CN102229658B

CN102229658B - Anti-rheumatoid arthritis polypeptide and its application in pharmacy

Info

Publication number: CN102229658B
Application number: CN2011101591096A
Authority: CN
Inventors: 常晓天; 韩金祥
Original assignee: Shandong Provincial Pharmaceutical Biological Tech Research Center
Current assignee: Shandong Provincial Pharmaceutical Biological Tech Research Center
Priority date: 2009-10-23
Filing date: 2009-10-23
Publication date: 2012-05-23
Anticipated expiration: 2029-10-23
Also published as: CN102229658A

Abstract

The invention discloses an anti-rheumatoid arthritis polypeptide and its application in pharmacy. An amino acid sequence of the polypeptide is shown in SEQ ID NO.2. In the invention, the polypeptide is injected into rats and then an artificial arthritis induction process on the rats is carried out. A result of the induction process shows that corresponding antibodies are produced in the rats injected with the polypeptide; a concentration of a vascular endothelial growth factor is reduced; more than 90% of the rats injected with II type collagen have not rheumatoid arthritis symptoms. A histopathological analysis shows that the vascular proliferation of diseased joints of experimental animals is reduced obviously and a vascular endothelial growth factor (VEGF) level is reduced. The polypeptide can inhibit the capillary vessel proliferation of synovial membranes of joints suffering from rheumatoid arthritis through an immunization intervention way, thus block up a nutritional pathway of synovial cell proliferation and a penetration pathway of T cells and inhibit organizations and organization damages thereby inhibiting a pathology process of rheumatoid arthritis. Therefore, the polypeptide can be developed as an anti-rheumatoid arthritis drug.

Description

The polypeptide of resisting rheumatoid arthritis and the application in pharmacy thereof

The application is dividing an application of publication number CN101696234A application, the applying date of original application: 2009.10.23, and application number: 200910019389.3, denomination of invention: the polypeptide of resisting rheumatoid arthritis and the application in pharmacy thereof.

Technical field

The present invention relates to one type of polypeptide and pharmaceutical applications thereof, relate in particular to the polypeptide of one type of resisting rheumatoid arthritis and the application in pharmacy thereof.

Background technology

Trx family is positioned on the endocytoplasmic reticulum, has protein disulfide isomerase (protein disulfide isomerase, PDI) function.The PDI pair cell breaks up unusually, and blood capillary proliferation and anti-oxidative damage play an important role ^[1](thiredoxin domain containing 5 TXNDC5) is positioned on No. 6 karyomit(e)s of people (6p24.3) Trx 5, and the disulphide isomerase of its coding contains the PDI structure, high expression level under the anoxybiotic situation, catalytic proteins disulfide linkage isomate process ^[1]Have the human proteomics method to find TXNDC5 in hepatocellular carcinoma, expression of tumor tissue such as colorectal cancer play the effect of protection to endotheliocyte under the environment of tissue hypoxia ^[2]

(rheumatoid arthritis RA) is a kind of self-Immunological diseases to rheumatoid arthritis, and clinical manifestation is that chronic, many synovial joints are scorching.The RA pathology show as the serious inflammation of synovium of joint, and tissue thickens, and the T cell infiltrates in a large number in the synovial membrane, B cell and synovial membrane fibrous cell paraplasm, and capillary vessel generates in a large number, is referred to as pannus on the anatomy.The inventor utilizes proteomic techniques relatively RA, osteoarthritis (osteoarthritis; OA) and ankylosing spondylitis (ankylosing spondilitis; AS) protein expression profiles of pathology synovial tissue is found TXNDC5 significance high expression level in the synovial tissue of RA.Immunohistochemical methods, western blotting and quantifying PCR method transcribe with protein level on all confirmed the specificity overexpression of TXNDC5 in RA synovial tissue of joint cell.In addition, the inventor finds with enzyme immunoassay side (ELISA), compares the expression amount of TXNDC5 increase twice or more than the twice in 53.3%RA patient's blood and 73.3%RA patient's the knuckle synovia with OA, AS patient ^[3], show that TXNDC5 maybe be relevant with the RA pathogenic process.

Based on above-mentioned discovery, the inventor studies the mechanism of causing a disease of RA with regard to TXNDC5.Many researchs prove that RA pathology joint is a low-oxygen environment, and RA has low oxygen characteristic in essence ^[5], and show response to oxidative stress ^[5,6]The inventor finds, induces synovial membrane TXNDC5 high expression level under the RA hypoxia condition.The TXNDC5 of high density can raise RA synovial membrane VEGF vegf expression, and VEGF stimulates synovial tissue's blood capillary proliferation, forms pannus.Pannus is that the vital tissue of RA The lesions of synovium contributed is learned characteristic.Just because of the generation of pannus, the T cell is able to cause inflammation to a large amount of infiltration of synovial tissue, and simultaneously, pathological tissues obtains sufficient nutrient and supports B cell and fibrous cell to breed in a large number.Given this, TXNDC5 can become potential RA treatment target spot.Through suppressing the TXNDC5 enzymic activity, can suppress RA synovial tissue capillary vessel and generate, thereby the nutrition approach of blocking lesion synovial cell proliferation and T cell transfer approach reach the treatment RA purpose of killing and wounding RA synovial cell and inflammation-inhibiting process.

Reference

1.Sullivan DC，Huminiecki L，Moore JW，et al.EndoPDI，a novel protein-disulfide isomerase-Like protein that is preferentially expressed in endothelial cells acts as a stress survival factor.J Biol Chem，2003，278：47079-47088.

2.Wang Y，Ma Y，LüB，et al.Differential expression of mimecan and thioredoxin domain-containing protein 5in colorectal adenoma and cancer：a proteomic study.Exp Biol Med(Maywood)，2007，232：1152-9.

3.Chang X，Cui Y，Zong M，et al.Identification of proteins with increased expression in rheumatoid arthritis synovial tissues.J Rheumatol，2009，36：872-880.

4Distler JH，Wenger RH，Gassmann M，et al.Physiologic Responses to Hypoxia and Implications for Hypoxia-Inducible Factors in the Pathogenesis of Rheumatoid Arthritis.Arthritis Rheum，2004，50：10-23.

5.Tak PP，Zvaifler NJ，Green DR，et al.Rheumatoid arthritis and p53：how oxidative stress might alter the course of inflammatory diseases.Immunol Today，2000，21：78-82.

6.Hitchon CA，El-Gabalawy HS.Oxidation in rheumatoid arthritis.Arthritis Res Ther，2004，6：265-78.

Summary of the invention

Based on the keying action of above-mentioned TXNDC5 in the RA pathogenic process, the purpose of this invention is to provide the polypeptide of one type of resisting rheumatoid arthritis and the application in pharmacy thereof.

The polypeptide of resisting rheumatoid arthritis according to the invention is characterized in that: it is the aminoacid sequence shown in the SEQ ID NO.2; Concrete sequence description is:

Arg Asp Gly Lys Lys Val Asp Gln Tyr Lys Gly Lys Arg Asp

1 5 10 。

The compound method of the polypeptide of above-mentioned resisting rheumatoid arthritis is conventional artificial synthesis.According to immunosuppressant principle; The small peptide of the different sections of corresponding TXNDC5; The applicant has designed 9 sections TXNDC5 polypeptide (being the aminoacid sequence shown in SEQ ID NO.1～SEQ ID NO.9); By conventional artificial synthesis 9 peptide species have been implemented to synthesize, the polypeptide of the aminoacid sequence shown in the SEQ IDNO.2 promptly wherein.

The polypeptide of above-mentioned resisting rheumatoid arthritis generates the application in the medicine in the pannus that preparation suppresses the rheumatoid arthritis joint.Further, the application of the polypeptide of above-mentioned resisting rheumatoid arthritis in resisting rheumatoid arthritis medicines such as preparation inhibition rheumatoid arthritis synovium of joint blood capillary proliferation, the nutrition approach of blocking synovial cell's propagation and T cell infiltration approach.

The application of the polypeptide of above-mentioned resisting rheumatoid arthritis in preparation Trx 5 (TXNDC5) polyclonal antibody or monoclonal antibody.

It is to adopt the approach of immunotherapy that the present invention studies thinking; The artificial RA patient's immunity system that stimulates produces anti-TXNDC5 antibody; Utilize antigen, antibodies specific combination principle,, thereby suppress this enzymic activity with the TXNDC5 of anti-TXNDC5 antibodies high expression level; The blocking-up capillary vessel forms, and reaches the purpose of the treatment RA course of disease.At present, the effective biotechnological formulation of existing several RA is like benefit plug spectrum (Etanercept), type gram etc.; All be antibody with RA inflammation-related factor TNF (tumour necrosis factor); Through in patient's body, injecting anti-TNF antibodies,, suppress the biological activity of TNF in conjunction with the TNF of RA high expression level; The development of blocking-up RA inflammation obtains the RA result of treatment.This research by this RA treatment thinking, is model with the RA laboratory animal at first also, through the mode of immunologic intervention treatment, through suppressing the TXNDC5 biological function, reaches treatment RA purpose.

Basic implementation process is following: uses 9 kinds of small peptides (being the aminoacid sequence shown in SEQ ID NO.1～SEQ ID NO.9) of each section of TXNDC5 for preparing by routine according to the invention, be injected into respectively in the big white mouse body, make animal produce anti-TXNDC5 antibody.Then; By RA sacroiliitis method for building animal model commonly used, injection II Collagen Type VI induced animal performance RA clinical symptom (injecting collagen-induced animal general arthritis to rat is RA pathological study and the most frequently used animal model of drug screening) in animal body.Compare with control group, whether the laboratory animal that observation has been injected after the injection of TXNDC5 small peptide shows the RA arthritis.If the RA symptom does not appear in animal; Then anti-TXNDC5 antibody and TXNDC5 antigen-specific combine in the animal body after the explanation injection; Through the PDI catalysis that suppresses this enzyme stoped effectively TXNDC5 downstream pathology by way of; Block the hormesis that TXNDC5 generates capillary vessel, thereby reached the RA therapeutic purpose.

Experimental result shows: anti-TXNDC5 antibody horizontal obviously raises after having injected the TXNDC5 small peptide, shows that TXNDC5 antibody and its antigen specificity take place in the rat body combines, and can form immunosuppression to the TXNDC5 enzymic activity.The animal of before collagen-induced RA, having injected the TXNDC5 small peptide no longer shows RA clinical symptom and histologic characteristics, and the pathology process that can suppress RA to the immunosuppression of TXNDC5 effectively is described.After control group comprised that α hair Keratin sulfate small peptide or BSA substitute the TXNDC5 small peptide, the RA phenomenon was not suppressed, and confirmed that it is not that immunological tolerance does not cause that the TXNDC5 small peptide suppresses the result to RA.TXNDC5 small peptide injection back does not show RA with the rat of BSA immunity, this means that TXNDC5 small peptide and polypeptide itself do not influence RA.In experiment, the TXNDC5 small peptide is terminal to C-that should enzyme respectively, positions such as calcium ion land, PDI reactive site, and therefore, the antibody of its generation can combine suppress effectively the physiological function of TXNDC5 enzyme through immunity.

Meaning of the present invention and potential use: the biotechnological formulation of treating RA now mainly be benefit match general with Ying Fuli former times; Be the monoclonal antibody of TNF; The beginning of the nineties is used abroad, plays the use in China, and has received good result of treatment in 2005.Said preparation mainly suppresses the morbidity process through inflammation-inhibiting.Another biotechnological formulation of bringing into use at present is Rituximab (Rituximab), is the monoclonal antibody to the B cell, through killing the B cell of a large amount of propagation, suppresses the self-immunoreation of RA.More than these biotechnological formulations all to a large amount of outgrowth synovial cells of RA or inflammatory factor, none generates to the important pathological characters of another RA-pannus.And whether all the medicine of China treatment RA no matter biotechnological formulation inscience property right.Test-results of the present invention shows, with TXNDC5 short peptide sequence prepared polypeptide drugs or antibody drug, can directly suppress the pannus generation of RA through immunosuppression TXNDC5 activity.Through cutting off required nutrition channel and the T cell route of entry of a large amount of propagation of synovial cell, fundamentally reach the purpose of treatment RA.The essence of RA pathology joint tissue is hypoxemia, and low-oxygen environment is induced the pathology process.Suppressing the biological activity of the hypoxemia inductive TXNDC5 of institute, is the important channel of treating RA on the whole.

Description of drawings

Fig. 1 laboratory animal RA time of occurrence figure.

Inflammation time (inflammation index) figure appears in Fig. 2 laboratory animal.

Anti-TXNDC5 antibody horizontal figure in Fig. 3 blood.

VEGF level view in Fig. 4 blood.

The immunohistochemical section photo of the synovium of joint of Fig. 5 animal.

Embodiment

Embodiment 1

According to immunosuppressant principle, the small peptide of the different sections of corresponding TXNDC5, the applicant has designed 9 sections TXNDC5 polypeptide.

It is the aminoacid sequence shown in the SEQ ID NO.1; Concrete sequence description is:

Arg Gly Gly Lys Lys Val Ser Glu His Ser Gly Gly Arg Asp

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.2; Concrete sequence description is:

Arg Asp Gly Lys Lys Val Asp Gln Tyr Lys Gly Lys Arg Asp

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.3; Concrete sequence description is:

Arg Thr Glu Thr Gly Ala Thr Glu Thr Val Thr Pro Ser Glu

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.4; Concrete sequence description is:

Pro Val Thr Pro Glu Pro Glu Val Glu Pro Pro Ser Ala Pro

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.5; Concrete sequence description is:

Thr Trp Asn Asp Leu Gly Asp Lys Tyr Asn Ser Met Glu Asp

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.6; Concrete sequence description is:

Lys Pro Gly Gln Glu Ala Val Lys Tyr Gln Gly Pro Arg Asp

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.7; Concrete sequence description is:

Pro Pro Ala Ala Asp Gly Glu Asp Gly Gln Asp Pro His Ser

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.8; Concrete sequence description is:

Thr Leu Ala Pro Thr Trp Glu Glu Leu Ser Lys Lys Glu Phe

15 10; Or

It is the aminoacid sequence shown in the SEQ ID NO.9; Concrete sequence description is:

Ser Leu His Ser Phe Val Leu Arg Gln Ala Lys Asp Glu Leu

1 5 10 。

Above-mentioned 9 peptide species are implemented synthetic, subsequent use by conventional artificial synthesis.

Embodiment 2

The pharmaceutical use of the polypeptide of resisting rheumatoid arthritis according to the invention

Experimental technique

60 of male Lewis rats, mean body weight 120 grams, 8 ages in week.

9 kinds of small peptides (being the aminoacid sequence shown in SEQ ID NO.1～SEQ ID NO.9) by each section of TXNDC5 of conventional synthetic.

Inject the small peptide of aminoacid sequence shown in the SEQ ID NO.1～SEQ ID NO.9 of 100 micrograms respectively to every rat through the tail vein.Small peptide respectively with carrier proteins keyhole limpet hemocyanin (KLH) coupling.This aminoacid sequence is that TXNDC5 people is mated aminoacid sequence fully.The people is almost completely consistent with the TXNDC5 aminoacid sequence of mouse.The freund's adjuvant of above small peptide and equal volume concentration (5ug/ml) thorough mixing, the 1st, 14 and 28 day to the laboratory animal immunization, make it to produce anti-TXNDC5 antibody by ordinary method.Then, after injection for the first time the 42nd, 56 and 70 day to these animals injection ox II Collagen Type VIs (Sigma company), set up RA animal model method induced animal by routine and showed RA.The limbs of daily inspection rat are rubescent, swelling and stiff degree.RA inflammation degree is by research standards of grading judge in used 12 minutes usually, and standards of grading are following: 0: normal, and no swelling/rubescent, 1: slightly rubescent, 2: redden and whole foot swelling; 3: strong inflammation and sufficient gross distortion.Every mouse four limbs RA degree largest cumulative 12 minutes.Simultaneously, the 0th, 35, gathered the animal blood sample, and measured anti-TXNDC5 antibody and VEGF VEGF level in the blood in 77 and 102 days with conventional ELISA method.The capillary vessel development degree is organized in the VEGF representative.The VEGF level is accomplished with rat VEGF ELISA test kit (Invitrogen company).The 102nd day execution of rat after injection for the first time.Dissecting the synovial tissue of coming out organizes lesion degree and TXNDC5 to express situation with the inspection of routine immunization organization method.

Experiment is provided with a series of control groups simultaneously, and every group of rat is 60, and concrete group is following: (1), and α hair Keratin sulfate small peptide (AAVGSRPIHCGVRFC peptide) substitutes the TXNDC5 small peptide; (2) the replacement II collagen type of bovine serum albumin (BSA, Sigma company) is induced; (3) BSA replaces TXNDC5 small peptide and II Collagen Type VI to induce; (4) it is collagen-induced only to inject II; (5) only use the BSA immune animal, replace the injection of small peptide and collagen immunization; (6) do not inject any small peptide and protein.

Experimental result

The experimental group rat is used collagen-induced RA by routine behind immune TXNDC5 small peptide.Experimental result is that example is set forth as follows with SEQ ID NO.9 small peptide: with after injecting collagen protein for the first time the 50th day (after promptly injecting for the first time the 92nd day), in 60 rats (97%) 58 do not show any inflammation and extremities joint stiff.Have only two after for the first time injecting collagen the 56th day limbs slight inflammation (inflammation scoring=3) appears.Substitute with α hair Keratin sulfate small peptide in the rat of TXNDC5 small peptide injection, 55 rats (92%) obvious swelling occurs in the 34.5th ± 3.6 joint, collagen-induced back.Substituting with BSA in the rat of TXNDC5 small peptide, 50 (83%) rats were at the 39.7th ± 4.3 day that injects for the first time collagen protein, and obvious inflammation appears in the joint.All 60 (100%) substitute the clinical manifestation that the rat of TXNDC5 small peptide, polypeptide and collagen does not all show RA with BSA.56 (93%, wherein 4 dead at experimental session) rats showed arthritis on the 37.3rd ± 2.1 day behind injection collagen separately.Injection TXNDC5 small peptide, 60 rats (100%) of injecting the alternative II Collagen Type VI of BSA then all do not show the RA symptom.Have no the rat of injection also all not show any joint inflammation symptom.

Laboratory animal RA time of occurrence and inflammation degree are seen Fig. 1, Fig. 2 and table 1.

Table 1:RA situation occurs and TXNDC5 expresses situation

Processing mode (respectively organize rat and be 60)	The RA number of elements appears	The RA time of occurrence	Inflammation index	Anti-TXNDC5 level in the blood	TXNDC5 expresses in synovial membrane
						TXNDC5 small peptide+collagen	0	Do not occur	Do not occur	High	High expression level
α hair Keratin sulfate small peptide+collagen	50	37.2 ± 3.6 days	High	Low	High expression level
						TXNDC5 small peptide+BSA	0	Do not occur	Do not occur	High	The low expression
BSA+ collagen	55	39.7 ± 3.3 days	High	Low	High expression level
						Collagen	50	39.3 ± 3.1 days	High	Low	High expression level
BSA+BSA	0	Do not occur	Do not occur	Low	The low expression
						There is not any processing	0	Do not occur	Do not occur	Low	The low expression

Detect anti-TXNDC5 level in the rat blood with the ELISA method in the experiment.

The result shows, rat 35 and 77 days after the TXNDC5 small peptide injection first time, and the high-caliber anti-TXNDC5 antibody of appearance dropped to normal level (Fig. 3) in the blood after the 102nd day.ELISA detects animal blood and also shows, the 102 day obviously risings of VEGF level after injection for the first time.But by contrast, the VEGF level obviously is reduced in the rat blood centering of having injected the TXNDC5 oligopeptides and but obviously reduces, even than the rat low (Fig. 4) that does not have treatment, explains that capillary vascular development degree is also low relatively in this group rat synovial tissue.

With immunohistochemical method variation of laboratory animal synovium of joint pathologic structure and TXNDC5 expression are studied in the experiment.In the rat synovial tissue behind the collagen injection; The obvious thickening of synovial membrane also contains a large amount of T cells; B cell and fibrous cell; Observe a large amount of capillary vesseies simultaneously and generate, this situation has been present in all injections in the rat of collagen, no matter whether they have also been injected other small peptide and or BSA simultaneously.But before collagen-induced, injected in the rat of TXNDC5 small peptide according to the invention at those, their synovial membrane is thinner, and is similar with non-RA inductive control group, and simultaneously, the synovial membrane capillary vessel also is starkly lower than collagen RA inductive rat.Immunohistochemistry detects the expression of TXNDC5 in the synovium of joint of all animals, this enzyme is many expresses at the cells of superficial layer that is positioned at synovial membrane.Yet in the rat synovium of joint of injection TXNDC5 small peptide according to the invention, the intensity of TXNDC5 immunity signal is starkly lower than the level (Fig. 5) that collagen RA induces control group.

Small peptide with the aminoacid sequence shown in other SEQ ID NO.1 according to the invention～SEQ ID NO.8 all obtains and the similar result of SEQ ID NO.9 small peptide.

Laboratory animal RA time of occurrence is seen table 2.

Situation appears in table 2:RA

Processing mode	The RA number of elements appears	The RA time of occurrence
			SEQ ID NO.1 small peptide	1	46 days
SEQ ID NO.2 small peptide	2	47.2 ± 2.0 days
			SEQ ID NO.3 small peptide	4	43.7 ± 3.1 days
SEQ ID NO.4 small peptide	2	47.7 ± 2.3 days
			SEQ ID NO.5 small peptide	3	39.3 ± 1.1 days
SEQ ID NO.6 small peptide	5	40.7 ± 4.5 days
			SEQ ID NO.7 small peptide	3	47.2 ± 2.7 days
SEQ ID NO.8 small peptide	0	52 days
			SEQ ID NO.9 small peptide	0	56 days

Claims

1. the polypeptide of a resisting rheumatoid arthritis, it is characterized in that: it is the aminoacid sequence shown in the SEQ ID NO.2; Concrete sequence description is:

Arg Asp Gly Lys Lys Val Asp Gln Tyr Lys Gly Lys Arg Asp

1 5 10 。

2. the polypeptide of the described resisting rheumatoid arthritis of claim 1 generates the application in medicine or Trx 5 antibody in the pannus that preparation suppresses the rheumatoid arthritis joint.