WO2021093551A1 - Cd81 aptamer and application thereof - Google Patents

Abstract

Disclosed in the present invention are a CD81 aptamer and an application thereof, the CD81 aptamer having (a) the nucleotide sequence shown in SEQ ID NO. 1 or (b) a sequence after deletion or replacement of one or more bases in the nucleotide sequence shown in SEQ ID NO. 1. The CD81 aptamer of the present invention can be used for preparing a reagent or reagent kit for the detection and/or diagnosis of cancer.

Description

A CD81 aptamer and its application Technical field

The present invention belongs to the field of biotechnology, and specifically relates to CD81 aptamers and applications thereof.

Background technique

An aptamer is a small piece of single-stranded oligonucleotide sequence (DNA/RNA) obtained after screening, which can bind with the corresponding ligand with high affinity and strong specificity. Aptamers can be combined with large molecules such as proteins and cells; they can also be combined with a small number of peptides, small molecule drugs, carbohydrate compounds and ions. Aptamers can be rapidly synthesized, identified and modified, and have the advantages of high reproducibility, high purity and low price, making aptamers an important role in the early diagnosis, monitoring and treatment of diseases, especially those based on exosomes. diagnosis.

Exosomes are a type of vesicle structure (30-150nm) that contains small RNA, DNA and protein. Exosomes are widely found in biological fluids, including blood, saliva, urine, cerebrospinal fluid, and milk, and most of the cultured cells can secrete exosomes. Exosomes have always been regarded as specifically secreted membrane vesicles, which can participate in the regulation of many biological functions such as cell-to-cell communication and cell microenvironment regulation, including immune response, neurotransmitter transmission, tumor growth and metastasis. More and more studies have shown that a large number of exosomes exist in the circulatory system of organisms, which is very suitable for the diagnosis and development of related diseases. Public studies have shown that GPC1 on exosomes can be used for the early diagnosis of pancreatic ductal carcinoma; and the EGFR T790M mutation on plasma exosomes can be used as a companion diagnostic marker for non-small cell lung cancer.

CD81 is a member of the four-span membrane protein family with a molecular weight of 26kD. CD81 has a highly conserved amino acid sequence in the intracellular and across cell membrane regions, while the extracellular region is volatile, and the extracellular amino acid sequence is different in different species. A large number of studies have shown that CD81 is related to many biological functions, including cell transfer, cell adhesion, cell proliferation and differentiation. Recent public reports have confirmed that CD81 is also abundant in exosomes, especially in serum exosomes related to various tumors, including breast cancer, colorectal cancer, liver cancer, and lung cancer. In addition, compared to other markers of exosomes, such as CD63, TSG101, Rab-5b, etc., CD81 is a broader spectrum of exosomes markers. Therefore, aptamers targeting CD81 are very suitable for the extraction and purification of exosomes and subsequent molecular diagnosis based on exosomes.

The current exosomes separation and purification kits are mainly based on particle size, density gradient and special exosomal surface markers, including density gradient centrifugation, ultracentrifugation, chromatography, and antibody immunobinding methods. However, the purity, completeness and process maturity of the exosomes obtained by these methods are not perfect, and the disadvantages such as time-consuming and high price are unavoidable.

Summary of the invention

The purpose of the present invention is to provide a DNA aptamer for CD81 and its application.

A nucleic acid aptamer, which is any of the following single-stranded oligonucleotide molecules:

A1. The nucleotide sequence is the single-stranded oligonucleotide molecule of SEQ ID NO.1 in the sequence list,

A2. The nucleotide sequence shown in SEQ ID NO.1 in the sequence list is substituted and/or deleted and/or added to the single-stranded oligonucleotide molecule shown in A1). Single-stranded oligonucleotide molecules with more than 50% identity and specific binding to CD81;

In the above-mentioned nucleotide sequence, the identity of more than 90% may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Wherein, the nucleic acid aptamer is directed against the human CD81 protein and can specifically bind to the human CD81 protein.

Further, the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.2.

Further, the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.3.

Further, the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.4.

Further, the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.5.

Further, the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.6.

The application of any of the above-mentioned aptamers in the preparation of reagents or kits for detecting CD81 should also fall within the protection scope of the present invention.

The application of any of the above-mentioned aptamers in the preparation of reagents or kits for extracting CD81 should also fall within the protection scope of the present invention.

Wherein, the CD81 is human CD81 protein.

The application of any of the above-mentioned aptamers in the preparation of reagents or kits for detecting exosomes should also fall within the protection scope of the present invention.

The application of any of the above-mentioned aptamers in the preparation of a kit for extracting exosomes should also fall within the protection scope of the present invention.

Wherein, the exosomes are human exosomes.

Wherein, the human exosomes are exosomes containing human CD81.

The application of any of the above-mentioned aptamers in the preparation of reagents or kits for detecting and/or diagnosing cancer should also fall within the protection scope of the present invention.

Wherein, the diagnostic marker of cancer includes human CD81.

Wherein, the cancer is a solid cancer, such as breast cancer, colorectal cancer, liver cancer and/or lung cancer.

The conjugate obtained by coupling any of the aforementioned nucleic acid aptamers to the solid support should also fall within the protection scope of the present invention.

The cross-linked product obtained by cross-linking any of the above-mentioned nucleic acid aptamers to the solid carrier should also fall within the protection scope of the present invention.

Wherein, the solid phase carrier is magnetic beads.

In the present invention, 6 kinds of CD81 DNA aptamers are screened, and their binding to CD81 has extremely high selectivity and specificity. It is conducive to the advancement and clinical application of follow-up CD81-related research.

Description of the drawings

Figure 1 shows the SELEX technology of the CD81 recombinant protein and CD81 positive expression cell line of the present invention;

Figure 2 is a schematic diagram of the secondary structure of 6 subcloned sequences of CD81-2 of the present invention;

Figure 3 is a comparison diagram of HepG2 cells expressing CD81 and HEK293T cells expressing CD81 of the present invention;

Figure 4 is a comparison diagram of the binding ability of the CD81-2 aptamer of the present invention with CD81-positive HEK293T and HepG2;

Figure 5 is a comparison of the binding ability of the full-length CD81-2 aptamer of the present invention to CD81 overexpressing HEK293T cells and negative control HepG2 cells.

Figure 6 is a comparison diagram of the binding ability of the 8 subtypes of CD81-2 aptamer and CD81 when the CD81 aptamer is used at a concentration of 200nM; among them, the CD81 aptamer is used at a concentration of 200nM and the FAM fluorescence obtained by flow cytometry The intensity value is expressed as mean±standard deviation (Mean±SD), the number of experiments is 3 (n=3), **p＜0.1,***p＜0.01;

Fig. 7 is a comparison diagram of the binding ability of 8 subtypes of CD81-2 aptamer and CD81 when the concentration of CD81 aptamer of the present invention is 600nM; wherein, the concentration of CD81 aptamer is 600nM, obtained by flow cytometry The FAM fluorescence intensity value of FAM is expressed as mean±standard deviation (Mean±SD), the number of experiments is 3 (n=3), *p＜0.5, **p＜0.1;

Figures 8 and 9 are schematic diagrams of the binding power of several subtypes of CD81-2J of the present invention; among them, the concentration of CD81 aptamer is 200nM and 600nM respectively, and the FAM fluorescence intensity values obtained by flow cytometry are averaged ±standard deviation (Mean±SD) means that the number of experiments is 3 (n=3), **p＜0.1,***p＜0.01;

Figures 10 and 11 are schematic diagrams of the binding power of several subtypes of CD81-2F of the present invention; among them, the concentration of CD81 aptamer is 200nM and 600nM, and the FAM fluorescence intensity values obtained by flow cytometry are averaged ±standard deviation (Mean±SD) means that the number of experiments is 3 (n=3), **p＜0.1;

Figure 12 is a two-dimensional structure diagram of the CD81-2J-1 of the present invention;

Figure 13 is a two-dimensional structure diagram of the CD81-2J-6 of the present invention;

Figure 14 is a two-dimensional structure diagram of the CD81-2F-2 of the present invention;

Figure 15 is a comparison diagram of the affinity of CD81-2J-1 of the present invention and HEK293T cells overexpressing CD81 or HEK293T cells that interfere with down-regulation of CD81 expression;

Figure 16 is a comparison diagram of the affinity of CD81-2J-2 of the present invention and HEK293T cells overexpressing CD81 or HEK293T cells that interfere with down-regulation of CD81 expression;

Figure 17 is a comparison diagram of the affinity of the CD81-2F-2 of the present invention and HEK293T cells that overexpress CD81 or HEK293T cells that interfere with down-regulation of CD81 expression;

Figure 18 is a Western blot analysis of the CD81 overexpressed HEK293T and other transmembrane protein overexpressed HEK293T cells of the present invention, using an anti-histidine tag antibody;

Figure 19 is a graph showing the specific affinity of CD81-2J-1 to HEK293T cells overexpressing CD81 and the non-binding of other transmembrane proteins to HEK293T cells;

Figure 20 is a graph showing the specific affinity of CD81-2J-6 to HEK293T cells overexpressing CD81 and the non-binding of other transmembrane proteins to HEK293T cells;

Figure 21 is a graph showing the specific affinity of CD81-2F-2 of the present invention to HEK293T cells overexpressing CD81 and the non-binding of other transmembrane proteins to HEK293T cells;

Figure 22 is the best way for CD81 nucleic acid aptamer to incubate with serum when using CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention to capture human exosomes from human serum Experimental verification in the shortest time;

Figure 23 shows the time required for incubation with Agilent Technologies Co., Ltd. 2.7 micron magnetic beads when capturing human exosomes from human serum with the biotin-labeled CD81-2J-1 nucleic acid aptamer of the present invention Experimental verification;

Figure 24 shows that CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention can effectively capture human exosomes from human serum;

Figure 25 shows that the CD81 nucleic acid aptamers of CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention only recognize human exosomes and do not bind to bovine exosomes;

Figure 26 shows that the CD81 nucleic acid aptamers of CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention capture human serum in comparison with three other commercial sources of biotin-labeled CD81 antibodies. In the case of exosomes, the obtained exosomes contain less blood-borne contaminating proteins;

Figure 27 shows that the CD81-2F-2, CD81-2J-1 and CD81-2J-6 CD81 nucleic acid aptamers of the present invention are used to capture human serum in comparison with products that have been captured by affinity coupling with other commercial sources of exosomes. In the case of exosomes, the obtained exosomes contain less blood-borne contaminating proteins;

Figure 28 shows that compared with other widely used mainstream exosomes purification methods, the CD81 nucleic acid aptamer of the present invention can obtain relatively pure exosomes when capturing exosomes in human serum;

Figure 29 is a comparison with other widely used mainstream exosomes purification methods. The CD81 nucleic acid aptamer of the present invention is used to capture exosomes in human serum with a narrower size fraction;

Figure 30 is a graph of test results based on the nanoparticle tracking analyzer in Figure 29, Figure 30A is the total number of extracellular vesicles/exosomes captured from 500 microliters of human serum by various methods as shown in the figure; Figure 30B is The average number of particle sizes (nm) of extracellular vesicles/exosomes captured from 500 microliters of human serum by various methods as shown in the figure;

Figure 31 shows that the ratio of real extracellular vesicles excluded from non-vesicles in human serum captured by the CD81 nucleic acid aptamer of the present invention is comparable to the current gold standard preparation method (ultracentrifugation);

Figure 32 shows that most of the extracellular vesicles captured by the CD81 nucleic acid ligand of the present invention are larger exosomes in human serum; as shown in the figure, the extracellular vesicles captured from 500 microliters of human serum After the particles, count them with a nanoparticle tracking analyzer;

Figure 33 shows that most of the extracellular nanoparticles captured by the CD81 nucleic acid aptamer of the present invention in human serum are biofilm-encapsulated exosomes;

Figure 34 shows that most of the extracellular nanoparticles captured by the CD81 nucleic acid aptamer of the present invention in human serum are large exosomes wrapped in biofilms;

Fig. 35 shows that the exosomes captured by the CD81 nucleic acid aptamer of the present invention maintain the original biological activity and can promote the cell proliferation of human-derived cells in vitro;

Fig. 36 shows the detection of ultra-sensitive epithelial cell adhesion molecule-positive exosomes according to the present invention;

Figure 37 shows that the CD81 nucleic acid aptamer of the present invention can detect a single epithelial cell adhesion molecule-positive exosomes in the background of 2000 exosomes that do not express epithelial cell adhesion molecules in a simulated liquid biopsy;

Figure 38 shows the biophysical thermodynamic determination of the binding of the CD81 nucleic acid aptamer of the present invention to the human CD81 recombinant protein.

Detailed ways

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The following non-limiting examples may enable those skilled in the art to better understand the present invention.

Any person skilled in the art who makes substitutions or changes according to the technical solutions and concepts of the present invention within the scope of the flaws of the present invention belongs to the protection category of the present invention.

Example 1. Screening of aptamers targeting CD81

CD81 aptamer screening is based on the SELEX technology of CD81 recombinant protein and CD81 positive expression cell line (the schematic diagram is shown in Figure 1), and the specific method is: ^{target in a library of about 10 14} aptamers-single-stranded DNA Screening for CD81 aptamers. The DNA in the library contains the following core sequence (86nt, where N is any other base): 5'-TAG GGA AGA GAA GGA CAT ATG AT-40N-TTG ACT AGT ACA TGA CCA CTT GA-3'. The library contained 671 DNA sequences shown in Table 12. Aptamers bound to CD81 recombinant protein or CD81-positive cell lines are eluted and amplified by PCR. The sequences of the upstream and downstream primers for PCR amplification are: FITC-5'-TA GGG AAG AGA AGG ACA TAT GAT-3' and 5'-TTT TTT TTT TTT TTT TTT TT/iSp9/T CAA GTG GTC ATG TAC TAG TCA A-3'. The amplified DNA was then identified by high-throughput sequencing. Among them, 6 aptamers that bind best to CD81 were obtained. The aptamers were CD81-2, CD81-2F, and CD81-2F-2, respectively. The corresponding sequences of CD81-2J, CD81-2J-1 and CD81-2J-6 are shown in SEQ ID NOs. 1-6; the schematic diagrams of the above-mentioned six CD81 aptamers are shown in Figure 2. The above-mentioned aptamers all have the ability to bind to human CD81 protein.

Table 1. Subclone sequence of 6 CD81-2

Aptamer name	Number of bases	Sequence (5’-3’)
CD81-2	40	TCATTTAGCCGACATCCGGTTGGTTTATGGTTTCCCTAAA
CD81-2F	32	CCGACATCCGGTTGGTTTATGGTTTCCCTAAA
CD81-2F-2	twenty four	CCGACATCCGGGGTTGGTTTCCCA
CD81-2J	28	CATTTAGCCGACATCCGGTTGGTTTATG
CD81-2J-1	28	CGTTTAGCCGCCATCCGGGCGGCTTACG
CD81-2J-6	twenty two	CATTTGACCATCCGGGTCTATG

Example 2. Identification of the binding ability of CD81 aptamer and CD81 at the cellular level

1. Construction of recombinant animal cells overexpressing CD81

The specific construction method of the recombinant animal cell overexpressing CD81 (named the cell 293T/CD81) is as follows: The KpnI and XbaI of the plasmid pCMV3-C-His (Beijing Yiqiao Shenzhou Technology Co., Ltd., a product of Sino Biological Inc.) The sequence between the recognition sites is replaced with the cDNA sequence of human CD81, and other partial sequences of pCMV3-C-His remain unchanged, and the recombinant expression vector is named pCMV3-CD81. PCMV3-CD81 was introduced into HEK293T cells to obtain recombinant cells, which were named 293T/CD81. The nucleotide sequence of the human CD81 cDNA is the nucleotide sequence shown in SEQ ID NO. 7 in the sequence listing, and the encoded amino acid sequence is the his tag protein shown in SEQ ID NO. 8 in the sequence listing .

2. In order to select a suitable cell line for CD81 aptamer-cell binding assay, use HepG2 cell line that does not express CD81 and 293T/CD81 for CD81 antibody staining by flow cytometry.

In this experiment, three experimental groups were designed, including a blank control (shown as a control in Figure 3, that is, without the treatment of CD81 primary antibody and fluorescent secondary antibody), and only Brilliant Violet510 labeled fluorescent secondary antibody (flow cytometry fluorescence detection The channel is AmCyan) and the CD81 primary antibody and fluorescent secondary antibody were added at the same time. The measurement results are shown in Table 2 and Figure 3. The results confirm that only 0.13% (less than 1%) of HepG2 cells express CD81; on the contrary, 98.4% of 293T/CD81 express CD81. Therefore, HepG2 is a good negative control, and CD81 overexpressing HEK293T is suitable for positive selection of cell lines for human CD81 with natural conformation.

Table 2. Comparison table of results of flow cytometry determination of CD81 antibody staining using HepG2 and CD81-overexpressing HEK293T cells (293T/CD81)

HepG2	Positive cell%	Median fluorescence intensity	Net median fluorescence intensity
Control	0.092	1046	0
Secondary Antibodies	0.032	1034	-12
Primary antibody + secondary antibody	0.13	1097	51
293T/CD81	Positive cell%	Median fluorescence intensity	Net median fluorescence intensity
Control	0.04	780	0

Secondary Antibodies	0.05	765	-15
Primary antibody + secondary antibody	98.40	3061	2281

Then, 600 nM FAM-labeled CD81-2 aptamer was used to test its binding ability to CD81 in its natural conformation. By fully binding the FAM-labeled CD81-2 aptamer with 293T/CD81 (expressing CD81) or negative control HepG2 cells (not expressing CD81), the unbound CD81-2 is eluted and detected by flow cytometry FAM fluorescence intensity and number of FAM-positive cells. The results are shown in Figure 4. The results show that the binding ability of CD81-2 aptamer to 293T/CD81 is significantly higher than that of the negative control HepG2 (*p<0.5). In other words, the CD81-2 aptamer has a certain specific binding ability with CD81 in its natural conformation. In order to evaluate the equilibrium dissociation constants (K'd) of the full-length CD81-2 aptamers against HepG2 and 293T/CD81 cells, different concentrations of FAM-labeled CD81-2 aptamers (0μM, 0.1μM, 0.25μM) , 0.5μM, 1μM, 2μM and 5μM) were applied to the determination of aptamer-cell binding affinity. The results are shown in Table 3 and Figure 5. The results showed that the binding K'd of CD81-2 aptamer to HepG2 cells was 1072.3±141.0nM, and the binding K'd of 293T/CD81 was 520.8±4.0nM. The above results show that the binding ability of the full-length CD81-2 aptamer to 293T/CD81 cells is significantly higher than that of the negative control HepG2 cells, which further confirms that the CD81-2 aptamer is a CD81 conjugate.

table 3

In addition, the CD81-2 aptamer was transformed into the 8 subtypes shown in Table 4, and the binding ability of these 8 subtypes to CD81 was verified by the above methods. The HepG2 cell content in each treatment group was the same, 293T/ The content of CD81 is the same, and the content of FAM-labeled aptamers is the same.

The test results are shown in Figure 6 and Figure 7. It can be clearly seen from the results that the binding ability of CD81-2F and CD81-2J to CD81 overexpressing HEK293T cells is significantly higher than that of the negative control HepG2 cells (**p＜0.1,** *p<0.01).

Table 4. Sequences of 8 subtypes of aptamer CD81-2

Aptamer name	Number of bases	Sequence (5’-3’)
CD81-2C	28	CCGACATCCGGTTGGTTTATGGTTTCCC
CD81-2D	11	CCGACATCCGG
CD81-2E	17	TTTATGGTTTCCCTAAA
CD81-2F	32	CCGACATCCGGTTGGTTTATGGTTTCCCTAAA
CD81-2H	27	CCGACATCCGGTTGGTTTATGGTTTCC
CD81-2J	28	CATTTAGCCGACATCCGGTTGGTTTATG
CD81-2M	25	TTTAGCCGACATCCGGTTGCCTAAA
CD81-2N	28	TTTAGCGTTGGTTTATGGTTTCCCTAAA

Specific test method: Collect HEK293T-CD81 positive and negative control HepG2 cells, respectively fold and incubate 200nM and 600nM CD81 aptamers at 95°C for 5 minutes, then place on ice for 5 minutes and 37°C for 15 minutes; fold it well The CD81 aptamer and the cells were incubated on ice for 30 minutes, and then washed with PBS three times, and then the complex of the cells and the CD81 aptamer was used for flow cytometry detection.

Example 3. Identification of the binding ability of CD81-2J and CD81-2F subtypes

For CD81-2J and CD81-2F, we constructed 8 more subclones, whose sequences are shown in Table 5 and Table 6. Through the test method in Example 2, the comparison of the binding strength of the subtypes of CD81-2J and CD81-2F is shown in Figure 8-11. The results proved that the binding capacity of subtypes CD81-2J-1, CD81-2J-6 and CD81-2F-2 to CD81 overexpressing HEK293T cells (293T/CD81) was significantly higher than that of the negative control HepG2 cells (without CD81) (* *p<0.1, ***p<0.01), indicating that the above three subtypes have stronger binding ability to CD81 with a natural conformation. Therefore, CD81-2J-1, CD81-2J-6 and CD81-2F-2 were selected for follow-up verification and testing experiments.

Table 5 Sequences of 8 subtypes of aptamer CD81-2J

Table 6 Sequences of 8 subtypes of aptamer CD81-2J

Example 4. Selective and specific detection of CD81-2J-1, CD81-2J-6 and CD81-2F-2 binding

CD81 overexpressing HEK293T cells (293T/CD81) down-regulated the expression of CD81 by siRNA.

In this example, two siRNAs that down-regulate the expression of CD81 were selected, siRNA-CD81-1 and siRNA-CD81-2, and their corresponding sequences were: siRNA-CD81-1: 5'-GAACUUUCCUGUUACCUUUdTdT-3' (sense strand ), 5'-AAAGGUAACAGGAAAGUUCdTdT-3' (antisense strand); siRNA-CD81-2: 5'-CACCU UCUAU GUAGG CAUCU A dTdT-3' (sense strand), 5'-U AGAUG CCUAC AUAGA AGGUG dTdT-3' (Antisense strand). The siRNA control sequence is: 5'-UUCUCCGAACGUGUCACGUdTdT-3' (sense strand), 5'-ACGUGACACGUUCGGAGAAdTdT-3' (antisense strand). Transfection method: When HEK293T cells are pooled to 60% density in a 6-well plate, first transfect pCMV3-CD81 (4μg/well) with lipofectamine 2000 to overexpress CD81 protein; 24 hours later, transfect siRNA to down-regulate intracellular CD81 Protein expression level: Take 8μL of lipofectamine 2000 plus 192μL of serum-free DMEM and place it at room temperature for 5 minutes as solution A; at the same time, add 5μL of 20μM siRNA (100pmol) to 195μL of serum-free DMEM and mix it into solution B; , B solution mixed, placed at room temperature for 20 minutes, then added to the HEK293T medium containing 600μL serum-free DMEM; 6 hours later, replaced with 10% serum-containing DMEM medium; 48 hours later, the recombinant cells will be transfected siRNA-CD81-1 cells were named 293T/CD81siRNA-1 (CD81 down-regulated cells); cells transfected with siRNA-CD81-2 were named 293T/CD81siRNA-2 (CD81 down-regulated cells).

CD81-2J-1, CD81-2J-6 and CD81-2F-2 were transfected with cells (HEK293T cells, HEK293T overexpressing CD81, HEK293T overexpressing CD81 and transfected with siRNA-CD81-1 and HEK293T overexpressing CD81 after transfection Any cell of siRNA-CD81-1) was incubated, and the affinity of the aptamer was tested according to the CD81 aptamer-cell affinity experiment in Example 2. The results showed that after interference with down-regulation of CD81 expression, the affinity of the three CD81-2 aptamer subtypes CD81-2J-1, CD81-2J-6 and CD81-2F-2 were significantly reduced (***p＜0.01, ** **p<0.001) (Figure 15-17). The above results indicate that CD81-2J-1, CD81-2J-6 and CD81-2F-2 are aptamers that selectively and specifically bind to CD81. In Figure 15-17, 293T/WT are wild-type HEK293 cells; 293T/CD81 control is HEK293 overexpressing CD81; 293T/CD81 scrambled is HEK293 overexpressing CD81 with random sequence siRNA transfection; 293T/CD81 siRNA-1 is HEK293 After overexpressing CD81, it was transfected with siRNA-CD81-1; 293T/CD81 siRNA-2 was HEK293 after overexpressing CD81 and transfected with siRNA-CD81-1. The result in the figure is the mean±standard deviation, and the number of experiment repetitions=3. ***, P≤0.001.

The test results show that the k'd values of CD81-2J-1, CD81-2J-6 and CD81-2F-2 are shown in Table 7-9, respectively, and the two-dimensional structure is shown in Figure 12-14, respectively.

Table 7. Comparison table of k’d values between CD81-2J and CD81-2J-1

Table 8. Comparison table of k’d values of CD81-2J and CD81-2J-6

Table 9. Comparison table of k’d values of CD81-2 F and CD81-2F-2

Incubate CD81-2J-1, CD81-2J-6 and CD81-2F-2 with the above-mentioned HEK293T cells that overexpress CD81 or HEK293T cells that interfere with down-regulation of CD81 expression, and then detect the affinity of the aptamer by flow cytometry .

In addition, by overexpressing other transmembrane proteins labeled with histidine (-His) in HEK293T cells to detect the selection of aptamers CD81-2J-1, CD81-2J-6, CD81-2F-2 and CD81 binding Sex and specificity. Specific method: Prepare HEK293T, CD81 overexpressed HEK293T, CD9 overexpressed HEK293T, CD63 overexpressed HEK293T, CDH13 overexpressed HEK293T, CD40 overexpressed HEK293T and Her2 overexpressed HEK293T cells (see Example 1 for the preparation method) CD9: Genebank Accession Number: NM_001769; CD63: Genebank Accession Number: NM_001257390; CDH13: Genebank Accession Number: NM_001220490; CD40: Genebank Accession Number: NM_001250; Her2: Genebank Accession Number: NM_004448.2); Every 50000 Each cell is a group. Fold and incubate the 200nM FAM-labeled CD81 aptamer at 95°C for 5 minutes, then place it on ice for 5 minutes and 37°C for 15 minutes; incubate the folded CD81 aptamer with the cells on ice for 30 minutes, and then use After washing with PBS three times, the complex of the above-mentioned cells and CD81 aptamer was used for flow cytometry detection.

The histidine tag was detected by western blot experiment, and the results proved that HEK293T cells overexpressing CD81 and HEK293T cells overexpressing other transmembrane proteins have been successfully established (as shown in Figure 18). The CD81 aptamer-cell affinity experiment in Example 2 was performed using the above-mentioned cells. The results showed that the three CD81-2 aptamer subtypes CD81-2J-1, CD81-2J-6 and CD81-2F-2 all have extremely high affinity for CD81 overexpressing HEK293T cells (***p< 0.01, ****p<0.001); there is no significant change in the affinity of HEK293T cells overexpressing other transmembrane proteins (as shown in Figure 19-21). The above results reveal that the binding of the three CD81-2 aptamer subtypes to CD81 has extremely high selectivity and specificity.

Example 5. Ability of CD81 aptamer to capture human serum exosomes

In order to evaluate the best time for a single CD81 aptamer to capture human serum exosomes, biotin (biotin; Gemma gene synthesis, catalog number: F01001) was used to label CD81-2F-2, CD81-2J-1 and CD81-2J- 6. Incubate the biotin-labeled CD81 aptamers CD81-2F-2, CD81-2J-1 and CD81-2J-6 with human serum for 30 minutes, 1 hour and 4 hours, respectively. The biotin-CD81 aptamer-exosomal complex was captured by streptavidin-coated magnetic beads, and then the anti-human CD81 antibody was used for Western detection. As shown in Figure 22, the CD81-2F-2 aptamer can capture a large number of exosomes within 30 minutes, while extending the incubation time to 4 hours only slightly increases the capture volume. CD81-2J-1 and CD81-2J-6 aptamers can effectively capture exosomes after 30 minutes of incubation, and further incubation for 1 hour or 4 hours will not significantly increase the amount of captured exosomes. The results are shown in Figure 22. Figure 22 shows the CD81 nucleic acid aptamer and serum when human exosomes are captured from human serum with biotin-labeled CD81-2F-2, CD81-2J-1 and CD81-2J-6 Experimental verification of the best and shortest time required for incubation. Figure 22A shows CD81 Western blots of exosomes captured with different incubation times. Figure 22B shows the semi-quantitative CD81 protein derived from exosomes obtained after the nucleic acid ligand and serum are incubated for different periods of time according to CD81 western blot analysis. The result in the figure is the mean ± standard deviation, and the experiment is repeated 3 times. The results show that CD81-2J-1 and CD81-2J-6 aptamers can effectively bind to exosomes in human serum within 30 minutes. Therefore, the optimal time for CD81-2J-1 and CD81-2J-6 aptamers to capture human serum exosomes is determined to be 30 minutes, while CD81-2F-2 aptamers require a longer incubation time to reach the maximum The amount of exosomes captured.

Example 6. The ability of a CD81 aptamer-magnetic bead-based system to isolate exosomes during incubation

In order to verify the shortest time for the CD81 aptamer-magnetic bead-based system to capture human serum exosomes, the biotin-labeled CD81-2J-1 aptamer (indicated by CD81 in Figure 23) and The human serum was incubated for 1 minute, 20 minutes and 1 hour. Then, the above-mentioned CD81 aptamer exosome complex was captured by streptavidin-coated magnetic beads for 1 minute (bead 1 min) and 5 minutes (bead 5 min). Finally, the exosomes captured by CD81 aptamer-magnetic beads were lysed, and the classical marker CD81 of exosomes was detected by Western blotting. The results are shown in Figure 23. Figure 23 shows the difference between using the biotin-labeled CD81-2J-1 nucleic acid aptamer of the present invention to capture human exosomes from human serum. Experimental verification of the time required for bead incubation. Figure 23A is a CD81 Western blot of exosomes captured after incubation with 2.7 micron magnetic beads from Agilent Technologies Co., Ltd. for different times. FIG. 23B is a semi-quantitative result of CD81 protein derived from exosomes captured after incubation with 2.7 micron magnetic beads of Agilent Technologies Co., Ltd. after different times of incubation based on CD81 Western blot analysis. The result in the figure is the mean ± standard deviation, and the experiment is repeated 3 times. As shown in Figure 23A, CD81 protein can be detected in exosomal samples captured after one minute of incubation with CD81 aptamer, and this signal increases with the incubation time of CD81 aptamer and serum (Figure 23B) . The above results show that the CD81 aptamer of the present invention can capture human serum exosomes within two minutes: a CD81 aptamer-exosomal complex is formed in one minute, and the other minute is used for streptavidin magnetic beads. Take the CD81 aptamer-exosomal complex. The effective separation of exosomes from serum within 2 minutes is the fastest method for separating exosomes reported so far.

Example 7. The ability of CD81 aptamer to capture human serum exosomes was detected by flow cytometry.

CD81-2F-2, CD81-2J-1 and CD81-2J-6 were simultaneously labeled with biotin and CY5 fluorescent label (Q670, purchased from Suzhou Gemma Gene) to obtain CD81 aptamers labeled with biotin and Q670. The CD81 aptamer labeled with Q670 was incubated with human serum, and then the biotin-CD81 aptamer-exocrine was captured using streptavidin-coated magnetic beads with a size of 2.7 μm (from Agilent Technologies Co., Ltd.)体conjugates. After staining with phycoerythrin (PE) labeled CD9 antibody, the exosomes immobilized on magnetic beads were detected by flow cytometry. The scrambled DNA co-labeled with CY5 fluorescent label (Q670) and biotin was used as a negative control for the CD81 DNA aptamer. PE-labeled mouse IgG1 was used as a negative control for PE-labeled CD9 antibody. The results are shown in Figure 24. Figure 24 is a diagram of the results of verifying the ability of CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention to capture human exosomes from human serum. Figure 24A shows The invented nucleic acid aptamer is double-labeled with Q670 and biotin, and is detected by APC channel in a flow cytometer to prove that the nucleic acid aptamer is indeed bound to the streptavidin-coated magnetic beads. Figure 24B shows that only the CD81-2F-2, CD81-2J-1 and CD81-2J-6 CD81 nucleic acid aptamers of the present invention can be captured by PE-labeled CD9 antibody (for detecting exosomes) and flow cytometry Exosomes in human serum, other negative controls (magnetic beads themselves, represented by magnetic beads in the figure; random DNA fragments, represented by DNA in the figure; or control antibodies, immunoglobulin G1 control in the figure) have no effect. As shown in Figure 24A, in sharp contrast to magnetic beads containing only human serum, 99.9% of streptavidin magnetic beads in the Q670-labeled CD81-aptamer group or Q670-labeled scrambled DNA group are covered with Q670 label CD81-aptamers or scrambled DNA. As shown in Figure 24B, compared with the three control groups, at least 55.4% of the magnetic bead complexes in the CD81 aptamer group were PE (CD9 labeled) positive. The above evidence shows that about 55.4% of 99.9% Q670-labeled biotin-CD81 aptamer-coated magnetic bead complexes are positive for serum exosomes. Therefore, consistent with the results of Western blotting, the results of flow cytometry further confirmed that all three CD81 aptamers can effectively capture human serum exosomes.

Example 8. Specificity verification of CD81 aptamer

In order to determine the species specificity of the aptamer of the present invention, it is first determined whether the streptavidin-coated magnetic beads can capture bovine exosomes after incubation with biotin-labeled anti-bovine CD81 antibody and bovine serum. For this purpose, the complex of biotin-labeled anti-bovine CD81 antibody and bovine exosomes was incubated with streptavidin-coated magnetic beads, and then anti-bovine CD81 or CD9 antibodies were used for Western and flow cytometry detection. The expected results indicate that the magnetic bead system of the present invention can effectively capture bovine exosomes through the mediation of anti-bovine CD81 antibodies.

These antibodies that can bind to CD81/CD9 from human and bovine are used as positive controls to prove the specificity of capturing exosomes based on the CD81 aptamer-magnetic bead system. As shown in Figure 25A, the incubation group with biotin-labeled anti-bovine CD81 antibody can clearly detect bovine CD81 and CD9, but the biotin-labeled CD81 aptamer incubation group does not detect bovine CD81 and CD9, indicating that CD81 is suitable. The ligand does not bind to bovine exosomes.

In order to further confirm that the CD81 aptamer does not interact with bovine exosomes, flow cytometry analysis was further used, and its sensitivity was several orders of magnitude higher than that of western detection. As shown in Figure 25B, compared with the magnetic bead group containing only bovine serum, the fluorescence intensity of PE-CD9 in the three CD81 aptamers CD81-2F-2, CD81-2J-1 and CD81-2J-6 was not significant The difference indicates that our CD81 aptamer does not capture bovine serum exosomes. On the contrary, the fluorescence intensity of PE-CD9 in the samples treated with anti-bovine CD81 antibody shifted significantly to the right (49.0% positive magnetic beads), which was significantly positive compared with the magnetic beads containing only bovine serum and the three CD81 aptamer groups . Therefore, CD81-2F-2, CD81-2J-1 and CD81-2J-6 aptamers recognize and specifically bind to human exosomes and do not cross-react with bovine exosomes.

The results are shown in Figure 25, which proves that the CD81 nucleic acid aptamers of CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention only recognize human exosomes and not bovine exosomes. Exosomes binding. The biotin-labeled 215 nM nucleic acid aptamer was incubated with calf serum for 4 hours and captured with streptavidin-coated magnetic beads. Then use the anti-bovine CD81 antibody to detect whether the invention CD81-2F-2, CD81-2J-1 and CD81-2J-6 can capture bovine exosomes. Figure 25A used anti-bovine CD9 antibody and Western blot experiment to show that biotin-labeled anti-bovine CD81 antibody can be captured from calf serum by streptavidin-coated magnetic beads, but with biotin-labeled CD81-2F- 2. The CD81 nucleic acid aptamers of CD81-2J-1 and CD81-2J-6 cannot capture bovine exosomes. Figure 25 Ba double-labeled CD81 nucleic acid aptamer with Q670 and biotin was detected by APC channel in a flow cytometer to prove that the nucleic acid aptamer did indeed bind to the streptavidin-coated magnetic beads. Figure 25Bb shows that the combination of biotin-labeled anti-calf-derived CD81 antibody (detection of exosomes) and streptavidin-coated magnetic beads can obtain exosomes in calf serum. However, the same biotin-labeled CD81-2F-2, CD81-2J-1 and CD81-2J-6 CD81 nucleic acid aptamers or magnetic beads of the present invention cannot capture exosomes in calf serum.

Example 9. Comparison of serum protein content in exosomes separated from CD81 antibody and CD81 aptamer

Common affinity exosome isolation kits use various antibodies. In order to evaluate the purity of the exosomes captured by the biotin-labeled CD81 aptamer, the serum protein content in the exosomes captured by the chemical antibody (CD81 aptamer) and the CD81 antibody were compared. Purchase three biotin-labeled anti-human CD81 monoclonal antibodies, namely antibody-1 (BioLegend, Cat#349514) (corresponding to CD81 Ab-1 in Figure 26), antibody-2 (R&D, Cat#RDSMAB4615) (corresponding to CD81 Ab-2 in Figure 26 and Antibody-3 (MyBioSource, Cat#MBS666563) (corresponding to CD81 Ab-3 in Figure 26). In the experiment, all CD81 aptamers and antibodies contained biotin label, and the same streptavidin-coated magnetic beads (Agilent, Cat#PL6827-1030) were used to separate CD81 aptamers-exosomes and CD81 antibodies -Exosomal complexes. After the exosomes captured by the magnetic beads are lysed, antibodies against exosomal markers (CD81 and CD9) and antibodies against common serum protein contaminants (IgG, serum albumin, IgM and ApoB) Western blot analysis. The results are shown in Figure 26. Figure 26 shows the CD81 nucleic acid of CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention in comparison with three other commercial sources of biotin-labeled CD81 antibodies. When the aptamer captures exosomes in human serum, the obtained exosomes contain less blood-borne contaminating proteins. Mix 215nM of biotin-labeled antibody or nucleic acid aptamer with 500 microliters of human serum and incubate for 30 minutes, then incubate with streptavidin-coated magnetic beads for 20 minutes and wash with PBS, then use antibodies respectively IgG, serum albumin (corresponding to serum albumin in Figure 26), IgM, ApoB, CD81 and CD9 were subjected to Western blot analysis, and the results are shown in Figure 26A. Figure 26B is a semi-quantitative analysis. The abundances of five different blood-derived proteins are all compared using the abundance of CD81 protein bands in their Western blots as a benchmark. The result in the figure is the mean ± standard deviation, and the experiment is repeated 3 times. As shown in Fig. 26A and Fig. 26B-a, through the analysis of the CD9/CD81 ratio detected by Western, the exosomal population captured by the CD81 aptamer of the present invention is slightly different from the three commercial CD81 antibodies. In order to compare the degree of contamination of serum protein when separating exosomes in different ways, the ratio of the signal of serum protein in Western to the signal of CD81 (as a loading control) was used as an index to judge the degree of contamination. As shown in Figure 21B-b, CD81-2F-2-(P≤0.001), CD81-2J-1-(P≤0.01), CD81-2J-6-(P≤0.0001) aptamer group) and CD81 The ratio of IgG/CD81 in the antibody-2 (P≤0.01) group was significantly lower than that in the CD81 antibody-1- and CD81 antibody-3 groups. In addition, compared with the CD81 antibody-2 group, the ratio of IgG/CD81 in exosomes captured by CD81-2F-2 (P≤0.05) and CD81-2J-6 (P≤0.05) aptamers was significantly reduced (e.g. Figure 26B-b). Therefore, the exosomes isolated using CD81-2F-2 and CD81-2J-6 aptamers have lower serum protein contamination (IgG) than those obtained with three CD81 antibodies.

As albumin is one of the most abundant serum proteins, the content of serum albumin in the exosome separation solution is an excellent indicator of the specificity and purity of the exosome separation method. As shown in Figure 21B-c, compared with CD81 antibody-2 and CD81 antibody-3, the ratio of serum albumin/CD81 in the exosomal separation solution of the three CD81 aptamers and CD81 antibody-1 was significantly reduced. Specifically, the amount of serum albumin in the exosomal separation solution of CD81-2F-2 and CD81-2J-6 aptamers was reduced by 12 times and 3 times compared with the use of CD81 antibody-2 or CD81 antibody-3, respectively.

Lipoprotein is the main contaminant in the exosomal separation solution. As many as 70% of the particles in the exosomal fraction separated by various methods are lipoproteins, not real exosomes. In most exosomes prepared from serum/plasma, apolipoprotein B (ApoB) is the main component in contaminating lipoprotein particles. Therefore, the present invention analyzes the content of ApoB in the exosomal separation liquid to assess the degree of lipoprotein contamination in the exosomes. As shown in Figure 26A and 26B-d, exocrine prepared with aptamers CD81-2F-2 (P≤0.001), CD81-2J-1 (P≤0.01) and CD81-2J-6 (P≤0.0001) The proportion of ApoB in the exosomes separated by the three anti-CD81 antibodies was 2 times lower. Therefore, an obvious advantage of the CD81 aptamers of the present invention is that they provide very rapid exosomes preparation, and compared with the use of CD81 antibody isolation methods, significantly reduce the content of contaminants, including serum proteins and non-exocrine体粒。 Body particles.

However, compared with the exosomes isolated using the CD81 antibody method, there is more immunoglobulin M (IgM) contamination in the exosomes isolated from the CD81 aptamer (Figure 26A and 26B-e).

Experimental results show that the CD81 aptamer of the present invention has high specificity in capturing CD81-positive exosomes in human serum, and can better reduce the contamination of serum albumin and lipoprotein particles.

Example 10

Compared with commercial affinity-based exosomes isolation kits, CD81 aptamers can produce higher purity exosomes

In order to further evaluate the purity of the exosomes captured by the biotin-CD81 aptamer, the present invention compares the extraction of exosomes based on the CD81 aptamer-magnetic beads with two commonly used commercial affinity-coupled magnetic The performance of the bead exosome extraction kit. The kits used are as follows: (1) MagCapture ^TM exosome isolation kit based on phosphatidylserine affinity (Novachem, Cat#293-77601), (2) exosome-human CD81 isolation kit (Life Technologies, Cat#) 10616D).

The results are shown in Figure 27. Figure 27 shows the CD81 nucleic acid of CD81-2F-2, CD81-2J-1 and CD81-2J-6 of the present invention in comparison with other commercial exosomes captured by affinity coupling. When the aptamer captures exosomes in human serum, the obtained exosomes contain less blood-borne contaminating proteins. The commercial source affinity capture products used in this experiment are MagCapture ^TM Exosome Isolation Kit PS (FUJIFILM Wako Chemicals, Cat No: 293-77601) and Exosome-Human CD81 Isolation Reagent (ThermoFisher Scientific, Cat. No. 10616D) . 500 microliters of 430nM nucleic acid aptamer was mixed with 500 microliters of human serum, incubated for 30 minutes, then incubated with streptavidin-coated magnetic beads for 20 minutes, and then captured by the antibody pair shown on the left of Figure 27A Western blot analysis of exosomes was performed. In the semi-quantitative analysis shown in Figure 27B, the abundances of five different blood-derived proteins were compared using the abundance of CD81 protein bands in the respective Western blots as a benchmark. The result in the figure is the mean ± standard deviation, and the experiment is repeated 3 times. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 compared with MagCapture ^TM kits in 5 experimental groups. #,P≤0.05;##,P≤0.01;###,P≤0.001 compared with the immunoglobulin G group and the albumin group in the ^{MagCapture TM kit experimental group.} Similar to the results obtained in Example 9, judging from the ratio of CD9 to CD81 in different exosomes preparation methods, the exosomes isolated by the CD81 aptamer population of the present invention are slightly different from the two commercial affinity separation kits. There are differences (Figure 27A and 27B-a). Compared with the two commercial kits, the contamination of serum proteins (immunoglobulin G (IgG), albumin, IgM and ApoB) in exosomes prepared from the three CD81 aptamers was significantly reduced. The above results indicate that, as an effective affinity separation tool for high-purity human serum exosomes, CD81 aptamers obviously have higher advantages (as shown in Figures 27A and 27B-b to 27B-e).

Example 11

Compared with the gold standard method for exosome isolation, CD81 aptamer-mediated exosome isolation contains less serum protein contamination

Among the many methods used to isolate exosomes, ultracentrifugation is still the gold standard, and more than 50% of the published papers in the field of exosomes use this method. In addition, physical methods such as ultrafiltration, chromatography and polymer-based precipitation (such as Exoquick) to separate exosomes constitute about 90% of all current exosomes separation methods. Therefore, it is imperative to compare the purity of CD81 aptamers with the current mainstream methods for preparing exosomes.

To this end, the present invention uses ultracentrifugation, ultrafiltration, cross-linked dextran G50 column, Exoquick kit (System Biosciences, Cat#EXOQ5A-1) and CD81 aptamer to separate exosomes from human serum. Then, through the experimental methods described in Figure 21 and Figure 22 above, the amount of serum protein contamination in the exosomes obtained by different methods was detected. The results are shown in Figure 28, which shows that compared with other widely used mainstream exosomes purification methods, the CD81 nucleic acid aptamer of the present invention can obtain relatively pure exosomes when capturing exosomes in human serum. body. Among them, the exosomes are made with 500 microliters of human serum. The methods used are ultracentrifugation, ultrafiltration, G-50 dextran gel filtration and ExoQuick kit. In the nucleic acid aptamer group, the magnetic beads and random sequence DNA were used as negative controls. Figure 28A is a Western blot analysis of exosomes obtained by various methods. The antibodies used are IgG, serum albumin (corresponding to serum albumin in Figure 28), Haptoglobin, IgM, ApoB, CD81 and CD9. In the semi-quantitative analysis of Figure 28B, the abundances of six different blood-derived proteins were compared using the abundance of the CD81 protein band in the respective western blots as a benchmark. The result in the figure is the mean±standard deviation, and the number of experiment repetitions=3. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 compared with ultracentrifugation samples in each experimental group. ^# , P≤0.05 compared with the exosome samples prepared by the Exoquik kit in the serum albumin group (28ABd). By Western analysis of the ratio of CD9 to CD81, it was found that the exosomes separated by four conventional methods and three CD81 aptamers were different.

In contrast to the ultrafiltration separation method, the exosomes separated from the CD81 aptamer of the present invention did not detect the presence of haptoglobin (Figure 28A). In addition, compared with the method of preparing exosomes by ultracentrifugation and cross-linked dextran G50 column, IgG contamination in exosomes separated by CD81 aptamer was significantly reduced (Figure 28B-c).

In terms of the most abundant serum protein in the serum, the exosomes isolated from the three CD81 aptamers have the least albumin contamination, and the CD81-2F-2 aptamer is the most excellent. Compared with the aptamers CD81-2J-1, CD81-2J-6 and the other four conventional methods, the aptamer CD81-2F-2 captures the least amount of serum albumin (Figure 28B-d).

In addition, compared with the gold standard ultracentrifugation method, the relative content of IgM in the exosomes separated from the three CD81 aptamers was significantly reduced (Figure 28B-e).

Secondly, compared with the gold standard ultracentrifugation method, the relative content of ApoB in the exosomes separated from the three CD81 aptamers also decreased significantly (P<0.0001, Figure 28B-f). ApoB is used to detect the contamination of prepared exosomes from blood apolipoproteins (low density apolipoprotein, very low density apolipoprotein and chylomicrons).

In short, compared with the mainstream exosomes separation and purification methods used by most researchers, the exosomes isolated by the CD81 aptamer-magnetic bead method have higher purity and lower protein contamination (including serum Albumin and apolipoprotein, etc.).

Example 12

Exosomes isolated using CD81 aptamers have a narrow size distribution that is significantly close to exosomes

The exosomes released by human cells are a heterogeneous population of membrane-coated vesicles, ranging in size from 30nm to 150nm. Among various types of exosomes, the size of exosomes is between 30 nm and 150 nm. After the present invention has proved that the exosomes isolated from the CD81 aptamer have less serum protein contamination, the present invention further analyzes the yield and size distribution of the isolated serum exosomes. Nanoparticle tracking analysis (NTA) is a widely used method for characterizing the concentration and size of exosomes in aqueous media. Figure 29 shows the use of conventional physical methods (ultracentrifugation, ultrafiltration, dextran G50 column, Exoquick kit), other commercially available affinity-based exosome separation kits, including targeted exosome membrane surface Lipid (MagCapture ^TM Exosome Isolation Kit, FUJIFILM Wako Chemicals, Cat#293-77601) and a separation kit targeting CD81 (CD81 Exo-Flow, System Biosciences, Cat#EXOFLOW400A-1) on the surface of the exosomal membrane , And the NTA curve of the exosomal size distribution obtained by the CD81 aptamer separation method of the present invention.

Compared with the currently popular exosomes isolation methods (Figure 29A-D), the exosomes separated by affinity-based purification methods have a narrower size distribution (Figure 29E-I). The size of exosomes isolated using commercial affinity methods (Figure 29E-F) ranged from 20 nm to 1000 nm. However, exosomes isolated from CD81 aptamers showed a very narrow size range of 50 nm to 150 nm. For CD81-2F-2, CD81-2J-1 and CD81-2J-6 aptamers, the most abundant exosomes populations are centered at 113nm, 89nm and 106nm, respectively.

In terms of yield, the total number of exosomes obtained by the current CD81 aptamer separation system of the present invention (determined by NTA) is lower than the ultracentrifugation method. Specifically, the total exosomal particle yields of CD81-2F-2 aptamer, CD81-2J-1 aptamer and CD81-2J-6 aptamer were 67%, 44% and 88% of the ultracentrifugation method, respectively. (Figure 30A). In addition, the average particle size of exosomes separated by CD81 aptamer was 130nm-147nm slightly smaller than the ultracentrifugation method (154nm) (Figure 30B).

It is crucial that NTA measures the number of all particles and does not represent the actual number of extracellular vesicles. In fact, the literature clearly records that about 30%-70% of the "external vesicle particles" separated from serum by the current general method are lipoprotein particles, not real exosomes. Therefore, the present invention first treats the sample with 0.5% Triton X-100 at room temperature for 15 minutes to lyse all the vesicle particles in the exosomes obtained by different extraction methods. During this process, the non-vesicle particles always maintain their integrity. . Secondly, by NTA analysis of the changes in the number of particles in the samples before and after 0.5% Triton X-100 treatment, the number and percentage of real exosomes in CD81 aptamers and exosomes prepared by other methods were determined. As shown in Figure 31A, the yield of total vesicles isolated using CD81-2J-6 aptamer was only slightly lower than that of ultracentrifugation. The total number of vesicles separated using CD81-2F-2 and CD81-2J-1 aptamers were 73% and 48% of the ultracentrifugation method, respectively. However, compared with the above four commonly used methods and two affinity separation-based kits, the percentage of true exosomes in the exosomes isolated by CD81 aptamer is similar (CD81-2J-6 aptamer) Or significantly increased (CD81-2F-2 and CD81-2J-1 aptamers) (Figure 31B). All in all, compared with the gold standard ultracentrifugation method, the serum exosomes separated by the CD81 aptamer affinity purification method have a narrower particle size distribution, the yield is the same order of magnitude, and the real exosomes are similar. It needs to be pointed out that the CD81 aptamer-magnetic bead-based system is currently the only one-step rapid exosome separation method, and the size of the exosomes captured by it is closest to the size range of the defined exosomes. Figure 29 shows that compared with other widely used mainstream exosomes purification methods, the CD81 nucleic acid aptamer of the present invention has a narrower size fraction of exosomes captured in human serum. The extracellular vesicles/exosomes in human serum are respectively adapted for ultracentrifugation, ultrafiltration, G-50 dextran gel filtration, ExoQuick, MagCapture ^TM CD81 Exo-Flow kit and the three CD81 nucleic acids of the present invention Body capture. Free extracellular vesicles/exosomes are analyzed by the Nanoparticle Tracking Analyzer to analyze the size, distribution and concentration of extracellular vesicles/exosomes captured by each method. The vertical dashed line in the figure marks the position of 150 nanometers. Figure 30 is a test based on the nanoparticle tracking analyzer in Figure 29, showing the total number of extracellular vesicles/exosomes captured from 500 microliters of human serum by various methods as shown in the figure (Figure 30A) and particle size ( Nanometers) (Figure 30B). Figure 31 shows that the ratio of the CD81 nucleic acid aptamer of the present invention in capturing real extracellular vesicles in human serum excluding non-vesicles is comparable to the current gold standard preparation method (ultracentrifugation). After displaying the extracellular particles captured from 500 microliters of human serum by various methods as shown in the figure, the total particle concentration was obtained by the counting of the nanoparticle tracking analyzer (Figure 31A). The sample was then treated with 0.5% polyethylene glycol octyl phenyl ether (Triton X-100) to dissolve the biofilm, and then counted with a nanoparticle tracking analyzer to obtain the concentration of non-vesicle particles. The total particle concentration of each sample minus its non-vesicle particle concentration is the concentration of true extracellular vesicles. Figure 31B shows the ratio of true extracellular vesicles to the total number of particles obtained by various capture/purification methods. The results in Figures 30 and 31 are mean ± standard deviation, and the number of experiment repetitions is 3 times. *,P≤0.05; **,P≤0.01 compared with ultracentrifugation samples; ^# ,P≤0.05; ^## ,P≤0.01 compared with two kits on the market for affinity separation with CD81 antibody Compare.

Example 13

CD81 aptamer can highly enrich and isolate exosomes within the size range of exosomes

Based on their biophysical properties, exosomes have recently been classified into small exosomes (Exo-S, 60-80nm) and large exosomes (Exo-L, 90-120nm). It has been determined that the CD81 aptamer affinity purification method can isolate exosomes with a narrow size distribution matching the characteristic size range of exosomes. We continue to study the exosomes in each subgroup of exosomes captured by the CD81 aptamer The abundance of the body. Focus the research on four size ranges, 50-80nm (small exosomes, Exo-S), 80-120nm (large exosomes, Exo-L), 120-150nm (large exosomes, Exo-L) And >150nm (for microbubbles). As shown in Figure 32B, compared with other physical or affinity separation methods, the size range of large exosomes (80-120 nm) in the total particles separated by CD81 aptamer is the highest. In addition, when the isolated exosomal suspension was treated with 0.5% Triton X-100 to remove vesicles, NTA analysis showed that the exosomes isolated by CD81 aptamers were in the size range of 80-120nm and 120-150nm Among the total particles, the percentage of exosomes was higher or similar to that obtained by ultracentrifugation (Figure 33B-C). Specifically, 82% of the total 80-120nm particles separated using CD81-2J-1 had exosomes (Figure 33B), and 79% of the total 120-150nm particles separated using CD81-2F-2 aptamer % Of exosomes (Figure 33C), compared with ultracentrifugation, the proportion of exosomes of the above two sizes is higher or comparable (corresponding values are 23% and 79%). It is worth noting that for all true exosomes isolated using CD81 aptamers, approximately 48%-57% are in the 80-120nm (large exosomes) size range (Figure 34B), and 23%-30% are in the 120-nm range (Figure 34B). 150nm (large exosomes) size range (Figure 34C). Therefore, most (71%-87%) true vesicles (exosomes) isolated using CD81 aptamers are large exosomes (80-150nm).

Among them, Figure 32 shows that most of the extracellular vesicles captured in human serum with the CD81 nucleic acid aptamer of the present invention are larger exosomes. After the extracellular particles are captured from 500 microliters of human serum by various methods as shown in the figure, they are counted with a nanoparticle tracking analyzer. Figure 32A shows the ratio of extracellular particles to total particles in the size range of 50-80 microns. Figure 32B shows the ratio of extracellular particles to total particles in the size range of 80-100 microns. Figure 32C shows the ratio of extracellular particles to total particles in the size range of 120-150 microns. Figure 32D shows the ratio of extracellular particles with a size greater than 150 microns to the total particles. The result in the figure is the mean ± standard deviation, and the experiment is repeated 3 times. *,P≤0.05; **,P≤0.01; ***,P≤0.001; ****,P≤0.0001 compared with ultracentrifugation samples; ^# ,P≤0.05; ^## ,P≤0.01; ^### , P≤0.001 compared with two commercially available kits for affinity separation with CD81 antibody. Figure 33 shows that most of the extracellular nanoparticles captured in human serum with the CD81 nucleic acid aptamer of the present invention are biofilm-encapsulated exosomes. After displaying the extracellular particles captured from 500 microliters of human serum by various methods as shown in the figure, the total particle concentration is obtained by the counting of the nanoparticle tracking analyzer. The sample was then treated with 0.5% polyethylene glycol octyl phenyl ether (Triton X-100) to dissolve the biofilm, and then counted with a nanoparticle tracking analyzer to obtain the concentration of non-vesicle particles. Figure 33A shows the ratio of extracellular particles and non-vesicular particles with a particle size of 50-80 microns in the total particles, respectively. Figure 33B shows the ratio of extracellular particles and non-vesicular particles with a particle size of 80-120 microns in the total particles, respectively. Figure 33C shows the ratio of extracellular particles and non-vesicular particles with a particle size of 120-150 microns in the total particles, respectively. Figure 32D shows the ratios of extracellular particles and non-vesicular particles with a particle size larger than 150 microns in the total particles, respectively. The result in the figure is the mean±standard deviation, and the number of experiment repetitions=3. P≤0.05; **,P≤0.01; ***,P≤0.001 compared with samples treated with 0.5% polyethylene glycol octyl phenyl ether (Triton X-100) in a specific particle size group . Figure 34 shows that most of the extracellular nanoparticles captured in human serum with the CD81 nucleic acid aptamer of the present invention are large exosomes encapsulated by biofilms. After displaying the extracellular particles captured from 500 microliters of human serum by various methods as shown in the figure, the total particle concentration is obtained by the counting of the nanoparticle tracking analyzer. Then the sample was treated with 0.5% polyethylene glycol octyl phenyl ether (Triton X-100) to dissolve the biofilm, and then counted by the nanoparticle tracking analyzer to obtain the concentration of non-vesicle particles to calculate the true extracellular The yield of vesicles. Figure 34A shows the ratio of extracellular vesicles (small exosomes) with a particle size of 50-80 microns obtained by various methods in the total extracellular vesicles obtained by that method. Figure 34B shows the ratio of extracellular vesicles (large exosomes) with a particle size of 80-120 microns obtained by various methods in the total extracellular vesicles obtained by that method. Figure 34 shows the ratio of extracellular vesicles (large exosomes) with a particle size of 120-150 microns obtained by various methods in the total extracellular vesicles obtained by that method. Figure 34D shows the particles obtained by various methods. The ratio of extracellular vesicles (microvesicles) with a diameter greater than 150 microns in the total extracellular vesicles obtained by that method. The result in the figure is the mean±standard deviation, and the number of experiment repetitions=3. *,P≤0.05; **,P≤0.01 compared with ultracentrifugation samples in this group. ^# ,P≤0.05; ^## ,P≤0.01 and the samples in this group purified by ultrafiltration, G-50 dextran gel filtration or with two commercially available CD81 antibody affinity separation kits Compare.

Example 14

Exosomes isolated from CD81 aptamers have the function of promoting cell proliferation

Exosomes can complete the intercellular communication function through the mediation of their intracellular substances, such as proteins, lipids and RNA (miRNA, lnRNA and snRNA). The above experiments have confirmed that the exosomes prepared by CD81 aptamers have the characteristics of high purity and narrow size distribution. This example will further determine whether the isolated exosomes still have cellular functions.

For this reason, in this example, human colon cancer HT29 cells were used as the donor of exosomes, 0.5% exosome-removed serum (EDS) was added to the culture medium, and then cultured in a carbon dioxide cell incubator at 37°C for 48 hours . Then use the biotin-labeled CD81 aptamer-magnetic bead system to separate the HT29 exosomes in the cell culture medium (CCM). The aptamer contains a built-in disulfide bond (SS in Figure 35B), which can be cleaved very gently using a reducing agent (100mM tris(2-carboxyethyl)phosphine hydrochloride/TCEP, incubated at 37°C for 5 minutes) And release the exosomes captured by CD81 aptamer-magnetic beads. As a parallel experiment, the present invention also uses a commercial CD81 Exo-Flow kit based on CD81 antibody affinity to separate and release HT29 exosomes. After analyzing the number of particles and vesicles of each sample using NTA, real vesicles instead of particles (5×10 ⁸ vesicles/well) were added to HEK293T cells that were previously cultured in 96-well plates and only cultured in DMEM. After 48 hours of incubation, the cell proliferation in each well was evaluated by MTT cell viability assay (Figure 35A). As shown in Figure 35C, the exosomes isolated from CD81 aptamer or antibody significantly promoted the proliferation of HEK293T cells. However, the exosomes isolated from CD81 aptamers have a more significant effect on promoting cell proliferation than exosomes isolated from CD81 antibodies (P≤0.05) (Figure 35C). One of the above factors restricting cell proliferation may be related to the buffer that releases exosomes. In other words, the TCEP used to release the exosomes captured by the CD81 aptamer-magnetic beads is likely to be milder than the elution buffer provided in the CD81 Exo-Flow kit. Therefore, exosomes isolated from CD81 aptamers preserve their original cellular functions better than antibody-based affinity separation methods.

In addition, compared with antibody-based CD81 Exo-Flow, CD81 aptamer-based exosome isolation is more effective and economical (see Table 10). Therefore, the CD81 aptamer-based exosome isolation kit will become a very potential substitute for other exosome isolation kits on the market.

Figure 35 shows that the exosomes captured by the CD81 nucleic acid aptamer of the present invention maintain the original biological activity and can promote the cell proliferation of human-derived cells in vitro. Figure 35A, the flow chart of the entire experiment. Figure 35B shows that there is a disulfide bond in the link between the base of the CD81 aptamer and the magnetic beads. After capturing the exosomes, adding a reducing agent (tris(2-carbonylethyl)phosphorus hydrochloride), the disulfide bond is opened, and the exosomes can be gently released from the magnetic beads. Figure 35C, in vitro cell proliferation experiment (using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, MTT). The CD81 nucleic acid aptamer of the present invention is used to capture exosomes from the HT29 cell culture supernatant. In every 2000 HEK293T cell culture wells, 5×10 ⁸ HT29 exosomes were added 48 hours later to detect MTT. Use HEK293T cells plus MTT reagent as the reference degree. The result in the figure is the mean±standard deviation, and the number of experiment repetitions=3. P ≤ 0.01; ***, P ≤ 0.001 compared with the benchmark. ^# , P ≤ 0.05 compared with the group of exosomes extracted with CD81 Exo-Flow kit.

Table 10. Comparison of CD81 Exo-Flow kit and CD81 aptamer (please give the Chinese translation of this table)

Example 15

The CD81 aptamer can detect one EpCAM-positive exosome in the background of 2000 epithelial cell adhesion molecule (EpCAM)-negative exosomes

One of the most promising applications of exosomes in cancer medicine is their application in liquid biopsy. However, one of the key challenges facing exosomal liquid biopsy is the detection of very small amounts of cancer-derived exosomes in a broad background of exosomes produced by blood cells. For example, the total vesicle count in human blood is 7.3-9.4×10 ¹⁰ /mL, of which 93.9% of vesicles are derived from platelets, 4.5% are derived from white blood cells, 1.8% are derived from red blood cells, 1% are derived from endothelial cells, and 0.7% It is derived from hematopoietic stem cells (PLoS One.13(12):e0207950,2018). It is known in the prior art that the plasma exosomes concentration of cancer patients and healthy donors of different individuals can fluctuate by 40 to 50 times, and the average plasma exosomes concentration of cancer patients is (0.9±1.2×10 ⁹ )/mL ) And healthy controls (1.2±1.2×10 ⁹ /mL) have no significant difference. On the other hand, the concentration of exosomes in plasma from lung cancer donors was 1.41±0.31×10 ¹⁰ /mL, while the concentration of exosomes in healthy donors was 3.37±0.39×10 ⁹ /mL, that is, exosomes in the blood of lung cancer patients The concentration was 3.4 times higher than that of healthy controls. It has been determined that all 200 or so human-derived cells can release exosomes, most of which may enter the blood circulation. Therefore, exosomes derived from tumor cells account for only a small proportion of the total exosomes in the blood. Therefore, trying to detect tumor cell-derived exosomes in the extensive exosomal background of a patient's blood sample is like "finding a needle in a haystack."

EpCAM is overexpressed in many types of solid cancers. In fact, the first and only FDA-approved liquid biopsy CellSearch uses a monoclonal antibody against EpCAM to detect circulating tumor cells (CTC) for the diagnosis of metastatic breast cancer and other solid tumors.

This embodiment hopes to establish the detection limit of the CD81 aptamer-based exosome detection system in the context of liquid biopsy. To this end, this example extracts exosomes from the human colon cancer HT29 cell line that highly expresses EpCAM (~1.2×10 ⁶ /cell) and the human embryonic kidney HEK293T cell line that does not express EpCAM. For the initial test, the present invention establishes an aptamer-based sandwich flow analysis system to determine the detection limit of EpCAM-positive exosomes in the background of EpCAM-negative exosomes.

In the blood of healthy people, EpCAM-positive cells or EpCAM-negative exosomes are almost undetectable. In order to establish a measurement condition similar to the clinic, a sample with a total exosome concentration of 4×10 ¹⁰ /ml was prepared in this example to simulate the count of exosomes in the patient's blood. For the determination of the detection limit, a mixture of EpCAM-positive and EpCAM-negative exosomes in different ratios ranging from 1:000 to 1:8000 was first prepared. For this 4×10 ¹⁰ /ml exosomal solution, there is a continuous titration of EpCAM-positive and EpCAM-negative exosomes. Second, the CD81 aptamer double-labeled with fluorescein isothiocyanate and biotin is used to capture all exosomes in the mixture of exosomes, and magnetic beads are used to fix the aptamer-exosomal complex. Subsequently, the captured EpCAM-positive exosomes in each group were stained with phycocyanin-labeled anti-human EpCAM antibody or Q670-labeled EpCAM aptamer, respectively. Finally, the magnetic beads-CD81 aptamer-exosomes complexes were detected and analyzed by flow cytometry. When the percentage of fluorescently positive magnetic beads exceeds 1%, it is considered positive. The results are shown in Figures 36 and 37, and Figure 36 shows that the present invention can be used for the detection of ultra-sensitive epithelial cell adhesion molecule-positive exosomes. Figure 36A shows the flow of this test based on flow cytometry. Figure 36B, the exosomes produced by the HT29 cells captured by the CD81 nucleic acid aptamer of the present invention were incubated with 100 microliters of 8.33 nM allophycocyanin-labeled epithelial cell adhesion molecule antibody. Figure 36C, the exosomes produced by the HT29 cells captured by the CD81 nucleic acid aptamer of the present invention and the epithelial cell adhesion molecule nucleic acid aptamer labeled with the same concentration of stellar body 670 (Quasar 670 phosphoramidite) and HT29 cells are produced The exosomes are incubated. Figure 37 The CD81 nucleic acid aptamer of the present invention can detect a single epithelial cell adhesion molecule-positive exosomes in the background of 2000 exosomes that do not express epithelial cell adhesion molecules in a simulated liquid biopsy. Flow cytometry detects the fluorescence of fluorescein isothiocyanate (FITC) to confirm that the CD81 nucleic acid aptamer labeled with fluorescein isothiocyanate is indeed immobilized on magnetic beads (2.7 microns). Flow cytometry detects the fluorescence of allophycocyanin (APC) to confirm that the allophycocyanin-labeled anti-epithelial cell adhesion molecule antibody or nucleic acid aptamer is the exocytosis captured by the CD81 nucleic acid aptamer of the present invention体合。 Body combination. The experimental group consisted of magnetic beads, a negative control of isotype matching antibodies or nucleic acid aptamers with random sequences, as well as exosomes secreted by HT29 cells with positive epithelial cell adhesion molecules and HEK293T with negative epithelial cell adhesion molecules. A series of limiting dilutions of exosomes secreted by cells, 1:8000, 1:5000, 1:2000 and 1:1000. Flow cytometry detected 1% allophycocyanin fluorescence-positive events in the total samples as epithelium The detection threshold for cell adhesion molecule positive. Figure 37A shows the isosulfide detected on the flow cytometer after the exosomes captured by the CD81 nucleic acid aptamer and immobilized on the magnetic beads are incubated with the epithelial cell adhesion molecule monoclonal antibody Double change point plot of fluorescence of fluorescein cyanide and allophycocyanin. Figure 37B shows the detection of fluorescein isothiocyanate and allophycocyanin on a flow cytometer after the exosomes captured by the CD81 nucleic acid aptamer and immobilized on magnetic beads are incubated with the epithelial cell adhesion molecule DNA aptamer Double change point plot of protein fluorescence.

As shown in Figure 37A, the percentage of positive signals from the phycocyanin-labeled EpCAM antibody (Q2) reaches 1% when the ratio of HT29 EpCAM positive exosomes to HEK293T EpCAM negative exosomes is 1:1000. In contrast, the signal from the Q670 EpCAM aptamer (Q2) was 1% at 1:2000 (Figure 37B). The above data shows that the detection limits of EpCAM antibody and EpCAM aptamer in the system of this embodiment are 1:1000 and 1:2000, respectively. The detection limit of 1 EpCAM-positive exosome from the background of 2000 EpCAM-negative exosomes is unprecedented. That is to say, the present invention further improves the liquid biopsy system based on CD81 aptamer-magnetic beads to achieve Higher sensitivity.

Example 16

Use isothermal titration to study the thermodynamic and kinetic curves of the interaction between the CD81 extracellular macrocyclic structure and two different CD81 aptamers (ie 2J-6 and 2F-2):

Although the knowledge of protein-aptamer binding is essential to assess the performance of aptamers, there are still few studies exploring the thermodynamics and kinetics of the interaction. In order to use aptamers as diagnostic tools, we first studied the thermodynamics and interactions between CD81 extracellular macrocycles (LEL) and two different CD81 aptamers (ie CD81-2J-6 and CD81-2F-2). Kinetic characteristics. Second, explore the molecular mechanism and binding site of the aptamer-CD81 protein interaction.

First, dissolve two aptamers in PBS containing 2.5 mM MgCl _2. Then, it was denatured at 95°C for 5 minutes, and then cooled on ice for 5 minutes. Finally, incubate at 37°C for 15 minutes for folding. In the experiment, NanoDrop ^™ 2000 (Thermo Scientific ^™ , United States) was used to measure the concentration of the aptamer.

The ITC experiment was performed with a Microcal PEAQ-ITC instrument (Malvern Instruments Limited, United Kingdom) at 25°C. The aptamer and CD81 extracellular macrocyclic protein were prepared in ITC buffer containing PBS pH 7.4, 2.5mM MgCl ₂ , 1.33% trehalose, 1.33% mannitol, and 0.0027% Tween 80. The aptamer solution (68 μM) in the syringe was injected into the dimer CD81 outer macrocyclic protein solution (3.33 μM) [7-10]. Except for the first injection (0.4 μL), the volume of the aptamer solution for each injection is 2 μL. All experiments were performed under the following conditions: each injection interval was 250 seconds, a total of 19 injections were performed, the syringe stirring speed was 750 rpm, and the reference power was 10.0 μcal s ^-1 . The stoichiometry of binding parameters (N), binding affinity (K _D ) and thermodynamic parameters (ΔH) were obtained from Micro PEAQ-ITC analysis software. Then use the following equations [11] to calculate free energy (ΔG) and entropy (ΔS):

ΔG=RTlnK _D

ΔG=ΔH-TΔS

Among them, R is the universal gas constant, and T is the temperature in Kelvin.

The kinetic curve was obtained from the AFFIINImeter software (Software 4 Science Developments S.L., Spain).

Each binding parameter is represented by the mean ± standard deviation of three independent measurements.

Figure 38 The biophysical thermodynamic determination of the CD81 nucleic acid aptamer of the present invention in binding to the human CD81 recombinant protein. Figure 38A shows the isothermal titration calorimetry (upper figure) and thermogram (lower figure) of the binding of human-derived CD81 large extracellular domain recombinant protein to the CD81-2J-6 nucleic acid aptamer. Figure 38A shows the isothermal titration calorimetry (upper figure) and thermogram (lower figure) of the binding of human-derived CD81 large extracellular domain recombinant protein to the CD81-2F-2 nucleic acid aptamer.

The results are shown in Figure 38, Figure 38 is a representative ITC thermal analysis diagram, where Figure 38A is the binding isotherm of the D81 LEL-2J-6 interaction, and Figure 38B is the binding isotherm of the CD81 LEL-2F-2 interaction line. The interaction between CD81 LEL and 2J-6 is exothermic, but the interaction with 2F-2 absorbs heat. In addition, the comprehensive data from the heat of binding between 2F-2 and CD81 LEL can be well fitted to a 1:1 binding model, but 2J-6 is suitable for a sequential binding model with two stepwise binding sites. This sequential nature is also clearly shown in the two isothermal lines of the interaction (Figure 38A). The reason is that CD81 LEL exists as an inseparable homodimer in the solution. The results of this study are consistent with the report of the binding site of CD81 LEL by Kong et al.

Table 11. Binding parameters between the two aptamers and CD81 LEL

Table 11 shows the thermodynamic and kinetic parameters of the two aptamers. The first binding of the 2J-6 aptamer to the CD81 protein is caused by a difficult entropy change (enthalpy change ₁ of-9.30±1.02kcal/mol and–( Enthalpy change-Gibbs free energy) ₁ of 2.08±0.83kcal/mol) and has a weak binding affinity (dissociation constant ₁ = 5.19±1.56μM). However, the first binding makes the second 2J-6 aptamer bind more tightly to the CD81 protein (dissociation constant ₁ >> dissociation constant ₂ ), and this phenomenon is essentially positively synergistic. The binding of 2F-2 aptamer to CD81 protein is also driven by entropy change, and it has a weak binding affinity (dissociation constant = 4.28±1.10μM) and a low binding site (N of 0.13±0.04). ). The kinetic curve further proves that the interaction between 2F-2 aptamer and CD81 protein has a general binding constant (association rate constant = 2.32×10 ⁴ M ^-1 s ^-1 ), but its dissociation constant is relatively high ( Dissociation rate constant = 2.63×10 ^-1 s ^-1 ), indicating that the interaction between 2F-2 aptamer and CD81 protein may be weak.

Similar to the flow cytometry measurement, the affinity of the two CD81 aptamers to the CD81 protein is about 70 nM. However, the experimental results of isothermal titration calorimetry show that the two aptamers have completely different thermodynamic properties. Specifically, the 2F-2 aptamer interacts with soluble CD81 LEL through a 1:1 binding model, and has a very low binding site and weak binding affinity. The 2J-6 aptamer interacts with CD81 through a sequential binding model with 2 stepwise binding sites. Among them, the first binding was weak, with a binding affinity of 5.19±1.56μM, but the second binding was much stronger, with a 42-fold increase in binding affinity. However, the two interactions between 2J-6 aptamer and CD81 protein are driven by enthalpy, indicating that 2J-6 aptamer has extremely high specificity.

Table 12. List of partial DNA sequences contained in the library

Numbering	DNA sequence
S1	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S2	TCATTTAGCCGACATCCGGTTGGTTTATGGTTTCCCTAAA
S3	GACGCACCGATCGCAGGTTCACTCACAAACACTCTTCCCC
S4	AACATATATGCTGTATTAGACAACGCACCTGGCGCAAGGT
S5	ACTCGCGGTTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S6	ACTCGCGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S7	TAAGGTTTCCCCACTAACAATTAATTTGGCTTGTGGCATA
S8	GAGCTCGGTCTTTGGACCAAGAATAAGGTTTCCCTCACAT
S9	CCTGGCTTCACTTTGAGATTAAGGTTTCCCCAGGATCTAC
S10	GGATTAAGGTTTCCCCGTCCTGCCGGGACGGCCACGCATA
S11	CATTAAAATTGAGATTGCCACCCGCCGCAGGGTTCGCGAT
S12	TTATGGACGGTTTGTTAAATGCCCTTTAGATAGCCTTCCC
S13	TAAGGTTTCCCTGCGGCGACGTCCATGCAGCGTATGGCCA
S14	GACGCACCGGTCGCAGGTTCAGTGTACGACCTTCTTTTCC
S15	ATGACGCCATCGGATTAAGGTTTCCCGTTATATTATGCTA
S16	TAAGGTTTCCCTGCGGCTACGTCCATGCAGCGTATGGCCA
S17	GCAGGCACCGCTGATCGCCGCAGATCTGACTGATCATTAT
S18	ACTCTCGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S19	TCATGGACGGTTTGTTAAATGCCCTTTAGATAGCCTTCCC
S20	ACTCGCTGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S21	GTACCGCTTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S22	TCCAATATAGCGACTTGGCTCGTTTATGGTTTCCCTATAA
S23	GTACCTCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S24	GTACCGCGTCTCCTCTTTGGTTGCTCTCAATCGCCGGATG
S25	TTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S26	CATTAAAATTGAGATTGCCCCCCGCCGCAGGGTTCGCGAT
S27	ACCGCTTTCTTCCCCATTGGACCCTCTTAATCGGCGGAGT

S28	GACGCACCGGTCGCAGGTTCACTCACAAACACTCTTCCCC
S29	TGTGAGTACCACAGGGACGCGGGCGACGCAGCCCTCACTG
S30	TAAGGTTTCCCTGGGCCACTCAGCATCTCGTTGTTTCGAT
S31	ACTCACGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S32	ATCACCCCACCAATTTCTACCACGCCACACCTCATTCGAT
S33	AACATATATTCTGTATTAGACAACGCACCTGGCGCAAGGT
S34	TAAGGTTTCCCTGCGGCGACGTCCATGCAGCATATGGCCA
S35	ACTCGCGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCCT
S36	GACGCACCGATCGCAGGTTCAGTGTACGACCTTCTTTTCC
S37	CCACCACCTACAACACTCTCGAACTCCCACATTCCTCTTT
S38	AACATATATGCTGTATTAGACAACGCACCTGGCTCAAGGT
S39	TAAGGTTTCCCCGTCTACGGCATATGTACGGCCCGTCTAC
S40	GACGCACCGATCGCAGGTTCACTCATAAACACTCTTCCCC
S41	TCATTTAGCCGACATCCTGTTGGTTTATGGTTTCCCTAAA
S42	GTACCGCGTCTCCTCTTGGGTTTCTCTCAATCGCCGGATG
S43	TAAGGTTTCCCCCGCATTTTTATGTGGCATTAGCCCTTCT
S44	AACATATATGCTGTATTAGACAACGCACCTGGCGCAAGGC
S45	ACTCGCGTTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S46	AACATATATGCTGTATTATACAACGCACCTGGCGCAAGGT
S47	ACTCGCTGTTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S48	CGACCACCCCCAACCCATTCACACGTTCTACCCACTCTCC
S49	TCATTTAGTCGACATCCGGTTGGTTTATGGTTTCCCTAAA
S50	GAGCTCGGTCTTTTGACCAAGAATAAGGTTTCCCTCACAT
S51	CATAAAAATTGAGATTGCCACCCGCCGCAGGGTTCGCGAT
S52	ACTCTCGGTTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S53	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCTGGATG
S54	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGCTG
S55	CATTAAAATTGAGATTCCCACCCGCCGCAGGGTTCGCGAT
S56	CATTAAAATTGAGATTCCCCCCCGCCGCAGGGTTCGCGAT
S57	CCACAACACAACCCAAGCCCAACTACAGTTTCCCTCTTTT
S58	AACATATATGCTTTATTAGACAACGCACCTGGCGCAAGGT
S59	TCATTTAGCCGACATCCGGTTGGCTTATGGTTTCCCTAAA
S60	GAGCTCTGTCTTTGGACCAAGAATAAGGTTTCCCTCACAT
S61	GTACCGCGTCTCCTCTTGGGCTGCTCTCAATCGCCGGATG
S62	ACCACCTACACACCTACTTTTTTTTCGATCCAACCTGCTT
S63	ACTCGCGGTTGTCAAAGGTTGCTCCCCGACGCAGGGGCGT
S64	CGACTACCACCACTACTGACAGCGCCCACCACTTTTTTTT
S65	CAACCTACCTCACTGACACCACCCCAGCCTTTCTCCTCTT
S66	GTATCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S67	CTGACCACTACCGCTCCCCATCCTCACCGAACACCTTGCT
S68	GTACCGCGTCTCCTCTTGTGTTGCTCTCAATCGCCGGATG
S69	CACCATCCACACCCATAAATTTCACACCAACATTTTCCTA
S70	TGATCCTCCCGCACCATCCTCGTTCTACCTCATCAATCAT
S71	TCATTTAGCCGACATCCGGTTGTTTTATGGTTTCCCTAAA
S72	TCATTTAGCCGACATCCGGTTGGTTTATGGCTTCCCTAAA

S73	CTGACCAACCCCCCGTAATCGCTTTTCTCCAGCCACTCAC
S74	CGATCCCCACCAACTCTCTCCGAACCCTCCTAACTCTCGC
S75	CGACCCGCTTTTCCGCTCCTGCCTCCATCCTCTCCAACCA
S76	CACATCACACTACCACCAACCATACACGCTATTTCTCCTT
S77	ACTCGCGTTTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S78	AACCACACCACACAACTACAGACACCCATCTACACAACGA
S79	CACCCCCAACCACTCTCTCTCTCCAGCCTCCTAATTCTAA
S80	TCCCCCTTTCAGCGTTCCCCCCACAGCTCCTCGACACTCC
S81	AAACATCCCACGTACAAAAAAAAAAAAACCACCTCACACTCCT
S82	TAAACACACCACACTACCCAGATCCCACACCGACTTTATT
S83	GTACTGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S84	CATTAAAATTGAGATTGCCACCCGCCGCAGGGCTCGCGAT
S85	AACACACCACCTCCACAACTTACAACCCCCTCACCTTTCT
S86	CCAAGACCAACAACACTACACCACACTAACCCCATTCTGA
S87	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATT
S88	CTTAGCCACAACCTCCACACCTCTACTGCTCCACATAACC
S89	GACGCACCGATCGCAGGCTCACTCACAAACACTCTTCCCC
S90	CGATCCAAAACCCCCCTCTCTGCGTTAGCCATCTCTCTCT
S91	GTCCCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S92	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCTCCGGATG
S93	CTCACACAACCCCCAACATCTCTCAGCTACCCCCTCGATT
S94	AACATATATGCTGTATTAGACAACGCACCTGGCGCAAGGA
S95	TTACAACCCGCCAGGCTCACCACCCCCCACCGTTTTATTT
S96	ACACACAGCAACCACACCACCAGCCACTCACTCTCTTTTT
S97	GACTCACCGATCGCAGGTTCACTCACAAACACTCTTCCCC
S98	AACATATATGCTGTATTAGACAACGCCCCTGGCGCAAGGT
S99	TCTCCCGCAACTCTCTCTCTCTCTCTCCGATCTATCTCTCGT
S100	ATAAACCCACCAACACCTCCACCTCAACACAACTGATTTT
S101	CACAACACACACAATCCCCCTTTTTATCCACCAACTCTAT
S102	CAACACCAACCTCCAGCCAGCTACCGCCCACCTCACCTCT
S103	CGCCCCCCCTCAACCACACTATTTCGCCCTCCATCCTCCT
S104	CGACCCGAATTCAACCTCACCCCTCTTTCGATCTCTCACT
S105	ACTCGCGGTTTTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S106	CGGACCGGACTCCATCCCCCCTCCAAACTCTCGCTCCATC
S107	CACACAATACCACACCACCTCCATCACCCCCTCGTTAGCT
S108	ATTCCCCATGCTCACACTCCCGTTCCCCTCACCTCTCTTT
S109	GTGCCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S110	AACATATATGCTGTATTAGACAACGCGCCTGGCGCAAGGT
S111	CTGACCAACGACACACACACATTCTTATACCCCCAGCTTT
S112	ACTCGCGGTTGTTAAATGTTGCTCCCCGACGCAGGGGCGT
S113	CATAAAAATTGAGATTGCCCCCCGCCGCAGGGTTCGCGAT
S114	TAAGGTTTCCCCAGCATTTTTATGTGGCATTAGCCCTTCT
S115	ACTCGCGGTTGTTAAACTTTGCTCCCCGACGCAGGGGCGT
S116	CGACCACCATTCCACACCTCCACCTCCATCTCGCCGACAC
S117	TAACTCCAACACCAACTACAACCCCCACTCCACGCTCGAT

S118	GTACCGCGTCTCCTCTTGGTTTGCTCTCAATCGCCGGATG
S119	ACTCGCGGTTGTTAAACGTTTCTCCCCGACGCAGGGGCGT
S120	GGATTAAGGTTTCCCCGTCCTTCCGGGACGGCCACGCATA
S121	TACAATAACTCCACCACCGATACACACAGCGACACCCTTC
S122	CGTAACCTCTCCACAACCCCTCTTATCCTCCTCGCTTTTT
S123	CACACCCACCCTGCCTCCACCAACCACCATCCTCTCTCTC
S124	AATTAAAATTGAGATTGCCACCCGCCGCAGGGTTCGCGAT
S125	CTTTCCCCCTCCGCTCGTTGATCCGACCCCTGATTTCTTT
S126	TCATTTAGCCGACATCCGGTTTGTTTATGGTTTCCCTAAA
S127	CTGATCCGTCACCTCCGCAATACCTCTCGACTCTCTCCTC
S128	CGACCAACCCCCCTCAACACCGACCACCCTCTACCTCTCC
S129	CGCTTAAACACATTCACCCCCACGTACACCACCCCTCTAC
S130	TAAGGTTTCCCTGCGGCCACGTCCATGCAGCGTATGGCCA
S131	AACACCCCGACTAACGCAACTCACCACCCCTCTCAGCTTT
S132	AACATATATGCTGTATTAGACAACGCTCCTGGCGCAAGGT
S133	AATCCACCACAACGCATCACACTCACCAGCACCTCCTAAT
S134	AATTCGGCCTCATTTACTTCCTAATTATCTTCGCTTGCGT
S135	TAATTTAGCCGACATCCGGTTGGTTTATGGTTTCCCTAAA
S136	TTACCACCCCCCCACTACCAGCTACACCGACACCTCATTA
S137	TTACAGCACCCACACCTCCACACCGCGAACGACTCCGATT
S138	TACACCACAACCACCTACAACTAACTTTATCCCTCTTTTA
S139	ACACAACAACACCAAACTCAAACAACGCCAACTCTACTTC
S140	CGACTAACCCCACCACGCCTTACTGCCTACCCCAACACAC
S141	AAACCACCACCACCCCAACAACATCTCCACGACCGACACT
S142	CAACCCCAACTCTAGACTACTCCACTCTCTCGCACATTTC
S143	CGACCGCCTTGACTCTCTCTCTCTGCTAACCTCCCCGTTTTA
S144	CAACACACACCCACACACAATAACCTCAAGCCTACCCTTT
S145	AACCACCACACCACCCACATACTCACCACTCCAGCTTTTT
S146	CGAACCGTGCCTCACCGCCTTTACCATCTACATCCACGCC
S147	ACCACCCCCACACCTCAACACCTCTGAACACCACCACTTT
S148	TAACACCACCACCAACCCTCCGATCAACACACACACCACT
S149	TTCCCTCCACATCTCCAGCCCAACTTTCTCGCTTTCTCTC
S150	GGATTAAGGTTTCCCCGTCCTGCCGGGACGGCCACTCATA
S151	CATCCCCACTTTCGTTACATTTCCACCCCCTCTCTCGACT
S152	TACACCCCACCACACCCTGCCACACTTTCACCTTCTGCTT
S153	GACGCACCGATCTCAGGTTCACTCACAAACACTCTTCCCC
S154	TCATTTAGCCGACATCCGTTTGGTTTATGGTTTCCCTAAA
S155	ATTTTACCGCAGACACCCCAGACACCGATCCACCCACTTT
S156	CTCGCCCCCCTCTCCTGTTTTGCCCCTCTATCGCTCTTTT
S157	CCAGAAAAAAAACAATGCATACCCCCTCACCACAGCTCTA
S158	CCTGGCTCCACTTTGAGATTAAGGTTTCCCCAGGATCTAC
S159	ACTCGCGGTTGTTAAACGTTGCTCCCCGACGCAGGGGCGC
S160	ATGACGCCATCGGATTAAGGTTTCCCGTTATATTATTCTA
S161	TACACCCAACACAAAACACCACCCGCACATTAACCCTTAA
S162	CGATCCCACACTACCTCTTTACCTCACCTGCAACACCCCT

S163	ACACACACAGCTAACCCCCATTTTACTACCCACACATTTC
S164	ACACCCCACTTACAACACCACGATTAAATTCACCACCCTT
S165	TTTTACACCCCCACTACAGACACCACACTGCACCACCAAA
S166	CGGACCACCTACACCCCCTCTTTCGCTACTCCACTCCACC
S167	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCCCCGGATG
S168	CATTAAAATGGAGATTGCCACCCGCCGCAGGGTTCGCGAT
S169	GTACCGCGTCTCCTATTGGGTTGCTCTCAATCGCCGGATG
S170	CTGACCCCCCATGCCCCTCACTCTCTACACTCCACTCCCA
S171	CAGAACCACCACCCACTCTACAACTCCTCTCCAACGACTT
S172	CACCTAGCACTACAATTACACCCACAACTCTACTCTCTCC
S173	CGACACCACTCTCTAGCTCTCTGTTTACCCCACCCCACCT
S174	ACAAAACAAAAAACCCGACACACAGCTAACCCTCTACTTA
S175	CACCCCACCAACGTCTCCAGCCCACCACCTGCCTCCGCCT
S176	AACCACACCACATCACACAAAACACCATCCACACACATAC
S177	TTCAACCCAGCCACACCTTTACAGACTCCACCTCCACACC
S178	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCTGATG
S179	CGAACCTAGCAACTCCACCCCTAACTCACTCTCCGCTCAC
S180	TCCCCCACCCTCTACTTTCGACTCAATCTCTCCAACCCAT
S181	AACTAAACCCCAACACTCTCTCCGACCACCTCCGTTTGCT
S182	TAACACACACAACAACACCTACCGAAAATAGCTCTCTCCA
S183	TCTTCCTGCAGTCTCGCCCACCGTTCCTCCACCTATCCTC
S184	TCTAATACAACCCAGCACACCACCACACGGATCCACCTAA
S185	AGATACCCACAAACACAACTTCAGACACACAACACCCAAC
S186	AATAACACCCACACCTCACACAACATCCACCTTCCATTTT
S187	TACACCAACCCACAACTCGACAACTCCACCGCTCCAAAAC
S188	CGACCACCATCCCAATCACCTCCACACAATCCCCTCACAC
S189	TCATTTAGCCTACATCCGGTTGGTTTATGGTTTCCCTAAA
S190	CCCACCACCTCACACATCCACATACCACAGCCGTTTTCCT
S191	GTACCGAGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S192	CGACCTCCCATCTCTCACCCCCTTTTACTCTCCCATCCGA
S193	CACACTACAATTTTATACAACCCCCAACTACACAACCCTT
S194	ACACTACCACCGCACACACTTTACAGTTCTTTAGCTTTTT
S195	TAGCTCGGTCTTTGGACCAAGAATAAGGTTTCCCTCACAT
S196	AACACCACCCAGCACACACAACATCCCCTTTGAGATATTC
S197	ACAAAACAGAAGATAACCCACAAAAAAAAGCACACTCCCA
S198	CTGAACGGCACCCACGCAACTCTCACTCCTCCGCCTTTTG
S199	CAACCGCTCCATCCCCCTCTTACACACCTATCCTCTACCA
S200	CACCACACCCCAGTTACTGTAACCACCACCCCATTATTCT
S201	ACACTAAACACACAAACCCACACCTATCCACACCGATTAA
S202	ACAACACACACAACCCATACCTCGTTAGCACCTCCACTTT
S203	ACCAACCCCACCACCGATACCACAACCTCCACCGTTATTT
S204	TACGCACCGATCGCAGGTTCACTCACAAACACTCTTCCCC
S205	CGACACACTACACCCACGCTCTACGCAACTCCTACACCGC
S206	ATACAACTACCCCCCAACTCCACCCCATCGATCTCACTCT
S207	CGACCGCAACTCCGCTTACTCACCTCTGCCGAACCTCTCC

S208	TCCCCATCCCTCTCTCTGTCTCCATCCTCTCCTCCGATTT
S209	ACCCCCTGCACACTCTGCGATACACACCTACCCCTCCTTT
S210	AAAAAAAACACTACCCCACACCCACCACGCGTATCACTAA
S211	ACCCACACCCACTAGACTCTCCGATTTACTCCATCCCCAC
S212	TAAATTTCGATACACACAACACCCCACACGTTCACCTCCT
S213	ATTACTCCCACCATCCTGCACCACCACTCCGTTTCCAGCT
S214	AAACCACAACTAACACCCTCGACAGCGACAACTCCACACT
S215	CACGCCAACAATTTACCACCTCAGCAATCTCTCACTCCCA
S216	CACACAACCCCATCGGATACACACCCACCTGCAGCTCTAC
S217	CAACCCACCACAGCTCCCCCCTTGACCTCTTTTTTGCAAC
S218	ACACCCCACCACACCTCCACTACCTTGTGAATAACCGAAT
S219	AACATATATGCTGTATTAGACAACGCACCTGGCGCAAGGG
S220	GTACAGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S221	CGACACCCACAACTGCTCCAACCCCCTCCTCAACCACACT
S222	CGACACAACCTCACACCCCATCTCCACTCTCCTAACACTT
S223	GCACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S224	CCACCACAAAAACCCAACTCCACCTCCGATACTCCACAGT
S225	AACATATATGCTGTATTAGACAACGCACCTGGCCCAAGGT
S226	GTACCGCGTATCCTCTTGGGTTGCTCTCAATCGCCGGATG
S227	ACCAATCCCAACACACTGCAACACTACACATTTTCCACCT
S228	CGCACCCAGTAACACTCCCATCCCACGCATCTTTCTCGCT
S229	CATTGCGCAAACCCACCACCGTTCCACCTCCATCTACCAC
S230	CTTTCCTCCCATTCTCACCGCTGACTCCCTCCTCCGATTT
S231	TTTTTACACCCACCAACCCCAACCCAAGCTGCATCCGCAA
S232	CACCACCACCCAAACGTAACATCTCAACTACACCCCAATC
S233	CATCCACCATTCACCTCACCAACCTCCCCCTCCTCTCTTT
S234	CGACCAACTTTAAGCCATCCGCTCTCACCAGCCCCCATTT
S235	CACCACACCAAACACAACACCTATCCACACAGATTTCTCC
S236	CAACACTACACCACTCTAACTCCACACTACCTCTCCTGTT
S237	ATAACACGCTAAAAGACCACCCCACCTCCACACTACCGAT
S238	TCCCCCTACAACTACCTCTACTTAACTCTACCCCTCTCTG
S239	CACCTACCCTCTACGTTTTCCACCTGCGCACCTCCACATC
S240	CTACTAACCACTCCATTCACCCTCGCACAGTTTCTCTCCT
S241	TCACCCCCCACATCTCACTCACCCTCACTCTCTGTTTCGA
S242	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGTATG
S243	ACTCGCGATTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S244	CGAACCTACCAGCCTCTACTACCGCTTACATACCACCCAC
S245	CACAACCAATACACACACCATCCATCTACCTCCATCATAC
S246	CACGAAAAAAAACCACCACCACACACACACCCTCGAATTTGC
S247	TTAACCATCCAGACACACCCCCTTTCACTCCATCGCTCAC
S248	GACGCACCGATCGCAGTTTCACTCACAAACACTCTTCCCC
S249	AACCACCCACGCTAAAGACCCCAACTACCTATCACACTCT
S250	TCATTTATCCGACATCCGGTTGGTTTATGGTTTCCCTAAA
S251	CGAACCACCCCCCGCTCCACCTCTCGCAGACCCACTCCAC
S252	CACACCCACCCCACGCCTGTTACACCACCACACAACATCC

S253	TACAACCCACACCAACAGACTCCACTTTCTTTATAACCAC
S254	TTAACCAGCTACCACCCCCACATTCACACAGCATCTGAAC
S255	CAACACAACACCAGCACCCCACTGCCTACACTACACCAAT
S256	CGACCGTGCTACCCCTCCATCCCTCGGCCCAACACACCCT
S257	GCTCGCGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S258	GCTCGCGGTTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S259	ACACGCTACACCCCCAAGAACCACCGCTCTCTCTCTTTAA
S260	CACATCCACCACACCACCCACATCACCTACTGCTACGATA
S261	AACATATATGCTGTATTAGACAACTCACCTGGCGCAAGGT
S262	CCACACCATCCACCCCGCTATACCACCATCTCCTATGACC
S263	ACTCGCGGTTTTTAAACGTTGCTCCCCGACGCAGGGGCGT
S264	CCACCTAAAACACTCACCGATGCACCACCCCAGCTCTTTT
S265	TTGTTCATAGAACTTAACTAGGCACGCATCTGCCGCAAGA
S266	TACCACCACCACCCACTCCTACCAACAACTCCCCTCCATT
S267	TTCAAACCCACCACCACAACACCAGCCAGCAGCATCCACC
S268	CGCTAACCCTCATTCCCCCCAACCGCTTTACCTCTCTCTA
S269	TAACCCACGACACACACCAGCAGCTACACATACCCAACAT
S270	AAACCCACACGACAATTTCCACCCACACAAACCCCTACTT
S271	CACCACACACCACACACATCCAAGATCCCCCTCCGCTATT
S272	ACTCGATCCACCCCCCGCTACCGCTCTCTCTATCTCCTTT
S273	ACTCGCGGTTGTTAAACGTTGCTCCCCGACGCAGGGGCTT
S274	TTTATCCAGACACACACAACACTCCACCCAACGCTCTCAC
S275	CGCCTCCAATCCATTCACCCCAGCCTAACACGCTCTACTT
S276	CGCTACCGTTAACCTCCCCTGTTTTCCTCCACTCTCCTAC
S277	TCCCCCCCTCCTACATGCTCACCTCCACCACTCTCTATAT
S278	GGATTAAGGTTTCCCCGTCCTGCCGTGACGGCCACGCATA
S279	ACAACCAACTCCATCCACACCTCACACTGCTATCCCATTC
S280	GAGCTCGGTCTTTGGACCAAGAATAAGGCTTCCCTCACAT
S281	CGACCAGCCACTCCTCACATCATCTCAATATATATCCCCT
S282	CCAACACCCACCACTACCTAGACACACCTCCGATCTCTTT
S283	CACCATCCACTACCACCTGTATACACCGCCCAACATCTCC
S284	CATCCACCCAAACACGCGCTATCCCACACTCCTCCAATCT
S285	CACTTAAAAACAAAACACACCCACATAACACTTACCCTGA
S286	TCCCACACCAAAACTACCTCCCCAACTCACCTCCCGCTTT
S287	TAAATCCACCCCACAGCTACAACTCGACTTTACCCCTACC
S288	CGACCACACTCCACTCACCTCCCTGCCGTAGACCCATCCG
S289	ACACTCCACAACTCAACACCTCACCACTTTACCCTCTCGC
S290	CGAACCGTAGAAATCTCACTCCCACCAACTCTCTCTCTCC
S291	GTACCGCATCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S292	TTTCCCCCTGTACAACCCCTCTACCTCTCTCTTTTTCATC
S293	GGATTAAGGTTTCCACGTCCTGCCGGGACGGCCACGCATA
S294	TCCCCCCTTTCTCCACCTCTCGCTCTTTACACACCCCACT
S295	ACTCGCGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCTT
S296	ACTCGCGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGC
S297	ACACAACATCCATACCACCATACAACACATAATTCTACCT

S298	CACACAACGACACCACCCCCGTACACACCGAACCCCTTTT
S299	CAATAAAAACACCCAATACTACTGCAACTCCACCCACTTT
S300	ACAACTCCACACCAACACCTTGCTCCACACCACCTCGACC
S301	CAACACCACACACAACTTGCGCCTCAACCACCACCCAACT
S302	CCCCCACTTAGACGCAACACCCATTACACCCTCCGCTCGT
S303	CTGACACACCACTCCACCACCACACTCCCATCCTGCCCTT
S304	TCAACCCCACATTCGCCCAAACCGATCCACCACTCTCCTC
S305	ACCACCAACACAACCAAACTCTTTAAACACCCAACACTTT
S306	GTACCGCGTCTCCTTTTGGGTTGCTCTCAATCGCCGGATG
S307	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGTTG
S308	TCCCCCCACGCCTCTCACCTCGCTTTCACTTTTTCCCTGC
S309	CTGATACCCCACTCCCACCTTTCGCTCTCACCTCTCTCGC
S310	AAAAACAACAACACACCCAGACACCACTCCTCTCACTCGA
S311	TACACCAACTCCACCACACATCGAACCATCCCTTGATCCT
S312	ACACCCCCAACACCAATCTCACCACCTGCCGCTCTATAAT
S313	TTTACACCACCCCACCATCGGCACATAACCCCACTTTACC
S314	TACCACCTACCACTCCATCCAGACATCACCTTTTATCGAC
S315	CCTGGCTTCACTTTGAGATTAAGGCTTCCCCAGGATCTAC
S316	AACGACAAACTACAACCACCACACTCCAACCACCCAATTC
S317	CGACCCCATCCCTCCATCTCCGGAAACTTGCTCCTCCACC
S318	TCACACACACATCCAACACAGCCACCCCACTCCGAACTTT
S319	TTAACCCACACACCCAGCACTATCTGTATACACCACCCCC
S320	ACACCCCCACACTCTCCAGAAACTCTCAGCTCCACCCTGT
S321	AACTCAACACACACAATTTACACCCCTACCACCGCCTTAC
S322	AACATATATGCTGTATTAGACAACGCACCTGGCGCAAGTT
S323	CATCCACACCAACTCCACTTACATCCAAGATCCCCTCCAC
S324	CACGAACACAGAACCACCTACCACACTTTCCACTCCGATA
S325	ACACCACCCTCACCACAACCATCCCTCTGATTCTCACGTT
S326	CTACCTCTACTACTTTTACTCCTCACCTCTCTTTCTCCGT
S327	CTCCCCCACTACCACCGCTTACGTCTCCACCGAACCATTT
S328	GTACCGCGTCTCCTCTTGGGTTCCTCTCAATCGCCGGATG
S329	CCATCCACTCCACCTCCTTTCTGGCCTTCTTCCATCTTTT
S330	GACGCACCGATCGCAGGTTCACTCACAAACGCTCTTCCCC
S331	TTTACACACCTACCACCACACACTGATACACATTCTCCAC
S332	TCATTTAGCCGACATCCGGTTGGTTTATGGTTTCCCTATA
S333	CTGACCTCGACACCACTCTCTCGCTCTACCCCTTCCTCTG
S334	CTGACCTCCGCCCCGCGTATTACCCCTCCTCTCGCATATC
S335	ACCACCCACACACTCCAGACGAACATACCCATAACTCCAT
S336	CACTACACCACCACACACCCAACTCTACGATCCCCCGATT
S337	CGACCCCTGTTATCCCATTATTTTTCGCCGGTTTTTTTTT
S338	TTAACCCATCCACCACAACGCCATCCAACCCAACATTGAT
S339	ACACCACCACAGCACCTCACCCAACCTTGATTTTTCCACT
S340	CTGACCACCATCCCACGCGAACTCCCTCCTCCTGTCCATT
S341	CACCTCACCGCAACACCAATACCCCCTCCACCCACTGACT
S342	CAACACCCGACGCGCACAACTCACCCCCATCCCGCTCTTT

S343	TCATTTAGCCGACATCCGGCTGGTTTATGGTTTCCCTAAA
S344	CGCACCAAACCACCTCCTTCAGCTCAACCTCTGCCTCTCC
S345	AATTCCCCGACCAGTTCTGCCATCCACCATCCCCTACCGT
S346	CGACCGGAAACACTCCCCCCACACTCTCCTCGCCCTCCCT
S347	AATCCACCACAGAAACACACAATATAACCCCACAATACAC
S348	ACTCGCGGTTGTTAAACGTTGCTCCCCGACTCAGGGGCGT
S349	ACCACCACCACACAAACTCTATACAGCATCCCCATCAGCT
S350	CACCCAACCAAATCCCACCGACTTTCTACACTCCTCCACT
S351	CACCACAACCAACACATACGCAATACCCACCCAACCCTGC
S352	CACAACAAAAATACACGGACACCCACCAACACACACACCTCC
S353	CTCTCCAGCATCCCCCTCACCATCCACCCTGTATCCACAC
S354	CACAACAACCCCCAGACCACTATAATATCACCACACCACC
S355	ACCAACCACAACACACCCCAATTCTTTTTTGCCACCATTA
S356	CGAACAAACAACCCCTCTCACTCTGCGACTGCCCCCGACT
S357	ACAACAACCTCAACCACTCTGCTCACTCCAACACTCCCAC
S358	ACAAACACACGATATAACCCCATCCACACTCCAACACTTT
S359	TACACACAACCCACCCACACCATCCAACTATATCTCAACC
S360	AACCCTACCGCACTCCACAACACAACTCCACCAGTTCCCT
S361	AAAAACCCAACACACAAAACATCCCCTCACATCTACCACT
S362	TAATACACTAAACACCTCCACAAACACACCTCGACTTTTT
S363	CGCACACGTCTGCTACCCACCACCGACACCCAACCCTTTA
S364	CGTACCCCTCACACCTGTCTCTATCCATCCTCTCCTCGCT
S365	CACACTACCACACACCCTCACCACTACCTCTCCTTTCTTA
S366	CAACACCACTACTACCTCACCACACACGAGCTGCTTCCAA
S367	AAAACCCCACACACAGAACTACACCTTACACACCAGCTCC
S368	CACAAAAGCGACACAATTAACCCACCCCCACACACACTTC
S369	AATTAAAATTGAGATTGCCCCCCGCCGCAGGGTTCGCGAT
S370	TACACCACCACAACCCATCCCCAGATCACCTCACCAACCT
S371	CACACACACAACACAAACTCTCCGACACCACCTGCTTTCC
S372	CACCATGACAACAACAACACCCATTCCACTCCACCTGCAT
S373	CGACCTAACCTTTTTTAGCACACCTCTTACACCACCCAGC
S374	GTATTAAGGTTTCCCCGTCCTGCCGGGACGGCCACGCATA
S375	ACAACACCACCACATCTCACAAACCAGCATCCAAATCACC
S376	TACACCCACACCAACACCTGCAGACAGACACACACTCTAC
S377	CAACTACCACCATTCACAGCTCCACCACCACGATTTTACC
S378	TATCCACCCACTCCATCTGACACACCCCTCAACTTTATCC
S379	CATTAAAATTGAGATTGCCACCCGCCGCAGGGGTCGCGAT
S380	GACGCACCGATCGCAGGATCACTCACAAACACTCTTCCCC
S381	GACGCACCGATCGCAGGTTCACTCACAAACCCTCTTCCCC
S382	TAACACACCACCAACCCACACGACTTTACCACCAACTTAT
S383	ACTCGCGGTTGTTAAAGGCTGCTCCCCGACGCAGGGGCGT
S384	TTAACCCCCAGACCACCCACCTTTTTTCACACAAAACCTG
S385	TAACCACACGCAACACCTCAACTCACTCCATCACACACTC
S386	CCCCACCACACTCAACACCACACTCCTTGCTCTCTGAAAT
S387	ATTCCCACCCGCCTACCTCTCACACCTCTCCTCGATCTTT

S388	CGGCAACACAACCCACAATTTCTTTACAAACCACCACAAC
S389	CAGCAGACACACACAGACACCACCACTACCTTTTTTTTAT
S390	AACACAACAACCTACCACCAAAACTCTAAATCGATCCTCC
S391	CCAACAGCTCCACACACACAACCCCACCCAATCCGAAATT
S392	ATCGACACACACAAATACCTCAACAACACCTCCCACATTC
S393	CGAAATACACCACCCAACCCCACCTACAACACCACTCTGC
S394	CACCTACAACCCCTCGACGAACTCCGCAACACACACTTCT
S395	CACCTACAGACCCACACGTTTCACCTCCAGCCTCCATTTT
S396	TTAAGCCACACCCAGCACACACAACTACACACACACTTTT
S397	ACAAAAACACAACTAACAACACACACCACCCAAACTACGAAA
S398	TTGCGACTACCGCTCTACGTTACACCCCCCCTGCACCACC
S399	TACAACACAACCAACGACACCACCTGAAACCCAACTCTCT
S400	CCACCACACCTAACCACCCAATTTCCACTACATTTTGCTT
S401	GTACCGCCTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S402	GACGCCCCGATCGCAGGTTCACTCACAAACACTCTTCCCC
S403	TCTCGCGGTTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S404	AACCACAATGCAACCCTCCCAGCTATACCACTCCCCGTTC
S405	CACAACACAACCCCCCCACCATCTTCGACCCAGTTAGCTA
S406	CACCTCCACCAACCTCCACGCCAGCATCTCACTCTCCATA
S407	AACCACCCACTACATCACCAGCCCCAAACACAACACATTA
S408	CGACCGGTAAACCACGCTCCCAAACTCTCTCCACACACTC
S409	CCTCAACCGGAAACCCTCGAACCTCCACTCTCCTGCTTTT
S410	ACCACTACACCACCTCACCATCCTCCAACCTCCGATCACT
S411	ACCCACCACACACGCAACTACCACCAGTACCCATCTTACA
S412	GGATTAAGGTTTCCCCTTCCTGCCGGGACGGCCACGCATA
S413	CCCAAACCAACACTCCACCACACCTCCTGCACCCTCCTCG
S414	CACCACCACCCCCTCCAATACTCTATCCACCGCTTACTCA
S415	TCATTTAGCCAACATCCGGTTGGTTTATGGTTTCCCTAAA
S416	ACTCGCGGCTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S417	TTTACACCCCACCCTCACAACGCCTTTCACCGCTCCATCT
S418	TTCCCCACAGCTCTCTCGGTACACTCCCCCACTCTCTCGC
S419	CGAACAACCACACTCACTCTCCATTTCGCTCCTCTACTTA
S420	CATCAACACCACCCCTCCGCCGAATATACATCTACCACCT
S421	AACACCAACTCCACAGCAGCAACAAACCTCCACACACTAT
S422	TCCCGTGCACCCCCTCTCTCACTCTCACTCCCCCAACTTA
S423	TCCCCAACCCTCAACTTCGAACCTCCACCATCTCCCACTA
S424	CATACCGCACCTCCCATCTCTCTCGATCTCTCACCCCCGT
S425	AAACAGCAACAACCCCCACCATAGACATCACCCAACACTA
S426	CAGAATACAAAACCCCCCATCCGCAACACTCTCGCCAGCT
S427	CGACTAAACCCCACTCCGCCTACCCAGCCTCCCACCATCT
S428	TACTACTCCCGAAGACCCACCCGTTTCCTTTTTACCATTT
S429	GATCTCGGTCTTTGGACCAAGAATAAGGTTTCCCTCACAT
S430	CTGACCGAGCCTCCAAGCCCACACTCCCTCTCTCTCTCTC
S431	ATCACCATCAACACACACACACACACCAATTACAGCACCCAT
S432	TCCCCCTCACTCTCAGCTCTCACCCCCGGATACACCCTCC

S433	AAGAACCACCGCAACACCACACCACCCCACACATTATGAA
S434	ATACACCCACCACCCGAACTACCAACTCTTGACCCCCTTT
S435	AATTAACACCACCAACCACAACGCAATACCCTCCAACTCT
S436	TTCACACACACAAAAGACACACTCTCCATCCACCTTACTT
S437	GTACCGCGTTTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S438	TCAACTACACTCCACCACCACGCTCCAACCGACTTACACT
S439	ACCACACAAAACAGCACACCCACATTACCCGACACATCTA
S440	CAACCCAAACTGCAACCCTCCGACTACACCACATTACACC
S441	CTCCACCTCCGCACACACAATCCCACTTTCTCCCTCGTTT
S442	GAGCTCGGTCTTTGGACCAAGAATAAGGATTCCCTCACAT
S443	TACCTCCAACACACACTGCCTACCCAGCCTCTCTCTCAGT
S444	CCCCATCACGCATCGCCATCCACACAAGCACCTCCGATTT
S445	ACCAATTGCAACTCACACACCAGCCCATCCACCACAATCC
S446	CACAACACCAGCCACCCATCAGCCTCTATAAAACACAAAC
S447	AACACCCACATCCAACTCCCAACTTAACTCCAACGATTTT
S448	CGCAACCAACTCACCACACATCACGCCCACCCCCAGAATT
S449	GAAAAAAAAACCACCCTCCGAGACACCAACCCCCGAACCC
S450	GTACCGCGTCTCCTCTTGGGTTGATCTCAATCGCCGGATG
S451	TACACCACACACACTTACAACACATCACCCAATATACCCA
S452	CGCAAATACCACCAACCACCCACACTTACAACCTCACCCA
S453	GTACCGCGTCTACTCTTGGGTTGCTCTCAATCGCCGGATG
S454	CACACCGTCTCCACATCACCAGCAACACCCCAATTCCTCC
S455	TCCCTCCCTGATCTCTCCAACTCGCTCTCTCCATCACCCT
S456	CACAACACCACCACCCCCCACGTCCAACAACGACTATAAC
S457	GACTGAGCGCCTTTCCACCTCCGACAGCCCGATTCCACCT
S458	ACCCCCAACACTTTCAACACCACCACCATTCCCCGTTACC
S459	AAACACAATACCAACCACACAACACTGACCCCATCCATGC
S460	ACCACCAACCCACCCAGCACGACTTTACACATACGACCCA
S461	CCCCACCAAACTCGCACTCAACGCAACACCTCACCCACTT
S462	AACCACCAACCACCACAGAAACACCTCGAGCGCTAACTCC
S463	CCTGGCTTCACTTTGAGATTAAGGTTTCCCCAGTATCTAC
S464	AACATATATGCTGTATTAGACAACGCACCTGTCGCAAGGT
S465	CTGACCACTCCTTACCATCTCTATCTCTCCACCCGAAATC
S466	TCCCCCCCGACTCGCTGCACACTCCACCACCTGCTTTACA
S467	TAACAACACAACCACACAGCACACCCGGTACACCCTATTT
S468	CGAACCACCTCCACTTTCTACCGCTGACACCCCACCACAA
S469	CACCACAATTATCGCTAAACATCACCACACCCATCACCTT
S470	CACCACACCCACCACTTTGCAACTCCGAAACCACCAGACT
S471	ACACAAACCACGACACGCCAACCAACCATTCCGCTACCTA
S472	ACACACCAATCCACAACACAACTCAGCATCCACCATATAT
S473	AATAAAAATTGAGATTGCCACCCGCCGCAGGGTTCGCGAT
S474	CACCAACAGACACCCCCGCAGACACCAATTTCCAATCACT
S475	GACGCACCGGTCGCAGGTTCATTGTACGACCTTCTTTTCC
S476	AACTGATAACACTCCCACCTACCACTCGCTCGCTCTCCTT
S477	TCCCCATCCACACATCCTCCAACCACATCTGCAGACCCCA

S478	GTACCGCGTCTCCTCTTGGGGTGCTCTCAATCGCCGGATG
S479	ACCACCGAACAATACACCCCCAAACTGTCTCTCCACAACC
S480	GACGCACCGATCGCAGGTTCACTCACAAACACGCTTCCCC
S481	TACCACCAGCCACACACCCCCCCACTCCCTCCTTTTTGAA
S482	CCATCCAACACACACACATACCCCTCCGCTATATCGACGA
S483	TTTTCCCCCGATCTACCACCCTCCGACACACCTCCGCTTT
S484	CACCACCAAACGCAAACCCGAGTACCCACCACCCCATACC
S485	AACCACACCACTTTAGACCCACCCATAACCACCGCTTTTA
S486	CTAAAACCAACACAGCACCCCTCACCACTCCAACCTCACT
S487	CTGACACAACTTTTCTCATTAGCCCACACCCAAACACACC
S488	TACAGCACCCACCACCAGCACTCACCTCACAACGCTCTTT
S489	ACAACATTCAACCCACCCACCACTCAACTCCATCCGTTTT
S490	TCCCCCACTGCTACTATTTCTCCACTAACCCACTCTCCTC
S491	AAACCACTAACCACGCAAGATACCACACATACCCCACTTA
S492	CGACCGAGACCTCAGCCCCGCCACCCCTACAATCCCAACT
S493	AACTCCACACACAAAACCAAACCTCTCCATCCACTCCGAC
S494	TCACACCCACGCACACACCCATCTCCCACACTCCGCTTCT
S495	TTACACACACCCACATCCTACCACATTATCACACTCATCT
S496	CGTAATCCCACACACCACCACAACACTGCCTCCCCCCTTA
S497	GTACCGCGTCGCCTCTTGGGTTGCTCTCAATCGCCGGATG
S498	CACACAAAACAGACACACACCAACCCAGCACATTATCACC
S499	TTACACCACCAACCAACAAACACCAATACTCAACCTCTCC
S500	AACAGACGCTCAAAACACCACCCAACACAACTCCACCTTC
S501	AACCCCACACACTTTACACACAGCCCACCCGACTACTTTT
S502	CATCCATTCAACACCACCATCACCTACATCTCCATCTACC
S503	GTACCGCGTCTCCTCTTGGGTTGCTTTCAATCGCCGGATG
S504	TAAACCCCACTCACTGACACCACCATCCGCTGAACAATTT
S505	ACCACCCCAACGAACACCCAACGATCCGCTCACCTCGCCT
S506	CGACCGACCCACTCAACATCCCACTCCCCCTCTCCACCGT
S507	CTGACCACACTGAAACTCTCTCTCCGGCCCACCCTCCCACGA
S508	CACAACACACACACACAAAACACACACCCCCTTATCCCATTT
S509	GACGCACCGATCGAAGGTTCACTCACAAACACTCTTCCCC
S510	CAAGCTGACACTCCCAACATACTCTCACCCCCTCTTCTTT
S511	ATTTAACACCACACCAACACCCTCCGACAGCTCCACCCCA
S512	CCACACACAATTTTAACCACACTACACCCACTTGACTTTT
S513	CTACCGCCTATCCACCTCCTATCCAATCTCTCTGCTCTCT
S514	ACAACACCCACACCAGCACTCCACGACACAATCTCCTTTA
S515	ATGACGCCATCGGATTAAGGCTTCCCGTTATATTATGCTA
S516	ACACCCCCCACTACCGACAACTCCACATCTAATTCGACAA
S517	CACCACTACACACAATACAGAATCCCGACCCCACCGACAC
S518	GTACCCCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S519	CACCCACCCCCCTCTCAACTCGCTGACTTCCACTCACTCC
S520	CACCACCAGCCTACACACTCAACTCAAACCTCACCCATCC
S521	ACTCGAGGTTGTTAAACGTTGCTCCCCGACGCAGGGGCGT
S522	ACACCTAACCACCCAACTACCAATCCTCTCCATCCTCGAT

S523	GGATTAAGGTTTCCCCGTCCTGCCGGTACGGCCACGCATA
S524	CCCCTGCAACTCTACTCTTATCCCCACGTTCACCCTGCTT
S525	GACGCACCGATCGCAGGTTCACTCACAAACACTCTTCCCA
S526	ACACCCAACCAACAACACTCCTTTCACATGCAGACCCCAA
S527	TAAGGTTTCCCTGCGGCGACGTCCATGCAGCGTATGTCCA
S528	CAAAGCCCACCCAACACATCCTAACCACCACACCGACTAC
S529	CGAACACTACAACACCTCACACCTCGTCCCCTCCGCTACC
S530	TTAAGACACACCAACCAAAAAACCCACCAACACCAACCCC
S531	ACACCATCCACACTAACTACCAACTGCATACCCCACCACC
S532	CACCTATTTAACCACACACTCCACCCCGGAATCCCCTCTA
S533	AAACACCCAAAGCCACACACCACAACCTCGCATCTCTTAT
S534	CGACCTATCTCACCAACTCCCCATCCACATCACCTCCATC
S535	GGCGCACCGATCGCAGGTTCACTCACAAACACTCTTCCCC
S536	TAACCCACCCACGCTCAACGAACACCAACATTTAACACAC
S537	GTACCGCGTCTCCTCTTGGGATGCTCTCAATCGCCGGATG
S538	CCCACACAACCCATCCACCTCCATCCACATCCACCCATTC
S539	CACCTCCAAGCCGCACACGCAATAACCTCCCACCGCCTGA
S540	GTACCGCGTCTCCTCTTGGGTTGCTCTCAAGCGCCGGATG
S541	CACCCACCCAAGTTCCCAGCTACACTCTCTCCATCTCCAC
S542	CGGACCATGCTACCCCCCTTCTAACTCTCTCCACTATCTC
S543	GTACCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGACG
S544	ACACCACACTACACTAGATTCTACCCCTGTTATCCTCTTT
S545	TATACACCACCACATCAACTCCAGCACATTATCACCACAA
S546	CAACCAAGCCATCCCACCAACTACCTCCCACTGCCTCCCC
S547	TTACCCAACCGCCAACCACTGACTCCGTTCCGCCACCACT
S548	AACACCAACACACAAGAACAGCCACCACCTTAGATAACAC
S549	ATCAATATAGCGACTTGGCTCGTTTATGGTTTCCCTATAA
S550	CACCCACCTACCTCCAACACCCAGCCACTTCCTCCACGTT
S551	TTAACCACACCAAAGACCCACCATCACCTCAGCGCTCCAA
S552	CGACTAACACCCCCAGCCTCCCCGCACATCCTAATTCCAC
S553	AACCAACCCACAACACCACAGCCCTTGCAACCTCTTCGAA
S554	ACCACCCACCAACATTTTTCACACCACCATTCCAGCTACC
S555	ACACTAACCACTCCGCCAGCATCCTCCGAACCACATCTCC
S556	AAACACCACACAGAACCACGCAAAATAACAAACACATCGC
S557	AACATATATGCTGTATTAGACAACGCACCTTGCGCAAGGT
S558	CTCCACAACCATCTACACCCCACTTACGGATACACACAAC
S559	GACGCGCCGATCGCAGGTTCACTCACAAACACTCTTCCCC
S560	CACCCCAGCACTCTCACTATCGACACCACCACTTTATTTA
S561	ACATAACAAACAACACAATTACACCCCACACCTTACGCTA
S562	CAACCCAACCTCCTCACACAGCTATCCCACGCTCCACACT
S563	TCATTTAGCCGACATCCGGTTGGTTTATGTTTTCCCTAAA
S564	CACCACTCTACAACACACACACCATCTACAACCCTTTTTACT
S565	CGAACTGTTAACCAACTCCCACTCCCTCTCCTATTTCTTT
S566	ACCACCTAACTATAAAAAAACACATTCACCATCAGCACCA
S567	CATAAAAATTGAGATTCCCACCCGCCGCAGGGTTCGCGAT

S568	TTTTTACACCACCCCCGAGATACTCTATACTACATCCACT
S569	ACTCGCGGTTGTTAAACGTTGCTCCCCGACGCCGGGGCGT
S570	ACACCACAACACCACACAACCATACAGCTCCCCATTACGT
S571	ACTCGCGGTTGTTAAAGGTTTCTCCCCGACGCAGGGGCGT
S572	CAGACACCCACCCAACTCCGCTTTCAGACCCAACTCACCG
S573	CAACAACCCACCACAAACATAACACCATTCCCATTTGCTT
S574	CCCGTAACCAGCCTCCACCTACCACACAGACTTGATCCCC
S575	TACCACAACCCCAGCACCACGCCAACACTCACCCCCATCT
S576	ACAACTACCACACCACCACCGACACATAACGCGCATTTTT
S577	ACACCAACACAAGACCACCACACACAACGCATCTCCTATT
S578	CAACCCGGACTCCACCACTGCTATACCTCCGCCCCACTTA
S579	ATATACCACTAAATACACACCAGCCCCACACAACCATCTT
S580	ACAGAACAACACCACACACAAACTCCAACAATACACCAAT
S581	GACGCACCGATCGCAGGTTCACTCACAAACACTCTGCCCC
S582	CACCCTACCAACTCTCTCTCACTCGCTTTCCCAATCTCAC
S583	AACTGACCAACTACACAGCACACCCTCCACCCCGCGCCTT
S584	CAACCGTCGACACGCTACACCCTCCCACTACCTCTCCTCT
S585	AATACCCACTGCATCCACTCACACACCACCCCCTGCCTTT
S586	TCAACCAACCCTCTATACACTCACCCCCACACTCTATCTC
S587	ACATTAAACCACATCCACATTCACATTTTTATCCACCATT
S588	ACCACCACTACGCTCAACCACACCTCCACTGATATCTCTT
S589	GTACCGCGTCTCCGCTTGGGTTGCTCTCAATCGCCGGATG
S590	TCCACACTACCACCAACTCCGACTAACGCTCTCCACTACT
S591	ACTCGCGGTTGTTAAACGTTGCTCCCCGCCGCAGGGGCGT
S592	ACACCCCCACCTACACCTACAGCCGATCCATCATCTCACT
S593	CATGCCAACACACACAACACTCACCTCCACACCAATCCTC
S594	ACTCGAGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S595	ATAACCCAGCACCAAGAGACTCACCTCCATACCTCTCCAA
S596	CACCAACACCTACAAATACGACACCCACCCCCACATTCTT
S597	AAACAACACAAACACTCACCGCGATACTACCCCCACCTTA
S598	CTGACCACCTGACGACTCGACTATCTCGCTCTCCATACCC
S599	ACTCGCGGCTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S600	GAGCTCGGTCTTTGGACCAATAATAAGGTTTCCCTCACAT
S601	AACCACTCCAGCAAACCATCCCCACACCGAATCCACACTT
S602	TTAACCACCCATCACCACCACATCAGACTTTTATATATCT
S603	CCACCCAACTCCATTCTACCAGCACTCCCCTCTCCTACCG
S604	TCAACCCAACACCAGCACCTCTACCTACACCGCATCTTTT
S605	GTACCGCGTCCCCTCTTGGGTTGCTCTCAATCGCCGGATG
S606	AACAACAACCCCCACACGATCACCATACATCACATCCACA
S607	ACACACAACACACGACTTAACCCAACTTTACACACCCTCC
S608	CACCACACTACAGACTCACCACCACACCACCCATCTAATA
S609	CACACACGCACAACACACACACCAGACACCCCCTACCCAGCC
S610	TAAGGTTTCCCCGCTAACAATTAATTTGGCTTGTGGCATA
S611	ACCACACACACCTACACAGACAAAATCCTCTCTCTCTAGC
S612	CACCATCCAACACCGCTAAAATACTCCCCGATTCACCTCT

S613	CAACACACACCACCCAACAAATTTTCACCGCCCCCGTATT
S614	AAAACCACCAACACACTCCAACTCACACCAGAACCTCGCT
S615	CACCAACAATCCACCTGCATGCAAAACACCAACACTCCAC
S616	CACAACACACACCACCACCAACATACCGACACAACACACT
S617	ACATCACACCCAACACCACATCACCCATCAGCAGACCAAC
S618	GTTCCGCGTCTCCTCTTGGGTTGCTCTCAATCGCCGGATG
S619	CATATAACACACAACACACACACCAACACCCTTCACATATTA
S620	AACAAAAAAAACCCGACACAATGCTCCCAACGTCTCCAAC
S621	CGACCGTTTCCACCGCCTCTCGCTCTAATACCTCTCCGAA
S622	ACCACACCACCCCGCACACACACAACCAAGATCCCCCCATTT
S623	TACACCACAACAGCTCTCGGCAACACCACACCAATCTATC
S624	ACACACAACTACAACACAACATCTGTTACACCCACCGCTT
S625	CCTTGCTTCACTTTGAGATTAAGGTTTCCCCAGGATCTAC
S626	TCACACACTTTACACGTTACACCCATTCCCCACGCCGATT
S627	AAATCCACCCACCACCAGCAACTCCACACACTACTACACA
S628	TCCCCCCTCCACACTCTCCAGTTACGCTTTCCCCTCGACT
S629	CATTAAAACCCACCCTCCACCACATTCCTACTGCTCCTTT
S630	ACCCACGCCACACACACACTCCCCCCTACACGCTCTTATT
S631	CAAACCTCACCACCACAGCTACTAACCCCTTATATCTACC
S632	CATAAAAATTGAGATTCCCCCCCGCCGCAGGGTTCGCGAT
S633	CTGACCTCTCCACCTCTCTACTCTCCCACCTCCGCTCCTA
S634	TATACACACACCAGACACCGCACATTTATACCCCACTTTA
S635	GACGCAACGATCGCAGGTTCACTCACAAACACTCTTCCCC
S636	TAAACACTCACCACACCCCATCCTCACAGCACCATAATTC
S637	CGCTCCCCAAACCACACCCACCTCACTGCAGTTCCATCCT
S638	CACAGCACACCAACCACACAGTACACCCCCGCGCCACACC
S639	CTTCCCGATTCGCCAACTCTATTCGCCACACCTCCCTCCT
S640	TTTTAGCCCCCCAGACACTCCTACCACTCCACAACCAACT
S641	AACCCACCACACCACTCTCTCTCGCGCTGCCTCCAACCTGAT
S642	CACAACCAAGCAACCTCACCTCCAGCCTGCCACACCCACA
S643	CGACCCATCCACACATCCACTCTCCATACACACTCCGATT
S644	TCGACCAACACCACTCTACCCCCTCCATCCTCACCGACTT
S645	CGACCGCTGAACTCACCTCACCACACTCCTCCTATCCGAT
S646	AACCACCACCAACCACCGCTATAACGCAATACACTCCTCC
S647	CGCTAACCACACACACCACACTACACCCCTCCAAACCCTA
S648	TACCCACCAGACTTACACCCACACTTACACCCACTCATTT
S649	TTAAAGAACCACCCACCAACCCACTCCTTTTGCAACATCC
S650	CCCCACCACTCCAGCGACAATTACCACACCGCCTCCATCT
S651	TAACGTTACAACACCACCTCCCACCACTTCAGCCTGCCTT
S652	ACGCGCGGTTGTTAAAGGTTGCTCCCCGACGCAGGGGCGT
S653	AACCACCACACACTGCAACTACACCACCTACTTACATCAC
S654	CCTGTCTTCACTTTGAGATTAAGGTTTCCCCAGGATCTAC
S655	TAACACACCGGATCCATTTTAGATTTACACCACACACCCA
S656	CGACCGTTCACACAACTACCCCTCGCACGAACTGCACATC
S657	GACGCACCGATCGCAGGGTCACTCACAAACACTCTTCCCC

S658	TACCCCCTACCTCACTACTCTAGCTCTCTCGCATCTCCAT
S659	CCACAAACACCAAAGCAACCCCACGATACACACTACATCT
S660	CGAAACGAACGCACTCAAGCACACTCCCACACCCTCCTTA
S661	TTCACACCACGCGACACAACCTGTTTACCACACCCATCTT
S662	ACGACCCATTACATCCACCCAACGATCACCACCTCCACGC
S663	ACACACACCCAGACCAGCCAGCCTCCACCACACGTTTCTT
S664	AAACTAACCCTCAATACTCACACACACACCCCATCTTCCT
S665	CGATACACCACCTCCCACCATCCCATAGCTCTGTTCCACC
S666	CACCAACCCTCCACCCACAAAACACCGACCCATCTCGACA
S667	CACCCACTATATTGCACCCCCGCTACATCTCACCTCTACC
S668	TACTCCCACCCACCGACTAACGCTTTTCGATCTCTCAACT
S669	TACTATCCCACCAAACCAACGACCACCGCCAACTCTCTCC
S670	TCAATCCTCCAACAACAGCACCACCCTCCAGCACATCTCC
S671	AACATTCCACCCCCCCCTCCGATACACTCCTCCTCCGTTT

Claims (20)

1. A nucleic acid aptamer, characterized in that the nucleic acid aptamer is any of the following single-stranded oligonucleotide molecules:

   A1. The nucleotide sequence is the single-stranded oligonucleotide molecule of SEQ ID NO.1 in the sequence list,

   A2. The nucleotide sequence shown in SEQ ID NO. 1 in the sequence table is substituted and/or deleted and/or added to obtain the same single-stranded oligonucleotide molecule shown in A1) A single-stranded oligonucleotide molecule that has more than 50% identity and specifically binds to CD81.
2. The nucleic acid aptamer of claim 1, wherein the nucleic acid aptamer binds to human CD81 protein.
3. The aptamer according to claim 1 or 2, wherein the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.2.
4. The aptamer according to claim 1 or 2, wherein the deleted or replaced nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.3.
5. The aptamer according to claim 1 or 2, wherein the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.4.
6. The aptamer according to claim 1 or 2, wherein the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.5.
7. The aptamer according to claim 1 or 2, wherein the deleted or substituted nucleotide sequence in (A2) has the nucleotide sequence described in SEQ ID NO.6.
8. Use of the aptamer of any one of claims 1-7 in the preparation of reagents or kits for detecting CD81.
9. Use of the aptamer of any one of claims 1-7 in the preparation of reagents or kits for extracting CD81.
10. The application according to claim 8 or the application according to claim 9, wherein the CD81 is a human CD81.
11. The use of the aptamer of any one of claims 1-7 in the preparation of reagents or kits for detecting exosomes.
12. The use of the aptamer of any one of claims 1-7 in the preparation of a kit for extracting exosomes.
13. The application according to claim 11 or the application according to claim 12, wherein the exosomes are human exosomes.
14. The exosomes according to claim 13, wherein the human exosomes are exosomes containing human CD81.
15. Use of the aptamer of any one of claims 1-7 in the preparation of reagents or kits for detecting and/or diagnosing cancer.
16. The application according to claim 15, wherein the diagnostic marker of cancer comprises human CD81.
17. The application according to claim 15, wherein the cancer is breast cancer, colorectal cancer, liver cancer and/or lung cancer.
18. A conjugate obtained by coupling the nucleic acid aptamer of any one of claims 1-7 to a solid support.
19. A cross-linked product obtained by cross-linking the nucleic acid aptamer of any one of claims 1-7 to a solid support.
20. The conjugate according to claim 19 or the cross-linked product according to claim 19, wherein the solid phase carrier is a magnetic bead.

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