阅读说明：本技术 一种多肽片段d及其应用 (Polypeptide fragment D and application thereof ) 是由 张彩华 常英 于 2021-02-02 设计创作，主要内容包括：本发明属于生物医药技术领域,具体涉及一种多肽片段D及其应用,所述多肽片段D具有SEQ ID No.1所示的氨基酸序列,其中,第9位氨基酸Xaa为Tyr、Val、Gly、Ser、Gln或不存在,第20位氨基酸Xaa为Ser、Gln、Glu、Tyr、Arg或不存在,第25位氨基酸Xaa为Asn、Thr、Ser、Pro、Leu或不存在。MP-D显著改善了IBD小鼠模型的结肠病理性形态,降低了结肠组织病理学评分和结肠干扰素-γ表达含量,具有干预小鼠炎症性肠病发生的能力。(The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide fragment D and application thereof, wherein the polypeptide fragment D has an amino acid sequence shown in SEQ ID No.1, wherein the 9 th amino acid Xaa is Tyr, Val, Gly, Ser, Gln or does not exist, the 20 th amino acid Xaa is Ser, Gln, Glu, Tyr, Arg or does not exist, and the 25 th amino acid Xaa is Asn, Thr, Ser, Pro, Leu or does not exist. MP-D significantly improves colon pathomorphology of IBD mouse model, reduces colon histopathology score and colon interferon-gamma expression content, and has the ability to interfere with the development of inflammatory bowel disease in mice.)

1. A polypeptide fragment D, characterized by having the amino acid sequence shown in SEQ ID No. 1.

2. The polypeptide fragment D according to claim 1, wherein the amino acid Xaa at position 9 of the amino acid sequence shown in SEQ ID No.1 is Tyr, Val, Gly, Ser, Gln or is absent, the amino acid Xaa at position 20 is Ser, Gln, Glu, Tyr, Arg or is absent, and the amino acid Xaa at position 25 is Asn, Thr, Ser, Pro, Leu or is absent.

3. Use of the polypeptide fragment D of claim 1 in the preparation of a medicament against inflammatory bowel disease.

4. Use of the polypeptide fragment D of claim 1 in the preparation of a food or food additive for anti-inflammatory bowel disease.

5. The use of the polypeptide fragment D of claim 1 in the preparation of a health product for treating inflammatory bowel disease.

6. Use according to claim 3, for the preparation of a medicament for ameliorating the pathological colon shortening of inflammatory bowel disease.

7. Use according to claim 3, for the preparation of a medicament for reducing the colon histopathological score of inflammatory bowel disease.

8. The use according to claim 3, for the preparation of a medicament for down-regulating the level of colonic interferon- γ expression in inflammatory bowel disease.

9. A pharmaceutical formulation comprising the polypeptide fragment D of claim 1 and a pharmaceutically acceptable carrier, excipient or diluent.

10. The pharmaceutical preparation according to claim 9, wherein the pharmaceutical preparation is in the form of injection, capsule, tablet, granule, suspension, enema, emulsion or powder.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide fragment D and application thereof.

Background

Inflammatory Bowel Disease (IBD) is an idiopathic chronic inflammatory bowel disease, the lesions of which are mainly located in the colorectal segment and involve mucous membrane and mucous membrane muscularis, and serious complications in liver, gallbladder, muscle skin and blood coagulation, and 20-30% of patients who relapse may be transformed into colorectal cancer, and the IBD is a very serious inflammatory bowel disease and can be divided into Ulcerative Colitis (UC) and Crohn's Disease (CD). IBD is considered to be an intestinal inflammatory reaction caused by abnormal intestinal mucosal innate immunity and acquired immunity under the interaction of multiple factors such as environment, heredity, infection and immunity, and the inflammatory reaction in the intestinal mucosal innate layer is considered to be a cornerstone of IBD pathogenesis. In recent decades, the incidence rate of IBD has been on the rise year by year, and traditional IBD treatment drugs, such as salicylic acids, steroid hormones, immunosuppressants and the like, mainly reduce inflammation and regulate immune disorders to effectively control the onset of disease, but these traditional methods cannot completely cure IBD, are often accompanied with serious adverse reactions, and cause serious damage to the quality of life of patients, so that a new IBD treatment method is urgently needed.

In recent years, probiotics are becoming a new approach for the treatment of IBD, and research shows that such preparations can improve dysbacteriosis of IBD patients to different degrees in intestinal tract. Lactobacillus L.plantarum is a common probiotic, and researches show that lactobacillus L.plantarum can inhibit the damage of pathogenic bacteria in intestinal tracts through adhesion and colonization, regulate the intestinal permeability of immunodeficient mice, and further intervene the development of colitis, and microfilmin (MIMP) is an active polypeptide fragment which is separated from L.plantarum CGMCC 1258 strain and can competitively adhere to intestinal epithelial cells with invasive pathogenic escherichia coli, and the sequence is shown in SEQ ID No. 2: THTVGSYFSVQNGYVGAFSQALGNSEYAMNSPLGSLDGRTTMYNLLGVKYLFAREDQLK KQ, the fragment can significantly improve the inflammatory state of intestinal tract and prevent IBD mice intestinal flora imbalance. However, MIMP is a biomacromolecule consisting of 61 amino acids, and the relatively large molecular weight is easy to generate immunogenicity and not easy to form a medicament, so that the clinical application of MIMP is limited; in addition, the higher molecular weight is not favorable for the industrial production of the drug. From the aspects of medicinal value and economic benefit, the MIMP is further structurally modified and reformed to improve the pharmacological activity or/and the patent drug property of the MIMP fragment, thereby being beneficial to the clinical application and the economic benefit of the active fragment.

Disclosure of Invention

In order to overcome the defects of easy immunogenicity and difficult drug development of MIMP (mimP) with an improvement effect on inflammatory bowel disease in the prior art, the invention aims to provide a polypeptide fragment D (MP-D for short) and application thereof.

The invention is realized by the following technical scheme:

the invention provides a polypeptide fragment D, which has an amino acid sequence shown in SEQ ID No. 1.

Preferably, the amino acid Xaa at the 9 th position of the amino acid sequence shown in SEQ ID No.1 is Tyr, Val, Gly, Ser, Gln or is absent, the amino acid Xaa at the 20 th position is Ser, Gln, Glu, Tyr, Arg or is absent, and the amino acid Xaa at the 25 th position is Asn, Thr, Ser, Pro, Leu or is absent.

The invention also provides application of the polypeptide fragment D in preparing anti-inflammatory bowel disease medicines.

The invention also provides application of the polypeptide fragment D in preparation of anti-inflammatory bowel disease foods or food additives.

The invention also provides application of the polypeptide fragment D in preparation of anti-inflammatory bowel disease health-care products.

Preferably, the polypeptide fragment D is used for preparing a medicament for improving pathological colon shortening of inflammatory bowel diseases.

Preferably, the use of said polypeptide fragment D for the preparation of a medicament for reducing the colon histopathological score of inflammatory bowel disease.

Preferably, the polypeptide fragment D is used for preparing a medicament for down-regulating the expression content of colon interferon-gamma in inflammatory bowel diseases.

The invention also provides a pharmaceutical preparation, which comprises the polypeptide fragment D and a pharmaceutically acceptable carrier, excipient or diluent.

Preferably, the pharmaceutical preparation is in the form of injection, capsule, tablet, granule, suspension, enema, emulsion or powder.

The invention has the beneficial effects that:

the invention establishes an acute Inflammatory Bowel Disease (IBD) mouse model by a Dextran Sodium Sulfate (DSS) chemical induction method, and explores whether polypeptide fragment D (MP-D) has an improvement effect on the IBD mouse model by means of colon morphology, histopathology and immune factor expression analysis. The research result shows that under the same dosage with MIMP, the intervention of the polypeptide fragment D obviously improves the colon pathophysiology morphology of an IBD mouse model, reduces the colon histopathology score of the IBD mouse model, and has the capability of improving and intervening the occurrence of the inflammatory bowel disease of the mouse. And compared with MIMP, the MP-D fragment has smaller molecular weight, is also beneficial to the pharmacy and the application of the MP-D fragment, and discloses the application potential of the polypeptide fragment D in the preparation of active products for preventing, intervening and treating inflammatory bowel diseases.

The abbreviations used in the present invention have the following specific meanings:

thr is threonine; his is histidine; val is valine; gly is glycin; ser is serine; phe is phenylalanine; asn is asparagine; tyr is tyrosine; ala is alanine; leu is leucine; glu is glutamic acid; met is methionine; pro is proline; asp is aspartic acid; arg is arginine; lys is lysine; gln is glutamine.

Drawings

FIG. 1 is a graph showing the body weight change trend of a model group mouse and a blank control group mouse,^#P<0.05，^##P<0.01，^###P<0.001, comparing with a blank control group, and carrying out significance test on two independent samples by t test;

FIG. 2 is a graph showing the comparison of colon lengths of mice in a placebo group, a model group, a MIMP positive control group and an MP-D experimental group,^*P<0.05,^**P<0.01，^***P<0.001, compared to model group;^###P<0.001, comparing with a blank control group, and carrying out significance test by single-factor analysis of variance;

FIG. 3 is a histopathological micrograph of colon of mice in blank control group, model group, MIMP positive control group and MP-D experimental group (HE staining 20 Xmicroscopy; A. blank control group; B. model group; C. MIMP positive control group; D.MP-D experimental group);

histopathological score comparison graph (A)^*P<0.05，^**P<0.01，^***P<0.001, compared to model group;^###P<0.001, single factor analysis of variance for significance test compared to blank control);

FIG. 4 is a comparison graph of IFN-gamma expression content in colon of mice in blank control group, model group, MIMP positive control group and MP-D experimental group,^*P<0.05,^**P<0.01，^***P<0.001, compared to model group;^#P<0.05, single-factor analysis of variance performed a significance test compared to the blank control group.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The reagents, materials and equipment used in the examples are shown in table 1:

TABLE 1

Name (R)	Manufacturer(s)
		Male C57BL6 mouse, clean grade	Shanghai Si Laike company (Shanghai, China)
Dextran Sulfate Sodium (DSS)	MP Biomedicals(CA,United States)
		Phosphoric acid buffer solution (PBS)	Shanghai Bo photo-biological technology Co., Ltd (Shanghai, China)
MIMP	Suzhou Qiangyao biological company (Suzhou, China)
		4% Paraformaldehyde	Shanghai Bo photo-biological technology Co., Ltd (Shanghai, China)
Tissue grinder	Shanghai Jing Shi Kogyo development Co., Ltd (Shanghai, China)
		Interferon-gamma enzyme-linked immunoreaction reagent kit	Shanghai Bo photo-biological technology Co., Ltd (Shanghai, China)

Example 1: experiment of intervention of MP-D on effect of DSS (direct sequence-derived) induced inflammatory bowel Disease (DSS) in mice

The polypeptide fragment D (MP-D) used in this example has a sequence in which Xaa at the 9 th amino acid position of the amino acid sequence shown in SEQ ID No.1 is Gln, Xaa at the 20 th amino acid position is Arg, and Xaa at the 25 th amino acid position is Asn, that is, THTVGSYFQVQNGYVGAFSRALGNNEYAMNS.

1. Experimental methods

1.1 establishment of mouse model of acute inflammatory bowel disease

When given to mice with a certain concentration of DSS solution, acute inflammatory bowel disease models characterized by diarrhea, fecal blood, ulcers, and granulocytic infiltration were induced. Experimental grouping the randomized grouping was done hierarchically according to the body weight of the mice, following the randomization principle. 40 healthy male C57BL6 mice were divided into four groups of 10 mice each:

blank control group: the stomach is irrigated with water every day, and the volume of the irrigated stomach is 0.4mL/20 g;

model group: performing intragastric administration with 2.5 wt% DSS aqueous solution, and continuously drinking for 7 days, wherein the DSS aqueous solution is prepared fresh and is replaced for 1 time every 1 day;

MIMP positive control group: the mice are firstly given a pre-administration process of one week, namely, the mice are gazed with a MIMP solution with the mass fraction of 50 mu g/kg in the first 7 days, and are gazed with a 2.5 wt% DSS aqueous solution (the gazed volume is 0.4mL/20g) every day and are gazed with a MIMP solution with the mass fraction of 50 mu g/kg (the gazed volume is 0.4mL/20g) starting from the day 8;

MP-D experimental group: firstly, the mice are subjected to a one-week pre-administration process, namely, the mice are subjected to intragastric administration by using an MP-D solution with the mass fraction of 50 mu g/kg for the first 7 days, and are subjected to intragastric administration by using a 2.5 wt% DSS aqueous solution (the intragastric administration volume is 0.4mL/20g) and an MP-D solution with the mass fraction of 50 mu g/kg (the intragastric administration volume is 0.4mL/20g) every day from the 8 th day;

the change in body weight of each group of mice was recorded daily to determine whether the mouse model of acute inflammatory bowel disease was successfully established.

1.2 sample Collection

Mice were sacrificed by dislocation of cervical vertebrae, placed on an operating table, the abdominal cavity was exposed, and intestinal conditions were observed, with or without hyperemia, ulceration, and adhesion. Simultaneously, the colon of the mouse is completely taken out from the anus end to the ileocecal end, after the length of the colon is measured, the intestinal tract is cut along the longitudinal axis, the excrement of the intestinal tract is washed, and the intestinal tract is preserved in 4 percent of methanol polymer or frozen at the temperature of minus 80 ℃.

1.3 histopathological evaluation

Histopathological sectioning of the colon samples stored in 4% paraformaldehyde in step 1.2, hematoxylin-eosin staining, dehydration, sealing of sections and examination under an optical microscope, histopathological scoring by two blind examiners:

evaluation criteria: 0 point, no obvious inflammation; 1 minute, moderate inflammation infiltration of the basal layer; 2 points, moderate hyperplasia or severe inflammatory infiltration of mucosa; 3 points, mucosa is heavily hyperplastic, goblet cells are absent; in score 4, crypts were absent or ulcerated.

1.4 enzyme-linked immunosorbent assay (ELISA) assay

And (3) selecting the colon sample which is frozen and stored at the temperature of-80 ℃ in the step 1.2, adding PBS and magnetic beads into an EP tube, placing the colon sample into a tissue grinder for ultrasonic homogenization, and centrifuging to obtain a supernatant. The expression level of the proinflammatory cytokine IFN-gamma (i.e. interferon-gamma) in colon samples was determined using a commercial ELISA kit, appropriate primary and secondary antibodies were used according to the instructions, OPD developing solution was developed, and after termination of the reaction, the microplate reader was read at 490nm wavelength, with triplicate wells per sample.

1.5 statistical analysis

Experimental data in the above experimental methodsIt is shown that the statistical tests were performed using GraphPad Prism (ver.8.0, GraphPad Software inc., San Diego, CA, USA), SPSS Program (ver.25.0, SPSS inc., Chicago, IL, USA), and the significance tests were performed using one-way anova or two independent sample t-tests when normality and homogeneity of variance were met. Let alpha equal to 0.05, P<A difference of 0.05 was statistically significant.

2. Analysis of Experimental results

2.1 intervention of MP-D significantly ameliorates pathological colon shortening in mice with inflammatory bowel disease

Fig. 1 is a graph showing the weight change trend of the model group mice and the blank control group mice, and it can be seen that the body weight of the model group mice significantly decreased one week after the induction of DSS administration (compared to the blank control group,^###P<0.001 with significant difference), indicating that the mouse model of acute inflammatory bowel disease is successfully established. FIG. 2 is a graph showing the comparison of the colon lengths of mice in the placebo group, the model group, the MIMP positive control group and the MP-D experimental group, and it can be seen that the colon lengths (5.3. + -. 0.6) of the mice in the model group are compared with the colon lengths (9.4. + -. 0) of the placebo group.6) Compared to a significant shortening (compared to the blank control,^###P<0.001), whereas the colon shortening of the MP-D experimental group (8.0 ± 0.9) mice was significantly different relative to the model group (compared to the model group,^***P<0.001, there is a significant difference), indicating that the intervention of MP-D can significantly reverse this shortening, comparable to the effect of MIMP positive control group (7.9 ± 0.5), improving the pathological colon morphology in mice with inflammatory bowel disease.

2.2 intervention of MP-D significantly reduced Colon histopathology scores in mice with inflammatory bowel disease

FIG. 3 is a histopathological micrograph of colon of mice in blank control group, model group, MIMP positive control group and MP-D experimental group (HE staining 20 Xmicroscopy; A. blank control group; B. model group; C. MIMP positive control group; D.MP-D experimental group); histopathological score comparison graph (A)^*P<0.05，^**P<0.01，^***P<0.001, compared to model group;^###P<0.001, single factor analysis of variance for significance test compared to blank control);

the colon histopathological score was: blank control group 0.0 + -0.0, model group 7.4 + -0.6, MIMP positive control group 2.4 + -0.8, and MP-D experiment group 1.3 + -0.6.

It can be seen that both MIMP and MP-D intervention in colon histopathology scores significantly reduced colon histology scores in mice with inflammatory bowel disease. The pathological condition is improved to a corresponding degree, the epithelial structure of a mucous membrane layer is relatively complete, the morphological structure of epithelial cells is normal, and no obvious inflammation occurs, which shows that the intervention of MP-D can improve the large-area ulcer of the mucous membrane layer of colon tissues caused by DSS induction, reduce the infiltration of lymphocytes and neutrophils to a certain degree, and further intervene the occurrence of intestinal inflammation.

2.3 intervention of MP-D significantly downregulated the expression of inflammatory bowel disease mouse Colon Interferon-Gamma (IFN-. gamma.)

ELISA method for detecting colon cytokine IFN-gamma expression, as shown in FIG. 4 for comparing colon IFN-gamma expression content of mice in blank control group, model group, MIMP positive control group and MP-D experimental group (for comparison of IFN-gamma expression content in colon of mice in blank control group, model group, MIMP positive control group and MP-D experimental group) ((for comparison of IFN-gamma expression content in colon of mice in colon^*P<0.05，^**P<0.01，^***P<0.001, compared to model group;^#P<0.05, significance test by one-way anova compared to blank control).

The IFN-gamma expression content is as follows: blank control group 1145.3 + -89.6, model group 1385.6 + -99.9, MIMP positive control group 1188.8 + -65.4, and MP-D experimental group 11048.6 + -139.9.

The result shows that the intervention of the MP-D can obviously inhibit the increase of the proinflammatory cytokine IFN-gamma of the mice with the inflammatory bowel diseases induced by the DSS, and the result is consistent with the result of the MIMP positive control group, which indicates that the MP-D has the effect of improving the intestinal inflammation of the mice with the inflammatory bowel diseases equivalent to the MIMP.

Example 2

The reagents, materials and equipment used in this example, as well as the experimental methods, were the same as in example 1, except that: the sequence of the polypeptide fragment D (MP-D) used in this example is a sequence in which Xaa at the 9 th amino acid position of the amino acid sequence shown in SEQ ID No.1 is Tyr, Xaa at the 20 th amino acid position is Glu, and Xaa at the 25 th amino acid position is Thr, that is, THTVGSYFYVQNGYVGAFSEALGNTEYAMNS.

Example 3

The reagents, materials and equipment used in this example, as well as the experimental methods, were the same as in example 1, except that: the sequence of the polypeptide fragment D (MP-D) used in this example is a sequence in which Xaa at position 9 is Gly, Xaa at position 20 is Ser, and Xaa at position 25 is Pro in the amino acid sequence shown in SEQ ID No.1, that is, THTVGSYFGVQNGYVGAFSSALGNPEYAMNS.

Example 4

The reagents, materials and equipment used in this example, as well as the experimental methods, were the same as in example 1, except that: the sequence of the polypeptide fragment D (MP-D) used in this example is the amino acid Xaa at position 9, the amino acid Xaa at position 20 and the amino acid Xaa at position 25 of the amino acid sequence shown in SEQ ID No.1, which are not present, are THTVGSYFVQNGYVGAFSALGNEYAMNS.

After testing the experimental methods of example 2-example 4 according to the experimental method of example 1, the analysis results are not much different from the results of example 1, which shows that the polypeptide fragment D of the present invention can significantly improve the colon pathological morphology of IBD mice, reduce the colon histopathological score and the colon interferon-gamma expression content of IBD mice.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shanghai Long Xin biomedical science and technology Limited

<120> polypeptide fragment D and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 3

<211> 31

<212> PRT

<213> Lactobacillus plantarum (Lactobacillus plantarum)

<400> 3

Thr His Thr Val Gly Ser Tyr Phe Xaa Val Gln Asn Gly Tyr Val Gly

1 5 10 15

Ala Phe Ser Xaa Ala Leu Gly Asn Xaa Glu Tyr Ala Met Asn Ser

20 25 30

<210> 2

<211> 61

<212> PRT

<213> Lactobacillus plantarum (Lactobacillus plantarum)

<400> 2

Thr His Thr Val Gly Ser Tyr Phe Ser Val Gln Asn Gly Tyr Val Gly

1 5 10 15

Ala Phe Ser Gln Ala Leu Gly Asn Ser Glu Tyr Ala Met Asn Ser Pro

20 25 30

Leu Gly Ser Leu Asp Gly Arg Thr Thr Met Tyr Asn Leu Leu Gly Val

35 40 45

Lys Tyr Leu Phe Ala Arg Glu Asp Gln Leu Lys Lys Gln

50 55 60