CN105693847A

CN105693847A - Aptamer NKXA9 specifically for Neutrokine-alpha protein and application of aptamer NKXA9

Info

Publication number: CN105693847A
Application number: CN201610294381.8A
Authority: CN
Inventors: 马海龙
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-07-13
Filing date: 2014-07-13
Publication date: 2016-06-22

Abstract

The invention provides an aptamer NKXA9 specifically for Neutrokine-alpha protein and application of the aptamer NKXA9.The aptamer NKXA9 has activity for combining Neutrokine-alpha protein and can be used for being prepared into a kit for specifically screening out and removing Neutrokine-alpha protein.

Description

One species specificity is for the aptamer NKXA9 of Neutrokine-α albumen and application thereof

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to the polypeptide for systemic lupus erythematosus。

Background technology

Systemic lupus erythematosus (sle) (SLE) is presently considered to be autoimmune disease, and wherein the abnormal superfunction of bone-marrow-derived lymphocyte and a large amount of abnormal generation of immunoglobulin γ (IgG) autoantibody play pivotal role。This pathological process causes the disappearance of the Ig cell being coated with and destruction, the fixing and cutting of complement protein and chemotaxin (chemotaxin), vasoactive peptide and destructive enzyme release in the tissue。

The concurrent atherosclerosis (AS) of systemic lupus erythematosus (sle) (SLE) is one of main cause of death of SLE。As far back as 1976, having it has been observed that SLE mortality curve is in " bimodal " pattern, one is the high rate in activeness lupus commitment septicemia, and another is the high rate of inactivity lupus late stage myocardial infarction。Although various diseases control measures substantially increase the survival rate of Patients with SLE, but AS remains the main cause causing SLE death。Case-control (the age of lupus erythematosus, race, matching with sex) research shows: at the age less than in the patient of the lupus erythematosus of 50 years old, 31-37% patients goes out the representative mark of AS pathological changes, and the crowd only having 9-15% in matched group shows and this represents mark；At the age more than or equal in the patient of 50 years old, the lupus patient of 70-80% shows the representative mark of atherosclerotic lesion, and in comparison crowd, only the people of 21-45% has this representative mark。

SLE is characterised by manifestations form。In disease process, the patient of 95% has told musculoskeletal disease altogether, and 80% shows skin injury, 85% has hematologic disease, and 60% has neurological disorders, and 60% has heart and lung diseases, 30%～50% has kidney disease, and 40% has gastroenteropathy, and 15% has thrombosis and 15% to have ocular disease。Most patient (95%) is also subject to the systemic conditions all existed most of time, such as tired, uncomfortable, fever, anorexia and losing weight。Most of patients experience burst and the alleviation disease phase alternately。Permanent alleviation (does not have symptom) when not treating extremely rare。Before 50 years, the patient that most diagnosis are SLE was survived less than 5 years。Now, within 10 years, survival is more than 90%, and this is mainly based upon early diagnosis, suit the medicine to the illness antiinflammatory and immunosuppressant therapy。The common cause of the death is the infection that immunosuppressant causes。

Result of study shows: includes the conventional risk factors such as hypertension, obesity, diabetes, smoking, hypercholesterolemia, postmenopausal state and all can not explain the reason that in patients with SLE, coronary heart disease increases the weight of completely, and systemic lupus erythematosus (sle) itself (when non-life-time service glucocorticoid) is still that an independent hazard factor。Although disease course is all the independent predictor promoting atherosclerosis (carotid atherosclerotic plaque) with systemic lupus erythematosus (sle) damage index, but how lupus increases atherosclerotic occurrence risk and is not yet fully apparent from。

Conventional use antimalarial drug, anti-inflammatory agent and immunosuppressive drug in SLE treats。When symptom is difficult to control to, non-steroidal anti-inflammatory agent is supplemented with 17-hydroxy-11-dehydrocorticosterone。Additionally, the active SLE that major organs is got involved needs to adopt the aggressive therapy of cyclophosphamide。

Up to now, but without can be used for curing SLE and/or improving the causal treatment of quality of the life of patient in long-term basis。But, the development in antibody technique recently and the qualification further to the factor causing this autoimmune disease have had been switched on the probability using monoclonal antibody as therapeutic choice。Especially, the specific treatment that the favorable method of SLE will be with cause that a large amount of pathologic immune responses excessively produced of polyclone autoantibody interact or correct it is treated。Owing to the pathogeny of SLE relates generally to the B cell of imbalance, the monoclonal antibody being able to targeting B cell concerned especially。Potential B cell surface antigen target is CD19, CD20, CD21 and CD22。Additionally, IL-10, IL-1ra, IL-12 are the important cytokine regulating immune response, and especially raise during the burst of SLE patient。The blood plasma level of the autoantibody of IL-10 and anti-double-chain DNA (dsDNA) often reflects the disease activity of the patient suffering from SLE。Raise IL-10 level relevant to the disease activity of SLE patient (Park etc., Clin.Exp.Rheumatol.1998 May-June;16 (3): 283-8)。But, immune system is had polyphenic cytokine by IL-10, and known its also participates in reducing proinflammatory response。

SLE patient has carried out adopting the clinical trial of monoclonal antibody。Especially, several tests relate to antibody rituximab (Rituximab), a kind of gomphosis mouse anti-CD-20 monoclonal antibody for treating non Hodgkin lymphom。Robak and Robak (2009) notices, the result of these tests demonstrates the activity that this antibody is higher in SLE patient, and has been developed for the antibody (Ofatumumab, IMMU-106 and GA-101) of several new targeting CD20。Other clinical trial reporting monoclonal antibody activity in SLE adopts anti-CD22 antibody, epratuzumab (Epratuzumab), anti-TNF alpha antibodies, infliximab (Infliximab), anti-IL-10 antibody, B-N10 (Llorente etc., ArthritisRheum.2000 August;43 (8): 1790-800), anti-CD40L antibodies, IDEC131 and BG9588, BLYS inhibitor, Baily monoclonal antibody (Belimumab), anti-IL6 receptor antibody, the appropriate gram of female monoclonal antibody (Toclimumab) of profit and anti-C5 antibody complete according to storehouse pearl monoclonal antibody (Eculizumab)。

Neutrokine-α albumen (SEQIDNO:1) is a member of tnf ligand family, it enjoys Amino acid sequence identity (Moore with APRIL (28.7%), TNF α (16.2%) and Lymphotoxin-α (LT α) (14.1%), etal., (1999) Science285:260-263)。The formal name of Neutrokine-α is tumor necrosis factor (part) superfamily member 13B (TNFSF13b)。285 amino acid whose polypeptide of total length Neutrokine-α gene code, its 47th is cross-film district between 73 amino acids, is the distinctive non-hydrophobic sequence of II type embrane-associated protein before cross-film district。The same with other members of TNF family, Neutrokine-α plays a role with the form of trimer protein。When Neutrokine-α is after cell surface expression, at the 134th amino acids place cell lysis outskirt, in order to discharge the trimer of biologic activity。

Known Neutrokine-α with from three kinds of tumor necrosis factor receptor super family not isoacceptor be combined。They are cross-film activator and CAM mutual effect body, B cell activating factor receptor, B cell maturation antigen。The expression of receptor is principally limited to bone-marrow-derived lymphocyte。Believe that the major part effect of Neutrokine-α is all mediated by BAFF-R, because expressing the major defect in the B cell component of deficient mice in TACI or BCMA deficient mice and inconspicuous at Neutrokine-alpha expression defect or BAFF-R。

When detecting Neutrokine-α albumen in vitro and in vivo, Neutrokine-α is shown to promote the propagation of B cell, differentiation and existence。It addition, Neutrokine-α also shows that some effects to T cell。The mice being melted into overexpression Neutrokine-α by genetic engineering has the periphery B cell increasing number and the immunoglobulin concentrations increased。It addition, Neutrokine-α transgenic mice shows autoimmune phenotype, it is similar with what see in people's systemic lupus erythematosus (sle), including autoantibody and glomerulonephritis related symptoms occur。The Neutrokine-alpha levels that research later is shown in the serum of autoimmune disease such as systemic lupus erythematosus (sle), rheumatoid arthritis and Patients with Sjogren Syndrome and/or synovial fluid is also raised。Therefore, be Neutrokine-alpha-2 antagonists in the view that scientific circles are general has therapeutical effect in treatment autoimmune disease。

Summary of the invention

It is an object of the invention to provide can the aptamer of specific binding Neutrokine-α albumen, thus realizing being suppressed combined for Neutrokine-α in human body, separation or function, thus reaching the effect of systemic lupus erythematosus, rheumatoid arthritis。

The described oligonucleotide sequence that can recognise that Neutrokine-α albumen, including SEQIDNo.2-15, and adopts an oligonucleotide sequence therein just can complete the recognition detection to Neutrokine-α albumen；

The preparation method of described one group of oligonucleotide sequence that can recognise that Neutrokine-α albumen comprises the following steps:

1, the synthesis ssDNA oligonucleotide library (5 '-TCAGTCGCTTCGCCGTCTCCTTC----for screening

N35----GCACAAGAGGGAGACCCCAGAGGG-3 '), wherein N35 is 35 random oligonucleotides；

2, SELEX screening is carried out after being mixed with Neutrokine-α albumen respectively by oligonucleotide library, it is thus achieved that aptamer enriched library；

3, after SELEX has screened, the aptamer enriched library obtained is carried out cloning and sequencing；

4, selecting the height copy ssDNA occurred in sequencing result, carry out affine specific checking, screening obtains the oligonucleotide sequence that can recognise that Neutrokine-α albumen。

Detailed description of the invention

Embodiment:

1, the preparation of Neutrokine-α albumen

The mode adopting yeast well known to those skilled in the art recombinant expressed obtains has bioactive Neutrokine-α albumen identical with Neutrokine-α albumen, and this protein sequence is such as shown in SEQIDNO:1；The concentration of protein solution is 10mg/ml。

2, the synthesis of library and primer

2.1, the synthesis ssDNA oligonucleotide library (5 '-AATTCACTTACTTAACCAATCCGG----N35----ACACAAGAGTGAGAATTAGAG CG-3 ') for screening, wherein N35 is 35 random oligonucleotides；

Primer P1:AATTCACTTACTTAACCAATCCGG;

Primer P2:CGCTCTAATTCTCACTCTTGTGT。

2.2, the SELEX screening of aptamer, concrete grammar is as follows:

2.2.1ssDNA with the combination of Neutrokine-α albumen, separate, concrete grammar is as follows:

Take the ssDNA oligonucleotide library 4 μ L of 100 μMs, it is diluted to 100 μ l with 2 × binding buffer liquid, 95 DEG C of degeneration 5min, 100 μ lNeutrokine-α albumen are added after ice bath 10min, shaking table is in conjunction with 50min, the more centrifugal 7min of 6000rpm, abandons supernatant, then wash precipitation with 1 × binding buffer liquid, abandon supernatant；Precipitation adds 1 × binding buffer liquid 100 μ L, 96 DEG C of heating 5min, the then centrifugal 10min of 15000rpm, take supernatant, precipitation is again heated and is centrifuged, merges supernatant, then separable obtaining has ssDNA level library of affinity with Neutrokine-α albumen；Described 2 × binding buffer liquid is the solution after 20 × binding buffer liquid distilled water dilutes 10 times, and described 1 × binding buffer liquid is the solution after 20 × binding buffer liquid distilled water dilutes 20 times；Described 20 × binding buffer formula of liquid is 1MNaCl, 50mMKCl, 500mMTris-HCl, 10mMMgCl2, pH7.4。

2.2.2ssDNA with the combination of Neutrokine-α albumen, separate, concrete grammar is as follows:

Step 2.2.1 is separated obtain can ssDNA protein bound with Neutrokine-α, again with 100 μ lNeutrokine-α albumen shaking tables in conjunction with 30min, subsequent step is with step 2.2.1, separable to ssDNA the level library having affinity with Neutrokine-α albumen。

2.2.3 asymmetric PCR amplification ssDNA, concrete grammar is as follows:

Step 2.2.2 being separated ssDNA the level library obtained and carries out asymmetric PCR amplification, the asymmetric PCR amplification system that cumulative volume is 25 μ l is: 10 × PCR buffer: 2 μ l；P1 (10 μMs): 1 μ l；P2 (0.2 μM): 1 μ l；DNTP (each 2.5mM): 0.4 μ l；MgCl₂(25mM): 1.2 μ l；SsDNA template (0.2 μ g/ μ l): 2 μ l；Taq DNA polymerase (5u/ μ l): 0.2 μ l；DdH2O:17.2 μ l；PCR response parameter: 94 DEG C of denaturation 4min, then carries out 94 DEG C of degeneration 30s of 40 circulations, 58 DEG C of annealing 30s, and 72 DEG C extend 20s, and last 72 DEG C extend 7min；

2.2.4 the mensuration of affinity, concrete grammar is as follows:

2.2.4.1 amplification: expanding, with the primer P1 asymmetric PCR with digoxigenin labeled, ssDNA the level library screened, amplification condition is identical with the asymmetric PCR amplification system of step 2.2.3 and parameter with parameter；

2.2.4.2 with protein binding: take step 2.2.4.1 and expand the PCR primer 100 μ L of gained, 95 DEG C of degeneration 5min, add after ice bath 10min in 100 μ L albumen, are sufficiently mixed, at room temperature in conjunction with 30min, then 6000rpm is centrifuged, and separates albumen and supernatant, includes and the ssDNA with digoxigenin labeled of combination in albumen in albumen, supernatant is unconjugated ssDNA, do a blank being not added with ssDNA simultaneously, namely replace PCR primer with 2 × binding buffer liquid, carry out aforesaid operations equally；

2.2.4.3 washing: being washed 1 time by albumen 1 × binding buffer liquid 500 μ L, 6000rpm is centrifuged, and abandons supernatant, takes albumen；

2.2.4.4 be combined with enzyme mark rabbit anti digoxin antibody: in albumen, add the excessive enzyme mark rabbit anti digoxin antibody of 100 μ L1: 900TBS dilutions, after being sufficiently mixed, react 10min, so as to the ssDNA of the digoxigenin labeled in albumen is combined；

Described TBS is 0.5MTris-NaCl solution, and compound method is: first water-soluble 8.5～9gNaCl, then adds Tris-HCl (0.5M, pH7.6) solution 100ml, finally adds water and is settled to 1L；0.5MTris-HCl (pH7.6,100ml) solution preparation method: weigh Tris6.06g, adds distilled water 40ml and dissolves, and drips dense HCl and adjusts pH to 7.6, is settled to 100ml。

2.2.4.5 washing: 6000rpm is centrifuged, and removes supernatant, then washs 3 times with 1 × binding buffer liquid 500 μ L, obtains albumen；

2.2.4.6TMB (tetramethyl benzidine) colour developing: add the 400 μ resuspended albumen of L distilled water, add 200 μ LTMB nitrite ions, after lucifuge colour developing 10min, terminate reaction with 2mol/LH2SO4200 μ L, measure the light absorption value OD450 at 450nm place, namely this value reflects the affinity of the ssDNA being combined with bacterium, namely OD combines, and blank carries out above-mentioned steps 2.2.4.3,2.2.4.4 equally, 2.2.4.5 and 2.2.4.6, blank corresponding absorbance OD is obtained blank；

Described TMB nitrite ion uses conventional compound method preparation。

2.2.4.7 the molar concentration of DNA in PCR primer is measured: take step 2.2.4.1 and expand the PCR primer of gained, with the initial ssDNA library of concentration known gradient for standard substance, with Bandscan software as image analysis software, adopt the DNA content in ethidium bromide agarose gel electrophoresis method quantitative assay PCR primer, obtain the molar concentration of corresponding DNA, and then the DNA molal quantity in 100 μ LPCR products can be calculated。

2.2.4.8 the affinity in corresponding library is calculated:

2.3 repeat screening, method particularly includes: using each product screening library as next round taking turns asymmetric PCR, repeat above-mentioned SELEX and screen step 2.2, until affinity no longer rises, eventually pass 14 screenings taken turns and obtain the aptamer enriched library of ssDNA。After asymmetric PCR expands, condition, with step above, is cloned and checks order, obtaining 18 effective ssDNA that copy number is the highest, 18 aptamers are carried out affine specificity verification respectively, wherein has 4 specificitys not unique, therefore gives up。Therefore obtaining 14 and Neutrokine-α albumen has better affine specific oligonucleotide sequence (aptamer), particular sequence is as follows:

Aptamer title	Adaptor sequence
		NKXA1	5′-AATTCACTTACTTAACCAATCCGG ATATTTCCAACACTCCAACTTTCCCTACAATTCTAACACAAGAGTGAGAATTAGAGCG-3′
NKXA3	5′-AATTCACTTACTTAACCAATCCGG TCACATTACAAATACATTCATCCCCAACACACCAAACACAAGAGTGAGAATTAGAGCG-3′
		NKXA4	5′-AATTCACTTACTTAACCAATCCGG TTCTCTTACCCAAATTCTTCCAACCTTACCTTCCAACACAAGAGTGAGAATTAGAGCG-3′ 4 -->
NKXA5	5′-AATTCACTTACTTAACCAATCCGG TCTATAAAATATTTCAATCTCTCTCTATATCATTCACACAAGAGTGAGAATTAGAGCG-3′
		NKXA6	5′-AATTCACTTACTTAACCAATCCGG CCTCTACTAATCAAACTCTTACCCACCCTCTATCTACACAAGAGTGAGAATTAGAGCG-3′
NKXA7	5′-AATTCACTTACTTAACCAATCCGG TCACATATATCTCAACTCTATCCACTCTCTCAACTACACAAGAGTGAGAATTAGAGCG-3′
		NKXA9	5′-AATTCACTTACTTAACCAATCCGG CAACTCAATATCTTCCCTTAACTTATCCCTACACCACACAAGAGTGAGAATTAGAGCG-3′
NKXA10	5′-AATTCACTTACTTAACCAATCCGG ATCTACACCCCCACCATCTACCAACCACCACCCAAACACAAGAGTGAGAATTAGAGCG-3′
		NKXA11	5′-AATTCACTTACTTAACCAATCCGG CACTCATACATATCAACACCTCACTCCTTATTCCCACACAAGAGTGAGAATTAGAGCG-3′
NKXA13	5′-AATTCACTTACTTAACCAATCCGG CATTATATCAATTACACATCTCCATTTAATCAACAACACAAGAGTGAGAATTAGAGCG-3′
		NKXA14	5′-AATTCACTTACTTAACCAATCCGG TTACTTCTTCATTCATAATACCATCCTAATAACTTACACAAGAGTGAGAATTAGAGCG-3′
NKXA15	5′-AATTCACTTACTTAACCAATCCGG ACCTACACAATCTATCACTTATAACTTCAAACTTTACACAAGAGTGAGAATTAGAGCG-3′
		NKXA17	5′-AATTCACTTACTTAACCAATCCGG AATTTTAATACAAATCTCTAAATAAACCACCCCAAACACAAGAGTGAGAATTAGAGCG-3′
NKXA18	5′-AATTCACTTACTTAACCAATCCGG AATATACTAATACCCATAAACTTAAATCTCAACTCACACAAGAGTGAGAATTAGAGCG-3′

The concrete data of affinity are as follows:

Aptamer title	Affinity	Aptamer title	Affinity
				NKXA1	0.49	NKXA10	0.49
NKXA3	0.51	NKXA11	0.48
				NKXA4	0.46	NKXA13	0.57
NKXA5	0.49	NKXA14	0.54
				NKXA6	0.52	NKXA15	0.48
NKXA7	0.54	NKXA17	0.42
				NKXA9	0.51	NKXA18	0.47

2.4, specificity and the affinity of 20 aptamers are analyzed

Fluorescently-labeled adaptor sequence and Neutrokine-α albumen are hatched, carry out flow cytometry detection, wherein 14 sequences show high fluorescent, GraphPadPrism5.0 software is used to do nonlinear regression curve for saturation curve, respectively 14 high-affinity adaptor sequence being adopted identical experimental implementation, obtaining every is the Kd value configured:

Aptamer title	Kd value (nM)	Aptamer title	Kd value (nM)
				NKXA1	20.5	NKXA10	19.5
NKXA3	21.2	NKXA11	21.2
				NKXA4	24.7	NKXA13	22.5
NKXA5	25.6	NKXA14	27.2
				NKXA6	26.5	NKXA15	25.1
NKXA7	23.1	NKXA17	26.4
				NKXA9	28.6	NKXA18	30.1

Wherein the Kd value of NKXA10 is minimum, illustrates can be quickly combined with target protein and Stability Analysis of Structures is not readily separated。

Adopting the secondary structure of 14 aptamers of DNAMAN software building and calculate their minimum free energy, its structure minimum free energy is also all less, and structure is also relatively stable。

2.5 aptamer specificity analyses

It is respectively adopted BSA, APRIL albumen, Neutrokine-α albumen and 14 aptamers and carries out specific detection, find through binding tests, these 14 sequences do not combine with BSA, APRIL albumen, and only keep higher specificity with Neutrokine-α protein binding。

3, the application of aptamer

By the 14 of the present invention aptamers, its 5 ' end adds the labelling with magnetic mark magnetic bead, can be prepared as test kit, pharmaceutical composition, by with contacting blood, can realize for the special separation of Neutrokine-α albumen in blood by magnetic screening, thus reaching the effect of systemic lupus erythematosus, rheumatoid arthritis。

Sequence table

< 110 > horse Solenognathus

< 120 > mono-species specificity is for the aptamer NKXA9 of Neutrokine-α albumen and application thereof

〈160〉16

〈210〉1

〈211〉285

〈212〉PRT

< 213 > people

〈400〉1

1MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCC

61LTVVSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAP

121GEGNSSQNSRNKRAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEE

181KENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETL

241PNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL

〈210〉2

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA1

5′-AATTCACTTACTTAACCAATCCGGATATTTCCAACACTCCAACTTTCCCTACA

ATTCTAACACAAGAGTGAGAATTAGAGCG-3′

〈210〉3

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA3

5′-AATTCACTTACTTAACCAATCCGGTCACATTACAAATACATTCATCCCCAACACA

CCAAACACAAGAGTGAGAATTAGAGCG-3′

〈210〉4

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA4

5′-AATTCACTTACTTAACCAATCCGGTTCTCTTACCCAAATTCTTCCAACCTTACC

TTCCAACACAAGAGTGAGAATTAGAGCG-3′

〈210〉5

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA5

5′-AATTCACTTACTTAACCAATCCGGTCTATAAAATATTTCAATCTCTCTCTATATCAT

TCACACAAGAGTGAGAATTAGAGCG-3′

〈210〉6

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA6

5′-AATTCACTTACTTAACCAATCCGGCCTCTACTAATCAAACTCTTACCCACCCTCT

ATCTACACAAGAGTGAGAATTAGAGCG-3′

〈210〉7

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA7

5′-AATTCACTTACTTAACCAATCCGGTCACATATATCTCAACTCTATCCACTCTCT

CAACTACACAAGAGTGAGAATTAGAGCG-3′

〈210〉8

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA9

5′-AATTCACTTACTTAACCAATCCGGCAACTCAATATCTTCCCTTAACTTATCCC

TACACCACACAAGAGTGAGAATTAGAGCG-3′

〈210〉9

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA10

5′-AATTCACTTACTTAACCAATCCGGATCTACACCCCCACCATCTACCAACCACC

ACCCAAACACAAGAGTGAGAATTAGAGCG-3′

〈210〉10

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA11

5′-AATTCACTTACTTAACCAATCCGGCACTCATACATATCAACACCTCACTCCTT

ATTCCCACACAAGAGTGAGAATTAGAGCG-3′

〈210〉11

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA13

5′-AATTCACTTACTTAACCAATCCGGCATTATATCAATTACACATCTCCATTTAA

TCAACAACACAAGAGTGAGAATTAGAGCG-3′

〈210〉12

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA14

5′-AATTCACTTACTTAACCAATCCGGTTACTTCTTCATTCATAATACCATCCTAATA

ACTTACACAAGAGTGAGAATTAGAGCG-3′

〈210〉13

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA15

5′-AATTCACTTACTTAACCAATCCGGACCTACACAATCTATCACTTATAACTTCAAA

CTTTACACAAGAGTGAGAATTAGAGCG-3′

〈210〉14

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA17

5′-AATTCACTTACTTAACCAATCCGGAATTTTAATACAAATCTCTAAATAAACCAC

CCCAAACACAAGAGTGAGAATTAGAGCG-3′

〈210〉15

<211〉82

〈212〉DNA

< 213 > artificial sequence

〈400〉NKXA18

5′-AATTCACTTACTTAACCAATCCGGAATATACTAATACCCATAAACTTAAATCTC

AACTCACACAAGAGTGAGAATTAGAGCG-3′

〈210〉16

<211〉23

〈212〉DNA

< 213 > artificial sequence

〈400〉P1

AATTCACTTACTTAACCAATCCGG

〈210〉17

<211〉24

〈212〉DNA

< 213 > artificial sequence

〈400〉P2

CGCTCTAATTCTCACTCTTGTGT

Claims

1., for a Neutrokine-α albumen for aptamer screening, its sequence is shown in SEQIDNO:1。

2. the oligonucleotide sequence identifying Neutrokine-α albumen, it is characterised in that shown in SEQIDNo.8。

3. the oligonucleotide sequence shown in claim 2 is for screening the application of Neutrokine-α albumen。

4. pharmaceutical composition, reagent or the test kit that the oligonucleotide sequence shown in claim 2 prepares。

5. the oligonucleotide sequence shown in claim 4 is preparing the application for treating in the pharmaceutical composition of lupus erythematosus, reagent or test kit。