CN106053831A - Kit for detecting autoimmune disease - Google Patents

Kit for detecting autoimmune disease Download PDF

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CN106053831A
CN106053831A CN201610393707.2A CN201610393707A CN106053831A CN 106053831 A CN106053831 A CN 106053831A CN 201610393707 A CN201610393707 A CN 201610393707A CN 106053831 A CN106053831 A CN 106053831A
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trem
autoimmune disease
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aptamer
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潘时辉
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
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    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

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Abstract

The invention relates to a kit for detecting autoimmune disease. SLE (Systemic Lupus Erythematosus) is a diffuse and systemic autoimmune disease, the main pathogenesis is the formation of autoantibodies and immune complexes, and multiple organs and systems are often involved in the development process of the disease. Detection methods in the prior art havecertain limitations and are not suitable for low-cost and large-area promotion. The kit can identify whether a patient suffers from SLE by detecting blood, thereby being capable of rapidly and efficiently detecting SLE. The kit is easy to prepare.

Description

A kind of test kit for autoimmune disease detection
Technical field
The present invention relates to biological technical field, particularly to a kind of test kit for autoimmune disease detection.
Background technology
Toll-like receptor (Toll-like receptors, TLR) is the classical mode identification receptor of inherent immunity cell, It identifies pathogen-associated molecular pattern, starts innate immune response, and determines follow-up adaptive immune response direction and reaction Intensity, thus by the strict regulation and control of body immune system.The immunoregulation of TLR signal includes negative regulation and is just regulating and controlling, and marrow Like cell expresses triggering receptor (Triggering receptor expressed on myeloid cells, TREM) family The member of the one newfound immunoglobulin superfamily of class, TLR signal immunoregulatory molecules, can be by amplifying or suppression TLR letter Number thus regulate and control immune response intensity, avoid the generation of excessive inflammatory response and autoimmune disease.TREM family at present Race contain TREM-1, TREM-2, TREM-3, TREM sample transcription factor 1 (TREM like transcript1, TLT-1) and Many Receptor member of TLT-2, wherein TREM-1 can strengthen inflammatory reaction, positive regulation autoimmune disease develops； In contrast, TREM-2 is main in autoimmune plays negative regulation effect, the differentiation and maturation of scalable various kinds of cell；And for TLT-1, the most more thinks that it is a Class Activation receptor, participates in platelet activation, the process of gathering；Similar, TLT-2 Leukocyte activation can be promoted, T cell is had stimulation.TREM-1 is the TREM family activated form receptor of report in 2000, and And along with the research to its mechanism of action, find that it plays important work in part autoimmune disease develops With, now a summary is made with regard to TREM-1 molecule and the progress in autoimmune disease thereof.
It has now been found that TREM gene mapping is in No. 6 chromosomes of people.Outside first Bouchon etc. find that TREM-1 is expressed in Neutrophilic granulocyte in week blood, the inherent immunity cell surface such as mononuclear cell, the transmembrane glycopeptide being made up of 234 aminoacid In vain, which includes extracellular region, cross-film district containing lysine residue and the shortage being made up of independent immunoglobulin V-structure territory The intracellular region in intrinsic signal territory.Increasing research display TREM-1 also can express at natural killer cell, dendron in recent years Shape cell, airway epithelial cell, hepatic endothelial cells etc..
TREM-1 is mainly with membrane surface molecule and shla molecule (Soluble TREM-1, sTREM-1) two kinds expression Form exists.After the TREM-1 molecule on film surface is combined with part according to its construction features, activate a series of signal path, adjust Control inflammatory reaction and induction immunne response.There are two kinds of hypothesis in source for sTREM-1 at present: one is that TREM-1 shears variation Lacking the secretion hypotype in cross-film district, two is the result under the TREM-1 Proteolytic enzyme mechanism of film surface, and tests proof on antibacterial Lipopolysaccharide (Lipopolysaccharides, LPS) stimulate under person monocytic cell and neutrophilic granulocyte cultivation in, metal egg White enzyme inhibitor can increase the stability of cell surface TREM-1, reduce the release [5] of sTREM-1.Although TREM-1's is natural Part determines the most completely, but it expresses at human blood platelets surface and Peritonitis, endotoxemia little to have research to speculate Surfaces of granulocytes in mouse model, but to obtain definite answer, still need to further study confirmation.
Prior art has had substantial amounts of evidence to show TREM-1 and systemic lupus erythematosus (sle) (Systemic lupus Erythematosus, SLE) there is great dependency.SLE is the autoimmune disease of a kind of diffusivity, general, Principal pathogenetic mechanism is the formation of autoantibody and immune complex, often involves multiple organ and system in disease progression. The cause of disease of SLE and pathogenesis are complicated, diverse clinical manifestations, and infection is one of reason of disease triggering, is also to fall ill simultaneously Journey increases the weight of the common factors of the state of an illness.There is research that SLE fever patient is divided into disease activity group and concurrent infection group, by detection In patients serum, sTREM-1 level finds that concurrent infection patient is apparently higher than disease activity patient.And up-to-date research report Road is pointed out, sTREM-1 is up-regulated in SLE patients serum.
Prior art detects the amount of TREM-1 albumen typically by being carried out by enzyme linked immunoassay after antibodies Detection by quantitative, but this detection method needs to provide corresponding antibody for oneself, owing to the preparation of antibody requires higher, and prepares multiple Miscellaneous, there is huge limitation in the detection.
SELEX technology is a kind of new combinatorial chemistry technique grown up early 1990s.Utilize this technology can The aptamer affine with target substance height to screen specificity from random single chain oligonucleotide library.Its basic ideas are One single stranded oligonucleotide storehouse of iii vitro chemical synthesis, mixes with target substance, forms target substance-nucleic acid complexes, and eluting is not associated with Nucleic acid, separate the nucleic acid molecules that is combined with target substance, and carry out PCR amplification with this nucleic acid molecules for template, enter back into lower whorl Screening.By the screening repeated and amplification, some are not combined or have with target substance low-affinity, middle affinity with target substance Nucleic acid molecules is washed away, and with leather G material have the nucleic acid molecules of strong affinity from the biggest with hangar point the most out, and pure Spend carrying out and increase with SELEX process, finally occupy the great majority (> about 90% in storehouse).From Tuerk and Ellington etc. First use this technology screening to specific adsorption phage T4DNA polymerase and the specific nucleic acid aptamers of organic dye molecule After, through the development of more than ten years, SELEX technology has become as a kind of important grinds the means of making internal disorder or usurp and instrument.Therefore, selex is used Technology, screening specifically combines the aptamers of TREM-1 and has the most important meaning.
Summary of the invention
Technical scheme is realized by following steps:
It is an object of the invention to provide the nucleic acid aptamer sequence of a kind of TREM-1.
In the present invention, the aptamer (sequence 1-24) of described TREM-1 can specific bond TREM-1.
The present invention further objective is that the purposes providing described nucleic acid aptamer sequence.According to this sequence in the present invention Row application, can be further used for preparing the test kit of specific binding TREM-1.
From the random oligo DNA library of external synthesis, (5'-CGTTAAGACGAGTTGGCCAG-N36- AATGACCAGTGAAGCTTGAC-3'), wherein N36 is 36 random oligonucleotides；Therefrom filter out and TREM-1 specific bond Aptamer；The sequence primer P1:CGTTAAGACGAGTTGGCCAG that will filter out；Primer P2: GTCAAGCTTCACTGGTCATT, carries out expanding and carry out TA and is cloned into pMD19-T carrier (winning photo bio company purchased from Shanghai), Convert DH5a antibacterial (purchased from Beijing Tian Gen biotech firm)；Choose white colony to carry out after PCR determines positive colony, extracting plasmid And sequencing reaction, upper sequencer.
The present invention uses in-vitro screening (SELEX) technology of aptamer, with TREM-1 for just sieve target sieving with The aptamer of TREM-1 specific bond, prepares the sequence with specific bond TREM-1, named aptamers in the present invention TREM-1-1～24.Sequence is as follows:
TREM-1-1:CGTTAAGACGAGTTGGCCAGCTATATCCTACCTTATACCTATATACTTTACTCCTAAATG ACCAGTGAAGCTTGAC；
TREM-1-2:CGTTAAGACGAGTTGGCCAGTAACCCCACAATTTAACAACTTCTTTTCTTATCAACAATG ACCAGTGAAGCTTGAC；
TREM-1-3:CGTTAAGACGAGTTGGCCAGCCTCTCACCTACCCCTATCTCTTACTACCCTCTCCTAATG ACCAGTGAAGCTTGAC；
TREM-1-4:CGTTAAGACGAGTTGGCCAGTCCATCTCCCCATCCCTAATATACTACTTTTACCACAATG ACCAGTGAAGCTTGAC；
TREM-1-5:CGTTAAGACGAGTTGGCCAGCATAAAAACTATTCATACTCCTACATACACCCCAACAATG ACCAGTGAAGCTTGAC；
TREM-1-6:CGTTAAGACGAGTTGGCCAGTAAATTTTTCCTTATACTCTTCCCATTCAACAATTTAATG ACCAGTGAAGCTTGAC；
TREM-1-7:CGTTAAGACGAGTTGGCCAGTCCACATCTATAAAATATTATCAATCTCTCTCTATAAATG ACCAGTGAAGCTTGAC；
TREM-1-8:CGTTAAGACGAGTTGGCCAGAAAACCACTTTATTACCCTTTCAATCACAACACCATAATG ACCAGTGAAGCTTGAC；
TREM-1-9:CGTTAAGACGAGTTGGCCAGTACTTCTAATCTTTCCCATACCTCAATTCCATCCATAATG ACCAGTGAAGCTTGAC；
TREM-1-10:CGTTAAGACGAGTTGGCCAGCCTCATCCATTCACTCTATTTTTATATCTTTCCTCCAAT GACCAGTGAAGCTTGAC；
TREM-1-11:CGTTAAGACGAGTTGGCCAGCATTACCACAATCTTCATTTTATACCATTATATAAAAAT GACCAGTGAAGCTTGAC；
TREM-1-12:CGTTAAGACGAGTTGGCCAGTTATATAAACCTTATACTCAACTTCTTCCCCATAAAAAT GACCAGTGAAGCTTGAC；
TREM-1-13:CGTTAAGACGAGTTGGCCAGTCACACTTTAACCTACATCCCCTCACATACCACTAAAAT GACCAGTGAAGCTTGAC；
TREM-1-14:CGTTAAGACGAGTTGGCCAGAATCCTCATTTTCTCAATTCCCAACTTCAACCCCACAAT GACCAGTGAAGCTTGAC；
TREM-1-15:CGTTAAGACGAGTTGGCCAGTCTTTTCCCTTTATCAACACATATCTTCTATCATTTAAT GACCAGTGAAGCTTGAC；
TREM-1-16:CGTTAAGACGAGTTGGCCAGTCCTCTCTATCAACTCTCAACACACTCTCCAAATCCAAT GACCAGTGAAGCTTGAC；
TREM-1-17:CGTTAAGACGAGTTGGCCAGCCTCAATTTCCCTAACAACTACAACCTCTATAATCTAAT GACCAGTGAAGCTTGAC；
TREM-1-1:CGTTAAGACGAGTTGGCCAGCATACTTAATATAAAATCCACTTTCCTCCTACATATAATG ACCAGTGAAGCTTGAC；
TREM-1-18:CGTTAAGACGAGTTGGCCAGCTCAAAACTCCATTCTCTACACACCTTTACATCCTCAAT GACCAGTGAAGCTTGAC；
TREM-1-19:CGTTAAGACGAGTTGGCCAGCTATTCTTTATTTTATTCATCCCTCTTACTCCAATCAAT GACCAGTGAAGCTTGAC；
TREM-1-20:CGTTAAGACGAGTTGGCCAGAATCTCTCTCCTCATATTCCACATCTCCATCATATAAAT GACCAGTGAAGCTTGAC；
TREM-1-21:CGTTAAGACGAGTTGGCCAGTACACATCTCTCATTTAATCAACACACCTTTTACCTAAT GACCAGTGAAGCTTGAC；
TREM-1-22:CGTTAAGACGAGTTGGCCAGACAATATACTATATACCCATAAACTTAAATCTCAACAAT GACCAGTGAAGCTTGAC；
TREM-1-23:CGTTAAGACGAGTTGGCCAGTATTAATCATCTCCATCTACTTAATTACCCCTCTCTAAT GACCAGTGAAGCTTGAC；
TREM-1-24:CGTTAAGACGAGTTGGCCAGCCAATCATCCCACATCAAAATACCAATAACTTATCAAAT GACCAGTGAAGCTTGAC；
The aptamer of the present invention may be used for building test kit, and this test kit may be used for specific separation and quantitatively inspection Survey TREM-1.
Beneficial effects of the present invention: obtain a kind of can the test kit of specific detection TREM-1, this test kit low cost Honest and clean, can use with spread.
Detailed description of the invention
Embodiment 1: the acquisition of albumen
Generation BWZ.36/hTREM-l:DAP12:NFAT-LacZ cell line (this of people's TREM-1:DAP12 stable cell lines Literary composition also known as " BWZ/hTREM-Ι reports cell ") derive from BW5147T cell (mice Ofes thymic lymphoma cells system, ATCC TIB-47, LGC Standards, Middelsex, UK), and report containing the LacZ regulated by 4 copy NFAT promoter elements Construct (sees Karttunen, J. and Shastri, N. (1991) Proc.Natl.Acad.Sci.USA 88,3972-3976 And Fiering, S, Northrop, J.P, Nolan, G.P, Matilla, P, Crabtree, G.R. and Herzenberg, L.A. (1990) Genes Dev.4,1823-1834).Use Superfect transfection reagent (catalog number (Cat.No.) 301305, Qiagen Nordic, Denmark), general use TREM-1 cDNA (Gene Bank Ref.ID:NM_018643.2, Sino Biological Inc., Beijing, China) as template and oligomerization 5' TAGTAGGGATCCGCTGGTGCACAGGAAGG and 5'TAGTAGGCGGCCGCTTCGTGGGCCTAG GGTAC is as primer gram Grand to pIREShyg carrier GenBank accession number U89672 (catalog number (Cat.No.) 6061-l, Clontech Laboratories, CA, USA) TREM/DAP12/pMX-IRES carrier (encoding 786bp TREM-1 from SmaI site to BamHI site) is transfected into PLAT-E package cell line (is provided by W.Yokoyama, Washington University；Or, catalog number (Cat.No.) RV-101, RV- 101, Cell Biolabs Inc, Bio-Mediator KY, Vantaa, Finland).Used as described below containing TREM/DAP12/ The PLAT-E supernatant of pMX-IRES virion infects BWZ.36 cell: by 2xl05Individual BWZ.36 cell is trained in 6 orifice plates Support, replace medium to I.5ml contain the supernatant of virion+8mg/ml polybrene (polybrene).After 6-8 hour, will 1.5ml normal incubation medium is added in plate, makes cell incubation 24 hours again.By the BWZ.36 cell line of stable expression TREM-I with anti- TREM-I monoclonal antibody (clone 21C7) dyeing, and separated by cell sorting.The cell of isolated is cultivated, Results obtain TREM-1 albumen, and rich protein makes protein concentration be 100mg/mL, 4 degree of storages.
Embodiment 2 aptamer screens
From the random oligo DNA library of external synthesis, (5'-CGTTAAGACGAGTTGGCCAG-N36- AATGACCAGTGAAGCTTGAC-3'), wherein N36 is 36 random oligonucleotides；
Primer P1:CGTTAAGACGAGTTGGCCAG；Primer P2:GTCAAGCTTCACTGGTCATT.
L () TREM-1 albumen 1mg adds in 96 hole elisa plates after being dissolved in 200 μ l PBS, 4 DEG C overnight.
(2) albumen is coated hole PBS and washs after 6 times, adds 200 μ l 3%BSA (purchased from the raw work in Shanghai), hatches 2 hours. Meanwhile, blank 96 hole elisa plates add 200 μ l 3%BSA 37 DEG C and hatch 2 hours.
(3) ssDNA pool (first run consumption is 800pmol) is dissolved in 200 μ l 1*SHCMK, 95 DEG C of degeneration 5min. It is immediately placed on 10min on ice so that it is be rapidly decreased to room temperature, it is to avoid ssDNA renaturation becomes double-strand.
(4) ssDNA is added in the blank ELISA hole that PBS washs 6 times, hatch 2 hours for 37 DEG C.
(5) supernatant proceeds to PBS and washs the albumen of 6 times and be coated in hole, 37 DEG C hatch 2 hours after PBS wash 6 times.
(6) the enzyme mark hole combining ssDNA is resuspended with 200 microliters Elution Buffer, 95 DEG C of heating 5min, takes supernatant, warp Cross ethanol precipitation DNA, be dissolved in Millipore water, as the template of next round screening.
(7) take 5 μ l PCR primer and be splined on 5wt% agarose (purchased from the raw work in Shanghai), observe pillar location the most correct. After the first run, the amount in ssDNA level storehouse of input gradually decreases, and 12 circulations are so repeated.
12nd amplification taking turns screening product and purification: take turns the ssDNA sequence that screening obtains by the 12nd, use primer P1, primer P2 expands.100 μ l PCR amplification after system add 600pl combine liquid BB (20mmol/L Hepes (pH7.35), 100mmol/L NaCl, 5.5mmol/L KCl, l.5mmol/L CaC12, lmmol/L MgC12, l%BSA), fully mix.Will Previous step gained solution adds in adsorption column EC (adsorption column is put in collecting pipe), and it is centrifugal that room temperature places lmin, 12000rpm 60s, outwells the waste liquid in collecting pipe.Adding 700pmol rinsing WB (in conjunction with liquid BB+0.05%Tween20), 12000rpm is centrifuged 30s, discards waste liquid.Adding 500pmol rinsing liquid WB, 12000rpm is centrifuged 30s, discards waste liquid.Adsorption column EC is put back to empty receipts In collector, 12000rpm is centrifuged 2min.Take out adsorption column EC, put in a clean centrifuge tube, at the pars intermedia of adsorbed film Position adds the elution buffer EB of 65 DEG C of water-bath preheatings of 20pmol, and room temperature places 2min, and 12, OOOrpm are centrifuged lmin.By obtain Solution rejoins in adsorption column, recentrifuge lmin.
The clone of PCR purified product: take 1 μ l amplified production, be connected under the effect of T4DNA ligase with carrier PGM-T, Be transformed into competent cell again, take proper volume be spread evenly across the LA containing IPTG, x-gal, antibiotic (Amp) put down Plate, is inverted culture dish, within 12-16 hour, carries out " indigo plant-white macula screening " in 37 DEG C of cultivations.Order-checking: screen flat board with inoculating loop picking On single white colony in the LB fluid medium containing Amp, 37 DEG C of overnight incubation.Take bacterium solution lml in centrifuge tube, after sealer Serve sea raw work order-checking.
Sequencing result finds wherein have 24 nucleic acid aptamer sequence and TREM-1 to have the strongest affinity, these 24 Sequence is respectively: shown in SEQ ID NO:1-24.
The TREM-1 of embodiment 3 specificity high-affinity combines the performance measurement of aptamer
Aptamer taking 1.5 μ g respectively, digests 1h with calf intestinal alkaline phosphatase (CIP) 37 DEG C, purification reclaims and removes phosphoric acid The RNA changed；By T4 polynucleotide kinase labelling [γ-32P] ATP in dephosphorylized adaptor molecules end.10nmol is put The aptamer of penetrating property labelling TREM-1 protein 37 DEG C with variable concentrations (1-200nM) respectively hatches 30min, each group reactant liquor warp Nitrocellulose filter filters, and washs filter membrane, is dried filter membrane, and liquid scintillation counter measures the exit dose of residual, same sample on filter membrane Parallel do twice mensuration.Calculate the dissociation constant of each aptamer and TREM-1 albumen.Result is as follows:
As can be seen from the above results, 24 aptamers of the present invention have the strongest binding characteristic, in prior art Also the aptamer not having described binding characteristic can be in conjunction with TREM-1 albumen.
Embodiment 4 degrading activity analysis
Be respectively adopted human albumin, TREM-1, TREM-2, TREM-3, TLT-1 and TLT-2 respectively with 24 aptamers Carry out specific detection, through binding tests find, these aptamers do not combine with these albumen, and only with TREM-1 egg White combination keeps higher specificity.
By described aptamer, take 0.2ug, be respectively placed in the serum of room temperature, aqueous solution, place surrounding.Pass through RT- PCR detects, and finds its Stability Analysis of Structures of placement of three weeks, is not degraded.
Embodiment 5 clinical trial analysis
Taking 50 groups of clinical serum samples, be respectively as follows: SLE fever diseases activity group 20 example, SLE generates heat concurrent infection group 20 Example, 10 examples are normal person.
24 aptamers are built standard product examine with standard substance TREM-1 albumen respectively by the way of immuno magnetic cell separation The standard curve surveyed.Then go 10 μ l to join in sterile centrifugation tube respectively in 50 groups of clinical serum samples, add PBS solution shake Swing dissolving.Coupling have 24 groups of aptamers of magnetic bead and the serum solution mixing of 50 groups hatch 20 minutes (a kind of aptamers respectively 50 groups of serum samples of corresponding detection), then Magneto separate, the TREM-1 albumen of corresponding separation can be obtained, pass through standard curve Concentration Testing obtains corresponding protein concentration result, and by detection, for same serum, the concentration that each aptamer detects is several There is no gap；And the concentration between each group exists obvious difference, using the meansigma methods of the protein concentration of normal person as comparison, Result is as follows:
TREM-1 albumen relative concentration (corresponding meansigma methods)
SLE fever diseases activity group 1.7
SLE generates heat concurrent infection group 3.2
Normal person 1
As can be seen from the above results, the TREM-1 protein concentration of SLE heating concurrent infection group significantly increases, and normally Can significantly make a distinction between people.Illustrate that the aptamer of the present invention may be used for detection and analyzes.
Embodiment 6TREM-1 molecule barrier detects
According to the method for prior art, block TREM-1 when identifying aptamer: include that (a) cultivates and express TREM-1, letter Number transducin and the first cell of report construct；B () is by the first cell, (such as TREM-1 joins with activity compound Body) or activate neutrophil cell incubation together time measure the activity of described cell；C () makes the first cell and activity chemical combination The coculture of the neutrophil cell of thing (such as TREM-1 part) or activation contacts with TREM-I aptamer；(d) record The activity of the first cell is less than the activity measured in (b).According to described method, applicant is found by detection, adaptive in 24 Son all can block the activity of TREM-1.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify, All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Sequence table
Brightness during ＜ 110 Pan ＞
＜ 120 ＞ mono-kind is for the test kit of autoimmune disease detection
〈210〉1
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-1
CGTTAAGACGAGTTGGCCAGCTATATCCTACCTTATACCTATATACTTTACTCCTAAATGACCAGTGAAGCTT GAC；
〈210〉2
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-2
CGTTAAGACGAGTTGGCCAGTAACCCCACAATTTAACAACTTCTTTTCTTATCAACAATGACCAGTGAAGCTT GAC；
〈210〉3
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-3
CGTTAAGACGAGTTGGCCAGCCTCTCACCTACCCCTATCTCTTACTACCCTCTCCTAATGACCAGTGAAGCTT GAC；
〈210〉4
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-4
CGTTAAGACGAGTTGGCCAGTCCATCTCCCCATCCCTAATATACTACTTTTACCACAATGACCAGTGAAGCTT GAC；
〈210〉5
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-5
CGTTAAGACGAGTTGGCCAGCATAAAAACTATTCATACTCCTACATACACCCCAACAATGACCAGTGAAGCTT GAC；
〈210〉6
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-6
CGTTAAGACGAGTTGGCCAGTAAATTTTTCCTTATACTCTTCCCATTCAACAATTTAATGACCAGTGAAGCTT GAC；
〈210〉7
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-7
CGTTAAGACGAGTTGGCCAGTCCACATCTATAAAATATTATCAATCTCTCTCTATAAATGACCAGTGAAGCTT GAC；
〈210〉8
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-8
CGTTAAGACGAGTTGGCCAGAAAACCACTTTATTACCCTTTCAATCACAACACCATAATGACCAGTGAAGCTT GAC；
〈210〉9
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-9
CGTTAAGACGAGTTGGCCAGTACTTCTAATCTTTCCCATACCTCAATTCCATCCATAATGACCAGTGAAGCTT GAC；
〈210〉10
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-10
CGTTAAGACGAGTTGGCCAGCCTCATCCATTCACTCTATTTTTATATCTTTCCTCCAATGACCAGTGAAGCTT GAC；
〈210〉11
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-11
CGTTAAGACGAGTTGGCCAGCATTACCACAATCTTCATTTTATACCATTATATAAAAATGACCAGTGAAGCTT GAC；
〈210〉12
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-12
CGTTAAGACGAGTTGGCCAGTTATATAAACCTTATACTCAACTTCTTCCCCATAAAAATGACCAGTGAAGCTT GAC；
〈210〉13
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-13
CGTTAAGACGAGTTGGCCAGTCACACTTTAACCTACATCCCCTCACATACCACTAAAATGACCAGTGAAGCTT GAC；
〈210〉14
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-14
CGTTAAGACGAGTTGGCCAGAATCCTCATTTTCTCAATTCCCAACTTCAACCCCACAATGACCAGTGAAGCTT GAC；
〈210〉15
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-15
CGTTAAGACGAGTTGGCCAGTCTTTTCCCTTTATCAACACATATCTTCTATCATTTAATGACCAGTGAAGCTT GAC；
〈210〉16
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-16
CGTTAAGACGAGTTGGCCAGTCCTCTCTATCAACTCTCAACACACTCTCCAAATCCAATGACCAGTGAAGCTT GAC；
〈210〉17
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-17
CGTTAAGACGAGTTGGCCAGCCTCAATTTCCCTAACAACTACAACCTCTATAATCTAATGACCAGTGAAGCTT GAC；
〈210〉18
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-18
CGTTAAGACGAGTTGGCCAGCTCAAAACTCCATTCTCTACACACCTTTACATCCTCAATGACCAGTGAAGCTT GAC；
〈210〉19
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-19
CGTTAAGACGAGTTGGCCAGCTATTCTTTATTTTATTCATCCCTCTTACTCCAATCAATGACCAGTGAAGCTT GAC；
〈210〉20
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-20
CGTTAAGACGAGTTGGCCAGAATCTCTCTCCTCATATTCCACATCTCCATCATATAAATGACCAGTGAAGCTT GAC；
〈210〉21
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-21
CGTTAAGACGAGTTGGCCAGTACACATCTCTCATTTAATCAACACACCTTTTACCTAATGACCAGTGAAGCTT GAC；
〈210〉22
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-22
CGTTAAGACGAGTTGGCCAGACAATATACTATATACCCATAAACTTAAATCTCAACAATGACCAGTGAAGCTT GAC；
〈210〉23
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-23
CGTTAAGACGAGTTGGCCAGTATTAATCATCTCCATCTACTTAATTACCCCTCTCTAATGACCAGTGAAGCTT GAC；
〈210〉24
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉TREM-1-24
CGTTAAGACGAGTTGGCCAGCCAATCATCCCACATCAAAATACCAATAACTTATCAAATGACCAGTGAAGCTT GAC；

Claims (4)

1. the test kit being used for detecting autoimmune disease, it is characterised in that: it comprises can specific binding TREM-1 albumen Aptamer.
2. test kit as claimed in claim 1, it is characterised in that: described aptamer is SEQ ID No.1-24 any bar sequence Shown in row, or the replacement of one or several nucleotide carried out on the basis of this sequence, and retain its activity.
3. the test kit described in any one of claim 1-2 is in the application of detection autoimmune disease damage.
4. the application in the reagent preparing detecting system lupus erythematosus of the test kit described in any one of claim 1-2.
CN201610393707.2A 2016-06-05 2016-06-05 Kit for detecting autoimmune disease Pending CN106053831A (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101687916A (en) * 2007-01-16 2010-03-31 惠氏公司 Inflammation treatment, detection and monitoring via trem-1
CN104073496A (en) * 2013-02-28 2014-10-01 湖南中医药大学 Sequence and use of aptamer of colibacillus outer membrane protein TolC
CN104204803A (en) * 2012-02-09 2014-12-10 米密德诊断学有限公司 Signatures and determinants for diagnosing infections and methods of use thereof
CN104220456A (en) * 2012-02-15 2014-12-17 诺和诺德A/S（股份有限公司） Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
WO2015018936A1 (en) * 2013-08-09 2015-02-12 Inotrem Methods and kits for predicting the risk of having a cardiovascular disease or event
CN104830867A (en) * 2015-06-07 2015-08-12 杨洋 Aptamer capable of being specifically combined with DKK1 protein in cancer cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101687916A (en) * 2007-01-16 2010-03-31 惠氏公司 Inflammation treatment, detection and monitoring via trem-1
CN104204803A (en) * 2012-02-09 2014-12-10 米密德诊断学有限公司 Signatures and determinants for diagnosing infections and methods of use thereof
CN104220456A (en) * 2012-02-15 2014-12-17 诺和诺德A/S（股份有限公司） Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
CN104073496A (en) * 2013-02-28 2014-10-01 湖南中医药大学 Sequence and use of aptamer of colibacillus outer membrane protein TolC
WO2015018936A1 (en) * 2013-08-09 2015-02-12 Inotrem Methods and kits for predicting the risk of having a cardiovascular disease or event
CN104830867A (en) * 2015-06-07 2015-08-12 杨洋 Aptamer capable of being specifically combined with DKK1 protein in cancer cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J.KIM, ET AL.: "Serum levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and pentraxin 3 (PTX3) as markers of infection in febrile patients with systemic lupus erythematosus.", 《CLINICAL AND EXPERIMENTAL RHEUMATOLOGY》 *
YAIR MOLAD, ET AL.: "Serum Soluble Triggering Receptor on Myeloid Cells-1 (sTREM-1) is Elevated in Systemic Lupus Erythematosus but does not Dishtinguish Between Lupus Alone and Concurrent Infection.", 《INFLAMMATION》 *

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Application publication date: 20161026