CN109251237A - It is a kind of inhibit mouse arthritis disease polypeptide and its application - Google Patents

Abstract

The invention discloses a kind of polypeptide for inhibiting mouse arthritis disease and its applications.Micromolecule polypeptide SJMHE1 in schistosoma japonice ovum and Adult Antigens is made of 24 amino acid, is people and the common t cell epitope of mouse HSP60 molecule, can be prevented and treated the arthritis of mouse collagen induction.Beneficial benefit of the invention is to can be used for treating rheumatoid arthritis, has potential new drug development value.

Description

It is a kind of inhibit mouse arthritis disease polypeptide and its application

Technical field

The invention belongs to drug fields, and in particular to it is a kind of inhibit mouse arthritis disease polypeptide and its application.

Background technique

Rheumatoid arthritis (rheumatoid arthritis, RA) influences the population in the whole world about 1%, China's disease incidence The disability rate of about 0.3-0.5%, RA are very high, and RA patient's 2-3 disability rate of untreated is up to 70%, thereby result in forfeiture The crowd of labour capacity is up to over thousands of ten thousand, has become a kind of disease for seriously threatening human health^[1].Currently, the clinic of RA is controlled It treats still based on nonsteroidal anti-inflammatory drug and slow effect antirheumatic, glucocorticoid and cell toxicant immunosuppressor are also more Common, these drugs have certain curative effect, but since toxicity is larger, patient is difficult to take for a long time.Therefore, it is badly in need of seeking new Specific effect target spot or new intervention means RA patient is treated, develop small toxicity and the drug that can effectively treat And its research of mechanism of action not only has important social benefit, but also will generate great economic benefit.

RA pathogenesis is explored, and researches and develops the hot spot that related new drug is medical field research according to this.It is now recognized that in RA disease In sick development process, there are the unbalance of peripheral immune tolerance exception, lymphocyte abnormality proliferation and T cell homeostasis.And Regulatory T cells (Tregs) are to maintain body self tolerance and immunologic balance institute required, largely studies have shown that targeting The immunological homeostasis of Serum of Patients With Autoimmune Diseases is rebuild in the treatment of Tregs, will be a kind of comparatively safe and permanently effective therapeutic strategy With method.In the joint of idiopathic arthritis patient, while the quantity of Th17 cell significantly increases, the quantity of Tregs is aobvious Writing reduces, and Tregs is in opposite relationship in patient's inflamed joint with the quantity of Th17 cell, and Tregs/Th17 cell is prompted to lose Weighing apparatus participates in the morbidity of autoimmune arthritis, therefore, activates Tregs, rebuilds the immune of the autoimmune disease patients such as RA Balance, is one of the hot issue studied at present.

Currently, using the immune homeostasis of Tregs reconstruction RA patient increasingly by the concern of domestic and foreign scholars, for treating Cytokine antagonist and costimulatory molecules such as anti-TNF- α, anti-IL-6, CTLA4-Ig of RA etc., response to treatment Performance is closely related with the induction of Tregs, but these drugs, while inhibiting the reaction of autoimmunity pathology, also killing is normally exempted from Epidemic disease cell, long-term administration will lead to the fatal adverse reaction such as severe infections.In recent years, using identification autoimmune response in cause The epitope peptide of characteristic of disease T cell, by inducing Tregs, secretion inhibitory cells factor IL-10, TGF-β etc. inhibit autoimmunity or The generation of anaphylactia, avoids killing of the cytokine antagonist to normal immunocyte, and few side effects become and exempt from now One completely new research field of epidemic disease treatment.For example, a segment polypeptide P277 of identification people HSP60 molecule, has been carried out 1 type of the mankind The III phase of diabetes is clinical, can significantly protect the function of beta Cell of islet, and without side reaction；Glatiramer (glatiramer), one A rondom polypeptide (GLAT) being made of glutamic acid (Glu), lysine (Lys), tyrosine (Tyr) and alanine (Ala), is phosphorus The small molecule mimetics of sour sphingol have been used for treatment multiple sclerosis patients, achieve good therapeutic effect.These are most New result of study prompt: these micromolecule polypeptides are developed, by causing in direct or indirect method suppression of autoimmune responses Characteristic of disease T cell will treat more economical, Small side effects than cytokine antagonist, and therapeutic domain is more extensive.

Polypeptide currently used for arthritis treatment is mainly vasoactive intestinal peptide and from people HSP60, mycobacterium The epitope peptide of HSP65, people's II Collagen Type VI.2009, the research discovery Escherichia coli HSP albumen and pathogenic T of Koffeman etc. The epitope peptide dnaJP1 of cell-isogenic has very strong anti-inflammatory effect, and has carried out the II of dnaJP1 treatment rheumatoid arthritis Clinical trial phase obtains preferable therapeutic effect.The same year, Root-Bernstein discovery are same with people's myelin basic protein Source, from the polypeptide of measles virus also there is very strong anti-inflammatory effect, single immunization can significantly protect rat adjuvant The arthritic generation of property.But the domestic report there is presently no about the blood fluke polypeptide for inhibiting mouse collagen induced arthritis Road.

Summary of the invention

The object of the present invention is to provide a kind of polypeptides for inhibiting mouse arthritis disease.

It is a further object of the present invention to provide the applications of the polypeptide.

A kind of micromolecule polypeptide SJMHE1 from Schistosoma japonicum, sequence is as shown in SEQ ID NO.1.

Micromolecule polypeptide of the present invention is treating or preventing the application in medicine for treating rheumatoid arthritis.

A kind of drug treated and/or prevent rheumatoid arthritis, includes the micromolecule polypeptide and auxiliary material.

The utility model has the advantages that

Present invention discover that the micromolecule polypeptide SJMHE1 from Schistosoma japonicum is immune can to substantially reduce CIA mouse arthritis Disease incidence, mitigate symptom, reduce clinical score.

Figure of description

Fig. 1 SJMHE1 processing reduces the generation of CIA mouse arthritis

Fig. 2 SJMHE1 processing reduces the arthroncus of CIA mouse

Fig. 3 SJMHE1 processing reduces the pathological change in CIA mouse joint

Fig. 4 SJMHE1 processing can reduce the generation of the anti-ox II Collagen Type VI IgG antibody of CIA mouse

Specific embodiment:

Embodiment 1: building Collagen-Induced Arthritis (collagen induced arthritis, CIA) mouse model:

1) 200 μ g ox II Collagen Type VIs (CII is purchased from BD Bioscience) and complete Freund's adjuvant (CFA) mixed in equal amounts, Emulsifier is made, in 0,21 day root of the tail portion subcutaneous inoculation DBA/1 mouse, is immunized 2 times altogether.CFA by incomplete Freund's adjuvant (IFA, Purchased from Sigma) in plus 4mg/ml Mycobacterium Tuberculosis H37RA (Difco) prepare.And through arthritis The clinical score and histological characterization of disease, the building success of verifying CIA mouse.

2) after the 21st day CII is immune, every other day, each claw situation of mouse is observed in two people's double blinds: 0=is without change；1 =slight swelling and/or erythema；The swelling of 2=moderate and erythema；The significant swelling of 3=and erythema；4 claw maximums of every mouse Clinical score be 12.In the 42nd day execution mouse, mouse fore paw and hind leg are taken, is fixed through 10% neutral formalin, carried out Histology signing.

Embodiment 2:SJMHE1 treats CIA mouse；

1) mouse is divided into 7 groups: CIA, PBS, 10 μ g SJMHE1,10 μ g OVA_323-339(control peptide group) andGroup (Normal group)；By PBS, SJMHE1, OVA_323-339With IFA mixed in equal amounts, emulsifier is made, the subcutaneous injection of tail spine is small Mouse, 100 μ l/ of injection dosage is only；It is treated within 7th day (d7) ox II Collagen Type VI (CII) is immune, is subcutaneously injected every 14d Treatment, co-injection 3 times；Clinical score, histological characterization of each treatment group mouse through arthritis, anti-CII in serum The therapeutic effect of SJMHE1 is verified in IgG, IgG1, IgG2a antibody test.

2) mice serum is collected, ELISA detects IgG, IgG1, IgG2a that CII is special in mice serum.That is: elisa plate 5 μ g/ml CII of middle coating, 4 DEG C of refrigerator overnights.Each reaction plate is closed with 0.3% BSA at 37 DEG C after PBS-T washes 3 times 1h；After closing plus 1:100 4 DEG C of refrigerator overnights of dilute serum, PBS-T wash 5 times after plus HRP combine rat anti-mouse IgG, 37 DEG C of incubation 1h of IgG1, IgG2a；After PBS-T washes 5 times, TMB colour developing, H₂SO₄After termination, the value of absorbance is surveyed at 450nm (OD value).

As a result as shown in Figure 1, DBA/1 mouse is after ox CII collagen immunization, general 24-28d of claw or so occurs red and swollen Symptom, 100% there is symptom in 50d or so.As time went on, there is the frequency and severity of red and swollen symptom in mouse claw It is more serious, it is in Time Dependent.But the immune disease incidence that can substantially reduce CIA mouse arthritis of SJMHE1, reduction clinical score (Fig. 1).

As shown in Fig. 2, OVA_323-339, PBS and CIA mouse claw go out to manage serious red and swollen inflammatory symptom, but SJMHE1 The red and swollen symptom for handling mouse joint is substantially reduced.

As shown in figure 3, OVA_323-339, PBS and CIA mouse claw pathology go out to manage infiltration and the cartilage of a large amount of inflammatory cells It destroys, but SJMHE1 handles mouse joint, the infiltration of inflammatory cell and cartilage destruction are most light.Each group mouse claw joint pathology Scoring is shown in Table inflammation, pannus, cartilage and the bone injury scoring that 1, SJMHE1 processing mouse substantially reduces claw joint.

1 collagen-induced arthritis mouse tissue pathological score of table

As shown in figure 4, in addition toMouse, SJMHE1, OVA_323-339, PBS processing mouse detected anti-CII's The generation of IgG, IgG1, IgG2a, but SJMHE1 processing significantly inhibits the generation of anti-CII IgG.

<110>Nanjing Medical University

<120>a kind of polypeptide for inhibiting mouse arthritis disease and its application

<160> 1

<210> 1

<211> 24

<212> DNA

<213>Schistosoma japonicum

<220>

<223>from the micromolecule polypeptide SJMHE1 of Schistosoma japonicum

<400> 1

Val Pro Gly Gly Gly Thr Ala Leu Leu Arg Cys Ile Pro Val Leu Asp

1 5 10 15

Thr Leu Ser Thr Lys Asn Glu Asp

20

Claims (3)

1. a kind of micromolecule polypeptide from Schistosoma japonicum, it is characterised in that sequence is as shown in SEQ ID NO.1.

2. the micromolecule polypeptide in Schistosoma japonicum source is treating or preventing the application in medicine for treating rheumatoid arthritis.

3. the drug of a kind for the treatment of and/or prevention rheumatoid arthritis, it is characterised in that include small point described in claim 1 Sub- polypeptide and auxiliary material.