CN102839181A - Nucleic acid aptamer sequence of gastric cancer cells, and uses thereof - Google Patents
Nucleic acid aptamer sequence of gastric cancer cells, and uses thereof Download PDFInfo
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- CN102839181A CN102839181A CN2011101725226A CN201110172522A CN102839181A CN 102839181 A CN102839181 A CN 102839181A CN 2011101725226 A CN2011101725226 A CN 2011101725226A CN 201110172522 A CN201110172522 A CN 201110172522A CN 102839181 A CN102839181 A CN 102839181A
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Abstract
The invention belongs to the technical field of cell biology, and relates to a nucleic acid aptamer sequence of gastric cancer cells, and uses thereof. The vitro SELEX screening technology of a nucleic acid aptamer is used, the gastric cancer cell lines HGC27 are used as a positive screening target, the gastric epithelial cell lines GES-1 are used as a negative screening target, and a nucleic acid aptamer specifically binding to the HGC27 is screened to obtain a characteristic sequence GGTTT specifically binding to the HGC27. The characteristic sequence is a basis of specific binding of the nucleic acid aptamer and the HGC27, and can be used for design of an anti-gastric cancer drug, preparing drugs or other products; and the nucleic acid aptamer containing the characteristic sequence can be used as an anti-gastric cancer molecular probe or drug target.
Description
Technical field
The invention belongs to the cellular biological technique field, relate to a kind of aptamer sequence and purposes of stomach cancer cell.
Background technology
Cancer of the stomach is one of China's common malignancy, also is modal malignant tumor of digestive tract.According to statistics, cancer of the stomach occupies second of all kinds of tumours in the morbidity of China; In the malignant tumour of all stomaches, gland cancer accounts for 95%.When making a definite diagnosis, most of clinically Patients with Gastric Cancer often have been in late period; Its 5 years survival rates are no more than 10%; Therefore early diagnosis is the key that improves the cancer of the stomach survival rate, and the tumor markers of searching early diagnosis then has crucial meaning for the diagnosis and the treatment of cancer of the stomach.
At present, known nucleic acid aptamers (aptamer) is oligonucleotide (DNA or the RNA) fragment of the ability specific combination metals ion, polypeptide, protein and even the whole cell that go out through the in-vitro screening technology screening; Its specificity has the avidity of strict recognition capability and height to combinative part as synantibody.The in-vitro screening technology of aptamer is called as Fas lignand system evolution (Systematic evolution of ligand by exponential enrichment, the SELEX) technology of index concentration; Described SELEX technical modelling nature evolutionary process applies selective pressure (combination target) to the random oligonucleotide library, elutriation and target high special binding fragment (as shown in Figure 1); Compare the aptamers (peptide aptamer) of polypeptide classes such as antibody; The advantage of aptamer is quite obvious, as: preparation simple and fast, chemical property be stable, do not appear in the newspapers so far have immunogenicity or toxicity, the target molecule scope is wide, avidity is high, high specificity, be easy to transform modification.
Many advantages of aptamer make it at aspects such as fundamental research, clinical detection, new drug developments purposes widely arranged.Aspect clinical detection, can substitute with all available aptamer of the technique almost that antigen antibody reaction detects at present, like a kind of aptamers molecule sensor---the RiboReporter of Archemix company exploitation
TMCan detected protein signal directly be changed into optical signal record and analysis, thereby realize direct rapid detection the high molecular weight protein in the biological mixed solution (like serum, cell extract).Aspect new drug development; Most typical example is first aptamer medicine Macugen that passes through the drugs approved by FDA listing; It is the aptamer of anti-vascular endothelial cell growth factor (VEGF), is used to treat wet age-related macular degeneration (wet AMD).The example of another aptamer medicine is that preclinical test shows that AGRO100 all has restraining effect to multiple cancer cells by the AGR0100 of Aptamera company development.
At present; The major part scientist and the biotech company that are engaged in aptamer research is primarily aimed at cardiovascular disorder in the world; And the Chinese common disease of less concern; Like hepatitis, liver cancer, cancer of the stomach etc., so the present invention pays the utmost attention to the major disease how aptamer is applied to above-mentioned serious threat China people ' s health, exploitation to the aptamer of stomach cancer cell as the characteristic mark; Provide powerful support for for the molecular diagnosis of cancer of the stomach and targeted therapy provide, have important clinic value.
Summary of the invention
The aptamer sequence that the purpose of this invention is to provide a kind of stomach cancer cell is specifically related to have in the aptamer of a kind of stomach cancer cell line HGC27 the characteristic sequence of specific combination HGC27.
Among the present invention, contain the characteristic sequence of specific combination HGC27 in the aptamer of described stomach cancer cell line HGC27 (sequence 1), described characteristic sequence is GGTTT.
Among the present invention, described stomach cancer cell is selected from stomach cancer cell line HGC27.
Further purpose of the present invention provides the purposes of said aptamer sequence.According to this sequential analysis, can further design the medicine or the goods of anti-cancer of the stomach, and prepare medicine or other goods of anti-cancer of the stomach among the present invention.
The present invention adopts in-vitro screening (SELEX) technology of aptamer; With stomach cancer cell line HGC27 for just sieving target; With gastric epithelial cell strain GES-1 is anti-sieve target, and the aptamer of screening and HGC27 specific combination makes the characteristic sequence GGTTT with specific combination HGC27; No. 2, called after Huashan aptamers among the present invention, are called for short HSST02.
The present invention realizes through following technical proposals:
The in-vitro screening technology that adopts known aptamer is the SELEX technology; With stomach cancer cell line HGC27 (available from the Shanghai cell bank) for just sieving target; With gastric epithelial cell strain GES-1 (available from the Shanghai cell bank) is anti-sieve target, filters out the aptamer with the HGC27 specific combination at random oligo DNA library (5 '-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNctcatggacgtgctggtgac-3 ') from external synthetic; Increasing and carrying out TA is cloned into pMD19-T carrier (available from the rich photo bio company in Shanghai), transforms DH5a bacterium (available from sky, Beijing root biotech firm) with primer Aptamer_L (5 '-FAM-acgctcggatgccactacag-3 ') and Aptamer_R (5 '-biotin-gtcaccagcacgtccatgag-3 ') with the sequence that filters out; Choosing white colony carries out after PCR confirms positive colony with primer RV-M (5 '-GAGCGGATAACAATTTCACACAGG-3 ') and M13 (47) (5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 '); The extracting plasmid and with M13 (47) (5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ') for sequencing primer carries out sequencing reaction, last sequenator checks order; According to sequencing result analytical characteristic sequence.
Among the present invention, described sequence is selected from the sequence of natural existence or synthetic, or the same sequence in any other source;
The sequence of described aptamer contains the whole identical sequence with the Nucleotide of said characteristic sequence, and promptly all aptamers all contain described characteristic sequence.
The present invention with stomach cancer cell line HGC27 for just sieving target; With gastric epithelial cell strain GES-1 is anti-sieve target; Through the in-vitro screening technology; Acquisition has the characteristic sequence GGTTT of the aptamer of specific combination HGC27, is aptamer and HGC27 specificity bonded basis with this characteristic sequence, is further used for medicinal design, preparation medicine or other goods of anti-cancer of the stomach; The described aptamer that contains this characteristic sequence can be used as the molecular probe or the drug target of anti-cancer of the stomach.
Description of drawings
Fig. 1 is the general flow figure of SELEX technology for the in-vitro screening technology of aptamer.
Embodiment
Embodiment 1: aptamer screening (takes turns)
First run oligo DNA library sequence: 5 '-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNctcatggacgtgctggtgac-3 '
The enrichment the primer:
Aptamer_L：5’-FAM-acgctcggatgccactacag-3’
Aptamer_R：5’-biotin-gtcaccagcacgtccatgag-3’
Behind the mensuration OD260 of oligo DNA library, whiz; With 300ul binding buffer liquid (containing 4.5g/L glucose among the PBS, 5mM MgCl2,2mg/mL BSA, 0.2mg/mL yeast tRNA) dissolving library, get 250pmol (first round 10nmol), 95 ℃ of 5min put rapidly on ice, and are centrifugal fast after a while.
Anti-sieve: the library of 250pmol is sucked GES-1 growth coverage reach in 70～80% the diameter 6cm petridish, supply 1mL with binding buffer liquid; 4 ℃ of shaking table vibration 1h; Get supernatant.
Just sieve: will instead sieve supernatant and add during HGC27 growth coverage reaches in 70～80% the diameter 6cm petridish 4 ℃ of shaking tables vibration 1h; Abandon supernatant; Wash the HGC27 cell three times with lavation buffer solution (containing 4.5g/L glucose among the PBS, 5mM MgCl2); Scrape the HGC27 cell with cell, in the 1mL lavation buffer solution HGC27 cell transfer being managed to EP, 95 ℃ of 5min put on ice rapidly.After waiting to turn cold, the centrifugal 5min of 10000rpm; Supernatant is transferred in the clean EP pipe.
Enrichment: configuration PCR system (TV 1000ul): H
2O 740ul, 10 * PCR damping fluid 100ul, dNTP 80ul, primer mixture 25ul (H
2O: 100uMAptamer_L: 100uMAptamer_R=4: 0.5: 0.5), dna profiling (just sieving supernatant) 50ul, Taq HS enzyme 5ul.
Increasing on the PCR appearance by following condition: behind 94 ℃ of preparatory sex change 2min of heating, 94 ℃ of sex change 30s, 58 ℃ of annealing 20s, 72 ℃ are extended 20s, 20 circulations, 72 ℃ are extended 4min, 4 ℃ of insulation＜1h.
Purifying: purification column is with MilliQ washing one time, and PBS washes twice, and PBS washes twice behind the Streptavidin Sepharose of adding 150ul GE, adds the PCR product; Shaking table vibration 20min emits liquid, after PBS washes twice; Add and emit liquid after 0.5mL NaOH leaves standstill 2-3min, desalting column on the relief liquor treats that liquid adds 1mL MilliQ water elution when desalting column almost flows to end; Begin to connect elutriant 1mL simultaneously, this elutriant be enrichment the oligo DNA library, can carry out next round screening.
Embodiment 2: aptamer TA clone
The following solution of preparation in Eppendorf tube, full dose is 5ul:pMD19-T Vector 1ul, aptamers PCR product 1ul, dH2O 3ul; The Solution I that adds 5ul; 16 ℃ were reacted 30 minutes.Full dose (10ul) is added in the 100ul DH5a competent cell, places 30 minutes in the ice; After 42 ℃ of 45 seconds of heating, in ice, placed 1 minute again; Add the 890ulLB substratum, 37 ℃ of shaking culture 60 minutes; Cultivate containing on the LB Agar Plating of X-Gal, IPTG, Amp, form single bacterium colony.
Embodiment 3: aptamer clone's bacterium colony PCR and order-checking
PMD19-T bacterium colony PCR and sequencing primer:
RV-M：5′-GAGCGGATAACAATTTCACACAGG-3′
M13(-47)：5′-CGCCAGGGTTTTCCCAGTCACGAC-3′
Join in 4ml LB (Amp 50ng/L) liquid nutrient medium with sterilization toothpick picking white mono-clonal bacterium colony, 180rpm, 37 ℃ shake bacterium spend the night (＜16h); Get 1ul bacterium liquid and make pcr template, carry out pcr amplification; With water as negative control.
Configuration PCR system: bacterium liquid 1ul, primer RV-M (10uM) 0.5ul, primer M13 (47) 0.5ul, 2 * Taq Mix7.5ul, water 5.5ul (total system 15ul); By following condition, on the PCR appearance, increase: 95 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 33 circulations; 72 ℃ of total elongation 10min.
Get 3-6ul bacterium liquid PCR product after reaction finishes and carry out agarose gel electrophoresis, 140V electrophoresis 20min; Negative control does not have band if bright purpose size strip occurs, shows the positive clone of this bacterium liquid.
The plasmid of going subsequently extracts: get the fresh bacterium liquid of 4ml incubated overnight, collect thalline, carry out plasmid according to the plasmid extraction kit operation instructions of Omega and extract; Be dissolved in water at last; Adopt Nanodrop that the plasmid that extracts is carried out concentration and purity testing.
Sequencing reaction: BDT v3.1 0.5ul, plasmid 100ng, primer M13 (47) 0.5ul adds water to 5ul.Behind 96 ℃ of preparatory sex change 1min of heating, 96 ℃ of sex change 10s, 50 ℃ of annealing 10s, 60 ℃ are extended 4min, 33 circulations, 4 ℃ of insulations.After reaction finishes 5ul product+1.25ul EDTA (125mM, the pH 8.0)+15ul absolute ethyl alcohol that checks order is sealed, shaken 4 times; Room temperature is placed 15min, and 3860rpm tips upside down on the paper handkerchief behind the centrifugal 40min of room temperature (25 ℃) immediately immediately; Centrifugal to 900rpm, stop at once; Add 60ul 70% ethanol, (need not shake) seals; 3860rpm under the room temperature subsequently, centrifugal 15min.Tip upside down on immediately on the paper handkerchief, centrifugal to 900rpm, stop at once.Lucifuge drying at room temperature 15min adds 10ul Hi-Di, seals lid; 95 ℃ of sex change 4min leave standstill 4min at once on ice, and are of short duration centrifugal, last 3730xl sequenator.Sequencing result is found all to have same characteristic sequence GGTTT among 18 clones.Therefore, obtained to have the characteristic sequence of specific combination HBV cAg, this section characteristic sequence is GGTTT.
SEQUENCE?LISTING
< 110>Huashan Hospital Affiliated To Fudan Univ
< 120>a kind of aptamer sequence and purposes of stomach cancer cell
<130>
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 80
<212> DNA
<213> Artificial
<400> 1
acgctcggat?gccactacag?ccgttagcgc?ggcgaggagt?ttgggtttgg?ttgggttggc 60
ctcatggacg?tgctggtgac 80
Claims (9)
1. the aptamer sequence of a stomach cancer cell is characterized in that, has the structure of sequence 1, and it contains the characteristic sequence GGTTT of specific combination stomach cancer cell line HGC27HGC27.
2. press the aptamer sequence of the described stomach cancer cell of claim 1; It is characterized in that; Make through following method: adopting the external SELEX triage techniques of aptamer, for just sieving target, serves as anti-sieve target with gastric epithelial cell strain GES-1 with stomach cancer cell line HGC27; The aptamer of screening and stomach cancer cell line HGC27 specific combination makes the characteristic sequence GGTTT with specific combination HGC27.
3. by the aptamer sequence of the described stomach cancer cell of claim 1, it is characterized in that described sequence is selected from the sequence of natural existence or synthetic, or the same sequence in any other source.
4. by the aptamer sequence of the described stomach cancer cell of claim 1, it is characterized in that the sequence of described aptamer contains the whole identical sequence with the Nucleotide of said characteristic sequence.
5. by the aptamer sequence of the described stomach cancer cell of claim 1, it is characterized in that the purposes of described characteristic sequence GGTTT in design medicine or goods.
6. by the aptamer sequence of the described stomach cancer cell of claim 1, it is characterized in that the purposes of described characteristic sequence GGTTT in preparation medicine or other goods.
7. by the aptamer sequence of claim 4 or 5 described stomach cancer cells, it is characterized in that described medicine or goods are the medicine or the goods of anti-cancer of the stomach.
8. the sequence of the aptamer of the stomach cancer cell of claim 1 detects the purposes in the molecular probe of cancer of the stomach in preparation.
9. the sequence of the aptamer of the stomach cancer cell of claim 1 detects the purposes in the drug target of cancer of the stomach in preparation.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN104561014A (en) * | 2015-01-12 | 2015-04-29 | 兰州大学 | Early gastric adenocarcinoma primary cells single-stranded DNA aptamer and preparation method thereof |
| CN105671050A (en) * | 2016-03-31 | 2016-06-15 | 浙江大学 | Efficient quick aptamer screening method |
| CN105802973A (en) * | 2016-03-31 | 2016-07-27 | 浙江大学 | Nucleic acid adapter of target medullary system differentiation antigen CD33 protein and application |
| CN105891480A (en) * | 2015-11-29 | 2016-08-24 | 卢美珍 | Stomach disease detection kit |
| CN106754935A (en) * | 2016-11-30 | 2017-05-31 | 吴冬 | The aptamer WYZ 5 of ovarian mucinous cancer cell 3AO and its screening technique and application |
| CN106754936A (en) * | 2016-11-30 | 2017-05-31 | 吴冬 | The aptamer WYZ 2 of ovarian mucinous cancer cell 3AO and its screening technique and application |
| CN108929873A (en) * | 2018-07-10 | 2018-12-04 | 安徽省昂普拓迈生物科技有限责任公司 | It is a kind of specifically bind metastatic gastric carcinoma cell aptamer and its application |
| CN113308475A (en) * | 2021-06-04 | 2021-08-27 | 燕山大学 | Aptamer for specifically recognizing gastric adenocarcinoma serum and application thereof |
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| 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 |
| CN104561014A (en) * | 2015-01-12 | 2015-04-29 | 兰州大学 | Early gastric adenocarcinoma primary cells single-stranded DNA aptamer and preparation method thereof |
| CN105891480A (en) * | 2015-11-29 | 2016-08-24 | 卢美珍 | Stomach disease detection kit |
| CN105671050B (en) * | 2016-03-31 | 2018-12-04 | 浙江大学 | A kind of efficient fast adaptation body screening technique |
| CN105671050A (en) * | 2016-03-31 | 2016-06-15 | 浙江大学 | Efficient quick aptamer screening method |
| CN105802973A (en) * | 2016-03-31 | 2016-07-27 | 浙江大学 | Nucleic acid adapter of target medullary system differentiation antigen CD33 protein and application |
| CN105802973B (en) * | 2016-03-31 | 2019-04-30 | 浙江大学 | It is a kind of target myeloid differentiation antigens c D33 albumen aptamer and application |
| CN106754936A (en) * | 2016-11-30 | 2017-05-31 | 吴冬 | The aptamer WYZ 2 of ovarian mucinous cancer cell 3AO and its screening technique and application |
| CN106754935B (en) * | 2016-11-30 | 2019-04-30 | 吴冬 | The aptamer WYZ-5 and its screening technique of ovarian mucinous cancer cell 3AO and application |
| CN106754935A (en) * | 2016-11-30 | 2017-05-31 | 吴冬 | The aptamer WYZ 5 of ovarian mucinous cancer cell 3AO and its screening technique and application |
| CN106754936B (en) * | 2016-11-30 | 2019-06-07 | 吴冬 | The aptamer WYZ-2 and its screening technique of ovarian mucinous cancer cell 3AO and application |
| CN108929873A (en) * | 2018-07-10 | 2018-12-04 | 安徽省昂普拓迈生物科技有限责任公司 | It is a kind of specifically bind metastatic gastric carcinoma cell aptamer and its application |
| CN108929873B (en) * | 2018-07-10 | 2021-10-29 | 安徽省昂普拓迈生物科技有限责任公司 | Aptamer specifically binding to metastatic gastric cancer cells and application thereof |
| CN113308475A (en) * | 2021-06-04 | 2021-08-27 | 燕山大学 | Aptamer for specifically recognizing gastric adenocarcinoma serum and application thereof |
| CN113308475B (en) * | 2021-06-04 | 2022-10-04 | 燕山大学 | Aptamer for specifically recognizing gastric adenocarcinoma serum and application thereof |
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