CN104450713B

CN104450713B - The sequence of oligonucleotides aptamers C6 8 of specific recognition heterogeneity ribonucleoprotein A2/B1 (hnRNPA2/B1) a kind of and application

Info

Publication number: CN104450713B
Application number: CN201410143208.9A
Authority: CN
Inventors: 邵宁生; 李慧; 李少华; 黄皑雪; 丁红梅; 李洁; 苏雪婷; 赵强
Original assignee: Institute of Basic Medical Sciences of AMMS
Current assignee: Institute of Basic Medical Sciences of AMMS
Priority date: 2014-04-11
Filing date: 2014-04-11
Publication date: 2017-09-15
Anticipated expiration: 2034-04-11
Also published as: CN104450713A

Abstract

The invention discloses a kind of oligonucleotides aptamers C6 8 for specific recognition heterogeneity ribonucleoprotein A2/B1 (hnRNPA2/B1) for belonging to molecular biosciences medicine technology field sequence and application.Single strand dna oligonucleotide aptamers C6 8 is obtained by the SELEX technologies for kinds of tumor cells, after flag F ITC, the cancer cell that the aptamers are capable of in the tumor cell line of a variety of epithelial origins of specific recognition and clinical liver cancer pathologic section is proved through fluorescent staining.Therefore, the sequences of C6 8 have wide application prospect in tumor research field, the discriminating of clinical tumor histotomy sample, knubble biological targeted therapy field.

Description

A kind of few core of specific recognition heterogeneity ribonucleoprotein A2/B1 (hnRNPA2/B1) Thuja acid aptamers C6-8 sequence and application

Invention field：

The present invention relates to the oligonucleotides aptamers C6-8 of specific recognition hnRNPA2/B1 albumen sequence and modification, and Applications of the C6-8 in terms of the kinds of tumor cells of recognition expression hnRNPA2/B1 albumen after modification.Exist the present invention relates to C6-8 Application in terms of biological medicine, C6-8 as kit part or Testing index, for clinical tumor histotomy The auxiliary diagnosis of sample, knubble biological targeted therapy.

Background technology：

At present, the immunohistochemistry or immunofluorescence method of application specific antibody test Research of predicting markers are still faced " goldstandard " of bed pathological diagnosis.For the tumour of epithelial origin, existing multiple markers antibody is in clinical practice.Such as angle egg (Keratin, Ker), cytokeratin (Cytokeratin, CK), epithelial cell membrane antigen (epithelial membrane in vain Antigen, EMA) and carcinomebryonic antigen (carcinoembryonic antigen, CEA) etc. be all important tumor associated antigen, It is widely present in various epithelial tumours, polytype glycoprotein tumor associated antigen (carbohydrate antigen, CA) also it is found in succession in epithelial tumour tissue and expresses increase, such as more than 80% human body gland cancer can be on its cell membrane CA72-4 is detected, rather than epithelial malignant tumour and benign proliferative lesion are without the antigen presentation.But in general, these The specificity and sensitiveness of tumor associated antigen be not high, is clinically realized often through a variety of related antigens of joint-detection pair Clinical assistant diagnosis, prognostic evaluation, curative effect guidance, recurrence and transfer monitoring of tumor patient etc..Therefore, more tumor-markers Thing and its antibody are urgently found and application and preparation.

High-throughout biological technique method is that the identification of tumor markers and its acquisition of probe are provided convenience approach.It is logical Often apply the method for proteomics to identify new tumor markers, animal is immunized using tumour cell film component in some scholars The specific antibody of tumor cell is directly obtained, and then utilizes the corresponding target antigen of Identification of the antibodies, i.e. tumor markers.Index The aglucon phyletic evolution technology of enrichment, abbreviation SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology is the high flux biological libraries screening nearly more than ten years risen and be developed rapidly Technology.Using the random oligonucleotide library (ssDNA libraries and RNA libraries) of Large Copacity, with reference to PCR Amplification Technologies, with Exponential enrichment and the oligonucleotides of target molecule specific bond, by in-vitro screening repeatedly, amplification, the few nucleosides finally obtained Sour aptamers (aptamer) are based on the combination that space structure and target molecule are in high specific and high-affinity.Due to aptamer tools Have the advantages that accurate identification, non-immunogenicity, easily external synthesis and modification, be also known as " artificial substituting antibody ", preclinical medicine, Clinical diagnosis in terms of new drug development with having broad application prospects.Especially composition target SELEX technologies of rising in recent years, To carry out unknown target molecule screening, and by introducing abatement screening step, result in specific recognition two In the compound target of group there is the aptamer of molecule in difference, and difference target molecule can be studied in turn using the aptamers, This is just that exploitation novel molecular probe and identification biomarker, especially tumor markers molecule open new approach.

The present invention is to obtain an aptamer C6-8 by the SELEX technologies using intact cell as target.It is identified, C6-8 being capable of specific recognition various tumor cell strains, including breast carcinoma cell strain MCF7, hepatoma Hep G 2 cells, lung cancer cell line H1299, laryngocarcinoma Hep2, oophoroma CAOV-3, cervix cancer HeLa；C6-8 can also the tested clinical liver cancer patient of specific recognition Cancerous tissue section in special-shaped cancer cell.Control nucleic acid sequence is with above-mentioned cell and tissue without combination.

The target for taking combination is fished using the C6-8 aptamers p μ ll-down of the coated magnetic bead of Streptavidin and biotin labeling Albumen, is identified through biological mass spectrometry and EMSA, it was demonstrated that the target protein that C6-8 is combined is heterogeneous ribonucleoprotein A2/B1 (hnRNPA2/B1).There is document report, include breast cancer, ED-SCLC, oophoroma and colon cancer group in kinds of tumors tissue In knitting, it is observed that the expression rise of hnRNPA2/B1 albumen, and it is primarily targeted for nucleus, therefore it is presumed that Specific biological mark of the hnRNPA2/B1 albumen possible as kinds of tumors.HnRNPA2/B1 albumen is as a kind of important RNA connection albumen, is the main component of heterogeneous cell nucleus glycoprotein core complex in nucleus.HnRNPA2/B1 albumen It may participate in mRNA montage, the transhipment of core-kytoplasm, post-transcriptional control, chromosome stability regulation and control etc..Recent research indicate that, HnRNPA2/B1 may play an important role during tumor development.

Because C6-8 can be directly used for fluorescent method detection tumour cell after FITC mark modifications, operate compared with traditional immunization Method is simpler.The invention can be used widely in clinical medicine and preclinical medicine.

The content of the invention：

Present invention aims at propose specific recognition hnRNPA2/B1 albumen oligonucleotides aptamers C6-8 sequence and Its application field, is included in auxiliary discriminating, basic research and the knubble biological targeted therapy of clinical tumor histotomy sample In application.

The present invention is achieved through the following technical solutions：

C6-8 sequences are synthesized by company first, and 5 ' ends or 3 ' are terminal modified, mark (such as FITC, biotin or it is high It is pungent), then carry out application study；Histochemistry or fluorescent method detection C6-8 recognize hnRNPA2/B1 albumen and express the egg White kinds of tumor cells.

Advantage of the present invention：

Compared with the antibody of protide, single-stranded oligonucleotides is more stablized；Aptamer directly can in vitro be synthesized, marked, Therefore secondary antibody need not be marked so that operation is more simple, rapid；Aptamer synthesis cost is low compared with Antibody preparation cost, week Phase is short.

The invention demonstrates that C6-8 specific recognition hnRNPA2/B1 albumen and can express the kinds of tumor cells of the albumen. Therefore, C6-8 sequences are respectively provided with the tumor research association area of clinical medicine and preclinical medicine is widely applied value and wide Wealthy market prospects.

Brief description of the drawings：

Fig. 1, EMSA experiment confirm that the target protein that C6-8 is combined is heterogeneity ribonucleoprotein A2/B1 (hnRNPA2/ B1)。

Fig. 2, C6-8 and kinds of tumor cells specific bond Laser Scanning Confocal Microscope and Flow Cytometry Assay result figure.

Fig. 3, C6-8 can specific recognition clinically liver cancer tissue section on tumour cell.

Embodiment：

Below by C6-8 to the cancer cell in various tumor cell strains and clinical liver cancer patient histotomy sample Recognize to further describe the present invention.

1. synthesize C6-8 sequences (SEQ ID No.1 in sequence table) and control GP30 nucleotide sequences (SEQ ID in sequence table No.2), in 5 ' end mark Bio and FITC (completion of Invitrogen companies).

Bio-C6-8：

5’-GTACTTCCATTTGTGTTTGCCCGGAGCCTTAGTCTGTTCAAAAGTGCA-3’

FITC-C6-8：

5’-GTACTTCCATTTGTGTTTGCCCGGAGCCTTAGTCTGTTCAAAAGTGCA-3’

Bio-GP30：

5’-GCAATGGTACGGTACTTCC(N)₃₀CAAAAGTGCACGCTACTTTGCTAA-3’

FITC-GP30：

5’-GCAATGGTACGGTACTTCC(N)₃₀CAAAAGTGCACGCTACTTTGCTAA-3’

Note：N represent A, T, G, C any one.

2.C6-8 the separation of aptamers binding target molecule

1) total protein of tetra- kinds of cells of HeLa, H1299, MCF-7, HepG2 is extracted respectively；

2) the coated magnetic bead of streptavidin is washed after 3 times with 0.5 × PBS, then is washed 3 times with 1 × PBS；

3) take 600pmolGp30 libraries and C6-8 to be dissolved in 500 μ l incubation buffers respectively, be incubated at room temperature with magnetic bead 30min；

4) 1 × PBS washs magnetic bead 5 times, removes uncombined free aptamers, puts into 600 μ g total protein of cell extracts With above-mentioned 37 DEG C of incubation 2h of magnetic bead；

5) 1 × PBS washs magnetic bead 6 times, adds 50 μ l1 × 95 DEG C of SDS PAGE sample-loading buffers and boils 10min elution of bound Albumen；

6) the above-mentioned eluent loadings of 15 μ l are taken, 10%SDS-PAGE electrophoresis chooses specific band and carries out Mass Spectrometric Identification.

3.EMSA verifies the combination of C6-8 aptamers and target molecule

1) 1.2pmolBio-Gp30 libraries and Bio-C6-8 is taken to be dissolved in 100 μ l incubation buffers (1 × PBS- respectively 1mmolMgCl₂) in 95 DEG C heating 5min after be immediately placed in cooled on ice 10min；

2) the heterogeneous ribose cores of 5 μ g bought the good Bio-Gp30 libraries of denaturation treatment and Bio-C6-8 with company respectively Albumin A 1 (hnRNPA1) and heterogeneity ribonucleoprotein A2/B1 (hnRNPA2/B1) are incubated 1h at 37 DEG C；

3) 6% natural PAGE glue electrophoresis 2h30min, unloads glue and turns after nylon membrane 70V90min, UV-crosslinked 2min with closing 30min is closed in fluid-tight, is developed the color according to kit specification.

4. prepare cell climbing sheet, prepare histotomy

1) cell climbing sheet：Collect breast carcinoma cell strain MCF7, hepatoma H22 cells respectively, lung cancer cell line H1299, Laryngeal cancer cell strain Hep2, Ovarian Cancer Cells CAOV-3, cervical cancer cell strain HeLa, adjustment cell concentration to 1 × 10⁵/ Mi, takes 200 μ l inoculations to be pre-placed in 24 orifice plates of cover glass, cell climbing sheet is stayed overnight.- 20 DEG C of fixations of absolute methanol of precooling 2h, 1 × PBS are rinsed 3 times.

2) histotomy：The cancerous tissue section and cancer beside organism's section of FFPE are respectively through dimethylbenzene dewaxing 30min, ladder Degree alcohol enters water (absolute ethyl alcohol, 10min；95% ethanol, 5min；90% ethanol, 5min；80% ethanol, 3min；70% ethanol, 1min；Distilled water, 2min；PBS, 5min), put in the citrate buffer of preheating, in micro-wave oven it is low fire (keep 95 DEG C, but Do not seethe with excitement) irradiation 20min, room temperature placement Slow cooling progress antigen retrieval.Frozen section room temperature balances 30min, and methanol is fixed 10min, 1 × PBS are rinsed 3 times.

3) close：50 μ l confining liquids (1 μ g/ μ l tRNA, 1 μ is added dropwise on cell climbing sheet or histotomy after above-mentioned processing G/ μ l salmon sperm dnas, 1%BSA, 0.02%Tween20,1 × PBS), room temperature closing 20min.

5. it is incubated, with reference to, copolymerization Jiao Liutu

1) certain density FITC-C6-8 or FITC-GP30 are dissolved in 50 μ l incubation buffers (1 × PBS- 1mmolMgCl₂) in, it is immediately placed in cooled on ice 10min after 95 DEG C of heating 5min；

2) FITC-C6-8 after denaturation treatment or FITC-GP30 are added dropwise on the histotomy after cell climbing sheet or processing, 37 DEG C of incubation 1.5h in wet box；

3) 1 × PBS is rinsed 3 times, washes away uncombined aglucon；

4) 0.25% her Wen's orchid lining contaminates, room temperature 10min.Ibid 1 × PBS is rinsed 3 times, anti-fluorescence decay mountant envelope Piece；

5) fluorescence microscope or confocal microscopy, stay figure.

6. flow cytometry C6-8 and tumour cell combination

2) tumour cell is collected, sterile 1 × PBS is washed once, adjustment cell concentration is 5 × 10⁵/ pipe single cell suspension, plus Enter confining liquid 1 × PBS-0.1 μ g/ μ l ytRNA-.0.1 μ g/ μ l salmon sperm dna -1%BSA-0.02%Tween20 to seal in room temperature Close 30min；

3) centrifugation removes confining liquid, by 37 DEG C of incubations of denaturation treatment good FITC-C6-8 or FITC-GP30 and tumour cell 1h；

4) centrifugation removes supernatant, and often pipe adds 500 μ l1 × PBS-1mmolMgCl₂Solution uses fluidic cell after cell is resuspended Instrument meter number determines FITC fluorescence intensities.

Experimental result：

C6-8 not only heterogeneous ribonucleoprotein A2/B1 (hnRNPA2/B1) albumen of specific recognition, and can also specifically knowing The kinds of tumor cells of the albumen is not expressed.

By Fig. 1 can with it is concluded that：C6-8 specific recognitions heterogeneity ribonucleoprotein A2/B1 (hnRNPA2/B1) albumen, With heterogeneous ribonucleoprotein A1 (hnRNPA1) without substantially combination, GP30 is not combined to both albumen.

By Fig. 2 can with it is concluded that：C6-8 specific bond kinds of tumor cells, and control sequence is without combination.

By Fig. 3 it is concluded that：Tumour cell in the section of C6-8 specific bonds liver cancer tissue.Control sequence does not have then With reference to.

In a word, C6-8 can recognize heterogeneous ribonucleoprotein A2/B1 (hnRNPA2/B1) and express many of the albumen Tumour cell is planted, with extensive clinical practice and base application prospect.

Claims

1. a kind of heterogeneous ribonucleoprotein A2/B1 of specific recognition oligonucleotides aptamers C6-8 sequence, it is characterised in that Described nucleotide sequence is as shown in SEQ ID No.1 in sequence table.

2. the oligonucleotides aptamers C6-8 described in claim 1 is preparing the discriminating of tumor research reagent, clinical tumor sample Application in reagent.