CN107083388B

CN107083388B - Aptamer wh3 specifically binding to annexin A2 and application thereof

Info

Publication number: CN107083388B
Application number: CN201710255793.5A
Authority: CN
Inventors: 刘静; 周卫华; 叶茂; 萧小鹃; 张毅彬; 曾亚悦
Original assignee: Hunan University; Central South University
Current assignee: Hunan University; Central South University
Priority date: 2017-04-19
Filing date: 2017-04-19
Publication date: 2020-05-08
Anticipated expiration: 2037-04-19
Also published as: CN107083388A

Abstract

本发明公开了特异结合膜联蛋白A2(Annexin A2,ANXA2）的核酸适体wh3及其应用，wh3序列如SEQ ID NO.3所示，该核酸适体能够与ANXA2特异结合，因此可以用于ANXA2的纯化和浓缩，也可以与治疗药物结合后作为靶向ANXA2的药物,进一步研究表明wh3能阻断ANXA2的作用，抑制多发性骨髓瘤（Multiple Myeloma，MM）细胞的生长和粘附且可作为ANXA2定量或定性检测试剂，应用前景广泛。The present invention discloses a nucleic acid aptamer wh3 that specifically binds to Annexin A2 (Annexin A2, ANXA2) and its application. The sequence of wh3 is shown in SEQ ID NO. 3. The nucleic acid aptamer can specifically bind to ANXA2, so it can be used for The purification and concentration of ANXA2 can also be combined with therapeutic drugs as a drug targeting ANXA2. Further studies have shown that wh3 can block the effect of ANXA2, inhibit the growth and adhesion of multiple myeloma (MM) cells, and can inhibit the growth and adhesion of multiple myeloma (MM) cells. As a quantitative or qualitative detection reagent for ANXA2, it has wide application prospects.

Description

Aptamer wh3 specifically binding to annexin A2 and application thereof

Technical Field

The invention belongs to the field of aptamers, and particularly relates to a nucleic acid aptamer specifically binding to annexin A2, and an application of the nucleic acid aptamer.

Background

Annexin A2(Annexin A2, ANXA2) is a protein consisting of 339 amino acids and having a molecular weight of 36 kDa, and is Ca²⁺The dependent phospholipid binding proteins can exist in three forms in cells, namely, monomers (p36), heterodimers (p36/p11) and heterotetramers (p362/p 112). It is highly conserved in structure and is expressed in almost all eukaryotes, mainly distributed in the cell membrane, the cytoplasm, and a small fraction in the nucleus. ANXA2 is mainly expressed in endothelial cells, monocytes, macrophages, nerve cells and some tumor cells, in brain cancer, liver cancer, pancreatic cancer,Expression is upregulated in breast, lung and colon cancers as well as hematological tumors. High expression of ANXA2 in tumors is closely related to tumor development, infiltration, metastasis and prognosis. ANXA2 is highly expressed in high-invasion and high-metastasis malignant tumors, which indicates that ANXA2 is expected to be a marker for judging tumor infiltration and metastasis capacity and prognosis of tumor patients, and can be used as a potential target molecule for tumor treatment, and the aptamer can be used for early detection and treatment of tumors.

At present, the detection methods of ANXA2 mainly include immunological detection methods such as immunohistochemistry and enzyme-linked immunosorbent assay. The antibody-dependent immunological detection method is simple to operate, high in sensitivity, free of large-scale expensive instruments and equipment and suitable for detection of a large number of samples, but the method is high in false positive rate of detection and inaccurate in quantification. And the stability of reagents such as antibodies used for detection is poor, and the storage condition is stricter than that of DNA aptamer. The aptamer can be combined with various organic or inorganic target molecules with high specificity and high affinity, and has the advantages of small molecular weight, low immunogenicity, good stability, convenient preparation and labeling and the like. Nucleic acid aptamers play an important role in the diagnosis and treatment of diseases as alternative molecules to antibody molecules. However, no DNA aptamer against ANXA2 has been reported or disclosed so far, and therefore it is necessary to develop a nucleic acid aptamer that can specifically bind to ANXA 2.

Disclosure of Invention

In view of this, the present invention aims to provide a nucleic acid aptamer that specifically binds to and acts on ANXA 2.

The present inventors have adopted a subtractive selection strategy, and have used GST-ANXA2 fusion protein as a selection target to obtain a DNA sequence bound to the fusion protein, and have removed the DNA sequence bound to the GST protein by using the GST protein as a negative selection to obtain a nucleic acid aptamer capable of specifically binding to ANXA2 protein, and have obtained a nucleic acid aptamer specifically binding to ANXA2 by selection (named wh 3). The nucleotide sequence of the aptamer is shown in SEQ ID NO. 3.

The invention provides application of the aptamer wh3 specifically binding to ANXA2 in preparation of a reagent for detecting ANXA 2. Specifically, the ANXA2 protein is combined with an enzyme label plate, and then the combination is incubated by using biotin-labeled wh3 aptamer; after washing the plate with PBS, the plate is incubated with peroxidase-labeled streptavidin, the substrate added with peroxidase is developed, and the concentration of ANXA2 is detected and calculated by a microplate reader.

The invention provides the use of a nucleic acid aptamer wh3 that specifically binds ANXA2 in the purification and concentration of ANXA 2. Specifically, ANXA2 protein was bound to biotin-labeled wh3 aptamer, which was then incubated with streptavidin magnetic beads, and ANXA2 was purified and concentrated by magnetic particles.

The invention provides an application of a nucleic acid aptamer specifically binding to ANXA2 in preparation of a targeted ANXA2 medicine. Specifically, ANXA2 protein on the surface of cell membrane can combine with various proteins to play physiological roles, wh3 can combine with ANXA2 protein, possibly block the interaction of ANXA2 protein and other proteins, and the function of wh3 is shown in that wh3 blocks ANXA2 protein to promote the growth of MM.1S and RPMI-8226 cells, and wh3 can inhibit the adhesion of MM1.S and RPMI-8226 cells to ANXA2 protein.

The invention has the beneficial effects that:

the invention discloses a nucleic acid aptamer specifically binding to ANXA2, which can be specifically bound with ANXA2, provides a target molecule for purifying and concentrating ANXA2, targeting ANXA2 drugs and quantitatively or qualitatively detecting ANXA2, and simultaneously provides a tool for detecting the level of ANXA2 in serum, reflecting the tumor load state of an organism, provides a strategy for clinically selecting an optimal treatment scheme, and provides help for observing treatment effect and judging prognosis.

Drawings

FIG. 1 spatial structure of wh3 aptamer;

FIG. 2 ELISA detects wh3 binding to ANXA2 protein;

FIG. 3, Aptamer-pulldown detected binding of wh3 to ANXA2 protein;

FIG. 4. wh3 inhibits ANXA2 from stimulating the growth of MM.1S and RPMI-8226 cells;

FIG. 5 wh3 inhibits HS-5 cells from stimulating the growth of MM.1S and RPMI-8226 cells;

FIG. 6 wh3 inhibited adhesion of MM.1S and RPMI-8226 cells to ANXA2 protein.

Detailed Description

Example 1

A nucleotide library sequence with the length of 80 nt (Shanghai biological engineering, Shanghai, Ltd.) was synthesized, and the specific sequence was as follows: 5'-ACCGAC CGT GCT GGA CTC A (N) 42A CTA TGA GCGAGC CTG GCG-3', both ends are fixed sequences, the middle 42N of the library represents 42 random bases, and the library capacity is ensured to be about 10¹⁴Sufficient library capacity to form different three-dimensional spatial structures to ensure that sequences with spatial structures that bind to the target are present and are screened in a subsequent screening process, the library sequences being the P80 ss library. The random oligomeric ssDNA library was then amplified with the following primers:

P80-SF：5'-FAM-ACC GACCGT GCT GGA CTC A-3' （SEQ ID NO.1）

P80-AP: 5'-biotin-CGC CAG GCT CGC TCA TAG T-3' （SEQ ID NO.2）

random ssDNA libraries (5000 pmol for the first round of screening, corresponding to 4 OD random pools) were dissolved in 1mL of 1 × selection buffer in EP tubes (1% BSA coated, 4 ℃ closed overnight), denatured at 95 ℃ for 10 min, and immediately placed at 0 ℃ for 10 min after denaturation. The GST protein-coated beads were mixed well, 100. mu.L of which was placed in a BSA-coated EP tube, and washed 3 times with 150. mu.L of binding buffer. The ssDNA library (5 nmol) was mixed with GST protein-coated beads and incubated at room temperature for 1 h to remove ssDNA sequences bound to the GST protein beads. The supernatant ssDNA library was mixed with ANXA2 coated magnetic beads and incubated for 1 h while adding yeast tRNA to compete for binding. The EP tube was placed on a magnetic stand for 2-3 min, the supernatant was discarded, and unbound ssDNA sequences were washed off with 1 XWash buffer, 3 min each time, and repeated 3 times. Placing on magnetic frame for 2-3 min, and discarding the supernatant. Adding 200 μ L deionized water into EP tube, heating at 95 deg.C for 5 min, placing on magnetic frame for 2 min, taking supernatant as PCR template, and performing PCR amplification.

Configuring a PCR system: mu.L template, 1. mu.L primer P80-SF (10. mu.M), 1. mu.L primer P80-AP (10. mu.M), 4. mu.L dNTPs (2.5 mM each), 5. mu.L 10 XBuffer, 0.25. mu.L Taq DNA polymerase, ddH₂O36.75. mu.L (total volume 50. mu.L). Amplification was performed on a PCR instrument under the following conditions: pre-denaturation at 95 ℃3 min, denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for 25 cycles, and extension at 72 ℃ for 5 min. After amplification, the PCR product was transferred to a 1.5 mL centrifuge tube. 200 u L1 x magnetic bead combined washing buffer heavy suspension streptavidin agarose beads, to the final concentration of 5 u g/L streptavidin agarose beads, adding 1 mLPCR product, room temperature shaking table incubation binding for 1 h, then 1000 rpm, centrifugation for 2 min, abandon the supernatant. The streptavidin agarose beads were washed with 1 × magnetic bead binding wash buffer, 5 min each time, 3 times. The supernatant was removed and 400. mu.L of 200mM NaOH was added and incubated with streptavidin agarose beads for 5 min to melt the dsDNA, which was then centrifuged at 1000 rpm for 2 min, leaving one ssDNA containing biotin still on the streptavidin agarose beads and the other ssDNA without biotin in the supernatant. Separating ssDNA in the supernatant to obtain the enrichment pool for the next round of screening. Repeat 9 cycles of screening.

Clone sequencing and sequence analysis

And amplifying the aptamer enrichment library obtained by the 9 th round of screening by using unmodified primers, sending the obtained product to Shanghai biological engineering Co., Ltd for high-throughput sequencing, and obtaining two aptamers combined with ANXA2 after the sequencing is successful, wherein one sequence is shown as SEQ ID NO.3 and is named as wh3 (the other sequence is named as wh6, and the application is made in another case). The specific sequence is as follows:

ANXA2 nucleic acid aptamer (wh3) was: 5'-ACCGACCGTGCTGGACTCAGGCCGATTGACTTTCCAACAAACTTAGGCCCACTAGACAGAAACTATGAGCGAGCCTGGCG-3' (SEQ ID NO.3)

The secondary structure of wh3 aptamer sequence was then analyzed by mfold software and is shown in FIG. 1.

Example 2

1×10⁷The ANBL-6 cells were lysed with Western blot and IP cell lysate (Biyunyan, P0013) on ice for 30 min, total cell protein was extracted, and 20. mu.L of protein solution was taken as input. On average, the mixture was divided into three EP tubes, 50pmol of the 5' -biotin-modified wh3 aptamer and DNA-Lib were added, respectively, to the tubes, and a negative control group of streptavidin-agarose beads was added, followed by shaking incubation at 4 ℃ for 1 hour. Then 20. mu.L of streptavidin agarose beads were added and incubated for 1 h at 4 ℃ in a shaker. Centrifuging at 1000 rpm for 2 minAnd discarding the supernatant, adding 1mL of DPBS for washing for 5 min, centrifuging at 1000 rpm for 2 min, and discarding the supernatant. The DPBS wash was repeated three times, 40. mu.L of Western blot loading buffer was added to the EP tube and heated at 95 ℃ for 5 min. Centrifuging at 1000 rpm for 2 min, taking supernatant, performing SDS-PAGE electrophoresis, transferring membranes, sealing milk, incubating with mouse monoclonal ANXA2 antibody at 4 ℃ overnight, washing with TPBS three times, adding HRP-labeled rabbit anti-mouse IgG, and developing by ECL reaction solution to detect the binding condition of wh3 aptamer, DNA-Lib and ANXA 2.

The results showed (see FIG. 2) that DNAl-lib and streptavidin agarose beads did not bind to ANXA2 protein and that wh3 aptamer was able to bind to ANXA2 protein.

Example 3

The ANXA2 recombinant protein was diluted to 1 μ g/mL with binding buffer and 200 μ L was added to the wells of the microplate. Incubate overnight at 4 ℃ and wash with DPBS four times for 3 min each. Blocking with 1% BSA at 37 ℃ for 2h, washing with DPBS three times for 3 min each time. Denaturing the 5' end biotin modified wh3 aptamer and DNA-Lib for 10 min at 95 ℃, and placing on ice for 10 min; then 20nM of each was added to a well of an enzyme-labeled plate coated with ANXA2 protein and incubated at room temperature for 2 h. DPBS washes three times, each for 3 min. 200 μ L of horseradish peroxidase-labeled streptavidin (Biyuntian, P0013, A0303) was added to the wells of the microplate and incubated at room temperature for 1 h. DPBS washes three times, each for 3 min. 100 μ L of color developing solution (EL-ABTS color developing kit, C510031, Shanghai Biotech) was added to the wells of the enzyme plate, and color development was carried out for 15 min. And detecting the absorbance by a microplate reader at 405nm/650nm double wavelength.

The results show (see fig. 3): the results of affinity analysis of binding of wh3 aptamer to ANXA2 indicated that wh3 aptamer was capable of specifically binding to ANXA 2.

Example 4

MM.1S and RPMI-8226 cells in logarithmic growth phase are subjected to 2X 10⁵one/mL was plated in 24-well plates. After overnight growth, 1. mu.g/mL ANXA2 protein was added the next day; simultaneously, 4. mu.M of DNA-lib and wh3 aptamer were added, respectively. The culture was continued for 72h, the cells were counted and the stimulation of cell growth by ANXA2 and the blocking of cell growth by wh3 aptamer were observed.

The results show (see fig. 4): the ANXA2 protein can obviously promote the growth of MM.1S and RPMI-8226 cells, DNA-lib has no influence on the growth of MM.1S and RPMI-8226 cells promoted by the ANXA2 protein, and wh3 aptamer blocks the growth of MM.1S and RPMI-8226 cells promoted by the ANXA2 protein.

Example 5

1X 10 of HS-5 cells⁵one/mL was plated in 24-well plates. After overnight growth, 1X 10 was added the next day⁶MM.1S and RPMI-8226 cells; and 4. mu.M of DNA-lib and wh3 aptamer were added, respectively, and the culture was continued for 72 hours. The suspension cells cultured in the upper layer were then pipetted into a 96-well plate and the cell viability was measured using the CCK-8 kit.

The results show (see fig. 5): the ANXA2 aptamer can block HS-5 cells from promoting the proliferation of MM.1S and RPMI-8226 cells.

Example 6

ANXA2 recombinant protein was diluted to 1 μ g/mL with binding buffer and 200 μ L was added to 96 well plates. Incubate overnight at 4 ℃ and wash with DPBS four times for 3 min each. Blocking with 1% BSA at 37 ℃ for 2h, washing with DPBS three times for 3 min each time. MM.1S cells and RPMI-8226 cells were stained with DiI (Biyunyan, C1036) for 20 min. mu.L of the mixture was added to wells of a 96-well plate in which ANXA2 protein had been incubated, and 4. mu.M of DNA-lib and wh3 aptamer were added, respectively, and incubated with the cells for 2 hours. Washed 3 times with DPBS for 2 min each time. Fluorescence was detected by a full-wavelength plate reader (excitation 549 nm, emission 635 nm).

The results in FIG. 6 show that wh3 aptamer inhibited adhesion of MM.1S and RPMI-8226 cells to ANXA2 protein.

<110> university of the south of the middle, Hunan university

<120> nucleic acid aptamer wh3 specifically binding to annexin A2 and application thereof

<160>3

<210>1

<211>19

<212>DNA

<400>1

ACC GACCGT GCT GGA CTC A 19

<210>2

<211>19

<212>DNA

<400>2

CGC CAG GCT CGC TCA TAG T 19

<210>3

<211>80

<212>DNA

<400>3

ACCGACCGTGCTGGACTCAGGCCGATTGACTTTCCAACAAACTTAGGCCCACTAGACAGAAACTATGAGCGAGCCTGGCG 80

Claims

1. An aptamer wh3 that specifically binds to annexin a2, characterized in that: the sequence of the aptamer wh3 is shown as SEQ ID NO. 3.

2. The nucleic acid aptamer wh3 that specifically binds to annexin a2 of claim 1, wherein: the aptamer wh3 was labeled with biotin.

3. Use of the nucleic acid aptamer wh3 as claimed in any of claims 1 to 2, which specifically binds to annexin a2, for the preparation of a reagent for the detection of annexin a 2.

4. Use of the nucleic acid aptamer wh3 as claimed in any of claims 1 to 2 that specifically binds to annexin a2 for purification and concentration of annexin a 2.

5. The use of claim 4, wherein: annexin A2 protein was bound to the biotinylated aptamer wh3, which was then incubated with streptavidin magnetic beads, and annexin A2 was purified and concentrated by magnetic plates.

6. Use of the nucleic acid aptamer wh3 as claimed in any of claims 1 to 2, which specifically binds to annexin a2, for the preparation of a medicament targeting annexin a 2.