CN106636107B - A kind of aptamer and kit of laryngeal cancer cell - Google Patents
A kind of aptamer and kit of laryngeal cancer cell Download PDFInfo
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- CN106636107B CN106636107B CN201710048445.0A CN201710048445A CN106636107B CN 106636107 B CN106636107 B CN 106636107B CN 201710048445 A CN201710048445 A CN 201710048445A CN 106636107 B CN106636107 B CN 106636107B
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Abstract
The present invention discloses a kind of aptamer, sequence such as SEQ ID NO：Shown in 1 or 2.The invention also discloses the screening techniques of the aptamer, synthesize random single-stranded DNA banks and primer first, and aptamer is obtained using the Cell SELEX screening steps more taken turns.The aptamer of the present invention can specifically bind laryngeal cancer cell, have high specific recognition capability.
Description
Technical field
The invention belongs to aptamer field more particularly to a kind of aptamers can be used for detecting human laryngeal cancer cell
And its screening technique and application.
Background technology
Laryngocarcinoma is common one of H/N tumors, and incidence occupies second in respiratory tumor.There is domestic and international application form
Bright, the incidence of laryngocarcinoma constantly increases, and increases about 25% every year.The incidence of male's laryngocarcinoma in the world in 2008 is estimated as
5.1/100000 the male patient death rate is about
2.2/100000.Surgical method, chemotherapeutics and more advanced radiotherapy means new in the past 30 years are
Applied in the treatment of laryngocarcinoma, as other head and neck cancers, the overall survival of patients with laryngeal carcinoma is not improved, in addition under
Drop.Therefore early diagnosis is the key that improve laryngocarcinoma survival rate, and find tumor markers the examining for gastric cancer of early diagnosis
Disconnected and treatment then has a very important significance.
Aptamer (aptamer is also translated into aptamer, aptamer) refers to from the artificial synthesized libraries DNA/RNA
It is middle screen obtain being capable of high affinity and the single stranded oligonucleotide that is combined with high specificity with target molecules.At present it is known that core
Sour aptamers (aptamer) are the energy specific bond metal ion gone out through in-vitro screening technology screening, polypeptide, protein or even whole
Oligonucleotide (DNA or RNA) segment of a cell.The in-vitro screening technology of aptamer is referred to as the ligand of index concentration
Phyletic evolution (Systematic evolution of ligand by exponential enrichment, SELEX) technology；
The SELEX technical modelling natural evolution processes apply selection pressure (in conjunction with target) to random oligonucleotide library, wash in a pan
Choosing and target high special binding fragment (as shown in Figure 1)；Compared to the aptamers (peptide of the polypeptides such as antibody
Aptamer), the advantage of aptamer is quite apparent, such as：Prepare that simple and fast, chemical property is stable, is not reported so far and deposits
In immunogenicity or toxicity, target molecule range is wide, affinity is high, high specificity, is easy to be transformed modification.
In order to obtain to identify the aptamer of memebrane protein under native conformation, develop using cell as target
SELEX technologies, i.e. cell-SELEX.Carry out for single molecule that screening is different, and the target of cell-SELEX is from traditional SELEX
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, liver cancer cells, small cell lung cancer cell and non-small
Cell lung cancer cell.
Invention content
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of with than protein antibodies higher
Affinity and specificity, non-immunogenicity, can chemical synthesis, molecular weight it is small, it is stable, be easy to preserve and what is marked can be used for
The aptamer and its derivative for detecting human laryngeal cancer cell strain Hep-2, correspondingly provide the screening side of aforementioned nucleic acid aptamers
Method and application.
The present invention uses in-vitro screening (SELEX) technology of aptamer, is positive sieve target with laryngeal cancer cell strain, with nose
Pharynx cancer cell strain is reversed Screening target, the aptamer of screening and Hep-2 specific bonds.
The present invention provides a kind of method of the aptamer of specific binding human laryngeal cancer cell, includes the following steps：
(1) random single-stranded DNA banks and primer shown in following sequence are synthesized：
Random library：5’-ACAATGATCGAGTCGAGCAC(40N)CTGCTTGCCTCATCCGTCCA-3’；Draw upstream
Object：5’-FAM-ACAATGATCGAGTCGAGCAC-3’；Downstream primer：5’-Biotin-TGGACGGATGAGGCAAGCAG-3’；
(2) SELEX screens specific nucleic acid aptamer library：
(3) PCR amplification library；
(4) preparation in the single-stranded libraries DNA；
(5) reversed screening：It is control with nasopharyngeal carcinoma cell 5-8F, is reversely screened；
(6) positive screening；
(7) multi-turns screen：The single-stranded libraries DNA in the single-stranded library alternative steps of DNA that step 6 is obtained 4, repeat above-mentioned
The operation of step 5-6；The high sequence of degree of enrichment finally can be obtained, as can be used for detecting the nucleic acid of human laryngeal cancer cell strain Hep-2
Aptamers.
The present invention provides a kind of aptamer of laryngeal cancer cell, the sequence such as SEQ ID NO of the aptamer：1-
Shown in 2.
Sequence 1：(SEQ ID NO：1)
5’-ACAATGATCGAGTCGAGCAC(TAGCACCGCTCGTAGGGGAATGCGTGTTGGACCACGGTGG)
CTGCTTGCCTCATCCGTCCA-3’
Sequence 2：(SEQ ID NO：2)5’-ACAATGATCGAGTCGAGCAC(ATCGTGACTGCGGGATCTGTGAACGTCGACTGATCGTACG)CTGCTTGCCTCATCCGTCCA-3’
In the present invention, the laryngeal cancer cell, which is selected from, waits cancer cell line Hep-2.
In above-mentioned aptamer, a certain position on the nucleotide sequence of the aptamer can be modified.
The present invention provides a kind of diagnostic kit of laryngocarcinoma, and it includes above-mentioned aptamers.
Further, the present invention provides the aptamer answering in preparing the reagent for detecting laryngeal cancer cell
With.
Preferably, wherein the reagent of detection laryngeal cancer cell is the reagent that laryngeal cancer cell is imaged in being sliced for laryngeal carcinoma tissue.
The wherein described laryngeal cancer cell is Hep-2 cells.
Further, the present invention provides application of the aptamer in preparing the drug for treating targeting laryngocarcinoma, institute
The carrier that aptamers are stated as antitumor drug uses.
The present invention also provides application of the aptamer in the tumor markers for preparing laryngocarcinoma.
Advantageous effect：Compared with prior art, the present invention has the following advantages：Aptamer tool according to the present invention
Have a high specificity, compatibility is high, molecular weight is small, stability is good, prepares it is simple, at low cost, easy modify, non-immunogenicity it is excellent
Point, and can realize quick molecule diagnosis.Each aptamers in the present invention can with target cell Hep-2 stable bonds, without
It is combined with other control cells.By modification appropriate or label, it can be used for the quick detect and diagnose of human laryngeal cancer.In addition,
The aptamer itself is also used as drug, and either pharmaceutical carrier can by encapsulating corresponding drug or treatment preparation
To realize the targeted therapy of human laryngeal cancer.The present invention has very important meaning to clinical detection, the diagnosing and treating of human laryngeal cancer
Justice.
Description of the drawings
Fig. 1 is the fluorescence intensity comparison in difference that aptamers shown in sequence 1,2 are sliced with larynx normal structure, laryngeal carcinoma tissue,
Respectively as shown in A, B.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
Embodiment 1：Specifically bind the screening of the aptamer of laryngeal cancer cell
1. random single-stranded DNA banks and primer shown in the following sequence of synthesis.
Random library：5’-ACAATGATCGAGTCGAGCAC(40N)CTGCTTGCCTCATCCGTCCA-3’；Library left end
Fixed sequence program be sense primer, the fixed sequence program of library right end and the sequence reverse complemental of downstream primer.
Sense primer：5’-FAM-ACAATGATCGAGTCGAGCAC-3’
Downstream primer：5’-Biotin-TGGACGGATGAGGCAAGCAG-3’.
2.SELEX screens specific nucleic acid aptamer library
1) cell culture：1640 culture mediums of RPMI add 10% fetal calf serum culture human laryngeal cancer cell strain Hep-2 cells to paving
Full bottom of bottle 95%, is washed after removing culture medium with 10mL washing buffer, the binding with 1mL random sequences containing 1nM
Buffer is incubated 15min on ice, abandons solution；
2) cell combination：With the above-mentioned random libraries of 500 μ L binding buffer dissolvings 20nmol, 95 DEG C of heating
It is put into ice rapidly after 5min；It is 1mL that binding buffer are supplemented in random library to final volume, is handled with step 2.1
Hep-2 cells afterwards are incubated 30min on ice.
3) it dissociates：The liquid being incubated in culture bottle is poured out after being incubated, and is washed with 4 DEG C of washing buffer of 10mL
The cell being incubated in culture bottle, is eventually adding the aqua sterilisa of 500 μ L, is gently scraped off cell with cell scraping, and collects extremely
In the EP pipes of 1.5mL, then adding the aqua sterilisa of 250 μ L under remaining cell scraper and will be collected into EP pipes, be eventually adding
The aqua sterilisa of 250 μ L washes bottle wall and cell scraping one time, and collects in cleaning solution to EP pipes, 1000 μ L of total volume.It is placed in boiling
Water-bath is immediately placed on cooled on ice 5 minutes, 12000rpm after ten minutes in water, and 4 DEG C centrifuge 5 minutes, and supernatant is taken to be labeled as " first
Wheel screening library ".
3.PCR expands library：The Hep-2 specific nucleic acid aptamers library of 100 μ L screening gained is taken to carry out Standard PCR expansion
Increase, amplification condition is：94 DEG C of 3min, 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 30sec, through properly recycling wheel number, last 72 DEG C
Extend 3min.Note that need to expand the Hep-2 specific nucleic acid aptamers library of full income into 10 cycles in advance after first round screening,
The amplification for carrying out this step again, obtains amplified production.
The preparation in the single-stranded libraries 4.DNA：The agarose microbeads 5000rmp centrifugations of 100 μ L Streptavidins modification are gone
Clearly, then with 500 μ L PBS it washs, supernatant is removed in centrifugation；Repeated washing is primary.By the double-stranded DNA and agarose microbeads obtained by step 3
It is incubated half an hour at normal temperatures, passes through the interaction of the Streptavidin on the biotin and agarose microbeads on double-stranded DNA
Double-stranded DNA is attached to agarose microbeads surface；Supernatant is removed in 5000rmp centrifugations, twice with PBS centrifuge washings；Then it is added
The denaturation treatment of double-stranded DNA, 37 DEG C of reactions 10min, 5000rmp are used in 500 μ L to agarose microbeads of 150mM NaOH solutions
5min is centrifuged, supernatant is collected；After the salt plug sterile water washings of 10mL, the supernatant collected after alkaline denaturation is added, it is natural
It drips off.1mL sterile waters are added, collect the solution to drip, this is the single-stranded libraries DNA.
5, reversed screening：It is control with nasopharyngeal carcinoma cell 5-8F, is reversely screened.Culture nasopharyngeal carcinoma cell 5-8F first
To being paved with bottom of bottle 90% or so, and ensure that growth conditions are good, is then cleaned three times with WB, each 3mL.The text that will have been pre-processed
Library addition culture bottle is placed in be incubated on ice.Cell supernatant is collected after 30min, wherein containing being not associated on control cell surface
SsDNA, for follow-up positive sieve.
6. forward direction screening：The supernatant obtained with step 5 replaces the random library in step 2.2, repeat the above steps 2~
4 the step of, obtains the single-stranded library of the special aptamers of Hep-2.
7. multi-turns screen：The single-stranded libraries DNA in the single-stranded library alternative steps of DNA that step 6 is obtained 4, repeat above-mentioned
The operation of step 5-6.It repeats to monitor identification of the single-stranded libraries gained DNA to Hep-2 cells with flow cytometry in screening process
Ability, until the single-stranded libraries DNA meet the requirements to the recognition capability of Hep-2 cells after 10 wheel screenings.By products therefrom through clone
4 high sequences of degree of enrichment finally can be obtained after sequencing result finishing analysis in sequencing analysis, as can be used for detecting people's larynx
The aptamer of cancer cell line Hep-2.
Sequence 1：(SEQ ID NO：1)
5’-ACAATGATCGAGTCGAGCAC(TAGCACCGCTCGTAGGGGAATGCGTGTTGGACCACGGTGG)
CTGCTTGCCTCATCCGTCCA-3’
Sequence 2：(SEQ ID NO：2)5’-ACAATGATCGAGTCGAGCAC(ATCGTGACTGCGGGATCTGTGAACGTCGACTGATCGTACG)CTGCTTGCCTCATCCGTCCA-3’
Sequence 3：(SEQ ID NO：3)5’-ACAATGATCGAGTCGAGCAC(GACGTTGCTGACGATTGTGTTATTGGCGTGTGATCCTAAG)CTGCTTGCCTCATCCGTCCA-3’
Sequence 4：(SEQ ID NO：4)5’-ACAATGATCGAGTCGAGCAC(GTCTTGACTACGAAGTGAGCTAATGTCGTATGATCGATCG)CTGCTTGCCTCATCCGTCCA-3’
Embodiment 2：Aptamer analyzes the binding specificity of laryngeal cancer cell
The binding specificity of aptamer with flow cytometry to screening is evaluated.Since target is thin in screening
Born of the same parents are Laryngeal cancer cell Hep-2 cell, therefore the control cell selected also is wrapped in addition to the nasopharyngeal carcinoma cell 5-8F for reversely screening
It includes：Normal laryngeal epithelial cell, HGC-27 cell lines (stomach cancer cell), A549 cell lines (NSCLC, lung cancer), Hela (cervical carcinoma),
TCA (tongue cancer), CNE-2 (nasopharyngeal carcinoma), SMMC-7721, HepG2 (liver cancer), EC9706 (cancer of the esophagus).
Given threshold makes 95% cells mean fluorescence value of negative sample be below the threshold value, then sets different letters
Number grade, such as less than 10% cells mean fluorescence value is then denoted as higher than threshold value-, the cells mean fluorescence value of 10%-35% is high
Then be denoted as in threshold value+, the cells mean fluorescence value of 35%-60% is then denoted as higher than threshold value ++, 85% note is less than more than 60%
For +++, it is denoted as more than 85% ++++.Testing result is as shown in table 1 below, it can be seen that sequence 1-2 can specifically bind laryngocarcinoma
Cell Hep-2, and sequence 3-4 with laryngeal cancer cell other than being combined, it is also nonspecific to combine other tumour cells.
Table 1：Combinations of the nucleic acid aptamer sequence 1-4 to different cells
Embodiment 3：The measurement of the dissociation constant of flow cytomery aptamer and Laryngeal cancer cell Hep-2 cell
The measurement of the dissociation constant of flow cytomery aptamer and Laryngeal cancer cell Hep-2 cell, concrete operations and stream
It is identical as the operation of target cell binding specificity that formula cell instrument detects aptamer.Above-mentioned 4 aptamers point will be synthesized
Other configured in parallel is incubated with the target cell of equivalent respectively at various concentration, and it is thin to measure target under different aptamer concentration
The fluorescence intensity of born of the same parents.With a concentration of abscissa of aptamer, corresponding fluorescence intensity level is ordinate, according to formula Y=
Bmax*X/ (Kd+X) matched curve, obtains the dissociation curve of 4 aptamers.Each nucleic acid adaptation is obtained by dissociation curve
The dissociation constant Kd sizes of body are as shown in table 2.The result shows that：4 aptamers all have Laryngeal cancer cell Hep-2 cell very strong
Binding ability.
Table 2：The dissociation constant of nucleic acid aptamer sequence 1-4
Embodiment 4：The combination of aptamer and laryngeal carcinoma tissue slice
By the laryngeal carcinoma tissue of paraffin embedding slice in 60 DEG C of oven for baking 20min, slice, which is dipped in dimethylbenzene, dewaxes two
It is secondary, each 10min；Immerse absolute ethyl alcohol twice, each 5min；Slice passes through graded ethanol (90%, 85%, 70% second successively
Alcohol is each primary, each 2min), it is finally immersed in PBS；Microwave method method carries out antigen retrieval to slice, is cooled to room temperature, uses
PBS is washed twice；Then 1h is closed using containing the combination buffer room temperature of 20% fetal calf serum and 1mg/ml salmon sperm dnas；It goes
Except confining liquid, it is separately added into the aptamers nucleic acid sequence 1 of biotin labeling, sequence 2 is incubated 1h in 4 DEG C；Washing buffer washes 3
It is secondary, each 5min；The quantum dot of Streptavidin modification is then added, is incubated at room temperature 0.5h；After washed, mounting, fluorescence is shown
Micro- microscopic observation.The results are shown in Figure 1, sequence 1, sequence 2 can specific recognition slice in laryngeal cancer cell, but with it is normal
Larynx tissue present it is weaker or without combination, to clinically can be used for the detect and diagnose of laryngocarcinoma.
By above example and investigate result it is found that by above-mentioned screening obtain the present invention two aptamers it is equal
Energy specific recognition human laryngeal cancer cell, and two aptamers identify high specificities, it is high to target affinity, for laryngocarcinoma
Diagnosis and detection are of great significance.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (9)
1. a kind of aptamer that can specifically bind human laryngeal cancer cell, it is characterised in that sequence such as SEQ ID NO：1 or SEQ
ID NO：Shown in 2.
2. aptamer as described in claim 1, wherein the laryngeal cancer cell is Hep-2 cells.
3. a kind of diagnostic kit of laryngocarcinoma, it includes claim 1-2 any one of them aptamers.
4. such as claim 1-2 any one of them aptamer answering in preparing the reagent for detecting laryngeal cancer cell
With.
5. application as claimed in claim 4, wherein the reagent of detection laryngeal cancer cell is that laryngocarcinoma is thin in being sliced for laryngeal carcinoma tissue
The reagent of born of the same parents' imaging.
6. such as claim 4-5 any one of them applications, the laryngeal cancer cell is Hep-2 cells.
7. such as application of the claim 1-2 any one of them aptamer in preparing the drug for treating targeting laryngocarcinoma, institute
The carrier that aptamers are stated as antitumor drug uses.
8. such as application of the claim 1-2 any one of them aptamer in the tumor markers for preparing laryngocarcinoma.
9. a kind of method of the aptamer of identification specific binding human laryngeal cancer cell described in claim 1, including it is as follows
Step：
(1) random single-stranded DNA banks and primer shown in following sequence are synthesized：
Random library：5’-ACAATGATCGAGTCGAGCAC(40N)CTGCTTGCCTCATCCGTCCA-3’；
Sense primer：5’-FAM-ACAATGATCGAGTCGAGCAC-3’；
Downstream primer：5’-Biotin-TGGACGGATGAGGCAAGCAG-3’；
(2) SELEX screens specific nucleic acid aptamer library：
(3) PCR amplification library；
(4) preparation in the single-stranded libraries DNA；
(5) reversed screening：It is control with nasopharyngeal carcinoma cell 5-8F, is reversely screened；
(6) positive screening；
(7) multi-turns screen：The single-stranded libraries DNA in the single-stranded library alternative steps of DNA that step 6 is obtained 4, repeat the above steps
The operation of 5-6；The high sequence of degree of enrichment is finally obtained, as is used to detect the aptamer of human laryngeal cancer cell strain Hep-2.
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| CN103374572A (en) * | 2012-04-28 | 2013-10-30 | 复旦大学附属华山医院 | Sequence of aptamer of hepatoma cell and application thereof |
| CN104450713A (en) * | 2014-04-11 | 2015-03-25 | 中国人民解放军军事医学科学院基础医学研究所 | Sequence and application of oligonucleotide aptamer C6-8 for specific recognition of heterogeneous ribonucleoprotein A2/B1 (hnRNPA2/B1) |
| CN105452466A (en) * | 2012-10-23 | 2016-03-30 | 卡里斯生命科学瑞士控股有限责任公司 | Aptamers and uses thereof |
| WO2017001491A2 (en) * | 2015-07-01 | 2017-01-05 | Immatics Biotechnologies Gmbh | Novel peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers |
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| CN103374572A (en) * | 2012-04-28 | 2013-10-30 | 复旦大学附属华山医院 | Sequence of aptamer of hepatoma cell and application thereof |
| CN105452466A (en) * | 2012-10-23 | 2016-03-30 | 卡里斯生命科学瑞士控股有限责任公司 | Aptamers and uses thereof |
| CN104450713A (en) * | 2014-04-11 | 2015-03-25 | 中国人民解放军军事医学科学院基础医学研究所 | Sequence and application of oligonucleotide aptamer C6-8 for specific recognition of heterogeneous ribonucleoprotein A2/B1 (hnRNPA2/B1) |
| WO2017001491A2 (en) * | 2015-07-01 | 2017-01-05 | Immatics Biotechnologies Gmbh | Novel peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers |
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