CN106636106A - Nucleic acid aptamer of tongue cancer cells and kit - Google Patents
Nucleic acid aptamer of tongue cancer cells and kit Download PDFInfo
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- CN106636106A CN106636106A CN201710048325.0A CN201710048325A CN106636106A CN 106636106 A CN106636106 A CN 106636106A CN 201710048325 A CN201710048325 A CN 201710048325A CN 106636106 A CN106636106 A CN 106636106A
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- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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Abstract
The invention discloses a nucleic acid aptamer capable of being specifically combined with tongue cancer cells. The sequence of the nucleic acid aptamer is shown as SEQ ID NO: 1. The invention further discloses a screening method of the nucleic acid aptamer. The screening method comprises the following steps: firstly, synthesizing a random single-chain DNA (Deoxyribonucleic Acid) library and a primer; carrying out a plurality of turns of a Cell-SELEX screening step to obtain the nucleic acid aptamer. The nucleic acid aptamer disclosed by the invention can be specifically combined with the tongue cancer cells and has a highly specific identification capability.
Description
Technical field
The invention belongs to aptamer field, more particularly to a kind of aptamer that can be used to detect people's Human Tongue Carcinoma Lines
And its screening technique and application.
Background technology
Oral cancer is one of modal malignant tumor of whole body, and the sickness rate of carcinoma of tongue accounts for first place in oral cancer.Majority research
Confirm that the sickness rate of oral cancer is in rising trend in the world, and the ill age tends to rejuvenation.Carcinoma of tongue grade malignancy height,
Local relapse is high, cervical metastasis rate is high, often jeopardize patient vitals, therefore needs row surgical radical treatment.Due to tongue be important pronunciation,
The Functional tissues such as chewing, so patient should be cured on the basis of dysfunction is reduced as far as possible.In the case, early diagnosiss
For therapeutic effect is improved, various complication are reduced, reduction case fatality rate is most important, finds the tumor markerses pair of early diagnosiss
Then have in the diagnosis and treatment of carcinoma of tongue and be of great significance.
Aptamer (aptamer is also translated into aptamer, aptamer) is referred to from the DNA/RNA libraries of synthetic
It is middle screen obtain being capable of high affinity and the single stranded oligonucleotide that combined with target molecules with high specificity.At present it is known that core
Sour aptamers (aptamer) are energy specific bond metal ion, polypeptide, the protein or even whole that Jing in-vitro screening technology screenings go out
Oligonucleotide (the DNA or RNA) fragment of individual cell.The in-vitro screening technology of aptamer is referred to as the part of index concentration
Phyletic evolution (Systematic evolution of ligand by exponential enrichment, SELEX) technology；
Described SELEX technical modelling natural evolution processes, to random oligonucleotide library selection pressure (with reference to target) is applied, and is washed in a pan
Choosing and target high special binding fragment (as shown in Figure 1)；Compare the aptamers (peptide of the polypeptides such as antibody
Aptamer), the Dominant Facies of aptamer work as obvious, such as：Simple and fast, stable chemical nature are prepared, is not reported so far and is deposited
Immunogenicity or toxicity, target molecule scope it is wide, affinity is high, high specificity, be easy to carry out transformation modification.
In order to obtain that the aptamer of memebrane protein under native conformation can be recognized, develop with cell as target
SELEX technologies, i.e. cell-SELEX.Carry out that screening is different for single molecule from traditional SELEX, the target of cell-SELEX is
One complete living cells.The aptamer of multiclass cell line is had been obtained for by cell-SELEX technologies, wherein cancer is thin
Born of the same parents system includes lymphocytic leukemia cells, myelogenous leukemia cells, hepatoma carcinoma cell, small cell lung cancer cell and non-little
Cell lung cancer cell.
The present invention goes out aptamer for Human Tongue Carcinoma Lines as characteristic mark by the use of cell-SELEX technological development
Thing, the searching of Clinical detection, tumor markerses, the discovery of tumor pathogenesis for carcinoma of tongue all has very important significance, and is
The molecular diagnosis of carcinoma of tongue and targeted therapy are provided and provided powerful support for, with important clinical value.
The content of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, there is provided one kind has higher than protein antibodies
Affinity and specificity, non-immunogenicity, can chemosynthesis, molecular weight is little, stable, be easy to preserve and labelling can be used for
The aptamer and its derivant of detection people longue carcinoma TCA-8113, correspondingly provides the screening of aforementioned nucleic acid aptamers
Methods and applications.
The present invention adopts in-vitro screening (SELEX) technology of aptamer, with longue carcinoma as target is just sieved, with nose
Pharyngeal cancer cell strain is the aptamer of reverse Screening target, screening and TCA-8113 specific bond.
The present invention provides a kind of method of the aptamer of specific binding people's Human Tongue Carcinoma Lines, comprises the steps：
(1) random single-stranded DNA banks and primer shown in following sequence are synthesized：
Random library：5’-TCAGTGATCGAATGGACCAC(40N)CTGCTTGCCTCATCCGTCCA-3’；Draw upstream
Thing：5’-FAM-TCAGTGATCGAATGGACCAC-3’；
Downstream primer：5’-Biotin-TGGACGGATGAGGCAAGCAG-3’；
(2) the fit library of SELEX screenings specific nucleic acid：
(3) PCR amplifications library；
(4) preparation in the single-stranded libraries of DNA；
(5) reversely screening：With nasopharyngeal carcinoma cell 5-8F as control, reversely screened；
(6) positive screening；
(7) multi-turns screen：The single-stranded libraries of DNA in the single-stranded library alternative steps 4 of DNA that step 6 is obtained, repetition is above-mentioned
The operation of step 5-6；1 high sequence of enrichment is finally obtained, as can be used to detect people longue carcinoma TCA-8113
Aptamer.
The present invention provides a kind of aptamer of Human Tongue Carcinoma Lines, the sequence such as SEQ ID NO of the aptamer：1-
Shown in 2.
Sequence 1：(SEQ ID NO：1)
5’-TCAGTGATCGAATGGACCAC(CCAGCATGGTGCATCACGCATTACCCTCCGAGCGTTGCTA)
CTGCTTGCCTCATCCGTCCA-3’
Sequence 2：(SEQ ID NO：2)
5’-TCAGTGATCGAATGGACCAC(CGATCGATCATACGACATTAGCTCACTTCGTAGTCAAGAC)
CTGCTTGCCTCATCCGTCCA-3’
In the present invention, described Human Tongue Carcinoma Lines are selected from longue carcinoma TCA-8113.
In above-mentioned aptamer, a certain position on the nucleotide sequence of the aptamer can be phosphorylated, first
Base, sulfhydrylation or isotopologue.
The present invention provides a kind of diagnostic kit of carcinoma of tongue, and it includes above-mentioned aptamer.
Further, it is of the invention that described aptamer answering in the reagent for detecting Human Tongue Carcinoma Lines is prepared is provided
With.
Preferably, wherein the reagent of detection Human Tongue Carcinoma Lines is the reagent for Human Tongue Carcinoma Lines imaging in tongue cancer section.
Wherein described Human Tongue Carcinoma Lines are TCA-8113 cells.
Further, the present invention provides application of the aptamer in the medicine for preparing treatment targeting carcinoma of tongue, institute
The carrier that aptamers are stated as antitumor drug is used.
The present invention also provides application of the described aptamer in the tumor markerses for preparing carcinoma of tongue.
Description of the drawings
Fig. 1 is the fluorescent staining intensity difference that the aptamers shown in sequence 1 are cut into slices with tongue normal structure (A), tongue cancer (B)
Different comparison.
Specific embodiment
Below in conjunction with the accompanying drawings the present invention is described in further detail, to make those skilled in the art with reference to description text
Word can be implemented according to this.
Embodiment 1：The screening of the aptamer of specific binding Human Tongue Carcinoma Lines
1. the random single-stranded DNA banks and primer shown in following sequence are synthesized.
Random library：5’-TCAGTGATCGAATGGACCAC(40N)CTGCTTGCCTCATCCGTCCA-3’；Library left end
Fixed sequence program be forward primer, the fixed sequence program of library right-hand member and the sequence reverse complemental of downstream primer.
Forward primer：5’-FAM-TCAGTGATCGAATGGACCAC-3’；
Downstream primer：5’-Biotin-TGGACGGATGAGGCAAGCAG-3’；
2.SELEX screens the fit library of specific nucleic acid
1) cell culture：The culture medium of RPMI 1640 adds 10% hyclone culture people's longue carcinoma TCA-8113 cells
To bottom of bottle 95% is paved with, removes and washed with 10mL washing buffer after culture medium, with 1mL 1nM random sequences are contained
Binding buffer are incubated 15min on ice, abandon solution；
2) cell is combined：The above-mentioned random libraries of 20nmol, 95 DEG C of heating are dissolved with 500 μ L binding buffer
It is put into rapidly in ice after 5min；It is 1mL to final volume that binding buffer are supplemented in random library, is processed with step 2.1
TCA-8113 cells afterwards are incubated 30min on ice.
3) dissociate：The liquid in incubation culture bottle is poured out after being incubated, is washed with 4 DEG C of washing buffer of 10mL
Incubation culture bottle in cell, be eventually adding the aquesterilisa of 500 μ L, cell is gently scraped off with cell sleaker, and collect to
In the EP pipes of 1.5mL, the cell of residual is scraped and collected in EP pipes by the aquesterilisa for then adding 250 μ L, is eventually adding
The aquesterilisa of 250 μ L washes bottle wall and cell sleaker one time, and collects cleaning mixture into EP pipes, the μ L of cumulative volume 1000.It is placed in boiling
Water-bath in water is immediately placed on cooled on ice 5 minutes after 10 minutes, 12000rpm, and 4 DEG C are centrifuged 5 minutes, take supernatant and are labeled as " first
Wheel screening library ".
3.PCR expands library：Taking the TCA-8113 specific nucleic acid aptamers library obtained by 100 μ L screenings carries out Standard PCR
Expand, amplification condition is：94 DEG C of 3min, 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 40sec, the suitable circulation wheel number of ECDC, last 72
DEG C extend 3min.Note, need the amplification 10 in advance of the fit library of TCA-8113 specific nucleic acids of full income after first round screening
Circulation, then the amplification of this step is carried out, obtain amplified production.
The preparation in the single-stranded libraries of 4.DNA：The agarose microbeads 5000rmp centrifugation that 100 μ L Streptavidins are modified is gone
Clearly, then with 500 μ L PBS wash, supernatant is removed in centrifugation；Repeated washing is once.By the double-stranded DNA and agarose microbeads of step 3 gained
Half an hour is incubated at normal temperatures, by the interaction of the Streptavidin on the biotin on double-stranded DNA and agarose microbeads
Double-stranded DNA is attached to into agarose microbeads surface；Supernatant is removed in 5000rmp centrifugations, with PBS centrifuge washings twice；It is subsequently adding
The μ L of 150mM NaOH solutions 500 are used for the degenerative treatments of double-stranded DNA, 37 DEG C of reactions 10min, 5000rmp into agarose microbeads
Centrifugation 5min, collects supernatant；Except salt plug is with after the aseptic water washings of 10mL, the supernatant for obtaining is collected after addition alkaline denaturation, it is natural
Drip off.1mL sterilized water is added, the solution for dripping is collected, this is the single-stranded libraries of DNA.
5th, reversely screening：With nasopharyngeal carcinoma cell 5-8F as control, reversely screened.Nasopharyngeal carcinoma cell 5-8F is cultivated first
To being paved with bottom of bottle 90% or so, and ensure that growth conditions are good, then cleaned with WB three times, each 3mL.By the good text of pretreatment
Storehouse adds culture bottle to be placed in being incubated on ice.Cell supernatant is collected after 30min, for subsequently just sieving.
6. positive screening：The random library that the supernatant obtained with step 5 replaces in step 2.2, repeat the above steps 2～
4 the step of, obtain the single-stranded library of the special aptamers of TCA-8113.
7. multi-turns screen：The single-stranded libraries of DNA in the single-stranded library alternative steps 4 of DNA that step 6 is obtained, repetition is above-mentioned
The operation of step 5-6.Repeat to monitor identification of the single-stranded libraries of gained DNA to Hep-2 cells with flow cytometry in screening process
Ability, until the single-stranded libraries of DNA meet the requirements to the identification ability of Hep-2 cells after 14 wheel screenings.By products therefrom Jing clones
Sequencing analysis, to sequencing result finishing analysis after, the high two sequences of enrichment are finally obtained, as can be used for detect people's tongue
The aptamer of JEG-3 TCA-8113.
Sequence 1：(SEQ ID NO：1)
5’-TCAGTGATCGAATGGACCAC(CCAGCATGGTGCATCACGCATTACCCTCCGAGCGTTGCTA)
CTGCTTGCCTCATCCGTCCA-3’
Sequence 2：(SEQ ID NO：2)
5’-TCAGTGATCGAATGGACCAC(CGATCGATCATACGACATTAGCTCACTTCGTAGTCAAGAC)
CTGCTTGCCTCATCCGTCCA-3’
Embodiment 2：Binding affinity assays of the aptamer to Human Tongue Carcinoma Lines
Evaluate with the binding specificity of aptamer of the flow cytometry to screening.Because screening hits carefully
Born of the same parents are Human Tongue Carcinoma Lines TCA-8113, therefore the compared with control cells selected is in addition to for the reversely nasopharyngeal carcinoma cell 5-8F of screening, also
Including：Normal tongue epithelial cell, HGC-27 cell lines (stomach cancer cell), A549 cell lines (NSCLC, pulmonary carcinoma), Hela (cervix uteri
Cancer), Hep-2 (laryngeal carcinoma), CNE-2 (nasopharyngeal carcinoma), SMMC-7721, HepG2 (hepatocarcinoma), EC9706 (esophageal carcinoma).
Given threshold causes 95% cells mean fluorescence value of negative sample to be below the threshold value, then sets different letters
Number grade, such as less than 10% cells mean fluorescence value is then designated as higher than threshold value-, the cells mean fluorescence value of 10%-35% is high
Then be designated as in threshold value+, the cells mean fluorescence value of 35%-60% is then designated as higher than threshold value ++, 85% note is less than more than 60%
For +++, it is designated as more than 85% ++++.Testing result is as shown in table 1 below, it can be seen that it is thin that sequence 1 can specifically bind carcinoma of tongue
Born of the same parents TCA-8113, and sequence 2 with Human Tongue Carcinoma Lines in addition to being combined, it is also nonspecific to combine other tumor cells.
Table 1：Affinity of nucleic acid aptamer sequence 1-2 to different cells
Embodiment 3：Aptamer and the combination of tongue cancer section
Paraffin-embedded tongue cancer is cut into slices in 60 DEG C of oven for baking 20min, section is dipped in dimethylbenzene and dewaxes two
It is secondary, each 10min；Immerse dehydrated alcohol twice, each 5min；Section sequentially passes through graded ethanol (90%, 85%, 70% second
Alcohol is each once, each 2min), it is finally immersed in PBS；Microwave method method carries out antigen retrieval to section, is cooled to room temperature, uses
PBS is washed twice；Subsequently using the closing 1h of the combination buffer room temperature containing 20% hyclone and 1mg/ml salmon sperm dnas；Go
Except confining liquid, the aptamers nucleotide sequence 1 for adding biotin labeling is incubated 1h in 4 DEG C；Lavation buffer solution is washed 3 times, each 5min；
The quantum dot of Streptavidin modification is subsequently added, 0.5h is incubated at room temperature；After scrubbed, mounting, fluorescence microscopy Microscopic observation.Knot
Fruit as shown in figure 1, sequence 1 can specific recognition section in Human Tongue Carcinoma Lines, but with normal tongue tissue present weaker or nothing
With reference to so as to clinically can be used for the detection and diagnosis of carcinoma of tongue.
From above example and investigation result, the aptamer of the invention obtained by above-mentioned screening can be special
Property identification people's Human Tongue Carcinoma Lines, and identification specificity is strong, high to target affinity, for the diagnosis and detection of carcinoma of tongue have important meaning
Justice.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in description and embodiment
With.It can be applied to completely various suitable the field of the invention.For those skilled in the art, can be easily
Realize other modification.Therefore under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (10)
1. a kind of aptamer that can specifically bind people's Human Tongue Carcinoma Lines, it is characterised in that sequence such as SEQ ID NO：Shown in 1.
2. aptamer as claimed in claim 1, wherein the Human Tongue Carcinoma Lines are TCA-8113 cells.
3. the aptamer as described in any one of claim 1-2, the wherein a certain position on the nucleotide sequence of aptamers
Can be phosphorylated, methylate, sulfhydrylation or isotopologue.
4. a kind of diagnostic kit of carcinoma of tongue, it includes the aptamer described in any one of claim 1-3.
5. the aptamer as described in any one of claim 1-3 is in answering that preparation is used in the reagent for detecting Human Tongue Carcinoma Lines
With.
6. application as claimed in claim 5, wherein the reagent of detection Human Tongue Carcinoma Lines is thin for carcinoma of tongue in tongue cancer section
The reagent of born of the same parents' imaging.
7. the application as described in any one of claim 5-6, the Human Tongue Carcinoma Lines are TCA-8113 cells.
8. application of the aptamer as described in any one of claim 1-3 in the medicine for preparing treatment targeting carcinoma of tongue, institute
The carrier that aptamers are stated as antitumor drug is used.
9. application of the aptamer as described in any one of claim 1-3 in the tumor markerses for preparing carcinoma of tongue.
10. a kind of method of the aptamer of the specific binding people's Human Tongue Carcinoma Lines described in identification claim 1, including as follows
Step：
(1) random single-stranded DNA banks and primer shown in following sequence are synthesized：
Random library：5’
-TCAGTGATCGAATGGACCAC(40N)CTGCTTGCCTCATCCGTCCA-3’；
Forward primer：5’-FAM-TCAGTGATCGAATGGACCAC-3’；
Downstream primer：5’-Biotin-TGGACGGATGAGGCAAGCAG-3’；
(2) the fit library of SELEX screenings specific nucleic acid：
(3) PCR amplifications library；
(4) preparation in the single-stranded libraries of DNA；
(5) reversely screening：With nasopharyngeal carcinoma cell 5-8F as control, reversely screened；
(6) positive screening；
(7) multi-turns screen：The single-stranded libraries of DNA in the single-stranded library alternative steps 4 of DNA that step 6 is obtained, repeat the above steps
The operation of 5-6；The high sequence of enrichment is finally obtained, as can be used to detect the nucleic acid adaptation of people Human Tongue Carcinoma Lines TCA-8113
Body.
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| US20050069910A1 (en) * | 2001-06-29 | 2005-03-31 | Turner John V. | Nucleic acid ligands to complex targets |
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| US20050069910A1 (en) * | 2001-06-29 | 2005-03-31 | Turner John V. | Nucleic acid ligands to complex targets |
| US20080286788A1 (en) * | 2001-06-29 | 2008-11-20 | Medimolecular Pty Ltd | Nucleic acid ligands to complex targets and uses thereof |
| CN103642810A (en) * | 2013-11-20 | 2014-03-19 | 中山大学附属第三医院 | Nucleic acid aptamer and screening method thereof, and application of nucleic acid aptamer in prostate cancer cell strain detection |
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