CN110090295B - Application of Ephrin-B1 in preparation of medicine for treating inflammatory bowel disease - Google Patents

Application of Ephrin-B1 in preparation of medicine for treating inflammatory bowel disease Download PDF

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CN110090295B
CN110090295B CN201810085585.XA CN201810085585A CN110090295B CN 110090295 B CN110090295 B CN 110090295B CN 201810085585 A CN201810085585 A CN 201810085585A CN 110090295 B CN110090295 B CN 110090295B
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inflammatory bowel
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CN110090295A (en
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王晨辉
张华芝
郭晓利
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Wuhan Kanglitong Technology Co ltd
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Abstract

The invention discloses an application of Ephrin-B1 in preparing a medicament for treating inflammatory bowel disease, belonging to the field of biological medicines. The invention constructs an Ephrin-B1 extracellular region coding sequence on an expression vector pINFUSE-hIgG2-Fc2, expresses a recombinant protein Ephrin-B1-Fc through mammalian cells, and intraperitoneally injects DSS-induced mice with colitis by using the Ephrin-B1-Fc to find that the Ephrin-B1-Fc can obviously reduce the DSS-induced enteritis of the mice, reduce the weight reduction of the mice and reduce the death rate of the mice, thereby indicating that the Ephrin-B1 can be used for preparing medicaments for treating inflammatory bowel diseases, and the Ephrin-B1 is human Ephrin-B1 (SEQ ID NO. 1) or mouse Ephrin-B1 (SEQ ID NO. 3). The invention provides a new direction for the treatment of inflammatory bowel diseases.

Description

Application of Ephrin-B1 in preparation of medicine for treating inflammatory bowel disease
Technical Field
The invention relates to the field of biological medicine, in particular to application of Ephrin-B1 in preparing a medicine for treating inflammatory bowel disease.
Background
Inflammatory Bowel Disease (IBD) is a chronic autoimmune disease that affects the entire colon, mainly including Ulcerative Colitis (UC) and Crohn's Disease (CD). In recent years, the incidence of inflammatory bowel disease in our country has increased year by year, and has become an important cause of common diseases of the digestive system and chronic diarrhea, and the patients are young and strong, so that the importance is increasing. The inflammatory bowel disease currently lacks specific and effective treatment methods, the disease condition is often not cured, and the chronic inflammatory bowel disease is greatly increasedIncrease the risk of the patient suffering from colorectal cancer 1 . Therefore, the research on molecular and cellular mechanisms of inflammatory bowel diseases is a hot spot in the research on autoimmune diseases. Dextran Sodium Sulfate (DSS) is widely used for building a model of inflammatory bowel disease in mice, has higher similarity with human inflammatory bowel disease, and is an ideal model for researching human inflammatory bowel disease 2 。
Eph (Erythropoietin-producing hepatocyte cell line) receptor is one of the largest members of tyrosine kinase receptor family, and the signal transduction between EPHB receptor and Ephrin-B ligand mediates numerous physiological and pathological functions and plays an important role in embryonic development, neuron migration, intestinal epithelial cell proliferation, intestinal epithelial cell positioning, angiogenesis and the like 3 . EPHB receptor signal pathway also plays an important role in inhibiting the occurrence and development of tumors, and is currently considered to be an important cancer suppressor gene 4 。
Reference documents:
1.de Souza HS1,Fiocchi.Immunopathogenesis of IBD:current state of the art.Nat Rev Gastroenterol Hepatol.2016Jan；13(1):13-27.
2.Whittem CG,Williams AD,Williams CS.Murine Colitis modeling using Dextran Sulfate Sodium(DSS).J Vis Exp.2010Jan 19；(35).
3.Boyd AW,Bartlett PF,Lackmann M.Therapeutic targeting of EPH receptors and their ligands.Nat Rev Drug Discov.2014Jan；13(1):39-62.
4.Pasquale EB.Eph receptors and ephrins in cancer:bidirectional signalling and beyond.Nat Rev Cancer.2010Mar；10(3):165-80.
disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a new application of Ephrin-B1 serving as a ligand of an EPHB receptor in preparing a medicament for treating inflammatory bowel diseases.
The invention constructs the coding sequence of the extracellular region of Ephrin-B1 to an expression vector pINFUSE-hIgG2-Fc2, expresses recombinant protein Ephrin-B1-Fc through mammalian cells, and uses Ephrin-B1-Fc to inject a DSS-induced colitis mouse in an abdominal cavity, so that Ephrin-B1-Fc can obviously reduce the enteritis of the DSS-induced mouse, reduce the weight reduction of the mouse and reduce the death rate of the mouse, which indicates that Ephrin-B1 can be used for treating inflammatory bowel disease.
The invention provides the following technical scheme:
use of Ephrin-B1 or a composition comprising Ephrin-B1 for the manufacture of a medicament for the treatment of inflammatory bowel disease. The Ephrin-B1 is human Ephrin-B1 or mouse Ephrin-B1. Wherein, the amino acid sequence of human Ephrin-B1 is shown as SEQ ID NO.1, and the coding nucleotide sequence is shown as SEQ ID NO. 2; the amino acid sequence of the mouse Ephrin-B1 is shown as SEQ ID NO.3, and the coding nucleotide sequence is shown as SEQ ID NO. 4.
Use of an Ephrin-B1 extracellular domain polypeptide or a composition comprising an Ephrin-B1 extracellular domain polypeptide in the manufacture of a medicament for the treatment of inflammatory bowel disease. The Ephrin-B1 extracellular region polypeptide is serine from 28 th site to 236 th site of human Ephrin-B1 and serine from 30 th site to 229 th site of mouse Ephrin-B1, and the coding nucleotides are nucleotides 82-708 of the sequence shown in SEQ ID NO.2 and nucleotides 88-687 of the sequence shown in SEQ ID NO. 4.
Use of a fusion polypeptide consisting of an extracellular domain of Ephrin-B1 and a heavy chain Fc domain of immunoglobulin IgG2, or a composition comprising said fusion polypeptide, for the preparation of a medicament for the treatment of inflammatory bowel disease.
The carrier of the skeleton of the carrier for expressing the fusion polypeptide is preferably pINFUSE-hIgG2-Fc2, and comprises the nucleotide for encoding the extracellular region of Ephrin-B1.
A host vector system for producing the above fusion polypeptide is a host cell comprising the above vector. The host cell is bacterial cell, yeast cell, insect cell or mammal cell, preferably 293F, COS7, CHO cell.
A method for producing the fusion polypeptide is to recover the fusion polypeptide produced by the above host vector system under conditions allowing the production of the fusion polypeptide, e.g., host cells containing the above vector are cultured, centrifuged and the supernatant is purified.
The inflammatory bowel disease includes Ulcerative Colitis (UC), crohn's Disease (CD); the route of administration of a drug for the treatment of inflammatory bowel disease is oral administration or intravenous administration.
The invention discovers that Ephrin-B1 can be used for treating inflammatory bowel diseases, and provides a new direction for treating the inflammatory bowel diseases.
Drawings
FIG. 1 is a photograph of running gel, coomassie brilliant blue staining after purification of FC protein and Ephrin-B1 protein.
FIG. 2 is a graph showing the change in body weight of DSS-induced mice injected with FC protein and Ephrin-B1 protein.
FIG. 3 is a graph showing H & E staining of colon tissue after injection of FC protein, ephrin-B1 protein into DSS-induced mice.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
(1) Laboratory mouse
C57BL/6 mice, purchased from Hubei province drug safety evaluation center, and in Hubei province drug safety evaluation center SPF animal laboratory feeding. And selecting male mice with the age of 8 weeks for animal experiments. Mice were randomly divided into 2 groups of 10 mice each, one group was injected with the control protein Fc, and the other group was injected with the recombinant protein Ephrin-B1-Fc.
(2) pINFUSE-hIgG2-Fc2-Ephrin-B1-Fc vector construction
Mouse Ephrin-B1 complete cDNA sequence (shown in SEQ ID NO. 4) is synthesized by a company, and is used as a template to construct a mouse Ephrin-B1 extracellular region expression vector (the amino acids of the extracellular region are from 30 th lysine to 229 th serine of mouse Ephrin-B1 protein) through PCR amplification, enzyme digestion and connection. The expression vector was pINFUSE-hIgG2-Fc2, purchased from Invivogen.
The upstream primer for constructing the vector is as follows: 5 'CATGCCATGGGCCACCTCTGCCAAGAACCTGGAGCC-3',
the downstream primer is: 5 'GGAAGATCTAGAGTCCCGCTCGCCACC-3'.
The PCR reaction system is as follows: 10 XPCR buffer 5. Mu.L, dNTPs 1. Mu.L, upstream/downstream primers 1. Mu.L each (10. Mu.M), template DNA 1ng, PFU DNA polymerase 1. Mu.L, add ddH 2 O to 50. Mu.L.
The PCR reaction procedure was as follows: 5 minutes at 95 deg.C, (1 minute at 95 deg.C, 30 seconds at 58 deg.C, 2 minutes at 72 deg.C) 30 cycles, 10 minutes at 72 deg.C, 4 deg.C.
And (4) running nucleic acid electrophoresis gel on the PCR reaction system after reaction, cutting the gel and recovering DNA fragments. The DNA fragment after gel cutting recovery and pINFUSE-hIgG2-Fc2 empty vector are cut for 2 hours at 37 ℃ (NCoI and BglII cutting), and the DNA fragment is recovered. The recovered DNA fragment and the vector were ligated overnight at 16 ℃. mu.L of the ligation product was used to transform DH 5. Alpha. Competent cells. And (3) putting the transformation plate into a 37 ℃ bacterial incubator overnight, taking a monoclonal colony the next day, carrying out colony PCR identification, extracting a plasmid from the correctly identified colony, and sending the colony to a company for sequencing. Sequencing the correct plasmid will be extracted from the plasmid and prepared to transfect cells. NCoI and BglII enzymes were purchased from NEB. DNA gel cutting recovery kit and DNA solution recovery kit were purchased from Omega, the goods numbers are D2500-01 and D2500-02, and the recovery methods refer to the product instructions. DNA T4 ligase was purchased from THERMO FISPEHER, cat # EL0014, and ligation methods were described in reference to the product instructions. DH 5. Alpha. Competent cells were purchased from Tiangen Biochemical technology Inc., cat # CB101.
(3) Preparation of recombinant protein Ephrin-B1-Fc
The constructed expression vectors pINFUSE-hIgG2-Fc2-Ephrin-B1 and pINFUSE-hIgG2-Fc2 empty vectors were transfected into 293F cells (1L cultured cells, 1X 10) 6 /mL), at 37 ℃ and 5% CO 2 Shaking at 130rpm for 6 days. The transfection reagent is PolyPlus transfection reagent from PolyPlus transfection company, the product number is FECTO PRO, and the specific loading method is shown in the product description. On the 6 th day after transfection, cell culture supernatant was collected, centrifuged to remove cells (4 ℃,1500rpm,5 minutes), and filtered through a 0.45 μm filter. The filtered supernatant was purified by protein G pre-packed column (pre-packed column from Wuhan Hui research, inc., cat. HZ 1012-1) according to the protocol described in the product. The purified eluted protein was ultrafiltered by an ultrafiltration strain of Milipore (Cat. UFC 501)096 Protein was fused in 1 XPBS after ultrafiltration, filtered through a 0.22 μm filter, and stored at-80 ℃.
(4) Establishment of DSS (direct sequence-derived signal) induced mouse colitis model
To generate a mouse model for acute colitis, DSS (36-50kD, DSS from MP Biomedicals) was added to mouse drinking water (3% concentration, mass percent) and the mice were exposed to DSS for 5 days.
(5) Treatment of DSS-induced enteritis by injection of recombinant protein Ephrin-B1-Fc
8 weeks old C57BL/6 mice were exposed to 3% DSS water for 5 days and then changed to plain water, and the two groups of mice were intraperitoneally injected with recombinant protein Ephrin-B1-Fc and control protein Fc (1 XPBS solution) 50. Mu.g/mouse, once every other day, 5 times in total, starting on day 0. The body weight of the mice was weighed every day, and a body weight change chart was drawn. Mice were sacrificed on day 10 and colon tissue was taken for H & E staining.
The results are shown in FIGS. 1, 2 and 3. FIG. 1 is a photograph of running gel, coomassie brilliant blue staining after purification of FC protein and Ephrin-B1 protein. FIG. 2 is a graph showing the change in body weight of mice, and it can be seen that the injection of the recombinant protein Ephrin-B1-Fc significantly reduced the DSS-induced weight loss of the mice, and the results of the two groups were very significantly different. The mice in the group injected with the recombinant protein Ephrin-B1-Fc begin to recover from later-period weight, and the state of the mice is obviously better than that of the mice in the group injected with the FC protein, which shows that the recombinant protein Ephrin-B1-Fc has obvious therapeutic action on the enteritis of the mice induced by DSS. FIG. 3 is a H & E staining graph of colon tissue, and it can be seen that recombinant protein Ephrin-B1-Fc significantly improved DSS-induced colonic inflammation. As can be seen from FIG. 3, the intestinal tract of the FC protein injection mice lost the crypt structure and had a large amount of inflammatory cell infiltration; the intestinal crypt structure of mice injected with the recombinant protein Ephrin-B1-Fc group is well preserved, and inflammatory cells infiltrate less. The results show that the injection of the recombinant protein Ephrin-B1-Fc has obvious treatment effect on the DSS-induced enteritis.
Sequence listing
<110> Wuhan Kanglishong science and technology Limited
Application of <120> Ephrin-B1 in preparation of medicine for treating inflammatory bowel disease
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Claims (3)

1. Use of a fusion polypeptide consisting of an extracellular domain of Ephrin-B1 and a heavy chain Fc domain of immunoglobulin IgG2, or a composition comprising said fusion polypeptide, for the preparation of a medicament for the treatment of inflammatory bowel disease.
2. Use according to claim 1, characterized in that: the extracellular region of Ephrin-B1 is leucine 28 to serine 236 of human Ephrin-B1 or lysine 30 to serine 229 of mouse Ephrin-B1; the amino acid sequence of human Ephrin-B1 is shown as SEQ ID NO.1, and the amino acid sequence of mouse Ephrin-B1 is shown as SEQ ID NO. 3.
3. Use according to claim 1 or 2, characterized in that: the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease.
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