a polypeptide or a salt thereof, the polypeptide comprising the following amino acid sequence: X1X2X3X4X5X6X7X8; wherein,

[0059] X1 is selected from W, P;

[0060] X2 is selected from N, F;

[0061] X3 is selected from W, D;

[0062] X4 is selected from E, A;

[0063] X5 is selected from R, G;

[0064] X6 is selected from A, Y;

[0065] X7 is selected from P, R;

[0066] X8 is selected from E, K.

[0067] In some embodiments, the polypeptide or a salt thereof according to any one of the preceding items, wherein the polypeptide comprises the following amino acid sequence: PX2X3X4X5X6PX8; wherein, X2 is V, X3 is P, X4 is Q, X5 is P, X6 is L, X8 is E; or X2 is E, X3 is W, X4 is V, X5 is G, X6 is Q, X8 is V; or X2 is Q, X3 is W, X4 is D, X5 is P, X6 is A, X8 is E; or X2 is T, X3 is L, X4 is V, X5 is F, X6 is E, X8 is E; or X2 is E, X3 is H, X4 is V, X5 is S, X6 is L, X8 is E; or X2 is F, X3 is D, X4 is E, X5 is T, X6 is F, X8 is K.

[0068] In some embodiments, the polypeptide or a salt thereof according to any one of the preceding items, wherein the polypeptide comprises the following amino acid sequence: X1X2X3VX5X6PX8; wherein,

[0069] X1 is W, X2 is Q, X3 is H, X5 is S, X6 is L, X8 is A; or X1 is W, X2 is N, X3 is Y, X5 is R, X6 is L, X8 is E; or X1 is L, X2 is V, X3 is W, X5 is K, X6 is Q, X8 is A; or X1 is L, X2 is E, X3 is H, X5 is R, X6 is L, X8 is K.

[0070] In some embodiments, the polypeptide or a salt thereof according to any one of the preceding items, wherein the polypeptide comprises the following amino acid sequence: X1X2WNX5X6X7X8; wherein,

[0071] X1 is W, X2 is E, X5 is P, X6 is A, X7 is R, X8 is K; or X1 is P, X2 is T, X5 is R, X6 is P, X7 is V, X8 is Y; or X1 is L, X2 is N, X5 is P, X6 is A, X7 is P, X8 is A.

[0072] In some embodiments, the polypeptide or a salt thereof according to any one of the preceding items, wherein the polypeptide comprises the following amino acid sequence: X1EX3X4SX6PD; wherein,

[0073] X1 is H, X3 is W, X4 is D, X6 is P; or X1 is R, X3 is P, X4 is L, X6 is L.

[0074] In some embodiments, the polypeptide or a salt thereof according to any one of the preceding items, wherein the polypeptide comprises the following amino acid sequence: YX2X3X4X5LX7K; wherein,

[0075] X2 is N, X3 is L, X4 is V, X5 is P, X7 is S; or X2 is T, X3 is P, X4 is L, X5 is W, X7 is V.

[0076] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide comprises the following amino acid sequence: X1X2X3X4X5X6X7X8; wherein,

[0077] X1 is W, X2 is N, X3 is W, X4 is E, X5 is R, X6 is A, X7 is P, X8 is K; or X1 is P, X2 is F, X3 is D, X4 is A, X5 is G, X6 is Y, X7 is R, X8 is E.

[0078] In some embodiments, there is provided a polypeptide or a salt thereof, the polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 1-19, or comprising an amino acid mutant sequence having no more than 3 amino acid differences (such as 3, 2, or 1 amino acid difference) from any one of the sequences of SEQ ID NO: 1-19;

[0079] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 1-19.

[0080] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1;

[0081] In some embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 3.

[0082] In some embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0083] In some embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0084] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO: 1-19;

[0085] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1;

[0086] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 3.

[0087] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2.

[0088] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1.

[0089] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the amino acids in the polypeptide are each independently selected from: D-amino acids and L-amino acids.

[0090] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the amino acids in the polypeptide are L-amino acids.

[0091] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the amino acids in the polypeptide are D-amino acids.

[0092] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide is a linear peptide or a cyclic peptide.

[0093] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide is a linear peptide.

[0094] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide is a cyclic peptide.

[0095] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the number of amino acids in the polypeptide is 8.

[0096] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide is a colipase inhibitory peptide;

[0097] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide or a salt thereof can specifically bind to colipase;

[0098] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide or a salt thereof can inhibit the functional activity of colipase.

[0099] In some embodiments, the polypeptide or a salt thereof as described in any one of the preceding items, wherein the polypeptide has at least one of the following characteristics (1) to (7):

[0100] (1) The polypeptide can inhibit the functional activity of co-lipase; in some embodiments, the inhibition rate of the polypeptide on co-lipase is greater than 10% (for example, greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or greater, or any value therebetween); in some embodiments, the in vitro co-lipase inhibition rate of the polypeptide is greater than 10% at a reaction concentration of 24 μM (for example, greater than 15%, 20%, 25%, 30%, 35%, 40%, 45% or greater, or any value therebetween); in some embodiments, the in vitro co-lipase inhibition rate of the polypeptide is greater than 20% at a reaction concentration of 24 μM; in some embodiments, the in vitro co-lipase inhibition rate of the polypeptide is greater than 30% at a reaction concentration of 24 μM; in some embodiments, the inhibition rate of the polypeptide on co-lipase is detected according to the method of Example 2 of the present application;

[0101] (2) The inhibition of the polypeptide on lipase activity is less than that on co-lipase activity; in some embodiments, the inhibition rate of the polypeptide on lipase is less than 10% (for example, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01% or less, or any value therebetween); in some embodiments, the in vitro lipase inhibition rate of the polypeptide is less than 10% at a reaction concentration of 24 μM (for example, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01% or less, or any value therebetween); in some embodiments, the in vitro lipase inhibition rate of the polypeptide is less than 0.1% at a reaction concentration of 24 μM (for example, less than 0.1%, 0.05%, 0.01% or less, or any value therebetween); in some embodiments, the in vitro lipase inhibition rate of the polypeptide is less than 0.1% at a reaction concentration of 24 μM; in some embodiments, the inhibition rates of the polypeptide on co-lipase and lipase are detected according to the method of Example 2 of the present application;

[0102] (3) The polypeptide has good stability; in some embodiments, when the polypeptide reacts in a pepsin solution or a trypsin solution at 37 °C for 1 hour, the change in the purity of the polypeptide does not exceed 5% (for example, does not exceed 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less, or any value therebetween); in some embodiments, the purity of the polypeptide is detected according to the method of Example 3 of the present application;

[0103] (4) The polypeptide has a lipid-lowering effect;

[0104] (5) The polypeptide has a weight loss effect;

[0105] (6) Each amino acid in the polypeptide is independently selected from: D-amino acid, L-amino acid; in some embodiments, the amino acids in the polypeptide are L-amino acids;

[0106] (7) The polypeptide is a linear peptide or a cyclic peptide; in some embodiments, the polypeptide is a linear peptide.

[0107] In some embodiments, for the polypeptide described in any one of the preceding items, the polypeptide is capable of inhibiting the functional activity of co-lipase; in some embodiments, the inhibition rate of the polypeptide on co-lipase is greater than 10%, for example, more than 20% (for example, greater than or equal to 20%, 25%, 30%, 35%, 40%, 45% or greater, or any value therebetween); in some embodiments, the in vitro co-lipase inhibition rate of the polypeptide is greater than 10% at a reaction concentration of 24 μM, for example, more than 20% (for example, greater than or equal to 20%, 25%, 30%, 35%, 40%, 45% or greater, or any value therebetween); in some embodiments, the in vitro co-lipase inhibition rate of the polypeptide is greater than 20% at a reaction concentration of 24 μM; in some embodiments, the in vitro co-lipase inhibition rate of the polypeptide is greater than 30% at a reaction concentration of 24 μM; in some embodiments, the inhibition rate of the polypeptide on co-lipase is detected according to the method of Example 2 of the present application.

[0108] In some embodiments, for the polypeptide described in any one of the preceding items, the inhibition of the polypeptide on lipase activity is less than the inhibition on co-lipase activity; in some embodiments, the inhibition rate of the polypeptide on lipase is less than 10%, for example, less than 1% (for example, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01% or less, or any value therebetween); in some embodiments, the in vitro lipase inhibition rate of the polypeptide is less than 10% at a reaction concentration of 24 μM, for example, less than 1% (for example, less than 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01% or less, or any value therebetween); in some embodiments, the in vitro lipase inhibition rate of the polypeptide is less than 0.1% at a reaction concentration of 24 μM (for example, less than 0.1%, 0.05%, 0.01% or less, or any value therebetween); in some embodiments, the in vitro lipase inhibition rate of the polypeptide is less than 0.1% at a reaction concentration of 24 μM; in some embodiments, the inhibition rates of the polypeptide on co-lipase and lipase are detected according to the method of Example 2 of the present application.

[0109] In some embodiments, for the polypeptide described in any of the previous aspects, the inhibition rate of the polypeptide against co-lipase is greater than 10%, and the inhibition rate of the polypeptide against lipase is less than 10%. In some embodiments, the in vitro co-lipase inhibition rate of the polypeptide is greater than or equal to 20% or more, and the lipase inhibition rate is less than or equal to 0.1% at a reaction concentration of 24 μM. In some embodiments, the inhibition rates of the polypeptide against co-lipase and lipase are detected according to the method of Example 2 of the present application.

[0110] In a second aspect of the present invention, there is provided a conjugate comprising a modifying moiety and the polypeptide of the first aspect of the present invention.

[0111] In some embodiments, the modifying moiety is located at the N-terminus and / or C-terminus of the polypeptide.

[0112] In some embodiments, the modifying moiety comprises at least one of a chemical modification, a targeting moiety, a fluorescent dye, and a protein tag.

[0113] In some embodiments, the chemical modification comprises at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, and ubiquitination.

[0114] In some embodiments, the targeting moiety comprises at least one of a ligand, a receptor, and an antibody.

[0115] In some embodiments, the fluorescent dye comprises at least one of fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, and rhodamine.

[0116] In some embodiments, the protein tag comprises at least one of His, Flag, GST, MBP, HA, Myc, GFP, and biotin.

[0117] In a third aspect of the present invention, there is provided a fusion protein comprising another polypeptide and the polypeptide of the first aspect of the present invention.

[0118] In some embodiments, the other polypeptide is located at the N-terminus and / or C-terminus of the polypeptide.

[0119] In some embodiments, the other polypeptide and the polypeptide of the first aspect of the present invention are linked to the N-terminus and / or C-terminus of the polypeptide through a linker.

[0120] In a fourth aspect of the present invention, there is provided a biological material related to the polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, and the biological material comprises any one of n1)-n9):

[0121] n1) A nucleic acid molecule encoding a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0122] n2) An expression cassette comprising the nucleic acid molecule of n1);

[0123] n3) A vector comprising the nucleic acid molecule of n1);

[0124] n4) A vector comprising the expression cassette of n2);

[0125] n5) A cell comprising the nucleic acid molecule of n1);

[0126] n6) A cell comprising the expression cassette of n2);

[0127] n7) A cell comprising the vector of n3);

[0128] n8) A cell comprising the vector of n4);

[0129] n9) A cell comprising a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention.

[0130] In some embodiments, there is provided a nucleic acid molecule encoding a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0131] In some embodiments, there is provided an expression cassette comprising a nucleic acid molecule encoding a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0132] In some embodiments, there is provided a vector comprising a nucleic acid molecule encoding a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0133] In some embodiments, there is provided a vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid molecule encoding a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0134] In some embodiments, there is provided a cell comprising a nucleic acid molecule encoding a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0135] In some embodiments, there is provided a cell comprising an expression cassette, wherein the expression cassette comprises a nucleic acid molecule encoding a polypeptide of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0136] In some embodiments, cells are provided that comprise a vector; wherein the vector comprises a nucleic acid molecule encoding the polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention;

[0137] In some embodiments, cells are provided that comprise a vector; wherein the vector comprises an expression cassette that comprises a nucleic acid molecule encoding the polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention;

[0138] In some embodiments, cells are provided that comprise the polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention; in some embodiments, for any of the biomaterials described in any of the preceding items, any of the cells in n5)-n9) does not contain propagation material.

[0139] In some embodiments, for any of the biomaterials described in any of the preceding items, any of the vectors in n3)-n4) includes a prokaryotic expression vector and a eukaryotic expression vector.

[0140] In some embodiments, for any of the biomaterials described in any of the preceding items, the eukaryotic expression vector includes a yeast expression vector, a mammalian expression vector, an insect expression vector, etc.

[0141] In some embodiments, for any of the biomaterials described in any of the preceding items, any of the cells in n5)-n9) is selected from prokaryotic cells and eukaryotic cells.

[0142] In some embodiments, for any of the biomaterials described in any of the preceding items, the prokaryotic cells include bacterial cells, Escherichia coli, Streptomyces.

[0143] In some embodiments, for any of the biomaterials described in any of the preceding items, the eukaryotic cells include yeast cells, mammalian cells, insect cells, etc.

[0144] In some embodiments, for any of the biomaterials described in any of the preceding items, the mammals are selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cows, pigs, dogs, cats.

[0145] In some embodiments, a method for preparing the polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention is provided, which comprises the following steps: culturing the host cell as described in any of the above under conditions allowing protein expression, and recovering the polypeptide, conjugate, or fusion protein from the cultured host cell culture.

[0146] In some embodiments, a method for preparing the polypeptide of the first aspect of the present invention by chemical synthesis is provided. In some embodiments, the chemical synthesis method is as described in Example 1 of the present invention.

[0147] The fifth aspect of the present invention provides a composition comprising the polypeptide of the first aspect of the present invention or a salt thereof, the conjugate of the second aspect of the present invention, and / or the fusion protein of the third aspect of the present invention.

[0148] In some embodiments, the composition further comprises other components that inhibit the activity of co-lipase and / or inhibit the activity of lipase (preferably pancreatic lipase).

[0149] The sixth aspect of the present invention provides the use of the polypeptide of the first aspect of the present invention or a salt thereof, the conjugate of the second aspect, the fusion protein of the third aspect, the biomaterial of the fourth aspect, and / or the composition of the fifth aspect in the preparation of co-lipase inhibitory peptides, foods, health products, or pharmaceuticals.

[0150] In some embodiments, provided are the polypeptide of the first aspect of the present invention or a salt thereof, the conjugate of the second aspect, the fusion protein of the third aspect, the biomaterial of the fourth aspect, and / or the composition of the fifth aspect for use as co-lipase inhibitory peptides, foods, health products, or pharmaceuticals.

[0151] In some embodiments, for any of the above-mentioned uses, co-lipase inhibitory peptides, foods, health products, or pharmaceuticals, wherein the food is for weight loss and / or lipid-lowering (preferably assisting in lipid-lowering).

[0152] In some embodiments, for any of the above-mentioned uses, co-lipase inhibitory peptides, foods, health products, or pharmaceuticals, wherein the health product is for weight loss and / or lipid-lowering (preferably assisting in lipid-lowering).

[0153] In some embodiments, for any of the above-mentioned uses, co-lipase inhibitory peptides, foods, health products, or pharmaceuticals, wherein the pharmaceutical is for weight loss, lipid-lowering, prevention and / or treatment of obesity-related diseases.

[0154] In some embodiments, for any of the above-mentioned uses, co-lipase inhibitory peptides, foods, health products, or pharmaceuticals, wherein the obesity-related diseases include obesity, hyperlipidemia, cardiovascular and cerebrovascular diseases, diabetes, hyperglycemia, and hypertension.

[0155] In some embodiments, a method for weight loss, reducing blood lipid levels, and / or preventing and / or treating obesity-related diseases is provided. The method includes administering a therapeutically effective amount of the polypeptide or its salt according to the first aspect of the present invention, the conjugate according to the second aspect, the fusion protein according to the third aspect, the biomaterial according to the fourth aspect, and / or the composition according to the fifth aspect to a subject in need thereof; in some embodiments, the subject is an obese and / or hyperlipidemic subject; in some embodiments, the subject is a patient with an obesity-related disease; in some embodiments, the obesity-related diseases include obesity, hyperlipidemia, cardiovascular and cerebrovascular diseases, diabetes, hyperglycemia, and hypertension.

[0156] In some embodiments, the present invention provides the application of the colipase inhibitory peptide or its salt in the preparation of products for weight loss, reducing blood lipid levels, and / or preventing and / or treating obesity-related diseases; in some embodiments, the colipase inhibitory peptide is the polypeptide described in any one of the first aspect of the present invention; in some embodiments, the colipase inhibitory peptide comprises the following amino acid sequence: X1X2X3X4X5X6X7X8; wherein,

[0157] X1 is selected from W, P, H, R, L, Y;

[0158] X2 is selected from Q, V, E, T, N, F;

[0159] X3 is selected from H, P, W, Y, D, L;

[0160] X4 is selected from V, Q, N, D, L, E, A;

[0161] X5 is selected from S, P, R, G, F, K, W, T;

[0162] X6 is selected from L, A, P, Q, Y, E, F;

[0163] X7 is selected from P, R, V, S;

[0164] X8 is selected from A, E, K, D, Y, V.

[0165] In some embodiments, in the amino acid sequence X1X2X3X4X5X6X7X8 comprised by the colipase inhibitory peptide, X1 is W or P, X2 is Q, V or E, X3 is H, P or W, X4 is V, Q or N, X5 is S or P, X6 is L or A, X7 is P or R, X8 is A, E or K;

[0166] In some embodiments, in the amino acid sequence X1X2X3X4X5X6X7X8 comprised by the colipase inhibitory peptide, X1 is W, X2 is Q or E, X3 is H or W, X4 is V or N, X5 is S or P, X6 is L or A, X7 is P or R, X8 is A or K;

[0167] In some embodiments, the amino acid sequence of the colipase inhibitory peptide is X1X2X3X4X5X6X7X8, where X1 is W, X2 is E, X3 is W, X4 is N, X5 is P, X6 is A, X7 is R, and X8 is K; or X1 is W, X2 is Q, X3 is H, X4 is V, X5 is S, X6 is L, X7 is P, and X8 is A; or X1 is P, X2 is V, X3 is P, X4 is Q, X5 is P, X6 is L, X7 is P, and X8 is E.

[0168] In some embodiments, the colipase inhibitory peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 1-19; in some embodiments, the colipase inhibitory peptide comprises the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1; in some embodiments, the colipase inhibitory peptide comprises the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1; in some embodiments, the amino acid sequence of the colipase inhibitory peptide is as shown in SEQ ID NO: 3.

[0169] In some embodiments, the obesity-related diseases include obesity, hyperlipidemia, cardiovascular and cerebrovascular diseases, diabetes, hyperglycemia, and hypertension.

[0170] The seventh aspect of the present invention provides a product comprising the polypeptide or its salt of the first aspect of the present invention, the conjugate of the second aspect, the fusion protein of the third aspect, the biomaterial of the fourth aspect, and / or the composition of the fifth aspect.

[0171] In some embodiments, the product is a food, a health product, or a drug.

[0172] In some embodiments, the product is a food.

[0173] In some embodiments, the product is a health product.

[0174] In some embodiments, the product is a drug.

[0175] In some embodiments, the product further comprises excipients acceptable on foods, health products, or drugs.

[0176] In some embodiments, the food is used for weight loss and / or reducing blood lipid (preferably assisting in reducing blood lipid).

[0177] In some embodiments, the health product is used for weight loss and / or reducing blood lipid (preferably assisting in reducing blood lipid).

[0178] In some embodiments, the drug is used for weight loss, reducing blood lipid levels, preventing and / or treating obesity-related diseases.

[0179] In some embodiments, the obesity-related diseases include obesity, cardiovascular and cerebrovascular diseases, diabetes, hyperglycemia, and hypertension.

[0180] The beneficial effects of the present invention are as follows:

[0181] In some embodiments, the present invention provides a polypeptide with colipase inhibitory activity. By inhibiting the activity of colipase, it can significantly reduce fat absorption, achieving the effects of weight loss and blood lipid reduction. It is safe and non-toxic, has good stability, and has a low blood absorption rate, which is beneficial for the polypeptide to better inhibit colipase in the digestive tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0182] Figure 1 Shows the colipase inhibition rate of BX-peptide A.

[0183] Figure 2 Shows the colipase inhibition rate of BX-peptide B.

[0184] Figure 3 Shows the colipase inhibition rate of BX-peptide C.

[0185] Figure 4 Shows the results of the OLTT test after the first administration on Day 1 in Example 4.

[0186] Figure 5 Shows the results of the OLTT test after the first administration on Day 8 in Example 4.

[0187] Figure 6 Shows the results of the OLTT test after the last administration on Day 14 in Example 4.

[0188] Figure 7 Shows the results of the blood routine test in Example 4.

[0189] Figure 8 Shows the results of the blood biochemical test and organ coefficients in Example 4.

[0190] Figure 9 Shows the results of the OLTT test after the first administration on Day 1 in Example 5.

[0191] Figure 10 Shows the results of the OLTT test after the last administration on Day 7 in Example 5.

[0192] Figure 11 Shows the results of the colipase inhibitory peptide enema PK experiment. DETAILED DESCRIPTION OF THE INVENTION

[0193] The following further elaborates on the content of the present invention through specific embodiments.

[0194] It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0195] Terms

[0196] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention pertains. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions in the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products obtained through commercial purchase. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0197] The terms "first" and "second" are only used for descriptive purposes and should not be construed as implying relative importance or implicitly indicating the quantity of the indicated technical features. The term "plurality" means at least two, such as 2, 3, etc., unless clearly stated otherwise in the text.

[0198] The terms "comprising" or "having" are understood in the sense of "including", rather than in an exclusive or exhaustive sense; that is, in the sense of "including but not limited to". For example, "comprising A" means including A, but not limited to A, such as including A and B.

[0199] The term "and / or" includes both the meanings of "and" and "or", and means each or any combination of specific features. For example, the phrase "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0200] The terms "optionally", "optionally", "optionally", "optionally" mean that the events or circumstances described after the word occur or do not occur, that is, including the two situations where the events or circumstances described therein occur and do not occur.

[0201] The term "about" as used herein represents a range of ±20% of the value thereafter. In some embodiments, the term "about" represents a range of ±10% of the value thereafter. In some embodiments, the term "about" represents a range of ±5% of the value thereafter.

[0202] In this text, the numerical range of "n to m" or "n - m" should be considered to specifically disclose all possible sub - ranges and each numerical value within this range. For example, the description of "1 to 4" or "1 - 4" should be regarded as having clearly disclosed sub - ranges such as 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, 3 to 4, etc., and individual numbers within this range: 1, 2, 3, or 4.

[0203] The three - letter and single - letter codes of amino acids used in this disclosure are as described in J.biol.chem, 243, p3558((1968)).

[0204] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and modified amino acids such as hydroxyproline, γ - carboxyglutamic acid, and O - phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α - carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Amino acid analogs usually have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds that have a structure different from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.

[0205] In this text,

[0206] A(Alanine, Ala): In a sequence, A represents an alanine residue; when used as a raw material or reaction product, A represents alanine.

[0207] R(Arginine, Arg): In a sequence, R represents an arginine residue; when used as a raw material or reaction product, R represents arginine.

[0208] N(Asparagine, Asn): In a sequence, N represents an asparagine residue; when used as a raw material or reaction product, N represents asparagine.

[0209] D(Aspartic acid, Asp): In a sequence, D represents an aspartic acid residue; when used as a raw material or reaction product, D represents aspartic acid.

[0210] C(Cysteine, Cys): In a sequence, C represents a cysteine residue; when used as a raw material or reaction product, C represents cysteine.

[0211] Q (Glutamine, Gln): Q in the sequence represents a glutamine residue, and when used as a raw material or reaction product, Q represents glutamine;

[0212] E (Glutamic acid, Glu): E in the sequence represents a glutamic acid residue, and when used as a raw material or reaction product, E represents glutamic acid;

[0213] G (Glycine, Gly): G in the sequence represents a glycine residue, and when used as a raw material or reaction product, G represents glycine;

[0214] H (Histidine, His): H in the sequence represents a histidine residue, and when used as a raw material or reaction product, H represents histidine;

[0215] I (Isoleucine, Ile): I in the sequence represents an isoleucine residue, and when used as a raw material or reaction product, I represents isoleucine;

[0216] L (Leucine, Leu): L in the sequence represents a leucine residue, and when used as a raw material or reaction product, L represents leucine;

[0217] K (Lysine, Lys): K in the sequence represents a lysine residue, and when used as a raw material or reaction product, K represents lysine;

[0218] F (Phenylalanine, Phe): F in the sequence represents a phenylalanine residue, and when used as a raw material or reaction product, F represents phenylalanine;

[0219] P (Proline, Pro): P in the sequence represents a proline residue, and when used as a raw material or reaction product, P represents proline;

[0220] S (Serine, Ser): S in the sequence represents a serine residue, and when used as a raw material or reaction product, S represents serine;

[0221] T (Threonine, Thr): T in the sequence represents a threonine residue, and when used as a raw material or reaction product, T represents threonine;

[0222] W (Tryptophan, Try): W in the sequence represents a tryptophan residue, and when used as a raw material or reaction product, W represents tryptophan;

[0223] Y (Tyrosine, Tyr): Y in the sequence represents a tyrosine residue, and when used as a raw material or reaction product, Y represents tyrosine;

[0224] V(Valine, Val): V in the sequence represents a valine residue. When used as a raw material or reaction product, V represents valine.

[0225] M(Methionine, Met): M in the sequence represents a methionine residue. When used as a raw material or reaction product, M represents methionine.

[0226] Unless otherwise specified, the amino acids (residues) mentioned herein can be of the D or L type. Unless otherwise specified, the amino acid sequences mentioned herein are in the order from the N-terminus to the C-terminus from left to right.

[0227] The term "amino acid mutation" means amino acid substitution (or replacement), deletion, insertion, and modification. Those skilled in the art can, as needed, perform substitution, deletion, insertion, and / or modification of amino acids to obtain new constructs, provided that the constructs have the desired functions. Amino acid deletions and insertions can be carried out at the amino terminus (N-terminus), middle, and / or carboxyl terminus (C-terminus) of the amino acid sequence. In some embodiments, the amino acid mutation is an amino acid substitution; in some embodiments, the amino acid mutation is a non-conservative amino acid substitution, that is, one amino acid is replaced with another amino acid having different structures and / or chemical properties. In some embodiments, the amino acid mutation is a conservative amino acid substitution, that is, one amino acid is replaced with another amino acid having similar structures and / or chemical properties. Amino acid substitutions can be replaced by non-naturally occurring amino acids or derivatives of natural amino acids (such as 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be accomplished using genetic or chemical methods well known in the art; for example, genetic methods: including site-directed mutagenesis, PCR, gene synthesis, etc., methods for changing amino acid side chain groups other than genetic engineering: including, for example, chemical modification, etc.

[0228] The term "conservative substitution" or "conservative replacement" or "conservative mutation" means an amino acid substitution that does not adversely affect or alter the expected properties of a polypeptide containing the amino acid sequence. Amino acids in the sequence can be replaced with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, backbone conformation and rigidity, etc.), without altering the biological activity of the protein. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacing an amino acid residue with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0229] The term "specifically binds" or "specifically bind" refers to non-random binding between two molecules. For example, a ligand binds to its corresponding receptor with a higher affinity than to other proteins. For example, a ligand (e.g., co-lipase inhibitory peptide) binds to its corresponding receptor (e.g., co-lipase) with an equilibrium dissociation constant (KD) of about 1×10 -6 M or less (e.g., about 1×10 -6 M, about 1×10 -8 M or less, about 1×10 -9 M or less). In some embodiments, the KD for the binding of the ligand to the receptor is 10%, 1% or less of the KD for the binding of the ligand to a non-specific receptor. KD values can be measured using methods well known in the art, such as by surface plasmon resonance assay.

[0230] The term "colipase inhibitory peptide" refers to a polypeptide that inhibits the functional activity of colipase in vitro or in a subject (such as a mouse or a human). For example, in some embodiments, the colipase inhibitory peptide inhibits at least 10% (such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more, or any value therebetween) of the functional activity of colipase.

[0231] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a biomolecule composed of amino acid residues linked by peptide bonds. A polypeptide can be a linear peptide or a cyclic peptide. A "linear peptide" refers to a straight-chain peptide in which the amino acid residues of the polypeptide are sequentially linked. A "cyclic peptide" refers to a peptide having a cyclic structure, which can be formed by connecting two non-adjacent amino acid residues (such as the amino-terminal amino acid residue and the carboxyl-terminal amino acid residue of a linear polypeptide sequence) in a linear polypeptide sequence through an amide bond or other chemical bonds (such as lactone, ether, thioether, disulfide bond, etc.).

[0232] The term "conjugate" refers to a polypeptide that is chemically or biologically linked to another substance (such as a chemical modification, a targeting moiety, a fluorescent dye, and a protein tag, etc.).

[0233] The terms "nucleic acid", "polynucleotide", and "nucleic acid molecule" are used interchangeably and refer to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, and nucleic acids can be synthetic, naturally occurring, and non-naturally occurring, such as non-natural nucleic acids having binding properties similar to those of a reference nucleic acid and being metabolized in a manner similar to that of a reference nucleotide. Including but not limited to, phosphorothioate, phosphoroamidate, methylphosphonate, chiral-methylphosphonate, 2'-O-methyl ribonucleotide, peptide-nucleic acid (PNA)-modified nucleic acids.

[0234] The term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. In some embodiments, the nucleic acid sequence also includes conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. For example, degenerate codon substitutions can be obtained by generating a sequence in which the third position of one or more codons is replaced with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0235] The term "vector" is a vehicle capable of transporting a genetic element (e.g., nucleic acid) to which it is linked. Vectors can be used to transform, transduce, or transfect a host cell so that the genetic element it carries can be expressed within the host cell. Exemplarily, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses, etc. In some embodiments, the vector is a "plasmid", which is a circular double-stranded DNA loop to which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), to which a DNA segment can be ligated into the viral genome. A vector can contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector can also contain an origin of replication. A vector can also include components that assist its entry into cells, including but not limited to, viral particles, liposomes, or protein coats. A vector can be an expression vector. In some embodiments, the vector (e.g., expression vector) contains a nucleic acid sequence encoding a polypeptide of the present disclosure, a promoter (e.g., SV40, CMV, EF-1α), and can also contain at least one selection marker.

[0236] The term "expression vector" or "expression construct" is a vector that is suitable for transforming a host cell and contains a nucleic acid sequence that directs and / or controls (along with the host cell) the expression of one or more coding regions operably linked thereto. Expression vectors can include, but are not limited to: sequences that affect or control transcription, translation, and RNA splicing of a coding region operably linked thereto in the presence of introns.

[0237] "Expression cassette" refers to a combination of nucleic acid sequences or elements that are expressed together or operably linked for expression. For example, an expression cassette can be a nucleic acid sequence comprising a promoter sequence, a start codon, a nucleic acid sequence encoding a protein of interest (POI) to be recombinantly expressed, a stop codon, and a terminator; the expression cassette can also comprise additional regulatory sequences and other sequences, such as enhancers, signal sequences, enhancers, introns, IRES sequences, etc. Host cells containing different expression cassettes can be host cells transfected with different vectors, where each vector contains the different expression cassettes.

[0238] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells (including progeny of such cells) into which exogenous nucleic acid has been introduced. Host cells include "transformants" and "transformed cells", which include primary transformed cells and their derived progeny cells, regardless of the number of passages. Progeny cells may not be completely identical to the parental cells in terms of nucleic acid content and may contain mutations. In some embodiments, host cells include mutant progeny cells having the same function or biological activity as the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include but are not limited to mammalian cells, insect cell lines, plant cells, and fungal cells. Exemplary host cells are as follows: Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei.

[0239] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers, or preservatives, etc. Those skilled in the art understand that other pharmaceutical carriers can be used in the present invention. If desired, the pharmaceutical composition can be present in a kit, vial, or dispenser, for example, and can contain one or more unit dosage forms of the polypeptide. The kit, package, or dispenser can be accompanied by instructions for administration.

[0240] The terms "subject" or "individual" include humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless specifically indicated, the terms "patient" or "subject" are used interchangeably in the present disclosure. Unless otherwise defined in the present disclosure. In certain embodiments, the subject is a human. In certain embodiments, the subject is a person related to obesity or hyperlipidemia.

[0241] "Administer" or "administered", when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition, etc. with the animal, the human, the subject, the cell, the tissue, the organ, or the biological fluid.

[0242] The term "treatment" refers to a clinical intervention that attempts to alter the natural course of the individual being treated and can be implemented either prophylactically or during the course of a clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis.

[0243] The term "effective amount" means an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the appearance of symptoms and / or their underlying causes, and improve the impairment (e.g., lung disease) caused by and associated with a disease state. In some embodiments, the effective amount can be a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptoms, particularly a condition or symptom associated with the disease state, or otherwise prevent, impede, delay, or reverse the disease state or the progression of any other undesirable symptoms associated with the disease in any way. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. A complete therapeutic or prophylactic effect does not necessarily occur after administration of a single dose and may occur after administration of a series of doses. Thus, a therapeutically effective amount or a prophylactically effective amount can be administered in a single or multiple doses. The "therapeutically effective amount" and the "prophylactically effective amount" can vary depending on a variety of factors: for example, the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual.

[0244] Materials and Reagents

[0245] 2-Cl-CTC-polymer resin: purchased from Xi'an LX Technology New Materials Co., Ltd.;

[0246] Piperidine: purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0247] Fmoc-L-alanine (Fmoc-Ala-OH), Fmoc-Pbf-L-arginine (Fmoc-Arg(pbf)-OH), Fmoc-N-trityl-L-asparagine (Fmoc-Asp(OtBu)-OH), Fmoc-S-trityl-L-cysteine (Fmoc-Cys(Trt)-OH), Fmoc-N-trityl-L-glutamine (Fmoc-Gln(Trt)-OH), Fmoc-O-tert-butyl-L-glutamic acid (Fmoc-Glu(OtBu)-OH), Fmoc-glycine (Fmoc-Gly-OH), N-Fmoc-N'-trityl-L-histidine (Fmoc-His(Trt)-OH), Fmoc-L-isoleucine (Fmoc-Ile-OH), Fmoc-L-leucine (Fmoc-Leu-OH), N-alpha-fluorenylmethoxycarbonyl-N-epsilon-tert-butoxycarbonyl-L-lysine (Fmoc-Lys(Boc)-OH), Fmoc-L-methionine (Fmoc-Met-OH), Fmoc-L-phenylalanine (Fmoc-Phe-OH), Fmoc-L-proline (Fmoc-Pro-OH), FMOC-O-tert-butyl-L-serine (Fmoc-Ser(tBu)-OH), Fmoc-O-tert-butyl-L-threonine (Fmoc-Thr(tBu)-OH), N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH), Fmoc-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH), Fmoc-L-valine (Fmoc-Val-OH): all purchased from GL Biochem (Shanghai) Ltd.;

[0248] Chemical reagents such as trifluoroacetic acid (TFA), HATU (2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), N,N-diisopropylethylamine (DIEA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1,2-ethanedithiol (EDT), triisopropylsilane (TIPS), N,N-dimethylformamide (DMF), dichloromethane (DCM), etc.: all purchased from Energy Chemical;

[0249] Lipase: purchased from Beijing InnoChem Science & Technology Co., Ltd.;

[0250] Colipase: purchased from Shanghai Titan Scientific Co., Ltd.;

[0251] PBS with 1×PH = 7.2 - 7.4: purchased from Guangzhou Jet Bio-Filtration Co., Ltd.;

[0252] Pepsin 1:3000: purchased from Solarbio Science & Technology Co., Ltd.;

[0253] Trypsin 1:250: purchased from Solarbio Science & Technology Co., Ltd.;

[0254] Hydrochloric acid 12N: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0255] For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. The materials, reagents, etc. used in this example, unless otherwise specified, are reagents and materials obtained through commercial channels. For the reagents with the manufacturer indicated, similar products from other manufacturers are substitutable.

[0256] Example 1. Preparation of colipase inhibitory peptide

[0257] Refer to the chemical synthesis method of the linear peptide compound in Patent Document CN117720621A to prepare the colipase inhibitory peptide in Table 1. Taking BX-peptide A as an example, its synthesis method is as follows:

[0258] Select 2-Cl-CTC-polymer resin. According to the characteristics of the amino acid sequence (WQHVSLPA) of BX-peptide A, first connect the carboxyl group of Fmoc-Ala-OH at the C-terminus to the resin in a covalent bond form, and then connect the amino group of Fmoc-Ala-OH and the carboxyl group of Fmoc-Pro-OH through a condensation reaction. After removing the unreacted raw materials, refer to the previous steps and sequentially add Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, and carry out a condensation reaction to make the polypeptide fragment on the resin extend in sequence from the C-terminus to the N-terminus of the polypeptide BX-peptide A, obtaining the target polypeptide connected to the resin. Finally, use the cleavage solution (TFA:EDT:TIPS:H2O = 95:2:1:2) to remove the resin to obtain the target polypeptide.

[0259] The product obtained after excision was purified by high performance liquid chromatography. The chromatographic column model was Phenomenex C18, with a size of 4.6×150 mm. Mobile phase A: water containing 0.1% (v / v) trifluoroacetic acid (TFA); Mobile phase B: acetonitrile solution containing 0.01% TFA (v / v); The proportion of phase B increased from 5.0% to 50.0% within 50 min, the flow rate was 3.0 mL / min, and the detection wavelength was 214 nm. The product purified by liquid chromatography was collected, quickly frozen in liquid nitrogen, and freeze-dried to obtain BX-peptide A. Identified by MS: The purity of BX-peptide A obtained by freeze-drying reached 98.5%, and the molecular weight was 936.48, which was consistent with the theoretical molecular weight of BX-peptide A (936.48), proving that the prepared BX-peptide A was consistent with the theoretical design.

[0260] Among them, the purity detection method is as follows: Detection instrument: Agilent ultra-high pressure liquid chromatography-mass spectrometry combined instrument 6545XT; Chromatographic column: ZORBAX RRHD SB-C18 2.1×50 mm, 1.8 um. Detection conditions: Mobile phase A: aqueous solution of 0.5% FA, Mobile phase B: acetonitrile solution of 0.5% FA, injection volume: 5 μL, flow rate: 0.4 mL / min. Gradient elution was carried out according to the program in Table 2.

[0261] The preparation methods of other colipase inhibitory peptides (BX-peptide B - BX-peptide S) were obtained by referring to the preparation method of BX-peptide A. Identified by MS, the results of colipase inhibitory peptides in Table 1 are shown in Table 3.

[0262] Table 1 Amino acid sequences of colipase inhibitory peptides

[0263] Polypeptide Name Amino Acid Sequence (from N-terminus to C-terminus) Sequence Number BX-Peptide A WQHVSLPA SEQ ID NO:1 BX-Peptide B PVPQPLPE SEQ ID NO:2 BX-Peptide C WEWNPARK SEQ ID NO:3 BX-Peptide D HEWDSPPD SEQ ID NO:4 BX-Peptide E REPLSLPD SEQ ID NO:5 BX-Peptide F PTWNRPVY SEQ ID NO:6 BX-Peptide G PEWVGQPV SEQ ID NO:7 BX-Peptide H WNWERAPK SEQ ID NO:8 BX-Peptide I LNWNPAPA SEQ ID NO:9 BX-Peptide J WNYVRLPE SEQ ID NO:10 BX-Peptide K PQWDPAPE SEQ ID NO:11 BX-Peptide L PFDAGYRE SEQ ID NO:12 BX-Peptide M PTLVFEPE SEQ ID NO:13 BX-Peptide N LVWVKQPA SEQ ID NO:14 BX-Peptide O LEHVRLPK SEQ ID NO:15 BX-Peptide P PEHVSLPE SEQ ID NO:16 BX-Peptide Q YNLVPLSK SEQ ID NO:17 BX-Peptide R YTPLWLVK SEQ ID NO:18 BX-Peptide S PFDETFPK SEQ ID NO:19

[0264] Note: The amino acids in the above polypeptides are all L-amino acids.

[0265] Table 2 Elution program

[0266] Time (min) Phase A (%) Phase B (%) 1 99 1 12 40 60 13 20 80 14 20 80 16 99 1 17 99 1

[0267] Table 3 MS identification results of synthesized colipase inhibitory peptides

[0268]

[0269] Example 2. Detection of in vitro lipase and colipase inhibitory activities of colipase inhibitory peptides

[0270] I. Detection of in vitro colipase inhibitory activity

[0271] Reagent preparation:

[0272] Substrate p-nitrophenyl phosphate (p-NPP): Prepared with isopropanol to 10 mM (i.e., 38 mg p-NPP + 10 ml isopropanol);

[0273] Lipase: 45 kDa, 1 mg / ml, with the solvent being PBS (containing Ca 2+ Mg 2+ ) solution, prepared 50 ml, stored at 4 °C, placed on ice;

[0274] Colipase: 11 kDa, dissolved in pure water to a stock solution of 2 mg / ml, aliquoted and sealed, stored at -20 °C, diluted to 0.2 mg / ml with pure water before use;

[0275] Bile salt (sodium taurodeoxycholate, CAS: 1180 - 95 - 6, purchased from Beijing Innochem Science & Technology Co., Ltd.): 100 mM, dissolved in water, 521 mg bile salt + 10 ml water;

[0276] Inhibitory peptide (colipase inhibitory peptide in Example 1, such as BX - peptide A, etc.): 0.1 mg / ml, with the solvent being Tris - HCl solution;

[0277] Tris - HCl: 50 mM, PH = 8;

[0278] Among them, the reaction concentration of lipase is approximately 13 μM; the reaction concentration of colipase is approximately 1.1 μM; the reaction concentration of the inhibitory peptide: 24 μM;

[0279] Operation:

[0280] Operate according to the reaction system in Table 4, specifically as follows:

[0281] ① Put (colipase + inhibitory peptide / Tris - HCl) into a 1.5 ml centrifuge tube, mix well, place it on a shaker at 37 °C, incubate for 15 min, then take it out and cool it down to room temperature;

[0282] ② Put (20 μl substrate + 30 μl bile salt) into a new 2 ml centrifuge tube, gently shake and mix well, and then quickly transfer the 150 μl system ① cooled down to room temperature to system ②, shake well to mix thoroughly, and place it on a shaker at 37 °C, incubate for 15 min;

[0283] ③ After taking out the reaction solution from the shaker, immediately add 300 μl Lipase, invert and mix well, place it on a shaker at 37 °C, incubate for 30 min;

[0284] ④ Terminate the reaction: The reaction system is at 98 °C for 5 min to terminate the reaction, cool it down with cold water, and then centrifuge at 12000 rpm for 5 min;

[0285] ⑤ Measure the OD value: Pipette 200 μl of the supernatant and measure the absorbance at 405 nm using a microplate reader.

[0286] Table 4 Reaction system

[0287]

[0288] Note: In Table, System ① refers to the incubation system of the sample (BX-peptide A / Tris-HCl) and Colipase; System ② refers to the incubation system of bile salt and fat, i.e., the substrate system; ① + ② refers to the mixture of System ① and System ②; The negative peptide in the negative control group is a polypeptide composed of 8 amino acids that does not bind to lipase and Colipase, and its amino acid sequence is: LEHLKLPY (SEQ ID NO: 20).

[0289] Colipase inhibition rate = (OD Colipase - OD 样品 ) / (OD Colipase - OD 胆盐 ) × 100%; where "OD Colipase " refers to the OD value of the Colipase group, "OD 样品 " refers to the OD value of the sample experimental group, and "OD 胆盐 " refers to the OD value of the bile salt control group.

[0290] The Colipase inhibition rate of the Colipase inhibitory peptide is shown in Table 5: The Colipase inhibitory peptide of the present invention has good Colipase inhibitory function, and the inhibition rate is above 20%.

[0291] Table 5 Colipase inhibition rate of Colipase inhibitory peptide (reaction concentration is 24 μM)

[0292]

[0293]

[0294] In addition, by repeating the above experiment on Colipase inhibitory peptides with different reaction concentrations (0.24 μM, 2.4 μM, 12 μM, 24 μM), the Colipase inhibition curve is obtained. Exemplarily, the Colipase inhibition curves of BX-peptide A, BX-peptide B, and BX-peptide C are shown in the appendix Figures 1 - 3 , and the experimental results show that the Colipase inhibitory peptides BX-peptide A, BX-peptide B, and BX-peptide C of the present invention have good Colipase inhibitory function.

[0295] II. Detection of in vitro Lipase inhibitory activity

[0296] Reagent preparation:

[0297] Substrate p-nitrophenyl phosphate (p-NPP): Prepared with isopropanol to 10 mM (i.e., 38 mg p-NPP + 10 ml isopropanol);

[0298] Lipase: 45 kDa, 1 mg / ml, solvent is D-PBS solution, prepare 50 ml, store at 4 °C, place on ice;

[0299] Inhibitory peptide (colipase inhibitory peptide in Example 1, such as BX-peptide A, etc.): 0.1 mg / ml, solvent is Tris-HCl solution;

[0300] Tris-HCl: 50 mM, pH = 8;

[0301] Among them, the reaction concentration of lipase is about 13 μM; the reaction concentration of the inhibitory peptide is about 24 μM;

[0302] Operation:

[0303] Operate according to the reaction system in Table 6, specifically as follows:

[0304] 1. Mix the inhibitory peptide / Tris-HCl + lipase in a thermostatic shaker at 37 °C, shake and mix evenly, and incubate for 10 min;

[0305] 2. Add the substrate, in a thermostatic shaker at 37 °C, shake and mix evenly, and incubate for 20 min;

[0306] 3. Terminate the reaction: At 98 °C for 5 min to terminate the reaction, cool with cold water, centrifuge at 12000 rpm for 5 min;

[0307] 4. Measure the OD value: Pipette 200 μl of the supernatant and measure the absorbance at 405 nm in an enzyme-linked immunosorbent assay (ELISA) reader.

[0308] Table 6 Reaction system

[0309]

[0310] Note: Negative peptide in the negative control group (a polypeptide composed of 8 amino acids that does not bind to lipase and colipase, its amino acid sequence: LEHLKLPY (SEQ ID NO: 20));

[0311] Lipase inhibition rate = (OD 对照 - OD 样品 ) / (OD 对照 - OD 空白 ) × 100%; Among them, "OD 对照 " refers to the OD value of the control experimental group, "OD 样品 " refers to the OD value of the sample group, "OD 空白” refers to the OD value of the blank control group.

[0312] The experimental results show that the colipase inhibitory peptide of the present invention has a weak inhibitory function on Lipase. Exemplarily, the Colipase inhibition rates of some colipase inhibitory peptides are shown in Table 7. The Lipase inhibition rate of the colipase inhibitory peptide of the present invention is <0.1%.

[0313] Table 7 Lipase inhibition rates of colipase inhibitory peptides

[0314] Polypeptide Name Lipase Inhibition Rate (24 μM) BX-Peptide A ＜0.1％ BX-Peptide B ＜0.1％ BX-Peptide C ＜0.1％ BX-Peptide F ＜0.1％ BX-Peptide I ＜0.1％ BX-Peptide J ＜0.1％ BX-Peptide K ＜0.1％ BX-Peptide L ＜0.1％ BX-Peptide M ＜0.1％ BX-Peptide N ＜0.1％ BX-Peptide P ＜0.1％ BX-Peptide Q ＜0.1％ BX-Peptide R ＜0.1％ BX-Peptide S ＜0.1％ Negative Control ＜0.1％

[0315] Example 3. Stability experiment of colipase inhibitory peptide

[0316] The stability test of the colipase inhibitory peptide of Example 1 was carried out according to the following method:

[0317] Preparation of standard solution: Weigh 20 mg of the colipase inhibitory peptide of Example 1 and dissolve it in 20 mL of PBS to make a standard solution; after diluting the standard solution 4 times with PBS, it is used as a control solution;

[0318] Preparation of simulated artificial gastric juice: Weigh 10 mg of pepsin, dissolve it in 4 mL of PBS, and adjust the pH to 4 - 5 with 1N hydrochloric acid, and make up the volume to 10 mL; after diluting the simulated artificial gastric juice 4 times with PBS, it is used as the simulated gastric juice background;

[0319] Preparation of simulated artificial intestinal juice: Weigh 10 mg of trypsin and dissolve it in 10 mL of PBS (pH = 7.4); after diluting the simulated artificial intestinal juice 4 times with PBS, it is used as the simulated intestinal juice background.

[0320] (1) Stability experiment of colipase inhibitory peptide in simulated gastric juice

[0321] Take 1 mL of the standard solution, add 1 mL of simulated artificial gastric juice and react at 37°C for 1 hour, take samples, and detect the purity of the colipase inhibitory peptide with a liquid chromatography - mass spectrometry (LCMS) instrument (the method is the same as in Example 1); subtract the LCMS detection result of the simulated gastric juice background to obtain the stability detection result of the colipase inhibitory peptide in the simulated gastric juice reaction solution.

[0322] (2) Stability experiment of colipase inhibitory peptide in simulated intestinal juice

[0323] Take 1 mL of the standard solution, add 1 mL of simulated artificial intestinal juice and react at 37°C for 1 hour, take samples, and detect the purity of the colipase inhibitory peptide with a liquid chromatography - mass spectrometry (LCMS) instrument; subtract the LCMS detection result of the simulated intestinal juice background to obtain the stability detection result of the colipase inhibitory peptide in the simulated intestinal juice reaction solution.

[0324] (3) Stability test of colipase inhibitory peptide in hydrochloric acid

[0325] 1 mL of the standard solution was taken, 1 mL of hydrochloric acid (0.1 M pH = 1) was added, and the mixture was reacted at 37° C. for 1 hour. Samples were taken, and the purity of the colipase inhibitory peptide was detected by liquid chromatography-mass spectrometry (LCMS); the LCMS detection results were subtracted from the simulated hydrochloric acid background (0.1 M pH = 1 hydrochloric acid was diluted 4 times with PBS) to obtain the stability test results of the colipase inhibitory peptide in hydrochloric acid.

[0326] (4) Stability test of colipase inhibitory peptide in sodium hydroxide

[0327] 1 mL of the standard solution was taken, 1 mL of sodium hydroxide (0.1 M pH = 14) was added, and the mixture was reacted at 37° C. for 1 hour. Samples were taken, and the purity of the colipase inhibitory peptide was detected by liquid chromatography-mass spectrometry (LCMS); the LCMS detection results of the simulated sodium hydroxide background (0.1 M pH = 14 sodium hydroxide was diluted 4 times with PBS) were subtracted to obtain the stability test results of the colipase inhibitory peptide in sodium hydroxide.

[0328] (5) Stability test of colipase inhibitory peptide in 60 degree aqueous solution

[0329] 1 mL of standard solution was respectively drawn into a sealed PE tube, and placed in a 60-degree aqueous solution for 1 h and 3 days, respectively. Samples were taken, and the purity of the colipase inhibitory peptide was detected by liquid chromatography-mass spectrometry (LCMS); the LCMS test results of the blank solvent were subtracted to obtain the stability test results of the colipase inhibitory peptide in a 60-degree aqueous solution.

[0330] The experimental results show that the colipase inhibitory peptide of the present invention has good stability. Exemplarily, the experimental results of BX-peptide A, BX-peptide B, and BX-peptide C are shown in Table 8: In a pepsin solution or a trypsin solution, the colipase inhibitory peptide of the present invention (BX-peptide A, BX-peptide B, BX-peptide C) can stably exist for 1 hour without degradation, which means that the colipase inhibitory peptide will not be decomposed under the action of pepsin or trypsin, and can maintain its chemical structure and active state; at the same time, the colipase inhibitory peptide of the present invention (BX-peptide A, BX-peptide B, BX-peptide C) also has good stability under acidic, alkaline, and high temperature conditions. Since LCMS is a highly sensitive analytical method, the results show that the experimental peptide has a high stability in a pepsin solution or a trypsin solution, and may have potential for oral administration.

[0331] Table 8 Stability test results

[0332] Treatment Method BX Peptide-A Purity BX Peptide-B Purity BX Peptide-C Purity Untreated (0 h) 98.62％ 98.89％ 99.33％ Pepsin (1 h) 98.49％ 98.82％ 99.27％ Trypsin (1 h) 98.55％ 98.78％ 99.01％ 0.1 M HCl, pH = 1 (1 h) 98.45％ 98.86％ 99.31％ 0.1 M NaOH, pH = 14 (1 h) 98.53％ 98.73％ 99.02％ Aqueous Solution at 60 °C (1 h) 98.59％ 98.80％ 99.12％ Aqueous Solution at 60 °C (3 days) 98.56％ 98.75％ 99.01％

[0333] Experimental Study on Enema of Colipase Inhibitory Peptide in Example 4

[0334] C57BL / 6J mice were raised in a SPF-class clean environment, fed ad libitum, with a light-dark cycle of (12h:12h). After 7 days of adaptive feeding, duodenal intubation surgery was performed on each mouse. After the surgery, anti-inflammatory treatment and postoperative care were carried out daily, and the mice recovered for one week after the surgery. One week after the mice recovered, the successfully operated animals were randomly grouped according to body weight, with 5 mice in each group, for a total of 12 groups, namely G1 (Vehicle group), G2 (D(15) group), G3 (D(75) group), G4 (A(15) group), G5 (A(75) group), G6 (A(500) group), G7 (B(15) group), G8 (B(75) group), G9 (B(500) group), G10 (C(15) group), G11 (C(75) group), G12 (C(500) group). For the specific grouping, see Table 9. The day of grouping was recorded as Day 0, and the high-fat diet was started.

[0335] Table 9

[0336]

[0337] Note: ALST refers to Orlistat (purchased from Beijing Innochem Science & Technology Co., Ltd., a chemotherapeutic agent for the treatment of obesity).

[0338] Starting from Day 1, duodenal administration was carried out 3 times a day, with a dosing volume of 0.1 mL / 10 g body weight, and continuous administration for 14 days. The body weight, general appearance, behavior, poisoning manifestations, and death conditions of the animals were observed and recorded daily. The fecal morphology was observed and photographed to check for diarrhea and whether the feces were greasy; feces were retained (before dosing (pre, D0), on the 1st day (D1), 7th day (D7), and 14th day (D14) after dosing), 3 feces were taken from each group and stored at -80 °C; the daily food intake and water intake were recorded during the dosing period.

[0339] OLTT experiments were carried out after the first dose on Day 1, after the first dose on Day 8, and after the last dose on Day 14: Before the OLTT experiment, the mice in each group were fasted for 12 h. Before gavage with soybean oil, blood was collected as the 0 h sample. Subsequently, each group of mice was gavaged with 5 mL / kg soybean oil, and blood was collected 1 h, 2 h, 3 h, and 4 h after gavage with soybean oil to detect serum TG and blood glucose, and the area under the serum TG curve (AUC) per hour was calculated.

[0340] At the end of the experiment, all animals were subjected to gross dissection to observe whether there were visible abnormalities in the main organs (heart, liver, spleen, lung, kidney). If there were abnormalities, samples were taken and fixed in 10% formalin; if there were no abnormalities, they were directly disposed of harmlessly. The heart, thymus, adrenal gland, liver, kidney, spleen, and testis were taken and weighed, and the relative weight (organ / body ratio) was calculated. The inguinal fat, perirenal fat, and peritesticular fat of the mice in each group were taken to calculate the body fat rate, and the body fat rate = total fat weight / mouse body weight × 100%.

[0341] At the end of the experiment, serum was collected from all animals for routine blood tests (5 - category excluding RET) and blood biochemical tests (alanine aminotransferase ALT, aspartate aminotransferase AST, γ - glutamyl transferase GGT, alkaline phosphatase ALP, urea UREA, creatinine CREA, blood glucose Glu, serum albumin ALB, total protein TP, total cholesterol CHOL, triglyceride TG, chloride CL, potassium K, sodium NA). Serum was retained at the end of the experiment for each test group and stored at -80 °C.

[0342] The results of the OLTT are as Figures 4 - 6 shown: Compared with the Vehicle group, the area under the curve (AUC) of serum triglyceride in the colipase inhibitory peptide group of the present invention decreased significantly.

[0343] Through the blood biochemical tests of the mice in each dose group, no abnormalities were found in the routine blood tests such as red blood cells, platelets, lymphocytes, monocytes, white blood cells (neutrophils, eosinophils, basophils), etc.; no abnormal expressions were found in the enzyme or protein markers related to liver and kidney functions; no abnormal expressions were found in other blood indicators( Figure 7 、 8 ). After administration, there were no significant differences in the relative weights of the organs of the heart, thymus, liver, adrenal gland, kidney, spleen, and testis of the mice( Figure 8 ).

[0344] During the administration period, the feces of the mice did not show diarrhea. The feces of the mice in each group were oily, and no obvious differences were found among the experimental groups. Fecal samples were collected before administration (pre), on the 1st day (D1), the 7th day (D7), and the 14th day (D14) after administration for detection, and no mass spectrometry signals of the colipase inhibitory peptide of the present invention were detected.

[0345] Example 5. Experimental study on intragastric administration of colipase inhibitory peptide

[0346] C57BL / 6J mice were housed in a SPF-class clean environment, with free access to food, and a light-dark cycle of 12 h:12 h. After 1 week of adaptive feeding, the animals were randomly grouped according to body weight, with 5 mice in each group, namely G1 (Vehicle group), G2 (D(500) group), G3 (A(500) group), G4 (B(500) group), and G5 (C(500) group). For the specific grouping, see Table 10. The day of grouping was recorded as Day 0, and the high-fat diet was started.

[0347] Table 10

[0348]

[0349] Note: ALST refers to orlistat (purchased from Beijing InnoChem Co., Ltd., a chemotherapeutic agent for the treatment of obesity).

[0350] Gavage administration started on Day 1, 3 times a day, with a dosing volume of 0.1 mL / 10 g body weight, and continuous administration for 7 days. The body weight, general appearance, behavior, poisoning manifestations, and death of the animals were observed and recorded every day. The fecal morphology was observed and photographed to check for diarrhea and whether the feces were greasy. Feces were retained (before dosing (pre, D0), on the 1st day after dosing (D1), and on the 7th day after dosing (D7)), 3 feces per group were taken and stored at -80 °C. The daily food intake and water intake were recorded during the dosing period.

[0351] The OLTT experiment was conducted after the first dosing on Day 1 and after the last dosing on Day 7: Before the OLTT experiment, the mice in each group were fasted for 12 h. Before gavage with soybean oil, blood was collected as the 0 h sample. Subsequently, the mice in each group were gavaged with 5 mL / kg soybean oil, and blood was collected at 1 h, 2 h, 3 h, and 4 h after gavage with soybean oil to detect serum TG and blood glucose, and the area under the serum TG curve (AUC) per hour was calculated.

[0352] After the last dosing, mice from the high-dose group were selected from each dosing group; at 0 h, 4 h, 24 h, and 48 h, 2 mice were selected from each group, blood was collected and stored at -80 °C; the mice were dissected, and the gastric tissue, intestinal (duodenum, jejunum, ileum, cecum, colon, rectum) tissue homogenates and contents were frozen at -80 °C.

[0353] The OLTT test results are as Figures 9 - 10 shown. The experimental results showed that, compared with the Vehicle group, the area under the serum TG curve on Day 1 and Day 7 in each dosing group decreased significantly.

[0354] C57bl / 6J mice (n = 2) were gavaged with the test drug at a dose of 500 mg / kg / Day. After administration, gastric tissues, intestinal (duodenum, jejunum, ileum, cecum, colon, rectum) contents and tissue homogenates were collected at 0, 4, 24, and 48 hours respectively for detection. The results are shown in Table 11: within 0 to 4 hours after administration, relevant mass spectrometry signals of the test drug could be detected in the stomach, duodenum, and jejunum.

[0355] Table 11

[0356] Detection Site BX Peptide-A BX Peptide-B BX Peptide-C Gastric Contents at 0 h √ √ √ Duodenal Contents at 0 h √ √ √ Jejunal Contents at 0 h √ √ √ Gastric Tissue Homogenate at 0 h √ √ * Duodenal Tissue Homogenate at 0 h √ √ * Jejunal Tissue Homogenate at 0 h √ √ * Gastric Contents at 4 h √ √ * Duodenal Contents at 4 h √ √ * Jejunal Contents at 4 h √ √ * Gastric Tissue Homogenate at 4 h * √ * Duodenal Tissue Homogenate at 4 h * √ * Jejunal Tissue Homogenate at 4 h * √ *

[0357] Note: √ indicates detectable, * indicates undetectable.

[0358] Example 6. Experimental study on PK of colipase inhibitory peptide by enema

[0359] C57BL / 6J mice were housed in a SPF-class clean environment, fed ad libitum, with a light-dark cycle of (12h:12h). After 7 days of adaptive feeding, duodenal intubation surgery was performed on each mouse. After the surgery, anti-inflammatory treatment and postoperative care were carried out every day, and the mice recovered for one week after the surgery. One week after the mice recovered, the successfully operated animals were randomly grouped according to body weight, with 3 mice in each group, for a total of 3 groups, namely G1 (Group A(500), administration: BX peptide-A at 500 mg / kg / day), G2 (Group B(500), administration: BX peptide-B at 500 mg / kg / day), G3 (Group C(500), administration: BX peptide-C at 500 mg / kg / day). Duodenal administration was started on the day of grouping, with a single administration, a dosing volume of 0.1 mL / 10 g body weight, and a dosing dose of 500 mg / kg / day. PK blood samples were collected before administration, and at 10, 30, 60, 120, 240, and 480 min after administration, and stored at -80°C.

[0360] The results are as Figure 11Shown: The highest concentration of BX-peptide A detected in plasma (233 nmol / L) was below the detection limit after 120 minutes. After calculation, the half-life of BX-peptide A was 14.8 minutes, and the plasma absorption rate was 0.60%; the highest concentration of BX-peptide B detected in plasma (2146 nmol / L) was below the detection limit after 120 minutes. After calculation, the half-life of BX-peptide B was 13.8 minutes, and the plasma absorption rate was 8.07%; the plasma concentration of BX-peptide C was below the detection limit throughout the detection process; it indicates that the colipase inhibitory peptide of the present invention enters the blood very little, and the colipase inhibitory peptide mostly stays in the front part of the digestive tract (stomach and the front part of the small intestine), which will be beneficial to inhibiting colipase in the digestive tract, thereby blocking the lipase / colipase interaction, reducing fat digestion and absorption, that is, the blood absorption rate of the colipase inhibitory peptide of the present invention is low, which will be beneficial for the colipase inhibitory peptide to better play its role on colipase in the digestive tract.

[0361] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A polypeptide or a salt thereof, wherein the polypeptide comprises the following amino acid sequence: X1X2X3X4X5X6X7X8; wherein, X1 is selected from W, P, H, R, L, Y; X2 is selected from Q, V, E, T, N, F; X3 is selected from H, P, W, Y, D, L; X4 is selected from V, Q, N, D, L, E, A; X5 is selected from S, P, R, G, F, K, W, T; X6 is selected from L, A, P, Q, Y, E, F; X7 is selected from P, R, V, S; X8 is selected from A, E, K, D, Y, V; Optionally, X1 is W or P, X2 is Q, V or E, X3 is H, P or W, X4 is V, Q or N, X5 is S or P, X6 is L or A, X7 is P or R, and X8 is A, E or K; Optionally, X1 is W, X2 is Q or E, X3 is H or W, X4 is V or N, X5 is S or P, X6 is L or A, X7 is P or R, and X8 is A or K; Optionally, X1 is W, X2 is E, X3 is W, X4 is N, X5 is P, X6 is A, X7 is R and X8 is K; or, X1 is W, X2 is Q, X3 is H, X4 is V, X5 is S, X6 is L, X7 is P and X8 is A; or, X1 is P, X2 is V, X3 is P, X4 is Q, X5 is P, X6 is L, X7 is P and X8 is E; Optionally, the polypeptide is a colipase inhibitory peptide.

2. The polypeptide or salt thereof according to claim 1, wherein the polypeptide is a polypeptide selected from any one of the following a) to f): a) The polypeptide comprises the following amino acid sequence: PX2X3X4X5X6PX8; wherein, X2 is selected from Q, V, E, T, F; X3 is selected from H, P, W, D, L; X4 is selected from V, Q, D, E; X5 is selected from S, P, G, F, T; X6 is selected from L, A, Q, E, F; X8 is selected from E, K, V; b) The polypeptide comprises the following amino acid sequence: X1X2X3VX5X6PX8; wherein, X1 is selected from W and L; X2 is selected from Q, V, E, N; X3 is selected from H, W, Y; X5 is selected from S, R, K; X6 is selected from L and Q; X8 is selected from A, E, K; c) The polypeptide comprises the following amino acid sequence: X1X2WNX5X6X7X8; wherein, X1 is selected from W, P, L; X2 is selected from E, T, N; X5 is selected from P and R; X6 is selected from A and P; X7 is selected from P, R, V; X8 is selected from A, K, Y; d) The polypeptide comprises the following amino acid sequence: X1EX3X4SX6PD; wherein, X1 is selected from H and R; X3 is selected from P and W; X4 is selected from D and L; X6 is selected from L and P; e) The polypeptide comprises the following amino acid sequence: YX2X3X4X5LX7K; wherein, X2 is selected from T and N; X3 is selected from P and L; X4 is selected from V and L; X5 is selected from P and W; X7 is selected from V and S; f) The polypeptide comprises the following amino acid sequence: X1X2X3X4X5X6X7X8; wherein, X1 is selected from W and P; X2 is selected from N, F; X3 is selected from W and D; X4 is selected from E and A; X5 is selected from R and G; X6 is selected from A and Y; X7 is selected from P and R; X8 is selected from E and K.

3. A polypeptide or a salt thereof, characterized in that: The polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-19, or comprises an amino acid mutant sequence having no more than 3 amino acid differences from any one of SEQ ID NOs: 1-19; alternatively, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-19; Optionally, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 1; Optionally, the amino acid sequence of the polypeptide is shown in SEQ ID NO:

3.

4. The polypeptide or salt thereof according to any one of claims 1 to 3, wherein The polypeptide has at least one of the following characteristics (1) to (7): (1) The polypeptide is capable of inhibiting the functional activity of colipase; optionally, the inhibition rate of the polypeptide on colipase is greater than 10%; optionally, the in vitro colipase inhibition rate of the polypeptide at a reaction concentration of 24 μM is greater than 10%; (2) The polypeptide has a lower inhibitory effect on lipase activity than on colipase activity; optionally, the polypeptide has an inhibitory rate on lipase of less than 10%; optionally, the polypeptide has an in vitro lipase inhibition rate of less than 10% at a reaction concentration of 24 μM; (3) The polypeptide has good stability; optionally, when the polypeptide is reacted in a pepsin solution or a trypsin solution at 37° C. for 1 hour, the purity of the polypeptide does not change by more than 5%; (4) The polypeptide has a lipid-lowering effect; (5) The polypeptide has a weight loss effect; (6) The amino acids in the polypeptide are independently selected from: D-amino acids and L-amino acids; optionally, the amino acids in the polypeptide are L-amino acids; (7) The polypeptide is a linear peptide or a cyclic peptide. Optionally, the polypeptide is a linear peptide.

5. A conjugate comprising a modified portion and the polypeptide of any one of claims 1 to 4; Optionally, the modification moiety comprises at least one of a chemical modification, a targeting moiety, a fluorescent dye, and a protein tag; Optionally, the modified portion is located at the N-terminus and / or C-terminus of the polypeptide. A fusion protein comprising the polypeptide according to any one of claims 1 to 4.

7. A biological material associated with the polypeptide of any one of claims 1 to 4, the conjugate of claim 5, or the fusion protein of claim 6, the biological material comprising any one of the following n1) to n9): n1) a nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 4, the conjugate according to claim 5, or the fusion protein according to claim 6; n2) an expression cassette comprising the nucleic acid molecule described in n1); n3) a vector comprising the nucleic acid molecule described in n1); n4) a vector comprising the expression cassette described in n2); n5) a cell comprising the nucleic acid molecule described in n1); n6) a cell comprising the expression cassette described in n2); n7) a cell comprising the vector described in n3); n8) a cell comprising the vector described in n4); n9) A cell comprising the polypeptide of any one of claims 1 to 4, the conjugate of claim 5, or the fusion protein of claim 6.

8. A composition comprising the polypeptide according to any one of claims 1 to 4, the conjugate according to claim 5, and / or the fusion protein according to claim 6; Optionally, the composition further comprises other ingredients that inhibit the activity of colipase and / or inhibit the activity of lipase.

9. Use of the polypeptide according to any one of claims 1 to 4, the conjugate according to claim 5, the fusion protein according to claim 6, the biomaterial according to claim 7, and / or the composition according to claim 8 in the preparation of a colipase inhibitory peptide, food, health product, or medicine.

10. A product comprising the polypeptide according to any one of claims 1 to 4, the conjugate according to claim 5, the fusion protein according to claim 6, the biomaterial according to claim 7, and / or the composition according to claim 8; Optionally, the product is food, health care product, or medicine; Optionally, the product further comprises excipients acceptable to food, health care products, or medicines; Optionally, the food is used for weight loss and / or lowering blood lipids; the health care product is used for weight loss and / or lowering blood lipids; or the medicine is used for weight loss, lowering blood lipids, preventing and / or treating obesity-related diseases.

11. Use of a colipase inhibitory peptide or a salt thereof in the preparation of a product for weight loss, lowering blood lipids, and / or preventing and / or treating obesity-related diseases; optionally, the colipase inhibitory peptide comprises the polypeptide according to any one of claims 1 to 4; Optionally, the colipase inhibitory peptide comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 1; Optionally, the amino acid sequence of the colipase inhibitory peptide is as shown in SEQ ID NO: 3; Optionally, the product is food, health care product, or medicine; Optionally, the product is a health product.

Images