CN-122011160-A - Swim bladder collagen anti-inflammatory polypeptide, screening method thereof and anti-inflammatory performance testing method

Abstract

The invention relates to the technical field of swim bladder collagen anti-inflammatory polypeptides, in particular to a swim bladder collagen anti-inflammatory polypeptide, a screening method thereof and an anti-inflammatory performance test method, wherein the polypeptide screened by the screening method is a natural peptide segment in two I-type collagen sequences of the swim bladder, and comprises a polypeptide 1, a polypeptide 2, a polypeptide 3 and a polypeptide 4, which have higher anti-inflammatory performance.

Inventors

• ZHANG YU
• HUANG DARONG
• ZHANG DEQUAN
• ZHONG CHAO
• LAI JIALIANG
• TIAN JIANXIN
• Zhong Biluan
• ZHAO YING

Assignees

• 广东官栈营养健康科技有限公司
• 中国科学院深圳先进技术研究院

Dates

Publication Date

20260512

Application Date

20260123

Claims (9)

1. 1. The swimming bladder collagen anti-inflammatory polypeptide is characterized in that the polypeptide is a natural peptide in two I-type collagen sequences of the swimming bladder, and the amino acid sequence of the natural peptide is any one of the following: A polypeptide 1:TRASIPLKNWYI;SEQ ID NO.1; A polypeptide 2:KTNPARMCRDLRM;SEQ ID NO.2; A polypeptide 3:NPTRASIPLKNWYI;SEQ ID NO.3; Polypeptide 4:QQNPVQTQPHPPQQ;SEQ ID NO.4.
2. 2. The method for screening a class of swim bladder collagen anti-inflammatory polypeptides according to claim 1, wherein the steps of: Step S1, screening polypeptide sequences: S11, constructing a swimming bladder collagen short peptide database, namely intercepting two I-type collagen sequences of the swimming bladder, and then removing the weight of all the sequences to obtain a swimming bladder non-redundant I-type collagen database; step S12, carrying out anti-inflammatory probability scoring on the collagen short peptide in the database by utilizing a PepFuncML program, wherein the scored score represents the anti-inflammatory probability of the sequence; step S13, screening the evolutionary tree of the sequences with high scores, namely firstly sequencing the sequences scored in the step S12, then selecting the sequences for evolutionary tree construction, and finally selecting a plurality of polypeptide sequences for chemical synthesis; step S2, chemical synthesis of the polypeptides, and synthesizing target polypeptides one by the polypeptide sequences obtained in the step S13.
3. 3. The method for screening anti-inflammatory polypeptides of swimming bladder collagen according to claim 2, wherein the sequence interception length in the step S11 is 2-50 amino acids, and the non-redundant type I collagen database of swimming bladder comprises 13-14 ten thousand sequences.
4. 4. The method for screening a class of swimming bladder collagen anti-inflammatory polypeptides according to claim 2, wherein in the step S13, sequences sequenced into 200 are selected to perform evolutionary tree construction through difference maximization, and the basis of the difference maximization is a difference matrix constructed after pairwise sequence comparison.
5. 5. The method for screening a class of collagen anti-inflammatory polypeptides according to claim 2, wherein the step S2 further comprises confirming the purity and structure of the target polypeptide during the synthesis of the polypeptide.
6. 6. The method for testing the anti-inflammatory performance of a swimming bladder collagen anti-inflammatory polypeptide according to claim 1, comprising the following steps: (1) Cell plating, namely taking mouse mononuclear macrophage leukemia cells (RAW 264.7) as a test object, taking frozen RAW264.7 cells for resuscitation, inoculating the RAW264.7 cells on a pore plate, and transferring the pore plate into an incubator for culture; (2) Sample group administration, namely directly diluting the sample to the working concentration by using a DMEM complete medium, and then administering the sample into an orifice plate; Positive control group dosing, namely, adding the DMEM complete medium containing dexamethasone into an orifice plate; the negative and blank control groups are dosed by directly adding DMEM complete medium into the well plate to cover the cells; Transferring the above groups into a cell incubator for culturing after the administration is finished; (3) Lipopolysaccharide (LPS) induction, namely adding DMEM complete culture medium containing LPS into the pore plates of a sample group, a positive control group and a negative control group, and adding DMEM complete culture medium into the pore plates of a blank group; Transferring the LPS samples of each group into a cell incubator after the sample addition is finished; (4) After the induction of the administration is finished, collecting cell culture supernatant and performing centrifugal treatment; (5) The inflammatory cytokine content was detected using enzyme-linked immunosorbent assay (ELISA).
7. 7. The method for testing the anti-inflammatory properties of a swimming bladder collagen anti-inflammatory polypeptide according to claim 6, wherein the well plate is a 24-well plate, RAW264.7 cells are inoculated on the well plate according to the density of 8 multiplied by 10 4 cells per well, the sample group administration, the positive control group administration and the negative and blank control group administration are carried out according to the volume of 400 mu L per well, and the dexamethasone concentration is 50 mu g/mL.
8. 8. The method for testing the anti-inflammatory performance of the collagen anti-inflammatory polypeptides of the swimming bladder according to claim 6, wherein the temperature in the incubator is 37 ℃, the incubator contains 5% carbon dioxide, the incubator is cultured for 16-24 hours to enable the cell fusion degree to reach 45% -60%, the incubator is used for feeding each group of drugs, and the cell incubator is used for transferring each group of LPS after the end of sample feeding, the temperature in the incubator is 37 ℃, the incubator contains 5% carbon dioxide, and the incubator is respectively cultured for 24 hours.
9. 9. The method for testing the anti-inflammatory properties of a swimming bladder collagen anti-inflammatory polypeptide according to claim 6, wherein the centrifugation is performed at a speed of 3000rpm for 5min.

Description

Swim bladder collagen anti-inflammatory polypeptide, screening method thereof and anti-inflammatory performance testing method Technical Field The invention relates to the technical field of biochemistry, in particular to a swimming bladder collagen anti-inflammatory polypeptide, a screening method thereof and an anti-inflammatory performance testing method. Background Swim bladder is an important byproduct of fish, is rich in collagen, and is widely studied and applied to the fields of food, health care products and medicines in recent years. Collagen short peptide, which is the degradation product of collagen, is attracting attention due to its remarkable biological activity, especially in anti-inflammatory and tissue repair. The collagen short peptide rich in the swim bladder has natural anti-inflammatory effect, can inhibit inflammatory reaction, reduce tissue injury caused by inflammation, and has potential curative effects on various inflammation-related diseases. Although much research has been directed to the discovery of polypeptide sequences with anti-inflammatory activity from swim bladders, the prior art is subject to several limitations. The existing polypeptide screening method generally relies on traditional experimental means, and functional tests are carried out after the polypeptide is obtained through extraction or chemical synthesis. However, these methods often have problems of low screening efficiency of polypeptide, high experimental cost, difficult precise grasping of active ingredients, etc., and if the anti-inflammatory performance of the polypeptide obtained by the experimental means is not obvious, how to accurately screen a polypeptide sequence with strong anti-inflammatory performance becomes a technical problem in the field. Disclosure of Invention Aiming at the problems, the invention mainly aims to provide the swimming bladder collagen anti-inflammatory polypeptide, the screening method and the anti-inflammatory performance testing method thereof, and the swimming bladder collagen anti-inflammatory polypeptide is obtained by the screening method, so that the screening efficiency is improved, the anti-inflammatory activity of a polypeptide sequence is ensured, and the method for accurately testing the anti-inflammatory performance of the swimming bladder collagen polypeptide is provided. In order to achieve the above purpose, the invention provides a swimming bladder collagen anti-inflammatory polypeptide, wherein the polypeptide is a natural peptide in two I-type collagen sequences of the swimming bladder, and the amino acid sequence of the natural peptide is any one of the following: A polypeptide 1:TRASIPLKNWYI;SEQ ID NO.1; A polypeptide 2:KTNPARMCRDLRM;SEQ ID NO.2; A polypeptide 3:NPTRASIPLKNWYI;SEQ ID NO.3; Polypeptide 4:QQNPVQTQPHPPQQ;SEQ ID NO.4. In addition, the invention also provides a screening method of the swimming bladder collagen anti-inflammatory polypeptide, which comprises the following operation steps: Step S1, screening polypeptide sequences: S11, constructing a swimming bladder collagen short peptide database, namely intercepting two I-type collagen sequences of the swimming bladder, and then removing the weight of all the sequences to obtain a swimming bladder non-redundant I-type collagen database; step S12, carrying out anti-inflammatory probability scoring on the collagen short peptide in the database by utilizing a PepFuncML program, wherein the scored score represents the anti-inflammatory probability of the sequence; step S13, screening the evolutionary tree of the sequences with high scores, namely firstly sequencing the sequences scored in the step S12, then selecting the sequences for evolutionary tree construction, and finally selecting a plurality of polypeptide sequences for chemical synthesis; step S2, chemical synthesis of the polypeptides, and synthesizing target polypeptides one by the polypeptide sequences obtained in the step S13. Preferably, in the step S11, the length of the sequence interception is 2-50 amino acids, and the swim bladder non-redundant type I collagen database comprises 13-14 ten-thousand sequences. Preferably, in step S13, the sequences ordered as 200 sequences are selected to construct the evolutionary tree through the maximization of the differences, and the basis of the maximization of the differences is the difference matrix constructed after the pairwise comparison of the sequences. Preferably, in the step S2, the process of synthesizing the polypeptide further includes confirmation of the purity and structure of the target polypeptide. In addition, the invention also provides a method for testing the anti-inflammatory performance of the swimming bladder collagen anti-inflammatory polypeptide, which specifically comprises the following steps: (1) Cell plating, namely taking mouse mononuclear macrophage leukemia cells (RAW 264.7) as a test object, taking frozen RAW264.7 cells for resuscitation, inoculating the RAW