CN104745590A - Nucleic acid aptamer capable of detecting ENOX2 protein in serum of cancer patient and application thereof - Google Patents

Abstract

The embodiment of the invention provides a nucleic acid aptamer capable of detecting an ENOX2 protein in the serum of a cancer patient. The nucleotide sequences of the nucleic acid aptamer are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4. The nucleic acid aptamer provided by the invention is higher in resolution ratio than that of an antibody, and quite high in specificity; the nucleic acid aptamer screened by an ENOX2 peptide chain only binds the ENOX2 protein and does not bind with other proteins. In addition, the nucleic acid aptamer provided by the invention is quite high in affinity; the dissociation constant of the aptamer screened by taking the protein as a target substance can achieve an nmol/L level, and even pmol/L level; moreover, once the nucleotide sequences of the aptamer are determined, the production cost is quite low, and the nucleic acid aptamer synthesised and produced every time keeps absolutely uniform consistent.

Description

Nucleic acid aptamer and the application thereof of ENOX2 albumen in Patients with Various Cancers can be detected

Technical field

The invention belongs to biomedical detection technical field, be specifically related to a kind of nucleic acid aptamer and the application thereof that detect ENOX2 albumen in Patients with Various Cancers.

Background technology

Reduced nicotinamide-adenine dinucleotide oxydase disulfide exchange albumen 2 (Ecto-Nicotinamide Adenine DinucleotideOxidase Disulfide-Thiol Exchanger 2 (ENOX2)) is the membranin regulating Growth of Cells and division.Malignant cell is in division and breeding, and ENOX2 albumen great expression is also cut to enter in blood, but not then there is not ENOX2 albumen in the serum of cancer patient.Cancer occur early stage, namely there is ENOX2 albumen in serum.Therefore, the ENOX2 albumen detected in blood can carry out the early diagnosis of cancer.In different tumour cells, ENOX2mRNA carries out alternative splicing, produces alternative splicing variant ENOX2.In different types of Patients with Various Cancers, can detect different alternative splicing variants, its molecular weight is different with iso-electric point.So, only need to detect the ENOX2 albumen in patients serum, can know which kind of cancer patient suffers from according to its molecular weight and iso-electric point.

The single-chain recombinant antibody (ScFv) of ENOX2 can be used for the early diagnosis of cancer, although ENOX2 single-chain recombinant antibody can not the ENOX2 alternative splicing variant of Direct Recognition various cancers cell, but, utilize two-dimensional electrophoresis different molecular weight can be separated with the ENOX2 albumen of iso-electric point.The recombinant antibodies transferring to i.e. available ENOX2 on film after separation detects.Detect iso-electric point and the molecular weight of the ENOX2 albumen in Patients with Various Cancers, thus determine which kind of cancer patient suffers from.Meanwhile, ENOX2 albumen also can be used as drug target, by expression or the activity of the ENOX2 albumen of anticancer, is then expected to the medicine developing effective Therapeutic cancer.

And prior art is with the single-chain recombinant antibody of anti-ENOX2 albumen in conjunction with this albumen, and the ENOX2 in serum is detected.

Prior art utilizes molecular biology method, and clone obtains ENOX2 recombinant single chain antibody ScFv and implanted colibacillus expression plasmid pET-11a.ScFv by IPTG abduction delivering, produces a large amount of ENOX2 recombinant single chain antibody in intestinal bacteria.Antibody expressed in intestinal bacteria does not have activated, and then by different Measurements for Biochemistry, comprise dialysis method and dilution method etc., antagonist carries out denature and renature process, obtains activated ENOX2 recombinant single chain antibody.

In order to detect the activity of the ENOX2 recombinant single chain antibody of escherichia coli expression, prior art has cloned mankind ENOX2 gene, and carries out great expression in intestinal bacteria.Then, we carry out Isolation and purification, for detecting the activity of ENOX2 recombinant single chain antibody the ENOX2 albumen of expressing from intestinal bacteria.First, with SDS-PAGE electrophoresis by ENOX2 albumen sepn in PAGE glue, then transfer on nitrocellulose membrane, then use ENOX2 recombinant single chain antibody as first antibody, anti-S-tag-AP, as second antibody, detects the ENOX2 on nitrocellulose membrane.

Prior art, then dyes with the substrate of AP on the S-tag of ENOX2 recombinant single chain antibody by anti-S-tag-AP antibodies.If containing ENOX2 albumen in patients serum, after adding AP substrate, film just can show blue spot.Detected result shows, ENOX2 recombinant single chain antibody has protein-bonded activity, can detect the ENOX2 albumen in Patients with Various Cancers, then can't detect the existence of ENOX2 albumen in the serum of normal people on film.

Although prior art also can detect ENOX2 albumen by ENOX2 recombinant antibodies, but there is shortcomings and deficiency in this technology: (1) under many circumstances, no matter monoclonal antibody, polyclonal antibody or recombinant single chain antibody, except in conjunction with except its specific antigens, also have keying action to other albumen etc., therefore specificity is strong not; (2) avidity of recombinant single chain antibody is not high; (3) production process of recombinant single chain antibody is complicated, and production cost is high, and after expressing in intestinal bacteria, must carry out denature and renature to recombinant antibodies.Therefore, different production batchs very easily changes, and the specificity of recombinant antibodies, avidity and resolving power etc. are changed.

Summary of the invention

The technical problem solved:

For above-mentioned defect of the prior art and problem, the object of the invention is to overcome the deficiencies in the prior art and a kind of nucleic acid aptamer and the application thereof that detect ENOX2 albumen in Patients with Various Cancers are provided, the sequence that the present invention is directed to ENOX2 albumen carries out the screening of nucleic acid aptamer, the aptamer filtered out both may be used for the detection of ENOX2, thus carry out the early diagnosis of various cancer, can suppress for ENOX2 albumen again, thus the new drug of research and development Therapeutic cancer, instant invention overcomes many shortcomings of recombinant single chain antibody, acquisition has ENOX2 protein-specific, resolving power and the high nucleic acid aptamer of avidity.

Technical scheme:

In order to achieve the above object, the embodiment of the present invention provides following technical scheme:

The nucleic acid aptamer of ENOX2 albumen in Patients with Various Cancers can be detected, its nucleotide sequence as SEQ ID NO.1, SEQID NO.2, SEQ ID NO.3 and or SEQ ID NO.4 shown in:

SEQ ID NO.1：GGATAAGCCG CATTAGCTTA CTTCAGTGAT GCTTAAGATC TAGTA；

SEQ ID NO.2：GCTTACGCGG CATTCGCTTA CTTCACTGTA GCTTCAGTTC TGCTA；

SEQ ID NO.3：GCAGTAGCCG CTTTCGCTTA GTTCATCCTA CTTTCAGATC TACTA；

SEQ ID NO.4：GGAGTACGGC TTATTCCTAT TGATACTGCA CTTACTGGTG TAGCC。

The nucleic acid aptamer of ENOX2 albumen in described detected Patients with Various Cancers, wherein, the conserved sequence of described ENOX2 albumen is as shown in SEQ ID NO.5:

SEQ ID NO.5：EEAKEK FKQALSGILI QFE

Namely

Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu。

1 5 10 15

The ENOX2 peptide chain of the conserved sequence synthesis of described ENOX2 albumen, its aminoacid sequence is as shown in SEQ ID NO.6:

SEQ ID NO.6：EEAKEKFKQALSGILIQFE

Namely

Ac Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu Amide。

1 5 10 15

For screening the above-mentioned DNA library detecting the nucleic acid aptamer of ENOX2 albumen in Patients with Various Cancers, its nucleotide sequence is as shown in SEQ ID NO.7, and wherein, N is any one in ATGC, totally 45 N:

SEQ ID NO.7：

GGT ATT GAG GGT CGC ATC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN GAT GGC TCT AAC TCT CCT CT。

Preferred as technique scheme, in described detected Patients with Various Cancers, the nucleic acid aptamer of ENOX2 albumen is applied at early diagnosis of cancer and by suppressing the activity of ENOX2 albumen.

Nucleic acid aptamer (Aptamer) is the nucleotide sequence of synthetic, can with very high avidity with various target molecule (small molecules, protein, even whole cell) specific binding.Its function class like antibody, but has more advantage than antibody.Nucleic acid aptamer is by SELEX technology, and namely the aglucon phyletic evolution technology (SystematicEvolution of Ligandsby Exponential Enrichment, SELEX) of index concentration screens.Utilize this technology can filter out specificity and the affine nucleic acid aptamer of target material height from random single chain nucleotide sequence storehouse.

The basic thought of SELEX technology is that iii vitro chemical synthesizes a single stranded oligonucleotide storehouse, mix with target material with it, the mixture of target material and nucleic acid is there is in mixed solution, wash the nucleic acid be not combined with target material off, be separated the nucleic acid molecule be combined with target material, with this nucleic acid molecule for template carries out pcr amplification, carry out the screening of next round.By screening and the amplification of repetition, some are not combined with target material or have the DNA of low-affinity, middle avidity or RNA molecule to be washed away with target material, and namely aptamer has DNA or RNA of high-affinity to separate from very large random library with target material, and purity carrying out and increase with SELEX process, from P mole to n mole, finally occupy the great majority (about >90%) in storehouse.

First, the present invention has synthesized single stranded DNA random oligonucleotide library, and library capacity is greater than 1010 single strand oligonucleotide sequences.The centre in library is stochastic sequence, and sequence length is 45 Nucleotide.In random library, strand random oligonucleotide acid molecule easily forms multiple three-dimensional space three-dimensional arrangement, almost can with the species effect of all existence of nature.Because DNA is low relative to RNA production cost, more stable in body, be not easily degraded, the aptamer that constructed library screening goes out is more suitable for in-vitro diagnosis and interior therapeutic, and therefore, the present invention has selected DNA library.

The conserved sequence that the present invention is directed to ENOX2 albumen carries out the screening of nucleic acid aptamer.First, the polypeptide of the conserved sequence of ENOX2 albumen is attached on magnetic bead, then the conserved sequence NOX2 peptide chain combination of the E on single stranded DNA storehouse and magnetic bead, removes unconjugated DNA, combining DNA wash-out out, increases with the DNA of PCR method by wash-out.A chain in PCR primer, with vitamin H (Biotin), removes the strand with vitamin H with Streptavidin MagneSphere, produces the screening that new single stranded DNA carries out next round.Screen 15 take turns after, PCR primer is cloned into carrier and carries out DNA sequencing, final obtain the specificity aptamer of combining closely with ENOX2.

Beneficial effect

The first, the resolving power of nucleic acid aptamer is higher than antibody: under many circumstances, no matter monoclonal antibody, polyclonal antibody or recombinant single chain antibody, and except in conjunction with except its specific antigens, also have keying action to other albumen etc., therefore specificity is strong not.Nucleic acid aptamer then has very high specificity, screens the nucleic acid aptamer that obtains only in conjunction with ENOX2 albumen, do not combine with other albumen with ENOX2 peptide chain.

The second, nucleic acid aptamer has very high avidity: be target material with protein, and sieve the dissociation constant of aptamer can reach nmo/L level, even pmol/L level, recombinant single chain antibody then can not reach so high avidity.

3rd, the practicality of nucleic acid aptamer is stronger: screening aptamer can timing, quantitatively and guarantee the quality, the denature and renature of aptamer is quick, reversible, reusable, long-term preservation and room temperature transport, Relative antibody, nucleic acid aptamer then has very strong practicality.

4th, the production process of recombinant single chain antibody is complicated, and production cost is high.And after expressing in intestinal bacteria, denature and renature must be carried out to recombinant antibodies.Therefore, different production batchs very easily changes, and the specificity of recombinant antibodies, avidity and resolving power etc. are changed.The nucleic acid aptamer that the present invention produces then does not have this defect, once the nucleotide sequence of aptamer is determined, production cost is very low, and synthesizes the nucleic acid aptamer produced at every turn and keep definitely consistent.

Accompanying drawing explanation

In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only embodiments more of the present utility model, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.

Fig. 1 be the embodiment of the present invention can detect nucleic acid aptamer SEQ ID NO.1 (Apt-1), the SEQ ID NO.2 (Apt-2) of ENOX2 albumen in Patients with Various Cancers, SEQ ID NO.3 (Apt-3) and or SEQ ID NO.4 (Apt-4) scheme in conjunction with the Western blot of ENOX2.

To be embodiment of the present invention nucleic acid aptamer SEQ ID NO.1 (Apt-1) that can detect ENOX2 albumen in Patients with Various Cancers scheme in conjunction with the Western blot of ENOX2 Fig. 2.

Embodiment

Below in conjunction with the accompanying drawing of invention, be clearly and completely described technical scheme of the present invention by embodiment, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.

It will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.

Below the aminoacid sequence of mankind ENOX2 albumen:

SEQ ID NO.11：Human ENOX2protein sequence(from AAB30428):

MQRDFRWLWV YEIGYAADNS RTLNVDSTAM TLPMSDPTAW ATAMNNLGMA

PLGIAGQPIL PDFDPALGMM TGIPPITPMM PGLGIVPPPI PPDMPVVKEI

IHCKSCTLFP PNPNLPPPAT RERPPGCKTV FVGGLPENGT EQIIVEVFEQ

CGEIIAIRKS KKNFCHIRFA EEYMVDKALY LSGYRIRLGS STDKKDTGRL

HVDFAQARDD LYEWECKQRM LAREERHRRR MEEERLRPPS PPPVVHYSDH

ECSIVAEKLK DDSKFSEAVQ TLLTWIERGE VNRRSANNFY SMIQSANSHV

RRLVNEKAAH EKDMEEAKEK FKQALSGILI QFEQIVAVYH SASKQKAWDH

FTKAQRKNIS VWCKQAEEIR NIHNDELMGI RREEEMEMSD DEIEEMTETK

ETEESALVSQ AEALKEENDS LRWQLDAYRN EVELLKQEQG KVHREDDPNK

EQQLKLLQQA LQGMQQHLLK VQEEYKKKEA ELEKLKDDKL QVEKMLENLK

EKESCASRLC ASNQDSEYPL EKTMNSSPIK SEREALLVGI ISTFLHVHPF

GASIEYICSY LHRLDNKICT SDVECLMGRL QHTFKQEMTG VGASLEKRWK

FCGFEGLKLT

That is:

Met Gln Arg Asp Phe Arg Trp Leu Trp Val Tyr Glu Ile Gly Tyr Ala

Ala Asp Asn Ser Arg Thr Leu Asn Val Asp Ser Thr Ala Met Thr Leu

Pro Met Ser Asp Pro Thr Ala Trp Ala Thr Ala Met Asn Asn Leu Gly

Met Ala Pro Leu Gly Ile Ala Gly Gln Pro Ile Leu Pro Asp Phe Asp

Pro Ala Leu Gly Met Met Thr Gly Ile Pro Pro Ile Thr Pro Met Met

Pro Gly Leu Gly Ile Val Pro Pro Pro Ile Pro Pro Asp Met Pro Val

Val Lys Glu Ile Ile His Cys Lys Ser Cys Thr Leu Phe Pro Pro Asn

Pro Asn Leu Pro Pro Pro Ala Thr Arg Glu Arg Pro Pro Gly Cys Lys

Thr Val Phe Val Gly Gly Leu Pro Glu Asn Gly Thr Glu Gln Ile Ile

Val Glu Val Phe Glu Gln Cys Gly Glu Ile Ile Ala Ile Arg Lys Ser

Lys Lys Asn Phe Cys His Ile Arg Phe Ala Glu Glu Tyr Met Val Asp

Lys Ala Leu Tyr Leu Ser Gly Tyr Arg Ile Arg Leu Gly Ser Ser Thr

Asp Lys Lys Asp Thr Gly Arg Leu His Val Asp Phe Ala Gln Ala Arg

Asp Asp Leu Tyr Glu Trp Glu Cys Lys Gln Arg Met Leu Ala Arg Glu

Glu Arg His Arg Arg Arg Met Glu Glu Glu Arg Leu Arg Pro Pro Ser

Pro Pro Pro Val Val His Tyr Ser Asp His Glu Cys Ser Ile Val Ala

Glu Lys Leu Lys Asp Asp Ser Lys Phe Ser Glu Ala Val Gln Thr Leu

Leu Thr Trp Ile Glu Arg Gly Glu Val Asn Arg Arg Ser Ala Asn Asn

Phe Tyr Ser Met Ile Gln Ser Ala Asn Ser His Val Arg Arg Leu Val

Asn Glu Lys Ala Ala His Glu Lys Asp Met Glu Glu Ala Lys Glu Lys

Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu Gln Ile Val

Ala Val Tyr His Ser Ala Ser Lys Gln Lys Ala Trp Asp His Phe Thr

Lys Ala Gln Arg Lys Asn Ile Ser Val Trp Cys Lys Gln Ala Glu Glu

Ile Arg Asn Ile His Asn Asp Glu Leu Met Gly Ile Arg Arg Glu Glu

Glu Met Glu Met Ser Asp Asp Glu Ile Glu Glu Met Thr Glu Thr Lys

Glu Thr Glu Glu Ser Ala Leu Val Ser Gln Ala Glu Ala Leu Lys Glu

Glu Asn Asp Ser Leu Arg Trp Gln Leu Asp Ala Tyr Arg Asn Glu Val

Glu Leu Leu Lys Gln Glu Gln Gly Lys Val His Arg Glu Asp Asp Pro

Asn Lys Glu Gln Gln Leu Lys Leu Leu Gln Gln Ala Leu Gln Gly Met

Gln Gln His Leu Leu Lys Val Gln Glu Glu Tyr Lys Lys Lys Glu Ala

Glu Leu Glu Lys Leu Lys Asp Asp Lys Leu Gln Val Glu Lys Met Leu

Glu Asn Leu Lys Glu Lys Glu Ser Cys Ala Ser Arg Leu Cys Ala Ser

Asn Gln Asp Ser Glu Tyr Pro Leu Glu Lys Thr Met Asn Ser Ser Pro

Ile Lys Ser Glu Arg Glu Ala Leu Leu Val Gly Ile Ile Ser Thr Phe

Leu His Val His Pro Phe Gly Ala Ser Ile Glu Tyr Ile Cys Ser Tyr

Leu His Arg Leu Asp Asn Lys Ile Cys Thr Ser Asp Val Glu Cys Leu

Met Gly Arg Leu Gln His Thr Phe Lys Gln Glu Met Thr Gly Val Gly

Ala Ser Leu Glu Lys Arg Trp Lys Phe Cys Gly Phe Glu Gly Leu Lys

Leu Thr)

Embodiment 1

1. synthesize peptide chain (peptide chain is synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.):

Ac-EEAKEKFKQALSGILIQFE-Amide:(namely

Ac-GluGluAlaLysGluLysPheLysGlnAlaLeuSerGlyIleLeuIleGlnPheGlu-Amide)(SEQ ID NO.5)

2. synthesis is for screening the DNA library (DNA library is synthesized by Jin Weizhi bio tech ltd, Suzhou) of ENOX2 peptide chain aptamer:

Apt-L:5 '-GGT ATT GAG GGT CGC ATC NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN NNN NNN NNN GAT GGC TCT AAC TCT CCT CT (SEQ IDNO.6), PAGE purifying.Wherein N can be any one in ATGC, totally 45 N.Therefore, this DNA library should have 445 kinds of different nucleotide sequences.The aptamer of peptide chain specificity to ENOX2 conserved sequence and high-affinity is filtered out from so large storehouse.

3. synthesize three short dna fragments, the primer for PCR:

Apt-F:5’-GGT ATT GAG GGT CGC ATC(SEQ ID NO.8)

Apt-R:5’-AGA GGA GAG TTA GAG CCA TC(SEQ ID NO.9)

Apt-RB:5’Biotin-AGA GGA GAG TTA GAG CCA TC(SEQ ID NO.10)

Apt-L, Apt-F, Apt-R and Apt-RB are dissolved in 10mM Tris, and pH 8.0,1mM EDTA (TE damping fluid), ultimate density 100 is micro-rubs.

4. be attached on magnetic bead by polypeptide, concrete grammar is:

(1) weigh 1 milligram of Dynabeads M-270Epoxy (Life Technologies), put in the test tube of 2 milliliters;

(2) in test tube, the C1 solution of 1 milliliter of Life Technologies company is added, mixing;

(3) test tube is inserted magnetic bead frame upper 1 minute, make magnetic bead be adsorbed onto on test tube wall, suck supernatant liquor;

(4) in test tube, add the polypeptide (1 milligram/100 Al of Solution of 15 microlitres) of 20 micrograms, then add the C1 solution of 235 microlitres;

(5) in test tube, add the C2 solution of the Life Technologies company of 250 microlitres;

(6) 37 DEG C of shaking table inside holding 24 hours, shaking table keeps per minute 200 turns, to ensure that magnetic bead is not attached on test tube wall all the time;

After (7) 24 hours, test tube is inserted magnetic bead frame upper 1 minute, make magnetic bead be adsorbed onto on test tube wall, suck supernatant liquor;

(8) HB eluant solution: HB (adding tween-20 to the ultimate density 0.05%) solution adding 800 microlitre Life Technologies companies in test tube, mixing;

(9) test tube is inserted magnetic bead frame upper 1 minute, make magnetic bead be adsorbed onto on test tube wall, suck supernatant liquor;

(10) LB eluant solution: LB (adding tween-20 to the ultimate density 0.05%) solution adding 800 microlitre Life Technologies companies in test tube, mixing;

(11) test tube is inserted magnetic bead frame upper 1 minute, make magnetic bead be adsorbed onto on test tube wall, suck supernatant liquor;

(12) SB eluant solution: the SB solution adding 800 microlitre Life Technologies companies in test tube, mixing.

(13) test tube is inserted magnetic bead frame upper 1 minute, make magnetic bead be adsorbed onto on test tube wall, suck supernatant liquor;

(14) SB eluant solution is repeated once;

(15) the long wash-out of SB solution: the SB solution adding 800 microlitre Life Technologies companies in test tube, mixing.Room temperature shakes 15 minutes;

(16) test tube is inserted magnetic bead frame upper 1 minute, make magnetic bead be adsorbed onto on test tube wall, suck supernatant liquor;

(17) in test tube, the SB of 500 microlitres is added, mixing.Insert 4 DEG C of refrigerators stand-by.

5. screening is for the nucleic acid aptamer of ENOX2 protein polypeptide

(1) in 1.5 milliliters of test tube, add the PBST of 50 microlitres, then add the ssDNA storehouse of 100 micro-molar concentrations of 10 microlitres, 95 DEG C 2 minutes, insert 10 minutes on ice;

(2) heat treated ssDNA storehouse is joined the PBST of 48 milliliters above, add 50 microlitre 1mg/ml BSA, 5 microlitre 1mg/ml poly dI:dC, 50 microlitre polypeptide-magnetic beads;

(3) the ssDNA storehouse of 48 milliliters is distributed into the test tube of two 50 milliliters, puts into shaking table room temperature shake insulation 30 minutes;

After (4) 30 minutes, the ssDNA storehouse of 48 milliliters is loaded the test tube of 24 2 milliliters, be put on magnetic bead frame, remove supernatant liquor;

(5) with 500 microlitre PBST, the magnetic bead in 24 test tubes is washed down, insert in the test tube of 2 milliliters;

(6) magnetic bead is washed twice with PBST;

(7), after removing supernatant, the water adding 138 microlitres mixes with magnetic bead;

(8) mixed solution of 138 microlitres is dispensed into 6 PCR pipe, each pipe 23 microlitre;

(9) add 1 microlitre Apt-F and 1 microlitre Apt-FR (100 M) at each test tube, ultimate density is 2 M, 25 microlitre Lucigen Taq98, Hot Start 2X Master Mix;

(10) PCR reaction: 95 DEG C, 4 minutes; 95 DEG C 30 seconds, 56 DEG C 30 seconds, 68 DEG C 30 seconds, repeat 30 times; 68 DEG C 4 minutes;

(11), after PCR reaction, the PCR primer of 6 test tubes is moved into a test tube, be put into by test tube on magnetic bead frame, supernatant is moved into (PCR (1)) in clean tube;

(12) run 2% agarose gel electrophoresis with 10 microlitre PCR (1) products, add GeneGreen, 1X TAE damping fluid, 100V, 45 minutes;

(13) PCR (1) product observed by UV lamp, has an obvious band at about 85bp place;

(14) in PCR (1) product of 250 microlitres, the 5M sodium-chlor of 64 microlitres is added, 2 microgram M-280Streptavidin magnetic beads (using front PBST to wash twice, each 1 milliliter), shaking table incubated at room 10 minutes;

(15) wash magnetic bead with PBST, each 1 milliliter, wash 3 times altogether;

(16) toward with the sodium hydroxide adding the freshly prepared 100mM of 140 microlitre in the test tube of magnetic bead, incubated at room is shaken 5 minutes;

(17) be put into by test tube on magnetic bead frame, ssDNA is moved in clean tube;

(18) in test tube, 1 milliliter of PBST is added, the 100mM phosphoric acid buffer of 10 microlitres, pH 7.5;

(19) test tube is heated 4 minutes at 95 DEG C, then put into immediately on ice, 10 minutes;

(20) ssDNA of sex change is joined PBST (the 1mg/ml BSA containing 50 microlitres, the poly dI of 5 microlitre 1mg/ml of 50 milliliters; DC, and the polypeptide-magnetic bead of 20 microlitres), be then divided in the test tube of two 50 milliliters;

(21) two test tubes are put into shaking table, shake incubated at room 30 minutes;

(22) on magnetic bead frame, put into 2 milliliters of test tubes, the ssDNA/ of 50 milliliters polypeptide-magnetic bead solution is dispensed in 25 2 milliliters of test tubes;

(23) remove supernatant liquor, the magnetic bead in 25 test tubes is merged in 2 milliliters of test tubes;

(24) magnetic bead is washed twice with 1 milliliter of PBST;

(25) with the deionized water of 93 microlitres, magnetic bead is mixed, be dispensed in 4 PCR test tubes, often pipe 23 microlitre, then add 1 microlitre Apt-F, 1 microlitre Apt-RB, 25 microlitre Lucigen Taq98, Hot Start 2X Master Mix;

(26) PCR reaction: PCR reacts: 95 DEG C, 4 minutes; 95 DEG C 30 seconds, 56 DEG C 30 seconds, 68 DEG C 30 seconds, repeat 25 times; 68 DEG C 4 minutes;

(27) be merged in 1 test tube by 4 pipe PCR primer, PCR primer is put on magnetic bead frame, and supernatant is moved into (PCR (2)) in clean tube;

(28) run 2% agarose gel electrophoresis with 10 microlitre PCR (2) products, add GeneGreen, 1X TAE damping fluid, 100V, 45 minutes;

(29) PCR (2) product observed by UV lamp, has an obvious band at about 85bp place;

(30) in PCR (2) product of 150 microlitres, the 5M sodium-chlor of 38 microlitres is added, the M-280Streptavidin magnetic bead (washing twice with the PBST of 1 milliliter before using) of 1 milligram, shake incubated at room 10 minutes;

(31) three times are washed with 1 milliliter of PBST;

(32) in test tube, the sodium hydroxide of the 100mM that 50 microlitres are newly prepared is added, room temperature 5 minutes;

(33) be put into by test tube on magnetic bead frame, ssDNA moves in clean tube;

(34) in test tube, 1 milliliter of PBST is added, the 100mM phosphoric acid buffer pH 7.5 of 10 microlitres;

(35) by heating test-tube 95 DEG C, 4 minutes, 4 DEG C of water are put into immediately, 10 minutes;

(36) ssDNA of sex change is joined PBST (the 1mg/ml BSA containing 50 microlitres, the poly dI of 5 microlitre 1mg/ml of 50 milliliters; DC, and the polypeptide-magnetic bead of 20 microlitres), be then dispensed in the test tube of two 50 milliliters;

(37) two test tubes are put into shaking table, shake incubated at room 30 minutes;

(38) on magnetic bead frame, put into 2 milliliters of test tubes, the ssDNA/ of 50 milliliters polypeptide-magnetic bead solution is dispensed in 25 2 milliliters of test tubes;

(39) remove supernatant liquor, the magnetic bead in 25 test tubes is merged in 2 milliliters of test tubes;

(40) magnetic bead is washed twice with 1 milliliter of PBST;

(41) with the deionized water of 93 microlitres, magnetic bead is mixed, be dispensed in 4 PCR test tubes, often pipe 23 microlitre, then add 1 microlitre Apt-F, 1 microlitre Apt-RB, 25 microlitre Lucigen Taq98, Hot Start 2X Master Mix;

(42) PCR reaction: PCR reacts: 95 DEG C, 4 minutes; 95 DEG C 30 seconds, 56 DEG C 30 seconds, 68 DEG C 30 seconds, repeat 20 times; 68 DEG C 4 minutes;

(43) be merged in 1 test tube by 4 pipe PCR primer, PCR primer is put on magnetic bead frame, and supernatant is moved into (PCR (3)) in clean tube;

(44) run 2% agarose gel electrophoresis with 10 microlitre PCR (3) products, add GeneGreen, 1X TAE damping fluid, 100V, 45 minutes;

(45) PCR (3) product observed by UV lamp, has an obvious band at about 85bp place;

(46) oppositely screen magnetic bead: the magnetic bead M-270 without polypeptide adding 50 microlitres in test tube, wash twice with the PBST of 1 milliliter;

(47) PCR (3) of past 150 microlitres adds the 5M sodium-chlor of 38 microlitres, 1 microgram M-280-Streptavidin magnetic bead (washing twice with 1 milliliter of PBST before using);

(48) room temperature shake hatches 10 minutes;

(49) three times are washed with 1 milliliter of PBST;

(50) in test tube, the freshly prepared 100mM sodium hydroxide of 50 microlitres is added, incubated at room 5 minutes;

(51) be put into by test tube on magnetic bead frame, ssDNA supernatant liquor moves in clean tube;

(52) in ssDNA test tube, 1 milliliter of PBST is added, the 100mM phosphoric acid buffer of 10 microlitres, pH 7.5;

(53) by ssDNA heating test-tube 95 DEG C, 4 minutes, 4 DEG C of water are put into immediately;

(54) ssDNA is joined in M-270 magnetic bead test tube (46), incubated at room 10 minutes;

(55) be put into by test tube on magnetic bead frame, the ssDNA oppositely after screening is moved into (ssDNA C1) in clean tube;

(56) in the magnetic bead test tube containing M-270, the water of 93 microlitres is added, mixing;

(57) PCR reaction: 23 microlitres contain the water of magnetic bead, 1 microlitre Apt-F, 1 microlitre Apt-RB, 25 microlitre LucigenTaq98, Hot Start 2X Master Mix.95 DEG C, 4 minutes; 95 DEG C 30 seconds, 56 DEG C 30 seconds, 68 DEG C 30 seconds, repeat 20 times; 68 DEG C 4 minutes;

(58) run 2% agarose gel electrophoresis with 10 microlitres, add GeneGreen, 1X TAE damping fluid, 100V, 45 minutes;

(59) PCR primer observed by UV lamp, has an obvious band at about 85bp place;

(60) 150 microlitre ssDNA C1 are heated 95 DEG C, 4 minutes, put in 4 DEG C of water immediately;

(61) the ssDNA C1 of sex change is joined PBST (the 1mg/ml BSA containing 50 microlitres, the poly dI of 5 microlitre 1mg/ml of 50 milliliters; DC, and the polypeptide-magnetic bead of 20 microlitres), be then dispensed in the test tube of two 50 milliliters;

(62) two test tubes are put into shaking table, shake incubated at room 30 minutes;

(63) on magnetic bead frame, put into 2 milliliters of test tubes, the ssDNA/ of 50 milliliters polypeptide-magnetic bead solution is dispensed in 25 2 milliliters of test tubes;

(64) remove supernatant liquor, the magnetic bead in 25 test tubes is merged in 2 milliliters of test tubes;

(65) magnetic bead is washed twice with 1 milliliter of PBST;

(66) with the deionized water of 93 microlitres, magnetic bead is mixed, be dispensed in 4 PCR test tubes, often pipe 23 microlitre, then add 1 microlitre Apt-F, 1 microlitre Apt-RB, 25 microlitre Lucigen Taq98, Hot Start 2X Master Mix;

(67) PCR reaction: 95 DEG C, 4 minutes; 95 DEG C 30 seconds, 56 DEG C 30 seconds, 68 DEG C 30 seconds, repeat 15 times; 68 DEG C 4 minutes;

(68) be merged in 1 test tube by 4 pipe PCR primer, PCR primer is put on magnetic bead frame, and supernatant is moved into (PCR (4)) in clean tube;

(69) run 2% agarose gel electrophoresis with 10 microlitre PCR (4) products, add GeneGreen, 1X TAE damping fluid, 100V, 45 minutes;

(70) PCR (4) product observed by UV lamp, has an obvious band at about 85bp place;

(71) screening is below identical with aforesaid method, and PCR is repetition 20 times.Wherein oppositely screen after PCR (6), PCR (9) and PCR (12);

(72) primer Apt-F and Apt-R of PCR (16);

(73) PCR (16) product cloning is to TOPO TA plasmid, then proceeds to One Shot Top 10E.coli, extracts plasmid and carries out determined dna sequence.

Sequencing result (1):

Altogether determined dna sequence is carried out to 40 samples, wherein 18 have identical sequence (Apt-1), 12 have identical sequence (Apt-2), 8 have identical sequence (Apt-3), two other has identical sequence (Apt-4), as follows:

Apt-1:GGA TAA GCC GCA TTA GCT TAC TTC AGT GAT GCT TAA GAT CTA GTA(SEQID NO.1)

Apt-2:GCT TAC GCG GCA TTC GCT TAC TTC ACT GTA GCT TCA GTT CTG CTA(SEQ IDNO.2)

Apt-3:GCA GTA GCC GCT TTC GCT TAG TTC ATC CTA CTT TCA GAT CTA CTA(SEQ IDNO.3)

Apt-4:GGA GTA CGG CTT ATT CCT ATT GAT ACT GCA CTT ACT GGT GTA GCC(SEQID NO.4)

Embodiment 2

As shown in Figure 1, for the embodiment of the present invention can detect nucleic acid aptamer SEQ IDNO.1 (Apt-1), the SEQ ID NO.2 (Apt-2) of ENOX2 albumen in Patients with Various Cancers, SEQ ID NO.3 (Apt-3) and or SEQ ID NO.4 (Apt-4) scheme in conjunction with the Western blot of ENOX2; The cDNA of mankind ENOX2 is cloned in pET-11a expression plasmid, proceeds to intestinal bacteria, with IPGE abduction delivering ENOX2 albumen, then extracts the total protein in inclusion body, measures the concentration of protein.Get 1 microgram inclusion body total protein albumen SDS-PAGE glue is separated according to molecular weight, then albumen is forwarded on nitrocellulose membrane and carry out WESTERN BLOT analysis.Apt-1, Apt-2, Apt-3 and Apt-4 biotin labeling, biotin labeled four nucleic acid aptamers and nitrocellulose membrane were incubation at room temperature one hour, after the unconjugated aptamer of wash-out, hatch with Streptavidin-alkaline phosphatase and film, room temperature is wash-out after one hour again, and the substrate adding alkaline phosphatase carries out color reaction.

As shown in Figure 2, the nucleic acid aptamer SEQ IDNO.1 (Apt-1) that can detect ENOX2 albumen in Patients with Various Cancers for the embodiment of the present invention schemes in conjunction with the Western blot of ENOX2; The cDNA of mankind ENOX2 is cloned in pET-11a expression plasmid, proceeds to intestinal bacteria, with IPGE abduction delivering ENOX2 albumen, then extracts the total protein in inclusion body, measures the concentration of protein.Get 1 microgram inclusion body total protein albumen SDS-PAGE glue is separated according to molecular weight, then albumen is forwarded on nitrocellulose membrane and carry out WESTERN BLOT analysis.Apt-1 biotin labeling, this nucleic acid aptamer biotin labeled and nitrocellulose membrane were incubation at room temperature one hour, after the unconjugated aptamer of wash-out, hatch with Streptavidin-alkaline phosphatase and film again, room temperature is wash-out after one hour, and the substrate adding alkaline phosphatase carries out color reaction.

The protein-bonded functional examination of nucleic acid aptamer, method is as follows:

(1) cDNA of mankind mankind ENOX2 albumen is cloned into colibacillus expression plasmid pET-11a, and plasmid is proceeded in intestinal bacteria;

(2) with IPTG process intestinal bacteria, abduction delivering ENOX2 albumen;

(3) in inclusion bodies of colibacillus, ENOX2 albumen is extracted;

(4) by 10%SDS polyacrylamide gel electrophoresis by ENOX2 albumen sepn, and by electricity turn by protein delivery on nitrocellulose membrane;

(5) nitrocellulose membrane is closed with the degrease milk powder of 5%, room temperature 1 hour;

(6) nitrocellulose membrane is hatched with the Apt-1 that vitamin H (Biotin) marks, room temperature 2 hours;

(7) film is washed four times, each 10 minutes with elution buffer (10mM Tris, pH 7.5,150mM NaCl, 0.05%Triton-100);

(8) nitrocellulose membrane is hatched with Streptavidin-alkaline phosphatase (Streptavidin-AP), room temperature one hour;

(9) film is washed four times with elution buffer, each 10 minutes;

(10) with AP nitrite ion (every milliliter of AP damping fluid adds 10 microlitre BCIP solution and 10 microlitre NBT solution), develop the color 3-30 minute.

Experimental result is shown in Fig. 1 and Fig. 2 shown in the embodiment of the present invention.

Embodiment 3Apt-1 is to the activity inhibition of ENOX2 albumen

The aminoacid sequence of mankind ENOX2 albumen is SEQ ID NO.11.

Whether can suppress the activity of mankind ENOX2 for measuring Apt-1, present invention employs the effect of reductase activity to thioredoxin of ENOX2.For measuring the generation of mercaptan, Regular Insulin and the rear turbidity reaction occurred of DTT mixing.Result of the present invention shows, after adding the recombinant human ENOX2 albumen of S-tag, turbidity obviously strengthens, and shows have a large amount of mercaptan to produce.After adding Apt-1 in the reaction, obviously inhibit the reductase activity of recombinant human ENOX2.Result is as shown in table 2, visible, and Apt-1 can effectively in conjunction with mankind ENOX2 albumen, and this specific nucleic acid aptamer can be used for detecting the ENOX2 albumen in serum, carries out the early diagnosis of cancer.Meanwhile, Apt-1 then can effectively suppress it active in conjunction with ENOX2, can be used for the treatment of cancer.

In the present invention, nucleic acid aptamer can be single stranded RNA, screens with the peptide chain of single stranded RNA storehouse to ENOX2, and the single stranded RNA filtered out can replace single stranded DNA nucleic acid aptamer of the present invention to carry out the detection of ENOX2 albumen;

Nucleic acid aptamer can also be double-stranded DNA, screens with the peptide chain of double-stranded DNA storehouse to ENOX2, and the double-stranded DNA filtered out can replace single stranded DNA nucleic acid aptamer of the present invention to carry out the detection of ENOX2 albumen;

Nucleic acid aptamer can also be double-stranded RNA, screens with the peptide chain of double-stranded RNA storehouse to ENOX2, and the double-stranded RNA filtered out can replace single stranded DNA nucleic acid aptamer of the present invention to carry out the detection of ENOX2 albumen.

Table 1 sequence table

Table 1 and SEQ ID NO.11 list nucleic acid aptamer storehouse, the aminoacid sequence of the DNA sequence dna of PCR primer, four nucleic acid aptamers, the aminoacid sequence of ENOX2 conserved sequence and mankind ENOX2 albumen.

Table 2 Apt-1 suppresses the reductase activity of ENOX2

	1	2	3	4
					Regular Insulin+DTT	100	100	100	100
Regular Insulin+DTT+ENOX2	378.21	377.98	365.84	346.92
					Regular Insulin+DTT+ENOX2+Apt-1	139.22	116.82	132.85	129.78

From table 2, restructuring ENOX2 albumen adds the turbidity after Regular Insulin+DTT, shows that ENOX2 albumen has the activity of reductase enzyme.After adding nucleic acid aptamer Apt-1 in reaction, inhibit the reductase activity of ENOX2 albumen, reacted turbidity is reduced.

Claims (5)

1. can detect a nucleic acid aptamer for ENOX2 albumen in Patients with Various Cancers, it is characterized in that, its nucleotide sequence as

Shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4:

SEQ ID NO.1：GGATAAGCCG CATTAGCTTA CTTCAGTGAT GCTTAAGATC TAGTA；

SEQ ID NO.2：GCTTACGCGG CATTCGCTTA CTTCACTGTA GCTTCAGTTC TGCTA；

SEQ ID NO.3：GCAGTAGCCG CTTTCGCTTA GTTCATCCTA CTTTCAGATC TACTA；

SEQ ID NO.4：GGAGTACGGC TTATTCCTAT TGATACTGCA CTTACTGGTG TAGCC。

2. the nucleic acid aptamer detecting ENOX2 albumen in Patients with Various Cancers according to claim 1, is characterized in that, the conserved sequence of described ENOX2 albumen is as shown in SEQ ID NO.5:

SEQ ID NO.5：EEAKEK FKQALSGILI QFE

Namely

Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu。

1 5 10 15

3. the ENOX2 peptide chain of the conserved sequence synthesis of ENOX2 albumen according to claim 2, it is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.6:

SEQ ID NO.6：EEAKEKFKQALSGILIQFE

Namely

Ac Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu Amide。

1 5 10 15

4. the DNA library detecting the nucleic acid aptamer of ENOX2 albumen in Patients with Various Cancers according to claim 1, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.7, and wherein, N is any one in ATGC, totally 45 N:

SEQ ID NO.7：

GGT ATT GAG GGT CGC ATC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN GAT GGC TCT AAC TCT CCT CT。

5. the nucleic acid aptamer detecting ENOX2 albumen in Patients with Various Cancers according to claim 1, is characterized in that, is applied in early diagnosis of cancer and passes through to suppress the activity of ENOX2 albumen in cancer therapy.