CN107164383A - One group of creatine kinase isozyme aptamer and its application - Google Patents
One group of creatine kinase isozyme aptamer and its application Download PDFInfo
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Abstract
The present invention relates to one group of creatine kinase isozyme aptamer and its application, belong to biological technical field.The nucleotide sequence of aptamer SEQ IDNo.1~SEQ ID No.10 in nucleotides sequence list, the binding specificity height of the aptamer and creatine kinase isozyme, affinity are high.The aptamer can be used for detecting creatine kinase isozyme, separating, purifying and immobilization, the analysis detection of preparation, target substance applied to biological inductor, be particularly suitable for use in the Test paper prepared in creatine kinase isozyme quick detection kit.Some advantages that detection does not possess with antibody are carried out to creatine kinase isozyme using the aptamer, such as in-vitro screening, the features such as synthesize, be easy to modification and high stability.
Description
Technical field
The present invention relates to one group of creatine kinase isozyme aptamer and its application, specifically, it is related to a kind of utilization
SELEX technologies in Protocols in Molecular Biology, i.e. phyletic evolution index concentration technology, prepare one group and have with creatine kinase isozyme
There is the aptamer of high specific and high-affinity, and applied.Belong to biological technical field.
Background technology
Creatine kinase isozyme (Creatine Kinase-MB) is a kind of clinically for diagnosing acute myocardial infarction
Biomarker.Creatine kinase isozyme enters blood on the 4th~6 hour what acute myocardial infarction AMI occurred, and peak was reached at the 24th hour
Value, normal level was returned to after 48~72 hours.The quick diagnosis of morbidity early stage is beneficial to formulate more excellent therapeutic scheme, rises
To preferable therapeutic effect.
At present, use colloidal gold immuno-chromatography test paper strip quick detection more.Colloidal gold immuno-chromatography test paper strip is exactly with glue
Body gold is colour developing medium, can specifically bind principle using antigen-antibody in immunology, this be completed in chromatography process anti-
Should, so as to reach the purpose of detection.Colloidal gold immunity chromatography is that with ribbon specific antigen or antibody are fixed on into film
On, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on pad, by the way that sample is added drop-wise to colloid gold immune
After in the sample pad of the test strips of chromatography, sample moves forward by capillarity, first passes around collaurum-antibody knot on pad
Compound, fully contacts with it and occurs corresponding reaction, by being advanced to forward up to antigen fixed position for continuation, and send out therewith
Raw reaction, the conjugate of thing and gold marked reagent to be checked occurs specific binding therewith again and is left, and can observe by the naked eye
The red of collaurum.
The appearance of aptamer provides new solution for the detection of Biological indicators.Aptamer is a kind of single
Chain DNA or RNA, specifically can be combined with target molecule.It is the discovery that in nineteen ninety earliest, Ellington and Tuerk
Their result of study has been delivered on Nature and Science respectively., can be by referring to for a given target molecule
Aglucon phyletic evolution technology (the Systematic Evolution of Ligands by Exponential of number enrichment
Enrichment, SELEX) from specific oligonucleotide library screening obtain with its have specificity interact nucleic acid be adapted to
Body.From nineteen ninety, researcher constantly has found new aptamer, and the molecular range that these aptamers are combined is covered
Albumen, cell and some small molecules.
Aptamer is used for test strips, the advantage of aptamer can be given full play to, because relative to antibody, nucleic acid
The scope for being adapted to physical efficiency generation affinity interaction is wider, and cost is lower, it is easy to modify and property is more stable.Importantly, nucleic acid
Aptamers not simply perform the function of antibody, the detection side being identified for the binding ability using itself and target molecule
Method.The application of aptamer also includes：Realize that signal is changed using the aptamer through modification, utilize aptamer
Crosslinking ability carries out fluid control, and signal amplification is carried out using the amplification ability of aptamer.
Up to the present, there is high-affinity and specific aptamer with creatine kinase isozyme and based on core
The quick determination method of the creatine kinase isozyme of sour aptamers is not reported.
The content of the invention
In order to overcome the shortcomings of existing detection method, an object of the present invention is to provide one group of creatine kinase isozyme
Aptamer, the aptamer has high specific and high-affinity with creatine kinase isozyme.
The second object of the present invention is the application for providing one group of creatine kinase isozyme aptamer, and the nucleic acid is fitted
The combination of part and creatine kinase isozyme has high specific and high-affinity, is examined available for creatine kinase isozyme
Survey, separate, purify and immobilization, the analysis detection of preparation, target substance applied to biological inductor.
To realize that the purpose of the present invention is as follows there is provided technical scheme.
One group of creatine kinase isozyme aptamer, described aptamer can be with protein of creatine kinase isoenzyme knot
Close, the nucleotide sequence of the aptamer numeric identifier in nucleotides sequence list<210>Represented sequence mark
Know the nucleotide sequence described in symbol 1~10, i.e., SEQ IDNo.1~SEQ ID No.10 in nucleotides sequence list.
Further, it is preferable to the nucleotide sequence of aptamer numeric identifier in nucleotides sequence list<
210>SEQ ID No.6~SEQ in nucleotide sequence described in represented sequence identifier 6~10, i.e. nucleotides sequence list
ID No.10.Nucleotide sequence in nucleotides sequence list shown in SEQ ID No.6~SEQ ID No.10 is by nucleosides
Preferably go out number of base respectively in every of nucleotide sequence in acid sequence table shown in SEQ ID No.1~SEQ ID No.5
Obtained by, the affinity to creatine kinase isozyme is remained, but because base is less, chain is shorter, and cost only has nucleotides
/ 8th of nucleotide sequence in sequence table shown in SEQ ID No.1~SEQ ID No.5.
Further, in the nucleotide sequence in nucleotides sequence list shown in SEQ IDNo.1~SEQ ID No.10,
5 ' one end or 3 ' one end of every nucleotide sequence are modified with sulfydryl or biotin, obtain one group and are modified with sulfydryl or biology
The creatine kinase isozyme aptamer of element, has essentially identical or similar molecule knot with aptamer of the present invention
Structure, physicochemical property and function, can be used in detecting creatine kinase isozyme.
The application of one group of creatine kinase isozyme aptamer, the application is by aptamer of the present invention
For being detected, being separated to creatine kinase isozyme, purified and immobilization, preparation, target applied to biological inductor
The analysis detection of material, and prepare the reagent for clinical diagnosis and the medicine of disease treatment；
Further, it is preferable to the application be the aptamer in creatine kinase isozyme detection kit is prepared
Application；
Further, 5 ' one end or 3 ' one end of the nucleotide sequence of aptamer of the present invention are modified with mercapto
Base or biotin, obtain being modified with the creatine kinase isozyme aptamer of sulfydryl or biotin applying.
A kind of creatine kinase isozyme detection kit, the detection kit includes Test paper, the Test paper
Including bottom plate, the sample pad for being bonded on bottom plate and overlapping successively, pad, nitrocellulose filter and adsorptive pads；The nitric acid
It is provided with cellulose membrane close to the side of pad on detection line, nitrocellulose filter and is provided with Quality Control close to the side of adsorptive pads
Line；5 ' one end or 3 ' one end are coated with described pad by colloid gold label, and 5 ' one end or 3 ' one end are connected with non-fit
The A aptamers of part nucleotide sequence；The B aptamers combined with Streptavidin, Quality Control are coated with detection line
The complementary nucleotide sequence combined with Streptavidin is coated with line, the complementary nucleotide sequence is and the non-aptamers
The complementary nucleotide sequence of nucleotide sequence.
Wherein, A aptamers are different with the nucleotide sequence of B aptamers, and are respectively selected from core of the present invention
SEQ IDNo.1~SEQ ID No.5 in nucleotide sequence table, or the SEQ being respectively selected from nucleotides sequence list of the present invention
IDNo.6~SEQ ID No.10；It is preferred that the SEQ IDNo.6~SEQ ID being respectively selected from nucleotides sequence list of the present invention
No.10；Wherein, it is the nucleotides sequence shown in the SEQ IDNo.2 in the nucleotides sequence list that should exclude A aptamers
During row, B aptamers are the situation of the nucleotide sequence shown in the SEQ IDNo.5 in the nucleotides sequence list, and A
When aptamer is the nucleotide sequence shown in the SEQ IDNo.7 in the nucleotides sequence list, B aptamers are institute
State the situation of the nucleotide sequence shown in the SEQ IDNo.10 in nucleotides sequence list.
A kind of preparation method of Test paper in detection kit of the present invention, methods described step is as follows：
(1) by 5 ' one end of A aptamers or 3 ' one end colloid gold labels, and 5 ' one end or 3 ' one end connection upper one
The non-aptamers nucleotide sequence of section, for hybridizing with the complementary nucleotide sequence on nature controlling line；
(2) sample pad, pad are put into immersion more than 30min in the phosphate buffer containing BSA and tween, drying
It is standby；
(3) the A aptamer solution for the colloid gold label for obtaining step (1) is dispersed in the treated knot of step (2)
Close on pad；
(4) the B aptamers and complementary nucleotide sequence that are combined with Streptavidin are fixed on nitrocellulose filter
On be used as detection line and nature controlling line, dry for standby；
(5) sample pad, pad, nitrocellulose filter and adsorptive pads are overlapped and are bonded on bottom plate successively, be prepared into
To the Test paper.
Wherein, after 5 ' one end of A aptamers or 3 ' one terminal modified sulfydryls, then colloid gold label is used；B aptamers
5 ' one end or 3 ' one end be modified with biotin, 5 ' one end or 3 ' one end of complementary nucleic acid sequences are modified with biotin.
Preferably, the A aptamer preparation methods of colloid gold label are as follows：
(1) the A aptamers that 5 ' one end or 3 ' one end are modified with into sulfydryl add TE buffer solutions (Tris-EDTA
Buffer) wiring solution-forming 1, takes solution 1 to add in three (2- carboxyethyls) phosphines (TCEP), reacts 1h~2h, obtains solution 2, wherein,
The mol ratio of TCEP and the A aptamers for being modified with sulfydryl is 5:1~50:1；
(2) collaurum is centrifuged into 10min~30min under 8000r/min~12000r/min, abandons supernatant, added ultrapure
Water is mixed, and is then mixed with solution 2, is finally shaken 1min, 4 DEG C, to ambient temperature overnight, obtain solution 3；
The volume ratio of the collaurum, ultra-pure water and solution 2 is 500:50:0.2~500:50:20.
(3) NaCl aqueous solution addition solution 3 is well mixed, the concentration for obtaining NaCl in solution 4, solution 4 is 20mM, will
Solution 4 shakes 0.5min~1min, 4 DEG C to room temperature reaction 6h~12h；
(4) the NaCl aqueous solution is added in the solution 4 after being handled to step (3), the concentration of NaCl in solution 5, solution 5 is obtained
For 40mM, solution 5 is shaken into 0.5min~1min, 4 DEG C to room temperature reaction 10h~24h；
(5) solution 5 after step (4) processing is centrifuged into 10min~30min under 8000r/min~1200r/min, abandoned
Supernatant, aggravates suspension to original volume, obtains the liquid of the A aptamers containing colloid gold label；
The re-suspension liquid is by the 0.005g of polyethylene glycol (PEG) 20000, sucrose 0.5g, the μ L of tween (Tween) -20 10
It is dissolved in 10mL Tris- hydrochloric acid solutions and is made with bovine serum albumin(BSA) (BSA) 0.1g；
(6) liquid for the A aptamers containing colloid gold label for obtaining step (5) directly with pipettor point pre-
On treated pad, dry, obtain being coated with the pad of the A aptamers of colloid gold label, it is standby；The pre- place
Manage and be：The phosphate that pad is immersed in into the 50mL added with 1g bovine serum albumin(BSA)s, 1.5g sucrose and 0.01g Sodium azides delays
30min in fliud flushing, is then rinsed using a large amount of phosphate buffers.
Preferably, the B aptamers combined with Streptavidin or the complementary nucleotide sequence combined with Streptavidin
The preparation method of row is as follows：
(1) raw material is dissolved with phosphate buffer, obtains the solution that concentration is 100 μM；
Raw material is 5 ' one end or 3 ' one end are modified with the B aptamers of biotin or 5 ' one end or 3 ' one end are modified with life
The complementary nucleotide sequence powder of thing element.
(2) the μ L of μ L of 1mg/mL Streptavidins 42~168 are added in the solution prepared to step (1), are mixed evenly,
1h~3h is placed in room temperature, mixed solution is obtained；
The mol ratio of raw material and Streptavidin is 4:1~1:1.
(3) mixed solution for obtaining step (2) with 30KD super filter tubes at 4 DEG C to being centrifuged at room temperature with 12000r/min
More than 10min, is inverted super filter tube, at 4 DEG C to more than 30min is centrifuged at room temperature with 1000g, obtains the solution after ultrafiltration, obtains
The B aptamers combined with Streptavidin or the complementary nucleotide sequence combined with Streptavidin.
Preferably, detection line and the preparation process of nature controlling line are as follows on nitrocellulose filter：
Concentration is drawn in cellulose nitrate for the 1OD/30 μ L~5OD/30 μ L B aptamers combined with Streptavidin
On plain film at pad side about 1/3, obtain being coated with the detection line of the B aptamers combined with Streptavidin,
Concentration is drawn in cellulose nitrate for the 0.025OD/30 μ L~1OD/30 μ L complementary nucleotide sequence combined with Streptavidin
On plain film at adsorptive pads side about 1/3, obtain being coated with the Quality Control of the complementary nucleotide sequence combined with Streptavidin
Line；During line, the flow velocity of detection line and nature controlling line is 1 μ L/cm；After line, place more than 2h in room temperature and fixation is dried, obtain
To the nitrocellulose filter for having marked detection line and nature controlling line.
Preferably, the preparation method of the Test paper is as follows：
Sample pad is pre-processed：Sample pad is immersed in added with 0.5g bovine serum albumin(BSA)s (BSA), 50 μ L
30min in Tween-20 50mL phosphate buffer, is then rinsed, drying for standby using a large amount of phosphate buffers.
By the sample pad by pretreatment, the pad for the A aptamers for being coated with colloid gold label, the good detection of mark
The nitrocellulose filter and adsorptive pads of line and nature controlling line are overlapped and are adhered on bottom plate successively, wherein, mark detection line and matter
The nitrocellulose filter of control line is fixed on bottom plate, and one end is pushed down by adsorptive pads, and the other end is combined pad and pushed down, and pad is by sample
Product pad is pushed down, and obtains the Test paper.
A kind of detection method of creatine kinase isozyme detection, the detection method passes through a kind of creatine of the present invention
Kinase isozyme detection kit is realized, is specially：
The μ L of μ L of phosphate buffer 60 containing protein of creatine kinase isoenzyme~100 are added dropwise in the detection kit
In the sample pad of middle Test paper, solution is moved to adsorptive pads under capillary action, after 5min~20min, and detection line presents clear
Red stripes, illustrate there is protein of creatine kinase isoenzyme, if detection line does not occur red stripes, illustrate that no creatine kinase is same
Work zymoprotein, detection terminates.
Beneficial effect
1. the invention provides one group of creatine kinase isozyme aptamer, the aptamer and creatine kinase are same
The binding specificity of work enzyme is high, affinity is high.
2. the invention provides the application of one group of creatine kinase isozyme aptamer, using the aptamer pair
Creatine kinase isozyme carries out some advantages that detection does not possess with antibody, such as in-vitro screening, synthesizes, is easy to modify and stably
The features such as property is high.
3. the invention provides the application of one group of creatine kinase isozyme aptamer, what is filtered out is same with creatine kinase
Work enzyme has the aptamer of high specific and high-affinity, can be with specific combination creatine kinase isozyme, for making
Test paper in standby creatine kinase isozyme quick detection kit.
Brief description of the drawings
Fig. 1 is each single stranded DNA and flesh for taking turns screening in creatine kinase isozyme aptamer screening process in embodiment 1
Relative affinity between acid kinase isodynamic enzyme.
Fig. 2 is the structural representation of obtained Test paper in 7 in embodiment.
In figure, 1-sample pad, 2-pad, 3-nitrocellulose filter, 4-adsorptive pads, 5-bottom plate, 6-detection line,
7-nature controlling line.
Embodiment
Following embodiment is easy to be better understood from the present invention, but is not limited to the present invention.
In following examples：
Experimental method is conventional method unless otherwise specified.
Experiment material used is to buy resulting from routine biochemistry reagent shop unless otherwise specified.
The present invention sieves target using the in-vitro screening SELEX technologies of aptamer using creatine kinase isozyme to be positive, with
Tosyl magnetic bead is counter-selection target, is filtered out and creatine kinase isozyme from the random oligo DNA library synthesized in vitro
The aptamer of specific bond.
Nucleic acid aptamer sequence of the present invention may be selected from naturally occurring or artificial synthesized sequence, or any other comes
The same sequence in source.
Heretofore described nucleic acid aptamer sequence contains and the nucleotides of the characteristic sequence all identical sequence
Row.Above-mentioned aptamer is combined after can using colloid gold label or biotin modification after sulfydryl modification with Streptavidin, in
Applied in subsequent detection kit.
Embodiment 1
1. the selection and synthesis of single-stranded DNA banks
Following 5 kinds of DNA are synthesized by Sangon Biotech (Shanghai) Co., Ltd.：One DNA containing 81 bases
Storehouse, its nucleotide sequence is：ATCCAGAGTGACGCAGCA(N 45)TGGACACGGTGGCTTAGT；" P1 biotins upstream "：
ATCCAGAGTGACGCAGCA；" P2 biotins downstream "：ACTAAGCCACCGTGTCCA；" P3 upstreams "：
ATCCAGAGTGACGCAGCA；" P4 downstreams "：ACTAAGCCACCGTGTCCA.Often pipe 1OD DNA are, it is necessary in use, before uncapping
12000rpm centrifuges 0.5min；100 μM of storing liquids are made into according to the explanation on tube wall.
2. tosyl magnetic bead is combined with creatine kinase isozyme
Tosyl magnetic bead (Dynal, 2mL) reagent bottle using preceding need carry out vortex concussion.Take 16.5 μ L (2 × 107
It is individual) magnetic bead, washed twice with Buffer B (0.1M Na-phosphate buffer pH=7.4), 1mL is added every time, after addition,
Be vortexed concussion, and 1.5mL centrifuge tubes are placed on magnetic collection device, after 1min, is treated that solution is clarified, is inhaled on magnetic frame with pipettor
Remove supernatant.Cleaning process removes the interference in magnetic bead solution.During the incubation of magnetic bead and protein of creatine kinase isoenzyme, protein content with
Magnetic bead amount has adjustment with experiment process：1 wheel~7 is taken turns, 16.5 μ L (2 × 107It is individual) magnetic bead and 5 μ g creatine kinase isozymes
(PROSPEC, 1mg/126.5 μ L) protein binding.Magnetic bead is resuspended with Buffer B to 75 μ L, then adds 0.63 μ L (5 μ g) flesh
Acid kinase isodynamic enzyme and 50 μ L Buffer C (3M ammonium sulphate in Buffer B), 4 DEG C of rotations are incubated 18h.
Magneto separate removes supernatant, adds Buffer D (PBS pH=7.4, phosphate buffer) 1mL, continues to shake 1h.Hereafter, Buffer is used
E (PBST pH=7.4) is washed twice, 1mL/ times, and be vortexed cleaning.It is final to be resuspended with 200 μ L Buffer D (PBS).8 wheels~9 are taken turns,
Magnetic bead amount is constant, and 0.63 μ L creatine kinase isozymes reduce to 0.5 μ L (4 μ g).10 wheels, 12.7 μ L magnetic beads, 0.5 μ L creatine kinases are same
Work enzyme.
3. positive screening and the counter-selection of empty magnetic bead are selected
It is preferred that 1OD DNA libraries are dissolved in 240 μ L PBS during positive screening, and the same work of creatine kinase after being resuspended with 500 μ L
Magnetic bead (the 16.5 μ L) mixing that enzyme is combined, carries out first round screening.In follow-up screening, for what is combined with creatine kinase isozyme
The storehouse that magnetic bead is incubated is to make single-stranded obtained DNA library after previous round is expanded.During~6 wheel screening of 1 wheel, DNA library swashs with creatine
The magnetic bead that enzyme isoenzyme is combined is incubated 120min in 37 DEG C of shaking tables.With wheel number increase, incubation time is reduced：7 wheels~8, which are taken turns, is
90min, the wheel of 9 wheel~10 is 60min.After the completion of often wheel library and albumen magnetic bead is incubated, PBS three times, last 40 μ L are used
TE buffer solutions are resuspended, and heat 15min in 95 DEG C, the aptamers of affinity and creatine kinase are same by having with creatine kinase isozyme
Work enzyme is separated, and is dissolved in TE buffer solutions, for expanding.
Often just screened by three-wheel, a counter-selection choosing will be carried out, its object is to removed and magnetic bead sheet from DNA library
Body has the DNA of affinity.When counter-selection is selected, previous round prepare it is single-stranded after obtained 240 μ L DNA libraries and 500 μ L be resuspended after magnetic bead
(33 μ L), after mixing, 37 DEG C of shaking tables are incubated after 120min, are collected the solution not combined with magnetic bead and are screened into next round.
4. the amplification and identification in storehouse
Screened since the storehouse of 1OD aptamers, the DNA solution that screening terminates is expanded as template, after amplification,
Identified using 4% agarose gel electrophoresis, deposition condition 110V, 50min, standard DNA applied sample amount is 2 μ L, Polymerization chain reaction
(PCR) sample applied sample amount is 5 μ L.
Performing PCR is entered to the TE DNA libraries being resuspended, 3 kinds are divided into, one is to be used to subsequently screen (P2, P3), and two be to be used to survey phase
To affinity (P1, P4), three be to be connected with carrier (P3, P4).In experiment, PCR reagent consumption is：Each 2.5 μ L of upstream and downstream primer,
The μ L of 0.5 25 μ L water of μ L, PCR mix of DNA library 19.
PCR programs are：95 DEG C of 5min pre-degenerations, then carry out 15 PCR cycles, each include：95℃30s；56.3℃
30s and 72 DEG C of 30s.After circulation terminates, 72 DEG C of 3min complete PCR programs.
The purifying of 5.PCR products
Explanation according to Sangon Biotech's post QIAquick Gel Extraction Kit is purified, and is preheated first
TE to 60 DEG C, Buffer B3, the 8000g 30s in 400 μ L kits are added in 2 pipe (80 μ L) PCR primers, ultrafiltration will be super
Solution in chimney filter outer side sleeve is repeated once to same method in sleeve pipe again, in order to allow DNA as much as possible to be adsorbed by filter membrane.
Then using 500 μ L Washing Buffer cleanings twice, condition is that 9000g centrifuges 30s, discards liquid.Last 9000g,
30s, empty centrifuge tube is centrifuged 1 time.40 preheated μ L TE are applied directly on filter membrane, 2min is stood, the DNA in face is removed in centrifugation
Solution.
6. prepare single-stranded DNA banks
When making single-stranded, the μ L of Streptavidin MagneSphere (Dynal, 2mL) 40 are taken, B＆W buffer (2 are used according to its specification
×) wash twice, 1mL is added every time, after addition, be vortexed concussion, and 1.5mL centrifuge tubes are placed on magnetic collection device, after 1min, treat molten
Liquid is clarified, and supernatant is sucked with pipettor on magnetic frame.Cleaning process removes the interference in magnetic bead solution, after cleaning, uses
Magnetic bead is resuspended to 80 μ L B＆W buffer (10mM Tris-HCl (pH=7.5), 1mM EDTA and 2M NaCl) (2 ×).
Using P2, two kinds of primers of P3 enter the DNA that performing PCR is obtained, and by purifying the double-stranded DNA that volume is 80 μ L, are added to
(40 μ L magnetic beads are fixed as after 3 wheels, no longer survey concentration) in 40 μ L magnetic beads, are mended cumulative volume to 160 μ L with 40 μ L TE.Room temperature
Place 10min.Using 1 × B＆W buffer cleanings twice, be vortexed concussion.200 μ L 100mM NaOH is added, 30min is denatured,
Take the NaH for resetting and adding 40 μ L 1M2PO4(pH 3.9) is neutralized.
7. relative affinity is determined
Use " P1 biotins upstream "：ATCCAGAGTGACGCAGCA and " P4 downstreams "：ACTAAGCCACCGTGTCCA two
Individual primer pair is respectively taken turns obtained DNA library in screening and expanded, the amplification can with prepare it is single-stranded before amplification remove primer not
With outside, method is identical.DNA after amplification is double-stranded DNA, and has with creatine kinase isozyme and be modified with a chain of affinity
Biotin.Aptamer surveys concentration (quantitative with micro ELISA Plate), normalization after purification.Diluted with phosphate buffer
It is configured to 37.5nM, 1mL.95 DEG C of water-bath 20min, are immediately placed on 20min on ice, to open double-strand.Before experiment, by creatine kinase
Isodynamic enzyme (PROSPEC, 1mg/126.5 μ L) and for verifying specific another albumen myoglobins (Abcam, 3.36mg/
ML) respectively with coating buffer solution (1.59g Na2CO3, 2.93g NaHCO3, it is dissolved in 1L ultra-pure waters) and it is diluted to 5 μ g/mL.According to every
96 orifice plates are placed in 4 DEG C of incubations by the volume coating creatine kinase isozyme of the μ L target proteins of hole 100, each three parallel holes of round
Overnight.Empty liquid and pat dry, with 200 μ L lavation buffer solutions (PBST：0.2g KH2PO4, 2.9g Na2HPO4·12H2O, 8.0g
NaCl, 0.2g KCl, are dissolved in 1L ddH2O, then add 0.5mL Tween-20) wash after 1 time, per the μ L confining liquids (0.05g of hole 300
BSA, is dissolved in 50mL coating buffer solution), 4 DEG C of incubation 1h.Empty liquid and pat dry, after being washed twice with 200 μ L lavation buffer solutions,
100 μ L are added to take turns DNA library, 37 DEG C of incubation 1h per hole.Empty liquid and pat dry, cleaned with 300 μ L lavation buffer solutions after three times, often
Hole adds horseradish peroxidase-Streptavidin (the green skies Bioisystech Co., Ltd in Shanghai) the 100 μ L of 2000 times of dilution, 37
DEG C be incubated 1h.Empty liquid and pat dry, lavation buffer solution is cleaned three times, then soaks 5min with lavation buffer solution, empties liquid
And pat dry, then washed twice with 300 μ L lavation buffer solutions, to remove free horseradish peroxidase.3,3 ', 5 are added per hole,
5 '-tetramethyl benzidine (TMB) nitrite ion (the green skies Bioisystech Co., Ltd in Shanghai) 100 μ L, lucifuge colour developing 30min, often
Hole adds at the μ L terminating reactions of sulfuric acid solution 50 that concentration is 2M, ELIASA 450nm and reads absorbance.Use Graphpad statistical numbers
According to and do figure, as shown in Figure 1.As a result show, with the increase of screening round, DNA library and creatine kinase isozyme affinity
It is being gradually increasing, while DNA library has declined with as the myoglobins and the affinity of bovine serum albumin(BSA) that compare, this table
Bright creatine kinase isozyme aptamers screening process is served after the effect being enriched with to high-affinity single stranded DNA, the wheel of screening 10
DNA library in contain creatine kinase isozyme aptamer.
8. clone and sequencing
Performing PCR is entered to the library first, 15 circulations identical with foregoing amplification program.Here unmodified biotin is used
Primer (P3, P4), each 2.5 μ L.It is identical with the amplification in screening process, it is necessary to by agarose gel electrophoresis identification and it is pure
Change.Enter according to carrier (pEASY-T1Simple Cloning Vector, Beijing Quanshijin Biotechnology Co., Ltd) specification
Row is connected, and linked system is：The μ L of PCR primer 1, the μ L of carrier 1 and the μ L of moisturizing 3, react at room temperature 1min.Then by 5 μ L linked systems
It is added in 50 μ L competent cells, flicks mixing, ice bath 30min.42 DEG C of water-bath heat shock 30s, are immediately placed on 2min on ice.Plus
250 μ L are balanced to the LB culture mediums of room temperature, 200rpm, 37 degree of culture 1h.The culture medium of all first times, 4000rpm centrifugations
1min, discards supernatant, leaves 150 μ L, flicks thalline, is all coated on ready ammonia benzyl flat board, and 37 DEG C overnight.Choose white single
It is cloned into 10 μ L sterilized waters, vortex mixed.1 μ L mixed liquors are taken in 25 μ L PCR systems, with M13Forward Primer and
M13Reverse Primer identify positive colony.Positive colony shakes and is sequenced after bacterium.
Obtained nucleotide sequence, the core of one group of creatine kinase isozyme aptamer as of the present invention is sequenced
Nucleotide sequence, it is specific as follows：
Nucleotide sequence described in SEQ ID No.1：
atccagagtg acgcagcacg gtggagtcgt tgggtcgtgg gggtggggtg gtgggattgg 60
tggtggacac ggtggcttag t 81
Nucleotide sequence described in SEQ ID No.2：
atccagagtg acgcagcagg ggtgggggtg ggtttgaagc acgtcgagtg ggatggcagg 60
gggtggacac ggtggcttag t 81
Nucleotide sequence described in SEQ ID No.3：
atccagagtg acgcagcagg gacacatcca tccatgcaca ggactgtcta catcgctatg 60
ttatggacac ggtggcttag t 81
Nucleotide sequence described in SEQ ID No.4：
atccagagtg acgcagcagg ggggtgggtg ggggatctcg gaggatgctt ttagggggtt 60
gggtggacac ggtggcttag t 81
Nucleotide sequence described in SEQ ID No.5：
atccagagtg acgcagcaca ttgagagggg gtggccgtag tcaggtgggt gggggtttga 60
gtggacacgg tggcttagt 79
By in every of the nucleotide sequence in nucleotides sequence list shown in SEQ ID No.1~SEQ ID No.5
Preferably go out number of base respectively, obtain the nucleotides sequence shown in SEQ ID No.6 in nucleotides sequence list~SEQ ID No.10
Row, it is specific as follows：
Nucleotide sequence described in SEQ ID No.6：
cggtggagtc gttgggtcgt gggggtgggg tggtgggatt ggtgg 45
Nucleotide sequence described in SEQ ID No.7：
ggggtggggg tgggtttgaa gcacgtcgag tgggatggca ggggg 45
Nucleotide sequence described in SEQ ID No.8：
gggacacatc catccatgca caggactgtc tacatcgcta tgtta 45
Nucleotide sequence described in SEQ ID No.9：
ggggggtggg tgggggatct cggaggatgc ttttaggggg ttggg 45
Nucleotide sequence described in SEQ ID No.10：
cattgagagg gggtggccgt agtcaggtgg gtgggggttt gag 43
The aptamer (including non-aptamers nucleotide sequence) of sulfhydrylation modification, by taking SEQ ID No.7 as an example：
HS-(CH2)6-tttttttttt tttttttttt ggggtggggg tgggtttgaa gcacgtcgag
tgggatggca gggg 65
Biotin modification aptamers, by taking SEQ ID No.9 as an example：
biotin-ggggggtggg tgggggatct cggaggatgc ttttaggggg ttggg 45
Complementary nucleic acid sequences：
aaaaaaaaaa aaaaaaaaaa-biotin 20
Above nucleotide sequence is synthesized by by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 2
Creatine kinase isozyme aptamer described in Example 1, carries out the measure of dissociation constant, specifically respectively
Assay method is as follows：
(1) in 96 orifice plates, per creatine kinase isozyme (PROSPEC, the 1mg/ that the μ L concentration of hole 100 is 2000ng/mL
126.5 μ L) solution, 4 DEG C are incubated overnight or 37 DEG C of incubation 2h；Creatine kinase isozyme solution coating buffer solution (1.59g
Na2CO3, 2.93g NaHCO3, it is dissolved in 1L ultra-pure waters) and dilution.
(2) liquid after emptying procedure (1) processing in 96 orifice plates is simultaneously patted dry, and two are washed with 200 μ L phosphate Tween buffers
It is secondary, each 1min.
(3) every hole after being handled to step (2) in 96 orifice plates adds 300 μ L confining liquids, 4 DEG C of incubation 1h；The confining liquid
Matching while using is needed, by 0.06g bovine serum albumin(BSA)s, is dissolved in 20mL phosphate buffer and being made；
(4) 96 orifice plates after cleaning step (3) processing, 200 μ L phosphate Tween buffers are washed three times, each 1min；
(5) using phosphate buffer by creatine kinase aptamer be diluted to 10nM, 20nM, 50nM, 100nM,
200nM and 400nM, the aptamers after dilution are added in 96 orifice plates after step (4) processing successively, and 100 μ L are per hole, Mei Genong
Spend three parallel holes；
(6) 37 DEG C of incubation 1h；
(7) washed after the completion of being incubated with phosphate Tween buffer three times；
(8) Streptavidin (the green skies Bioisystech Co., Ltd in Shanghai) of horseradish peroxidase-labeled uses phosphate
Buffer solution dilution presses 1:2000 ratio, is added in 96 orifice plates after step (7) processing, and per the μ L of hole 100,1h is incubated at 37 DEG C；
(9) emptying procedure (8) processing after 96 orifice plates in liquid and pat dry, washed with 300 μ L phosphate Tween buffers
Three times, then with phosphate Tween buffer immersion 5min, empty liquid and simultaneously pat dry, then with 300 μ L phosphate Tween buffers
Wash twice；
(10) every hole in 96 orifice plates after being handled to step (9) adds 3,3 ', 5,5 '-tetramethyl benzidine (TMB) and shown
Color liquid (the green skies Bioisystech Co., Ltd in Shanghai) 100 μ L, lucifuge colour developing 30min, if blueness is faded, are terminated anti-in time
Should；
(11) every hole in 96 orifice plates after being handled to step (10) adds 50 μ L 2M sulfuric acid, with ELIASA in 450nm
Read absorbance in place；
(12) amount using the aptamer of addition is abscissa, the relative quantity with protein bound aptamer
(being represented with absorbance, measured value button goes blank) is ordinate, with formula Y=BmaxX/ (Kd+X), carries out nonlinear regression；
In the formula, what y was represented is the amount of the aptamer combined, and Bmax represents maximal absorptive capacity, and what Kd was represented is that dissociation is normal
Number, X is the concentration of the aptamer added；What the X was represented originally is the concentration of the free aptamer, still
The aptamer concentration in this experiment due to addition is significantly larger than protein concentration, so X is added with the aptamer
Enter amount replacement.Regression coefficient and fitting degree nonlinear regression formula " One site in software Prism Graphpad 5.0
Binding (hyperbola) " is tried to achieve.
(13) calculate obtain nucleotide sequence described in SEQ ID No.1 in sequence table~SEQ ID No.10 dissociation it is normal
Number is followed successively by 11.95nM, 25.95nM, 56.01nM, 0.81nM, 47.77nM, 63.57nM, 35.21nM, 112nM, 14.74nM
And 24.04nM, illustrate that the nucleotide sequence and creatine kinase isozyme have preferable affinity, can be used for creatine kinase
The detection of isodynamic enzyme.
Embodiment 3
A kind of A aptamer preparation methods of colloid gold label, step is as follows：
(1) by sulfydryl modification and one end is connected with the nucleosides having described in SEQ ID No.2 of 20 thymidine sequences
The A aptamers of acid sequence add TE buffer solutions (Tris-EDTA buffer) wiring solution-forming 1, and three (2- carboxylics are added to solution 1
Ethyl) phosphine (TCEP), reacts 2h, the mol ratio of the aptamer of the present invention of TCEP and sulfydryl modification is 5：1, obtain molten
Liquid 2；
(2) 1mL colloidal gold solutions are taken, 30min is centrifuged under 8000r/min, supernatant is abandoned, the μ L of ultra-pure water 100, mixing 12 is mended
μ L solution 2, shakes 1min, overnight, obtains solution 3；
(3) the 40mM NaCl aqueous solution isometric with solution 3 is added into solution 3, obtains solution 4, concentration is 20mM, shaken
1min is swung, 8h is reacted at room temperature；
(4) the NaCl aqueous solution that concentration is 1M is added again, and the concentration for obtaining solution 5 in solution 5, centrifuge tube is 40mM,
1min is shaken, overnight；
(5) centrifuge tube is placed under 8000r/min and centrifuges 10min, abandon supernatant, plus 100 μ L re-suspension liquids (PEG 20000
μ L, the BSA 0.1g of 0.005g, sucrose 0.5g, Tween-20 10, are dissolved in 10mL Tris- hydrochloric acid solutions) to original volume, contained
There is the liquid of the A aptamers of colloid gold label；
(6) liquid for obtaining step (5) directly with pipettor point on pretreated pad 2, drying for standby, institute
Stating pretreatment is：Pad 2 is immersed in added with 1g bovine serum albumin(BSA)s (BSA), 1.5g sucrose and 0.01g Sodium azides
30min in 50mL phosphate buffer, is then rinsed using a large amount of phosphate buffers.
A kind of B aptamers combined with Streptavidin or the complementary nucleotide sequence that is combined with Streptavidin
Preparation method, step is as follows：
(1) raw material is dissolved with phosphate buffer, obtains the solution that concentration is 100 μM；
Raw material is the B aptamers or biology with the nucleotide sequence described in SEQ ID No.4 of biotin modification
It is complementary with non-aptamers nucleotide sequence on the aptamer with the nucleotide sequence described in SEQ ID No.2 that element is modified
Complementary nucleotide sequence；
(2) the μ L of 1mg/mL Streptavidins 42 are added, are mixed, room temperature places 3h, obtains mixed solution；Raw material and strepto- parent
Mol ratio is 4 between element:1.
(3) with 30KD super filter tubes, the 12000r/min under the conditions of 4 DEG C centrifuges 10min to the mixed solution for obtaining step (2),
Super filter tube 1000g under the conditions of 4 DEG C is inverted again and centrifuges 30min, is obtained the solution after ultrafiltration, is obtained the tool of marked by streptavidin
Have a B aptamers of the nucleotide sequence described in SEQ ID No.4, or marked by streptavidin with SEQ ID
DNA end thymidine (i.e. non-aptamers nucleotide sequence) where the aptamer of nucleotide sequence described in No.2 is mutually
The complementary nucleotide sequence of benefit.
Embodiment 4
A kind of A aptamer preparation methods of colloid gold label, step is as follows：
(2) 1mL collaurums are taken, 10min is centrifuged under 12000r/min, supernatant is abandoned, the μ L of ultra-pure water 100 are mended, 0.4 μ L are mixed
Solution 2, shakes 1min, 4 spend night, obtain solution 3；
(3) the 40mM NaCl aqueous solution isometric with solution 3 is added into solution 3, obtains solution 4, solution 4 is shaken
0.5min, 4 DEG C of 12h；
The A aptamer preparation methods of a kind of colloid gold label in remaining be the same as Example 3.
A kind of B aptamers combined with Streptavidin or the complementary nucleotide sequence that is combined with Streptavidin
Preparation method, step is as follows：
The SEQ ID No.4 in embodiment 3 are changed for SEQ ID No.1, it is a kind of affine with strepto- in remaining be the same as Example 3
B aptamers or the preparation method of the complementary nucleotide sequence combined with Streptavidin that element is combined.
Embodiment 5
A kind of A aptamer preparation methods of colloid gold label, are comprised the following steps that：
It is SEQ ID No.5 to change the SEQ ID No.2 in embodiment 3,
(4) concentration is added again and obtain solution 5 for the 1M NaCl aqueous solution, make the final concentration of 40mM of solution 5, shake
0.5min, 4 DEG C of 24h；
(5) solution 5 is centrifuged into 30min under 8000r/min, abandons supernatant, plus 100 μ L re-suspension liquids (PEG 20000
μ L, the BSA 0.1g of 0.005g, sucrose 2.5g, Tween-20 10, are dissolved in 10mL Tris- hydrochloric acid solutions) to original volume, contained
There is the liquid of the A aptamers of colloid gold label；
The A aptamer preparation methods of a kind of colloid gold label in remaining be the same as Example 3.
A kind of B aptamers combined with Streptavidin or the complementary nucleotide sequence that is combined with Streptavidin
Preparation method, step is as follows：
It is SEQ ID No.1 to change the SEQ ID No.4 in embodiment 3
(1) raw material is dissolved with phosphate buffer, obtains the solution that concentration is 100 μM；
Raw material is the B aptamers or biology with the nucleotide sequence described in SEQ ID No.1 of biotin modification
It is complementary with non-aptamers nucleotide sequence on the aptamer with the nucleotide sequence described in SEQ ID No.5 that element is modified
Complementary nucleotide sequence；
(2) the μ L of 1mg/mL Streptavidins 168 are added, are mixed, room temperature places 1h, obtains mixed solution；Raw material and strepto-
Mol ratio is 1 between Avidin:1.
(3) with 30KD super filter tubes, 12000r/min is centrifuged the mixed solution for obtaining step (2) at ambient temperature
10min, then super filter tube 1000g centrifugations 30min at ambient temperature is inverted, the solution after ultrafiltration is obtained, Streptavidin is obtained
Mark have SEQ ID No.1 described in nucleotide sequence B aptamers, or marked by streptavidin with
DNA end thymidine (i.e. non-aptamers nucleotides where the aptamer of nucleotide sequence described in SEQ ID No.2
Sequence) complementary complementary nucleotide sequence.
Embodiment 6
A kind of A aptamer preparation methods of colloid gold label, are comprised the following steps that：
Wherein, A aptamers are respectively selected from SEQ IDNo.1~SEQ ID No.10 in nucleotides sequence list, remaining
The A aptamer preparation methods of a kind of colloid gold label in be the same as Example 3.
A kind of B aptamers combined with Streptavidin or the complementary nucleotide sequence that is combined with Streptavidin
Preparation method, step is as follows：
Wherein, B aptamers are different with the nucleotide sequence of A aptamers, are respectively selected from nucleosides of the present invention
SEQ IDNo.1~SEQ ID No.10 in acid sequence table, and the SEQ in A aptamers are the nucleotides sequence list
During nucleotide sequence shown in IDNo.2, B aptamers are not shown in the SEQ IDNo.5 in the nucleotides sequence list
Nucleotide sequence, and A aptamers be the nucleotides sequence list in SEQ IDNo.7 shown in nucleotide sequence when,
B aptamers are not the nucleotide sequence shown in the SEQ IDNo.10 in the nucleotides sequence list.
A kind of B aptamers combined with Streptavidin or combined in remaining be the same as Example 3 with Streptavidin
The preparation method of complementary nucleotide sequence.
Embodiment 7
The preparation of detection line 6 and nature controlling line 7 on nitrocellulose filter 3, step is as follows：
Concentration is drawn for the 1OD/30 μ L B aptamers combined with Streptavidin and leaned on nitrocellulose filter 3
At the nearly side of pad 2 about 1/3, obtain being coated with the detection line 6 of the aptamer combined with Streptavidin；It is by concentration
The 0.025OD/30 μ L complementary nucleotide sequence combined with Streptavidin is drawn in close adsorptive pads 4 on nitrocellulose filter 3
At side about 1/3, obtain being coated with the nature controlling line 7 of complementary nucleotide sequence；During line, detection line 6 and the flow velocity of nature controlling line 7 are
1μL/cm；Room temperature places 2h and fixation is dried, and obtains having marked the nitrocellulose filter 3 of detection line 6 and nature controlling line 7.
The B aptamers combined with Streptavidin, which are respectively selected from embodiment 3~6, to be prepared and strepto- parent
The B aptamers combined with element；The complementary nucleotide sequence combined with Streptavidin is respectively selected from embodiment 3~6
In prepare the complementary nucleotide sequence combined with Streptavidin.
Embodiment 8
A kind of creatine kinase isozyme detection kit realization, wherein, the preparation method of Test paper is as follows：
By the sample pad 1 (material is the plain film of glass fibre) by pretreatment, (it is coated with selected from made from embodiment 3~6
The A aptamers of colloid gold label) mark for preparing of pad 2 (material be glass fibre plain film), embodiment 6 detects well
The nitrocellulose filter 3 and adsorptive pads 4 (material is absorbent filter) of line 6 and nature controlling line 7 overlap and bond PVC bottom plates 5 successively
On (80mm × 5mm), wherein, mark the nitrocellulose filter 3 of detection line 6 and nature controlling line 7 to be made by embodiment 7, be fixed on
On bottom plate 5, one end of nitrocellulose filter 3 is pushed down by adsorptive pads 4, and the other end is combined pad 2 and pushed down, and pad 2 is by sample pad 1
Push down, obtain the Test paper, as shown in Figure 2.
Embodiment 9
A kind of detection method of creatine kinase isozyme detection, the detection method passes through a kind of creatine of the present invention
Kinase isozyme detection kit is realized, is specially：
The μ L of phosphate buffer 1 00 containing protein of creatine kinase isoenzyme are added dropwise to the inspection prepared in embodiment 8
In the sample pad 1 of test paper, solution is moved to adsorptive pads 4 under capillary action, after 20min, and red stripes are presented in detection line, say
Bright to have detected protein of creatine kinase isoenzyme, detection terminates.
The present invention includes but is not limited to above example, every to carry out any completely replacing under spirit of the invention and principle
Change or local improvement, all will be regarded as within protection scope of the present invention.
Claims (10)
1. one group of creatine kinase isozyme aptamer, it is characterised in that：The nucleotide sequence of the aptamer is selected from
SEQ ID No.1~SEQ ID No.10 in nucleotides sequence list.
2. one group of creatine kinase isozyme aptamer according to claim 1, it is characterised in that：The nucleic acid adaptation
The nucleotide sequence of body SEQ ID No.6~SEQ ID No.10 in nucleotides sequence list.
3. one group of creatine kinase isozyme aptamer according to claim 1, it is characterised in that：In nucleotide sequence
In nucleotide sequence in table shown in SEQ ID No.1~SEQ ID No.10,5 ' one end of every nucleotide sequence or
3 ' one end are modified with sulfydryl or biotin, obtain one group of creatine kinase isozyme nucleic acid adaptation for being modified with sulfydryl or biotin
Body.
4. it is a kind of such as the application of one group of creatine kinase isozyme aptamer according to any one of claims 1 to 3, its spy
Levy and be：, should by described aptamer for being detected, being separated to creatine kinase isozyme, purified and immobilization
The analysis detection of preparation, target substance for biological inductor, and prepare the reagent for clinical diagnosis and disease treatment
Medicine.
5. the application of one group of creatine kinase isozyme aptamer according to claim 4, it is characterised in that：The core
Application of the sour aptamers in creatine kinase isozyme detection kit is prepared.
6. a kind of creatine kinase isozyme detection kit, the detection kit includes Test paper, the Test paper bag
Bottom plate (5) is included, the sample pad (1) overlapped on bottom plate (5) and successively, pad (2), nitrocellulose filter (3) is bonded in and inhales
Water cushion (4)；On the nitrocellulose filter (3) detection line (6), nitrocellulose filter (3) are provided with close to the side of pad (2)
The upper side close to adsorptive pads (4) is provided with nature controlling line (7)；It is characterized in that：Be coated with described pad (2) 5 ' one end or
3 ' one end are by colloid gold label, and 5 ' one end or 3 ' one end are connected with the A aptamers of non-aptamers nucleotide sequence；Detection
It is coated with to be coated with the B aptamers combined with Streptavidin, nature controlling line (7) on line (6) and is combined with Streptavidin
Complementary nucleotide sequence, the complementary nucleotide sequence is the nucleotides sequence complementary with the non-aptamers nucleotide sequence
Row；
A aptamers are different with the nucleotide sequence of B aptamers, and are respectively selected from such as any one of claims 1 to 3
Described in SEQ ID No.1~SEQ ID No.5 in nucleotides sequence list, or be respectively selected from as any in claims 1 to 3
SEQ ID No.6~SEQ ID No.10 described in nucleotides sequence list, and it is described that should exclude A aptamers
During nucleotide sequence shown in the SEQ ID No.2 in nucleotides sequence list, during B aptamers are the nucleotides sequence list
SEQ ID No.5 shown in nucleotide sequence situation, and A aptamers be the nucleotides sequence list in SEQ
During nucleotide sequence shown in ID No.7, B aptamers are shown in the SEQ ID No.10 in the nucleotides sequence list
The situation of nucleotide sequence.
7. a kind of a kind of preparation method of creatine kinase isozyme detection kit as claimed in claim 6, it is characterised in that：
The preparation method step of Test paper is as follows：
(1) by 5 ' one end of A aptamers or 3 ' one end colloid gold labels, and to connect the preceding paragraph non-for 5 ' one end or 3 ' one end
Aptamers nucleotide sequence；
(2) sample pad (1), pad (2) are put into immersion more than 30min in the phosphate buffer containing BSA and tween, dried
It is dry；
(3) the A aptamer solution of colloid gold label is dispersed on the treated pad (2) of step (2)；
(4) the B aptamers and complementary nucleotide sequence that are combined with Streptavidin are fixed on nitrocellulose filter (3)
It is used as detection line (6) and nature controlling line (7), drying；
(5) sample pad (1), pad (2), nitrocellulose filter (3) and adsorptive pads (4) are overlapped successively and is bonded in bottom plate
(5) on, the Test paper is prepared；
Behind 5 ' one end of A aptamers or 3 ' one terminal modified sulfydryls, then use colloid gold label；5 ' one end of B aptamers
Or 3 ' one end be modified with biotin, 5 ' one end or 3 ' one end of complementary nucleic acid sequences are modified with biotin.
8. a kind of preparation method of creatine kinase isozyme detection kit according to claim 7, it is characterised in that：
The A aptamer preparation methods of colloid gold label are as follows：
(1) the A aptamers that 5 ' one end or 3 ' one end are modified with into sulfydryl add TE buffer solutions wiring solution-forming 1, take solution 1 to add
Enter in TCEP, react 1h~2h, obtain solution 2, the mol ratio of TCEP and the A aptamers for being modified with sulfydryl is 5:1
~50:1；
(2) collaurum is centrifuged into 10min~30min under 8000r/min~12000r/min, abandons supernatant, added ultra-pure water and mix
Close, then mixed with solution 2, finally shake 1min, 4 DEG C, to ambient temperature overnight, obtain solution 3；
The volume ratio of the collaurum, ultra-pure water and solution 2 is 500:50:0.2~500:50:20；
(3) NaCl aqueous solution addition solution 3 is well mixed, the concentration for obtaining NaCl in solution 4, solution 4 is 20mM, by solution
4 concussion 0.5min~1min, 4 DEG C of extremely room temperature reaction 6h~12h；
(4) the NaCl aqueous solution is added in the solution 4 after being handled to step (3), the concentration for obtaining NaCl in solution 5, solution 5 is
40mM, 0.5min~1min, 4 DEG C to room temperature reaction 10h~24h are shaken by solution 5；
(5) solution 5 after step (4) processing is centrifuged into 10min~30min under 8000r/min~1200r/min, abandons supernatant,
Suspension is aggravated to original volume, the liquid of the A aptamers containing colloid gold label is obtained；
The re-suspension liquid is by PEG 20000 0.005g, sucrose 0.5g, the μ L of Tween-20 10 and bovine serum albumin(BSA)
0.1g, which is dissolved in 10mL Tris- hydrochloric acid solutions, to be made；
(6) by the liquid of the A aptamers containing colloid gold label with pipettor point on pretreated pad (2),
Dry, obtain being coated with the pad (2) of the A aptamers of colloid gold label；The pretreatment is：By pad (2) leaching
Bubble 30min in the phosphate buffer of the 50mL added with 1g bovine serum albumin(BSA)s, 1.5g sucrose and 0.01g Sodium azides, so
Rinsed afterwards with phosphate buffer；
The B aptamers or the preparation method of the complementary nucleotide sequence combined with Streptavidin combined with Streptavidin
It is as follows：
(1) raw material is dissolved with phosphate buffer, obtains the solution that concentration is 100 μM；
Raw material is 5 ' one end or 3 ' one end are modified with the B aptamers of biotin or 5 ' one end or 3 ' one end are modified with biotin
Complementary nucleotide sequence powder；
(2) the μ L of μ L of 1mg/mL Streptavidins 42~168 are added into solution, is mixed evenly, is placed 1h~3h in room temperature, obtain
Mixed solution；
The mol ratio of raw material and Streptavidin is 4:1~1:1；
(3) by mixed solution with 30KD super filter tubes 4 DEG C at room temperature with 12000r/min centrifuge more than 10min, be inverted ultrafiltration
Pipe, at 4 DEG C to more than 30min is centrifuged at room temperature with 1000g, obtains the solution after ultrafiltration, obtains the B combined with Streptavidin
Aptamer or the complementary nucleotide sequence combined with Streptavidin；
Detection line (6) and the preparation process of nature controlling line (7) are as follows on nitrocellulose filter (3)：
Concentration is drawn in nitrocellulose filter for the 1OD/30 μ L~5OD/30 μ L B aptamers combined with Streptavidin
(3) at pad (2) side about 1/3, obtain being coated with the detection line of the B aptamers combined with Streptavidin
(6), concentration is drawn in nitric acid for the 0.025OD/30 μ L~1OD/30 μ L complementary nucleotide sequence combined with Streptavidin
On cellulose membrane (3) at adsorptive pads (4) side about 1/3, obtain being coated with the complementary nucleotide combined with Streptavidin
The nature controlling line (7) of sequence；During line, the flow velocity of detection line (6) and nature controlling line (7) is 1 μ L/cm；After line, 2h is placed in room temperature
Fixation is dried above, obtains having marked the nitrocellulose filter (3) of detection line (6) and nature controlling line (7)；
The preparation method of the Test paper is as follows：
Sample pad (1) is pre-processed：Sample pad (1) is immersed in added with 0.5g bovine serum albumin(BSA)s, 50 μ L Tween-20s
50mL phosphate buffer in 30min, then rinsed with phosphate buffer, dry, obtain the sample by pretreatment
Pad (1)；
By the sample pad (1) by pretreatment, the pad (2) for the A aptamers for being coated with colloid gold label, the good inspection of mark
The nitrocellulose filter (3) and adsorptive pads (4) of survey line (6) and nature controlling line (7) are overlapped and are adhered on bottom plate (5) successively, wherein,
The nitrocellulose filter (3) of detection line (6) and nature controlling line (7) has been marked to be fixed on bottom plate (5), one end is pressed by adsorptive pads (4)
Firmly, the other end is combined pad (2) and pushed down, and pad (2) is pushed down by sample pad (1), obtains the Test paper.
9. a kind of detection method of creatine kinase isozyme detection, it is characterised in that：The detection method passes through such as claim 6
A kind of creatine kinase isozyme detection kit any one of~8 is realized, is specially：Creatine kinase isozyme will be contained
The phosphate buffer of albumen is added dropwise in the sample pad of the Test paper in the detection kit (1), and detection line (6) presents clear
Red stripes, illustrate there is protein of creatine kinase isoenzyme, if detection line (6) does not occur red stripes, illustrate that no creatine swashs
Enzyme isoenzyme albumen, detection terminates.
10. a kind of detection method of creatine kinase isozyme detection according to claim 9, it is characterised in that：It will contain
The sample of the Test paper in the detection kit is added dropwise in the μ L of μ L of phosphate buffer 60 of protein of creatine kinase isoenzyme~100
On product pad (1), solution is mobile to adsorptive pads (4) under capillary action, after 5min~20min, and clear red bar is presented in detection line (6)
Band, illustrates there is protein of creatine kinase isoenzyme, if detection line (6) does not occur red stripes, illustrates the same work of no creatine kinase
Zymoprotein, detection terminates.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108709994A (en) * | 2018-05-29 | 2018-10-26 | 吉林大学 | A kind of NeuGc ALPHA2-3Gal quick detection test paper and detection method |
| CN110714049A (en) * | 2019-11-12 | 2020-01-21 | 北京理工大学 | Microbial sensor for detecting biomarkers, detection method, culture and detection chip and detection system |
| CN111500586A (en) * | 2020-05-21 | 2020-08-07 | 昆明理工大学 | Aptamer specifically binding to cap region of rabies virus L protein and application thereof |
| CN113311161A (en) * | 2021-04-14 | 2021-08-27 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Microfluidic chip colorimetric detection method and kit for detecting creatine kinase isoenzyme |
| CN113804898A (en) * | 2021-09-18 | 2021-12-17 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isozyme |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108709994A (en) * | 2018-05-29 | 2018-10-26 | 吉林大学 | A kind of NeuGc ALPHA2-3Gal quick detection test paper and detection method |
| CN110714049A (en) * | 2019-11-12 | 2020-01-21 | 北京理工大学 | Microbial sensor for detecting biomarkers, detection method, culture and detection chip and detection system |
| CN110714049B (en) * | 2019-11-12 | 2021-07-02 | 北京理工大学 | Microbial sensor for detecting biomarkers, detection method, culture and detection chip and detection system |
| CN111500586A (en) * | 2020-05-21 | 2020-08-07 | 昆明理工大学 | Aptamer specifically binding to cap region of rabies virus L protein and application thereof |
| CN111500586B (en) * | 2020-05-21 | 2022-11-04 | 昆明理工大学 | Aptamer specifically combined with rabies virus L protein capping region and application thereof |
| CN113311161A (en) * | 2021-04-14 | 2021-08-27 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Microfluidic chip colorimetric detection method and kit for detecting creatine kinase isoenzyme |
| CN113311161B (en) * | 2021-04-14 | 2023-12-22 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Microfluidic chip colorimetric detection method and kit for detecting creatine kinase isoenzyme |
| CN113804898A (en) * | 2021-09-18 | 2021-12-17 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isozyme |
| CN113804898B (en) * | 2021-09-18 | 2023-07-07 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzyme |
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