CN102702326B - Epitope of systemic lupus erythematosus and application thereof - Google Patents

Abstract

The invention discloses an epitope of systemic lupus erythematosus and an application thereof, belonging to the technical field of immunology diagnosis. The epitope is a polypeptide of which the amino acid sequence is shown as a sequence table SEQ ID NO:1. As proved by ELISA (Enzyme-Linked Immunosorbent Assay) detection and the serum reaction condition of a patient, the epitope polypeptide can be specifically combined with IgG (Immunoglobulin G) in the serum of the patient without reacting with the serum of a healthy person. The epitope polypeptide can be used for preparing a medicament for diagnosing systemic lupus erythematosus.

Description

A kind of epitope of systemic lupus erythematous and application thereof

Technical field

The invention belongs to immunology diagnosis technical field, be specifically related to a kind of epitope and application thereof of systemic lupus erythematous.

Background technology

Systemic lupus erythematous (systemic lupus erythematosus, SLE) be a kind of autoimmune disorder that is formed as feature with autoantibody generation and immunocomplex, clinical manifestation is the symptom that constitutional symptom and the many organs of multisystem are got involved.The sickness rate of China SLE is 70.1/10 ten thousand, and has the trend rising year by year.The early diagnosis of SLE, early treatment are the keys of improving prognosis.But China's autoimmune disorder diagnostic level is lower, and the main dependence on import of diagnostic reagent, cause diagnosis somewhat expensive, increase country and patient's medical treatment cost, be also unfavorable for to basic hospital universal.Therefore, setting up detection method stable, quick, that be applicable to popularization, is current problem demanding prompt solution.

SLE clinical criteria comprises that autoantibody detects as antinuclear antibody (ANA), anti-dsDNA antibody (ds-DNA antibody) and anti-Sm antibody, but not high due to specificity and sensitivity is not also that desirable serology detects index.If can set up special " autoantibody repertoire " detection method of SLE, detect multiple specific antibody simultaneously, improve recall rate and accuracy, provide reliable foundation by the diagnosis for SLE, classification, observation of curative effect and prognosis judgement, there is important clinical meaning.Therefore, find the Serology test that a kind of susceptibility is strong, specificity is high and simple and easy to do, the early diagnosis to systemic lupus erythematous and treat significant.

The appearance of a large amount of autoantibodies is one of key characters of autoimmune disorder, but because its pathogenesis is still not clear, is difficult to directly utilize autoantigen to detect antibody.Epitope (epitope) is the basis of identification, binding antibody in antigen molecule, is therefore the actual active principle that detects antibody.There is following advantage using epitope peptide or its simulating peptide (mimotope) as diagnostic tool: 1) can improve sensitivity and the specificity of diagnosis, the non-specific binding (Deroo S et al (2001) Comb Chem High Throughput Screen 4:75 – 115) of having avoided biomacromolecule easily to produce; 2) scope of expansion medical diagnosis on disease, for studying autoantigen less or that be difficult to external application as antigen nucleic acid, polysaccharide antigen, lipid antigen, surrogate using simulating peptide as natural antigen is diagnosed, and also can obtain the result consistent with natural antigen epi-position (Bruce G (1997) Proc Natl Acad Sci USA 94:1955 – 1960; Jinaping Q et al (1999) Hyridoma 18 (1): 103 – 109); 3) cost of polypeptide preparation, application low, be easy to Quality Control.Therefore, the acquisition of auto-antigen epitope peptide or its simulating peptide (Meloen RH et al (2000) J Mol Recognit 13:352-359) is the basis of preparing the polypeptide detection reagent of autoantibody.Epitope based on phage peptide library is determined technology, only need to be taking patients serum's sample as target, and without natural autoantigen information, can realize quick and easy screening, and cost is low, credible result degree is high.Another outstanding feature of phage peptide library is, can obtain multiple epitope peptides of a certain disease specific by screening simultaneously, and they may represent the main immunodominant group of natural antigen.Natural biological macromole antigen is made up of multiple epitopes conventionally, wherein there are some in immune response, to play Main Function, being called as " immunodominant group ", is mainly multiple " monoclonal antibody " composition for these epi-positions for the antibody of complete antigen.Taking SLE patient serum sample as target, by phage display peptide library, obtain the specific epitope peptide of SLE, be the basis that obtains Novel diagnosis reagent.

Summary of the invention

The object of the present invention is to provide a kind of epitope of systemic lupus erythematous.

The present invention also aims to provide the nucleotide sequence of above-mentioned epitope.

The present invention also aims to provide a kind of test kit of detection system lupus erythematosus.

The purposes of the epitope that the present invention also aims to provide systemic lupus erythematous in the medicine of preparing diagnositc system lupus erythematosus.

Invent everybody and from 10 routine SLE patients serums, prepare and purifying obtains IgG, then with linear dodecapeptide storehouse and antibody incubation, obtained and the phage clone of antibody specific combination through screening.Determine positive phage clones by ELISA afterwards, measure positive phage clones displayed polypeptide sequence.One of them epitope peptide of chemosynthesis, called after SLEP, detects and patients serum's response situation by ELISA, shows that it can be combined by the special IgG in patients serum, and does not react with Healthy Human Serum.

An epitope for systemic lupus erythematous, this epitope is polypeptide, its aminoacid sequence is as shown in sequence table SEQ ID NO:1.

The nucleotide sequence of the epitope of coding said system lupus erythematosus.

A test kit for detection system lupus erythematosus, this test kit comprises: polypeptide described in claim 1, the antibody of polypeptide described in claim 1, solid enzyme target, bovine serum albumin, the goat anti-human igg of horseradish peroxidase-labeled, tetramethyl benzidine.

The purposes of the epitope of described systemic lupus erythematous in the medicine of preparing diagnositc system lupus erythematosus.

Beneficial effect of the present invention: the antigen epitope polypeptide of systemic lupus erythematous that the present invention obtains, detects and patients serum's response situation by ELISA, show that the special IgG in patients serum of its energy is combined, and do not react with Healthy Human Serum.

Brief description of the drawings

The ELISA result of the positive phage clone of Fig. 1.

Fig. 2 is the combination of polypeptide SLEP and SLE patient serum sample, Healthy Human Serum sample.

Fig. 3 is the ROC curve of SLEP antibody test.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention will be further described.

Following examples do not limit the present invention, and in embodiment, unspecified operation steps please refer to " molecular cloning experiment guide " third edition corresponding section (J. Pehanorm Brooker E.F. is Ritchie etc. not, Science Press) or consults the specification sheets of used kit.

Embodiment 1 preparation of SLE patients serum total IgG and purifying

Get appropriate Protein A resin suspension (GE company product), pack chromatography column into, with the PBS damping fluid washing balance chromatography column of 10 times of column volumes.By after Serum in Patients with SLE high speed centrifugation, add 1/10 volume 10 × PBS damping fluid to mix, adjust after pH and ionic concn, slowly add chromatography column.PBS with 10 times more than column volume washing, detects without albumen to effluent liquid.Add 6 times of column volumes, 0.1 M glycine (pH 3.0) wash-out, collect and pass liquid, with the 1M Tris-HCl(pH 9.0 of 1/10 volume) neutralization.To obtain antibody PBS is dialysed, and quantitatively after antibody concentration, add 50% glycerine, packing is also kept at-20 DEG C.

The specific antibodies peptide of embodiment 2 phage display peptide library screening SLE

Ph.D.-12 peptide storehouse test kit is purchased from NEB company, and storage capacity is 2.7 × 109, and titre is 1.5 × 1013/ μ l, and Escherichia coli ER2537 is the Host Strains in peptide storehouse, and screening process is referring to phage display peptide library working instructions.Often take turns screening coated (g), 1 × 1011 phage is dropped in every hole to every hole 10 μ with the total IgG in SLE patients serum.Phage enrichment degree calculates by " input/output ratio ".Through three-wheel screening, the order-checking of picking positive colony.The specific binding of positive phage clones and total IgG antibody is measured by ELISA.(every hole 10 μ g) are coated with 4 DEG C of 96 orifice plates (Nunc company) and spend the night total IgG antibody.Uncombined antibody that inclines, 3% 37 DEG C of BSA sealing 1 hour.Every hole is dropped into 37 DEG C, 1 × 109 phage and is hatched 2 hours.Washings (PBS-0.05% Tween 20) is washed plate 5 times, totally 3 minutes.37 DEG C of anti-M13 monoclonal antibodies of horseradish peroxidase (HRP) mark (1:1000 dilution, Amersham Biosciences company) are hatched 1 hour.Washing methods is the same, detects the anti-M13 monoclonal antibody of combination taking TMB (Sigma) as substrate, and 450 nm detect photoabsorption, the results are shown in Figure 1.As shown in Figure 1, these 12 clones are positive colony.Sequencing result shows, these sequences have good consistence, and the sequence frequency of occurrences is high, conservative property good wherein one.Contriver has synthesized this peptide section accordingly, and its concrete aminoacid sequence is: FKSGTGPQQYSY(SEQ ID NO:1), called after SLEP.

Embodiment 3 SLEP are combined detection with SLE patients serum IgG

Every hole 0.5 μ of synthetic peptide SLEP(g) is coated with 4 DEG C of 96 hole solid enzyme targets (Nunc company) and spends the night.Uncombined peptide that inclines, 37 DEG C of sealings of 3% bovine serum albumin (BSA) 1 hour.SLE patient or Healthy Human Serum sample 1:400 dilution, add in different holes, hatches 1 hour for 37 DEG C.Washings (PBS-0.05% Tween 20) is washed plate 5 times, totally 3 minutes.37 DEG C of the goat anti-human iggs of the HRP mark of 1:1000 dilution are hatched 1 hour.Washing methods is the same, detects the anti-M13 monoclonal antibody of combination taking tetramethyl benzidine (TMB, Sigma) as substrate, and 450 nm detect photoabsorption.

The results are shown in Figure 2, in patients serum, SLEP antibody horizontal, apparently higher than Healthy People, utilizes One-way ANOVA method to carry out statistical analysis, and result shows that the two has significant difference (P<0.001).

Sensitivity and the specific assay of embodiment 4 SLEP antibody tests

By the SLEP polypeptide of chemosynthesis and the coupling of SulfoLink coupling resin, pack chromatography column into, with the PBS damping fluid washing balance chromatography column of 10 times of column volumes.By after rheumatoid arthritis patients serum high speed centrifugation, add 1/10 volume 10 × PBS damping fluid to mix, adjust after pH and ionic concn, slowly add chromatography column.PBS with 10 times more than column volume washing, detects without albumen to effluent liquid.Add 6 times of column volumes, 0.1 M glycine (pH 3.0) wash-out, collect and pass liquid, with the 1M Tris-HCl(pH 9.0 of 1/10 volume) neutralization.To obtain antibody PBS is dialysed, and quantitatively after antibody concentration, add 50% glycerine, packing is also kept at-20 DEG C.Every hole 0.5 μ of synthetic peptide SLEP(g) is coated with 4 DEG C of 96 orifice plates (Nunc company) and spends the night.Uncombined peptide that inclines, 3% 37 DEG C of BSA sealing 1 hour.Using the SLEP antibody of affinity purification as standard substance, serial dilution concentration is respectively 0,10, and 25,50,100 μ g/ml add in different holes, hatch 1 hour for 37 DEG C.Washings (PBS-0.05% Tween 20) is washed plate 5 times, totally 3 minutes.37 DEG C of the goat anti-human iggs of the HRP mark of 1:1000 dilution are hatched 1 hour.Washing methods is the same, detects combination taking tetramethyl benzidine (TMB, Sigma) as substrate, and 450 nm detect photoabsorption, draw out typical curve., by RA patient or Healthy Human Serum sample 1:100 dilution, also add in coated hole, method is the same meanwhile.Utilize typical curve to calculate SLEP anti-body contg in different samples detected result, and utilize SPSS16 Software on Drawing ROC curve.

The detection that the results are shown in Figure 3, SLEP antibody has high specific (99.0%) and certain susceptibility (29.0%), and one that can be used as RA patient is detected index.

The test kit design of embodiment 5 systemic lupus erythematouses

The test kit of detection system lupus erythematosus, this test kit comprises: SLEP polypeptide 1.0 mg, SLEP antibody 1.0 mg, negative control sera 100 μ l, positive control serum 100 μ l, 96 hole solid enzyme target 10 plates, bovine serum albumin 10.0 mg, the goat anti-human igg 100 μ l of horseradish peroxidase-labeled, nitrite ion 100 ml(are containing tetramethyl benzidine), stop buffer 50ml.

The operation steps of application mentioned reagent box is as follows:

[1] (every hole 0.5 μ is coated 96 orifice plates (Nunc company) g), and 4 DEG C are spent the night for SLEP polypeptide;

[2] 3% 37 DEG C of BSA sealings 1 hour;

[3] SLEP antibody carries out serial dilution as standard substance, and concentration is respectively 0,10,25,50,100 μ g/ml; Negative control sera, positive control serum and test serum 1:100 dilution, add in different holes; Hatch 1 hour for 37 DEG C;

[4] washings (PBS-0.05% Tween 20) is washed plate 5 times, totally 3 minutes;

[5] goat anti-human igg 1:1000 dilution, hatches 1 hour for 37 DEG C;

[6] repeating step [4];

[7] every hole adds nitrite ion 100 μ l, room temperature lucifuge reaction 20 minutes;

[8] every hole adds stop buffer 50 μ l, and room temperature leaves standstill 5 minutes;

[9] enzyme connection instrument detects 450nm absorption value;

Drawing standard curve, calculates the SLEP anti-body contg of each testing sample, and result is judged to be the positive higher than three times of negative controls.

Claims (4)

1. an epitope for systemic lupus erythematous, is characterized in that, this epitope is polypeptide, and its aminoacid sequence is as shown in sequence table SEQ ID NO:1.

2. the nucleotide sequence of the epitope of systemic lupus erythematous described in coding claim 1.

3. a test kit for detection system lupus erythematosus, is characterized in that, this test kit comprises: polypeptide described in claim 1, the antibody of polypeptide described in claim 1, solid enzyme target, bovine serum albumin, the goat anti-human igg of horseradish peroxidase-labeled, tetramethyl benzidine.

4. the purposes of the epitope of systemic lupus erythematous in the medicine of preparing diagnositc system lupus erythematosus described in claim 1.