CN112779261B - Aptamer combined with human B7-H4 protein, application thereof and detection method using aptamer - Google Patents
Aptamer combined with human B7-H4 protein, application thereof and detection method using aptamer Download PDFInfo
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Abstract
The invention provides 2 nucleic acid aptamers combined with human B7-H4 protein, application thereof and a detection method using the same, and relates to the technical field of biomedicine. The aptamers 4s-7 and 4s-2 combined with the human B7-H4 protein can be specifically combined with the tumor protein B7-H4, have a sequence shown as SEQ ID NO.1 or NO.2, and have the advantages of precise identification, no immunogenicity, easiness in-vitro synthesis and modification and the like. The technical problem of lacking an aptamer capable of targeting tumor proteins B7-H4 in the prior art is solved.
Description
Technical Field
The invention relates to the technical field of molecular biomedicine, in particular to a nucleic acid aptamer combined with human B7-H4 protein, application thereof and a detection method using the same.
Background
Malignant tumors seriously affect people's health and life, and are the second leading cause of death worldwide. The risk of cancer increases with the growing population and the unhealthy lifestyle of people. Most malignant tumors are found already in the middle and late stages, missing the optimal treatment opportunity. The tumor can obtain better treatment effect by means of operations, medicines and the like in the early disease stage, so that the early diagnosis and the accurate treatment of the tumor can be realized, and the treatment effect and the survival rate of patients can be improved. Accumulation of intracellular gene mutations, abnormal cell division and increased anti-apoptotic phenomena are known factors in carcinogenesis, and these changes allow mutant cells to acquire the ability to express certain proteins that normal cells do not express. These tumor-specific proteins are important targets for tumor diagnosis and precision therapy. Traditional tumor diagnosis lags the disease, and most patients have advanced to the middle and advanced stage when tumors are found. Therefore, in recent years, much attention has been paid to the improvement of tumor treatment methods, and the study of early detection and diagnosis methods of tumors. At present, the molecular biology research of tumors is concerned and becomes a research hotspot. Wherein, the aptamer with high specificity and high affinity to the tumor target is searched by a molecular biology method, and a new thought and method are provided for early diagnosis and accurate treatment of the tumor.
B7-H4, also known as B7x, B7S1 or VTCN1, belongs to a B7 family member, is firstly discovered by bioinformatics analysis in three independent laboratories, and then proved to be present in human serous ovarian cancer and breast cancer by analyzing the expression of B7-H4 mRNA and B7-H4 protein, but not B7-H4 protein in normal tissues. The B7-H4 protein is a transmembrane protein of 282 amino acids, comprising an amino-terminal extracellular domain, a hydrophobic transmembrane domain and a very short intracellular domain (consisting of only two amino acid residues). Like other B7 family members, B7-H4 proteins contain 1 IgV and 1 IgC region in their extracellular domain, with the complete structure of a type I transmembrane protein.
The B7 family members bind to the corresponding receptors on the surface of target cells and activate or suppress the T cell immune response. Research shows that the B7-H4 protein as a new member of a B7 family can be combined with a T cell surface receptor, inhibit the proliferation, activation and cell cycle process of T cells and realize negative regulation of T cell immune response, thereby promoting the immune escape of tumor cells and causing the tumor to be worsened and metastasized. The current research shows that the human tumor cell surface over-expresses B7-H4 protein mainly comprises breast cancer, ovarian cancer and the like, while the B7-H4 protein is not expressed in normal cells. Over-expression of the B7-H4 protein inhibits T cell immune response, so that the tumor escapes immune monitoring and killing of the body.
Aptamers (aptamers) are a class of single-stranded DNA or RNA with a specific structure, typically 20 to 80 bases in length. By using a systematic evolution of ligands by evolution (SELEX) technology of exponential enrichment, aptamers that specifically bind to a target can be screened from a random single-stranded nucleic acid sequence library. The aptamer has small molecular mass, is easy to penetrate cell membranes, has stable property, is easy to prepare and modify, has no immunogenicity, has low cost and is convenient to store and transport; the combined target range is wide, and the combined target range comprises ions, small molecules, proteins, cells, microorganisms and the like; the affinity of the antibody can be equivalent to that of an antibody, and the affinity constant can reach a nanomolar or picomolar level. The aptamer has similar action with the antibody, but is superior to the antibody, and can provide a brand new and effective method and means for early diagnosis and accurate treatment of malignant tumors. Therefore, in recent years, nucleic acid aptamers have been receiving wide attention from scientists, and research on basic, clinical and drug development has been increasing.
Compared with the traditional treatment means, the molecular targeted therapy has better molecular targeting property, can selectively kill tumor cells, reduces the damage to normal tissues, is not easy to generate drug resistance, and has good safety and tolerance.
Targeted therapy utilizes biospecific interactions such as antigen-antibody binding or ligand-receptor binding to achieve targeted delivery of drugs. The existing ligand or 'targeting carrier' which can be used as active targeting is mainly antibody, polypeptide, folic acid and polysaccharide. Antibodies generally have high affinity for the target, but are highly immunogenic; the polypeptide has small molecular weight and is easy to synthesize, but the polypeptide is easy to carry out enzymolysis in systemic circulation and is not suitable for in vivo application; small molecule compounds, such as folic acid, have small molecular weight and good stability, but have low targeting to tumors. Therefore, screening an aptamer capable of being used for cancer cells and applying the aptamer to cancer cell detection, tumor research and preparation of a drug targeting tumor cells is a problem to be solved urgently.
Therefore, the B7-H4 protein which is over-expressed in the tumor and is not expressed in normal cells is used as a target for aptamer screening, and the aptamer which has high affinity and high specificity with the B7-H4 protein is obtained by screening through the SELEX technology, so that the possibility is provided for early diagnosis and treatment of the tumor which is targeted by the B7-H4 protein.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide an aptamer which binds to tumor/cancer cell specific protein B7-H4, and alleviate the technical problem of lacking an aptamer capable of targeting cancer cell B7-H4 in the prior art.
The second purpose of the present invention is to provide the application of the aptamer binding to cancer cells B7-H4 in tumor research or in the preparation of a kit for detecting cancer cells, so as to alleviate the problem of the prior art that a technical method for targeting a cancer cell aptamer that can be applied in tumor research or in the preparation of a kit for detecting cancer cells is lacking.
The third purpose of the invention is to provide a cancer cell targeting drug containing the aptamer capable of binding to the cancer cell B7-H4, which alleviates the technical problem of the prior art that a cancer cell targeting drug containing the aptamer capable of binding to the cancer cell is lacked.
The aptamers, 4s-7 and 4s-2, binding to cancer cell B7-H4 have the sequences shown in SEQ ID No.1 or No. 2.
Further, the 5 'end and/or 3' end of 4s-7 and 4s-2 may be labeled. Including fluorescent, biotin, drug, or chemical group labels.
Furthermore, 4s-7 and 4s-2 can specifically bind to cancer cells expressing B7-H4, including breast cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, pancreatic cancer and the like.
Further, the aptamers 4s-7 and 4s-2 are aptamers having homologous sequences that are artificially synthesized or derived from any other source.
Further, the artificial synthesis comprises in vitro chemical synthesis or molecular biological method synthesis;
the molecular biological synthesis is asymmetric PCR synthesis.
The method for detecting the cancer cells B7-H4 by using the aptamers 4s-7 and 4s-2 comprises the following steps:
synthesizing the aptamer, incubating the sample with the aptamer, and detecting binding of the sample to the aptamer;
preferably, the sample comprises cells or tissue sections or serum.
Therefore, the aptamers 4s-7 and 4s-2 can be applied to tumor research or preparation of a kit for detecting cancer cells.
4s-7 and 4s-2 can also carry anti-tumor drugs, become carriers of targeting cancer cells, and are applied to tumor targeting treatment.
The aptamer 4s-7 and 4s-2 combined with the cancer cell B7-H4 provided by the invention have the advantages of accurate identification, no immunogenicity, easiness in-vitro synthesis and modification and the like. Compared with protein antibodies, the single-chain oligonucleotide is more stable, the synthesis cost is lower than the preparation cost of the antibody, and the period is short. The aptamer can be directly synthesized in vitro and labeled, and a labeled secondary antibody is not needed to directly detect the cancer cells, so that the detection operation is simpler and quicker; or carrying antitumor drugs or connecting other nucleic acids, such as siRNA, and applying to targeted tumor treatment; the aptamers 4s-7 and 4s-2 can specifically identify various tumor cells, including breast cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, pancreatic cancer and the like; and can specifically recognize B7-H4 in the serum of the tested clinical cancer patient, and the control nucleic acid sequence is not combined with the cells and the serum B7-H4.
The method for detecting the cancer cells B7-H4 provided by the invention can be used for directly detecting the cancer cells in various modes. Detecting the fluorescence-labeled aptamer, for example, using flow cytometry or confocal microscopy; the digoxigenin-labeled aptamer is detected by immunochemiluminescence, for example, using digoxigenin antibody with alkaline phosphatase, and developing color using NBT or BCIP as a substrate, thereby detecting cancer cells bound to the digoxigenin-labeled aptamer. Therefore, 4s-7 and 4s-2 are suitable for various detection methods and systems, the applicability is wide, and most laboratories can use the method for detection.
The 4s-7 and 4s-2 combined cancer cell B7-H4 provided by the invention can be applied to tumor research or preparation of a kit for detecting cancer cells, has a wide application range, can be applied to the field of cancer detection or cancer research, and is simple and convenient to use.
The 4s-7 and 4s-2 provided by the invention can be coupled with tumor drugs, and then combined with B7-H4 of cancer cells, so that the targeting property of anticancer drugs is enhanced.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the OD 450nm values of the binding of different aptamers to B7-H4 protein in example 1 of the present invention.
FIG. 2 is a secondary structure diagram of aptamers 4s-7 and 4s-12 according to example 2 of the present invention.
FIG. 3 is a graph showing the affinity of aptamers 4s-7 and 4s-12 to B7-H4 in example 3 of the present invention.
FIG. 4 shows the case where the cellular immunofluorescence verification aptamers 4s-7 and 4s-12 specifically recognize the target B7-H4 in example 4 of the present invention
FIG. 5 is a graph showing the OD values of aptamers 4s-7 at various concentrations in example 3 of the present invention after binding to a quantitative B7-H4 protein.
FIG. 6 is a graph showing the OD values of aptamers 4s-12 at various concentrations in example 3 of the present invention after binding to a quantitative B7-H4 protein.
Fig. 7 is a flowchart of SELEX screening in embodiment 1 of the present invention.
A. Human normal mammary cells MCF 10A as negative control (no B7-H4 expression)
B. Human breast cancer cells as target cells (with B7-H4 expression)
C. Humanized immortalized normal liver cell line L-02 (No B7-H4 expression)
D. Human hepatoma cell line HepG2 (No B7-H4 expression)
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments and the accompanying drawings, and it is to be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides aptamers 4s-7 and 4s-2 capable of binding to cancer cells B7-H4, and sequences of the aptamers are shown in SEQ ID NO.1 and NO. 2.
In recent years, the research of using an aptamer (aptamer) as a carrier for active targeted drug delivery has become a new topic in the field of active targeted drug delivery system research. The aptamer is a section of oligomeric single-stranded DNA or RNA, generally composed of 20 to 80 bases, has a molecular weight of 6 to 25kD, can be folded into a special three-dimensional structure, and can specifically recognize and bind molecules such as proteins, sugars, nucleotides and the like. Because the aptamer has the advantages of accurate identification, no immunogenicity, easy in vitro synthesis and modification and the like, the aptamer is also called as an artificial substitute antibody. By designing the combination of the aptamer and the drug, the targeted delivery of the drug or drug carrier can be realized, the curative effect of the drug is improved, the adverse reaction is reduced, and a new direction is provided for the development of an active targeted drug delivery system. Therefore, the method has wide application prospects in the aspects of basic medicine, new drug research and development and the like, particularly, a composite target SELEX technology which is started in recent years enables screening of unknown target molecules to be possible, and by introducing a subtractive screening step, an aptamer which can specifically identify molecules which are different in two groups of cells or composite targets can be obtained, and the aptamer can be used for reversely and differentially guiding drugs in a targeted manner, so that a new way is opened for developing novel targeted drugs, particularly for specifically and targetedly killing tumor cells.
The exponential enrichment ligand system evolution technology (SELEX) is based on the knowledge of molecular biology: the specific interaction between nucleic acid molecule and protein molecule in vivo is the guarantee of orderly progress of a series of basic life activities such as gene transcription, nucleic acid replication, protein expression, etc. Basic principle of SELEX technology: artificially synthesized single-stranded nucleic acid library (ssDNA or RNA) containing about 40nt random sequences, and the possible sequences of the library can reach 4 in theory 40 And (4) seed preparation. The diversity of the primary sequence also results in the diversity of the secondary and tertiary structures of ssDNA or RNA. Through multiple cycles of screening, various target substances, such as organic molecules of proteins, nucleic acids, small peptides, amino acids and the like, even metal ions, can theoretically find the nucleic acid sequence specifically and firmly combined with the different sequences and the nucleic acids with different spatial structures. After multiple rounds of circular screening and PCR/RT-PCR amplification, and cloning, sequencing and identification, the nucleic acid chain-ligand of the section of specific sequence combined with the target substance, also called aptamer, can be produced in large quantities and purified.
In an alternative embodiment, the 5 'end and/or the 3' end of the aptamer may be modified with a modifier.
Because the aptamer can be artificially synthesized, different chemical groups can be modified at the tail end of the aptamer to connect the aptamer with a drug carrier to prepare drug delivery systems with different functions, for example, the aptamer can be connected with polymers, inorganic nanoparticles, dendritic molecules, liposomes and micelles to form different drug delivery systems. In addition, the aptamer can also be connected with various markers and applied to detection and identification of sample cells, tissues or biomacromolecules.
In an alternative embodiment, the modifier comprises a fluorescent label, a biotin label, a drug label, or a chemical group label.
After labeling the aptamer at the 5 'end and/or 3' end, the labeled aptamer is introduced into a sample to be detected, and the binding of the aptamer and the sample to be detected can be detected by various means, for example, a flow cytometer or a confocal microscope is used to detect the aptamer labeled with fluorescence, and an immunochemiluminescence detection method is used to detect the aptamer labeled with digoxin, so that the aptamer is suitable for various detection methods and systems. Furthermore, the insertion of drugs into aptamers is a simple and effective means of targeted drug delivery, and drugs can also be chemically modified to form stable ester, amine and disulfide bonds bound to the aptamers or covalently bound through linkers.
In an alternative embodiment, the protein to which the aptamer specifically binds is B7-H4. Therefore, the aptamer can specifically recognize a plurality of cancer cells expressing B7-H4, including breast cancer, ovarian cancer, bladder cancer, gastric cancer, pancreatic cancer and the like. The nucleic acid aptamer can also specifically recognize cancer cells in a cancer tissue section of a clinical cancer patient to be tested and B7-H4 in serum, and a control nucleic acid sequence is not combined with the cells and the serum.
In an alternative embodiment, the aptamer is artificially synthesized, or any other source of aptamers having homologous sequences.
In an alternative embodiment, the artificial synthesis comprises in vitro chemical synthesis or molecular biological synthesis, and a modifier may be added or linked to the siRNA during in vitro synthesis.
In a preferred embodiment, the molecular biological synthesis described above is an asymmetric PCR synthesis.
Asymmetric PCR (asymmetric PCR) uses unequal amounts of a pair of primers to generate large amounts of single-stranded DNA (ssDNA) after PCR amplification. A large amount of nucleic acid aptamers can be obtained by using a common PCR instrument and reagents, the operation is convenient and simple, and the cost is low.
The invention provides a method for detecting cancer cells or monitoring cancer by using the aptamer, which comprises the following steps: synthesizing the aptamer, incubating the sample with the aptamer, and detecting the binding between the sample and the aptamer.
Such samples include cells or tissue sections or serum.
The aptamers 4s-7 and 4s-12 can specifically recognize B7-H4, so that various tumor cells including breast cancer, ovarian cancer, bladder cancer, gastric cancer, pancreatic cancer and the like can be recognized. Such as cancer cells in cancer tissue sections of clinical cancer patients and B7-H4 in serum. 4s-7 and 4s-12 can be directly detected in various ways after being modified, for example, a fluorescence-labeled aptamer is detected by adopting a flow cytometer and a confocal microscope, and a digoxin-labeled aptamer is detected by adopting an immunochemiluminescence detection method, so that the aptamer is suitable for various detection methods and kits, and is simple and convenient to use.
The invention provides a targeted drug delivery carrier which can be applied to targeted therapy of tumors.
The incorporation of chemotherapeutic drugs into aptamers is a simple and effective means of targeted drug delivery, and such incorporation conditions are generally mild and do not require any chemical modification of the drug or ligand, and both the drug and the aptamer retain biological activity and achieve high drug loading. The drug may also be chemically modified to form stable esters, amines and disulfide bonds bound to the aptamer or covalently bound through a linker.
Antitumor drugs generally play a role in cells, and the key to the effectiveness is to improve the drug intake. The nanoparticles can enter cells through a cell endocytosis way, if the aptamer is connected to the surface of the nanoparticles, the endocytosis can be mediated after targeting tumor cells, the drug uptake can be improved, and the drug administration mode becomes a hotspot of the current research.
Research proves that inhibiting the expression of cancer genes and improving the expression of cancer inhibiting genes are basic strategies for treating tumors. In cells and animals, small double-stranded RNA has been shown to be the best method for inhibiting gene expression, and small amounts of double-stranded RNA introduced into cells can control the expression of the corresponding gene, which is also known as RNA interference (RNAi). Small double-stranded RNA can be synthesized in vitro. However, when small interfering RNA (siRNA) is introduced into a body as a drug, the targeting ability of the siRNA to tissues or cells is not provided, so that the targeting ability of the siRNA is a main obstacle influencing the application of the technology to clinical treatment. The siRNA is coupled with the aptamer, so that the siRNA has targeting property, and the technical problem that the siRNA does not have the targeting capability on tissues or cells is solved.
B7-H4 is a protein which is specifically expressed by tumor cells and inhibits the activation of T cells. Aptamers 4s-7 and 4s-12, which target and block B7-H4, can themselves act as drugs, because aptamers can block the inhibition of T cells by B7-H4, resulting in the killing of cancer cells by T cells.
The invention will now be further described with reference to the preferred embodiments
Example 1 screening of aptamers that bind to B7-H4
The screening of aptamers is based on SELEX technology. A random oligonucleotide ssDNA library H4-35 (5 '-gcaatggtac ggtactgtcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnaatcag tgcacgctac tttgctaa-3', n represents any of a, t, c, and g) was synthesized. The specific sequences at the two ends are PCR primer sequences, and the middle sequence is a core sequence.
Human B7-H4 protein is used as a target to screen an aptamer specifically bound with the human B7-H4 protein. After incubation and combination of the denatured oligonucleotide library and the target protein, the sequences which are not combined with the target B7-H4 protein are removed, and the sequences which can be combined with the target are collected and amplified for recovery, and are used as a secondary library in the next round of screening. The specific screening procedure is shown in FIG. 7.
After several rounds of screening, the enriched library was sequenced in high throughput, and ligated with T vector to transform Ecoli and establish clones. And selecting monoclonal sequencing to obtain an aptamer sequence in the enrichment library. After secondary structure and homology analysis of each sequence, appropriate ssDNA is selected for synthesis and Biotin (Biotin) labeling, and the affinity of the ssDNA is verified by an ELISA method. In the verification process, uncoated B7-H4 is used as a blank group, an original library H4-35 screened by an aptamer is used as a negative control, a biotin-labeled aptamer is combined with a certain amount of target B7-H4 protein, then horseradish peroxidase-labeled avidin is combined with the biotin-labeled aptamer, and after a color reaction, the absorbance OD value of each hole with the wavelength of 450nm is read on a microplate reader (the average value is obtained by measuring 3 times). The binding force is stronger when the OD 450nm value is larger, and the binding force between the aptamer 4s-7 and 4s-12 and B7-H4 is stronger by comparison (figure 1). The sequence is shown in SEQ ID NO.1 and N0.2.
Example 2 Secondary Structure prediction of aptamers
The RNA structure 4.6 software is used for predicting and analyzing the secondary structure, and different sequences are found to have different secondary structures, mainly stem-loop structures, but the sizes, the number and the positions of stem loops of different sequences are different. And selecting a nucleic acid sequence which can be used as an aptamer from the sequence to be sequenced according to the principle that the energy level is kept low, the secondary structure of the sequence is stable, and simultaneously, the GC structure stable region rich in each family consensus motif is kept. The result is matched with the detection result of the microplate reader. The 4s-7 and 4s-12 secondary structure prediction results are shown in FIG. 2.
EXAMPLE 3 determination of dissociation constants for binding of aptamers 4s-7 and 4s-12 to B7-H4
The 2 aptamer sequences selected were labeled with Biotin (Biotin) and diluted to 6 different concentrations, and their affinity was verified by ELISA. In the verification process, uncoated B7-H4 is used as a blank group, an original library H4-35 screened by an aptamer is used as a negative control, a biotin-labeled aptamer is combined with a certain amount of target B7-H4 protein, then horseradish peroxidase-labeled avidin is combined with the biotin-labeled aptamer, and after a color reaction, the absorbance OD value of each hole with the wavelength of 450nm is read on a microplate reader (the average value is obtained by measuring 3 times). The larger the OD 450nm value, the stronger the binding force. The results are shown in FIGS. 5 and 6.
When the concentration of the aptamer is lower, the target B7-H4 protein is relatively excessive, the aptamer can recognize more binding sites, and the OD value and the aptamer concentration are in a linear relation; when the aptamer concentration is increased to a certain amount, there is an excess of aptamer compared to the target, both bind into the plateau and the OD value remains constant. Nonlinear regression analysis using graphpad. Prism.5.0 software gave the following results: the dissociation constant Kd value of the affinity of the aptamer 4s-7 and the target B7-H4 protein is 14.61 +/-2.371 nmol/L, and the dissociation constant Kd value of the affinity of the aptamer 4s-12 and the target B7-H4 protein is 6.928 +/-3.152 nmol/L. As shown in fig. 3.
Example 4 binding assay of aptamers that bind B7-H4 to Breast cancer target cells
In order to detect the combination condition of the aptamer and a target B7-H4 protein on a cell level, a human breast cancer cell MDA-MB-231, a human liver cancer cell line HepG2, a human normal breast cell MCF 10A and a human immortalized normal liver cell line L-02 are selected.
The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ and 5% CO 2 . The cells are adherent cells, the growth state of the cells is observed every day, the culture medium is replaced in time and passage is carried out on time, and the cells are ensured to be in a good growth state.
a. The selected cells in good growth state were counted on a cell counting plate, and a cell suspension (1X 10) was seeded in a 24-well plate 5 Individual cells/well), preculture in an incubator;
b. the medium was aspirated off, 1 XPBS-3 mmol/L MgCl 2 Rinsing for 5min for 3 times;
c, fixing the 4% paraformaldehyde for 20min;
d.1×PBS-3mmol/L MgCl 2 rinsing for 5min for 3 times;
e.0.5% Triton punch for 15min;
f.1×PBS-3mmol/L MgCl 2 rinsing for 5min for 2 times;
g.1% BSA blocked at room temperature for 1h;
h.1×PBS-3mmol/L MgCl 2 rinsing for 5min for 3 times;
i. 100pmol of FAM-labeled aptamer (4 s-7, 4s-12 or stock) was added to each well and incubated at 37 ℃ for 2h in the absence of light. Adding each component of the mother liquor of the screening buffer, FAM-labeled aptamer, 10 μ l of 10 XPBS (after boiling for 5 min), 300mM MgCl 2 (after boiling water for 5 min) 1. Mu.l, appropriate amount of water, denature for 5min at 100 ℃ and immediately put on ice for 5min (put on ice until used). Then 10. Mu.l of 1. Mu.g/. Mu.l of ytRNA and 10. Mu.l of 1. Mu.g/. Mu.l of salmon sperm DNA were added to give a final buffer concentration of 1 XPBS-3 mmol/L MgCl 2 -0.1 μ g/μ l ytRNA-0.1 μ g/μ l salmon sperm DNA in a total volume of 100 μ l;
j.1×PBS-3mmol/L MgCl 2 rinsing for 5min for 2 times;
k.5. Mu.g/ml DAPI staining protected from light for 10min;
l.1×PBS-3mmol/L MgCl 2 rinsing for 5min for 2 times;
m. sealing the anti-quenching sealing agent;
and n, observing the combination of each group of cells and the aptamer by using a fluorescence microscope.
The results are shown in FIG. 4.
No green fluorescence was observed when aptamer or the original library was added to the negative control group of human normal breast cells MCF 10A (fig. 4A); after addition of the aptamer to human breast cancer MDA-MB-231, a more pronounced green fluorescence (FAM-labeled aptamer) was observed, and fluorescence mainly appeared around the cells, with little green fluorescence being observed when the original library (H4-35, library) was added (FIG. 4B). This also indicates that aptamers 4s-7, 4s-12 are specific for binding to breast cancer cells that highly express B7-H4 protein, and that the targets bound to aptamers are predominantly distributed on the cell surface, consistent with the distribution of B7-H4 protein in tumor cells.
When the aptamer or the original library was added to the human hepatoma cell line HepG2 not expressing the B7-H4 protein and the control human immortalized normal liver cell line L-02, no green fluorescence was observed, which also indicates that the nucleic acid aptamers obtained by screening were specific to the B7-H4 protein (FIG. 4C, D).
As can be seen from FIGS. 1-4, the 4s-7 and 4s-12 aptamers can specifically bind to B7-H4 and specifically recognize B7-H4-expressing human cancer cells, such as breast cancer MDA-MB-231, while not recognizing other human immortalized normal cell lines and other B7-H4-non-expressing tumor cell lines.
The conclusion is drawn from fig. 1: aptamers 4s-7 and 4s-12 can specifically bind to human B7-H4 protein as compared to the control library H4-35.
The conclusion is drawn from fig. 2: the secondary structure of aptamers 4s-7 and 4s-12 is mainly stem-loop structure.
The conclusions are drawn from fig. 5, 6 and 3: the aptamers 4s-7 and 4s-12 have strong binding force to B7-H4, and the binding force of 4s-12 to B7-H4 is greater than 4s-7.
The conclusion is drawn from fig. 4: compared with the sequence H4-35 of the control library, the aptamers 4s-7 and 4s-12 can specifically recognize the cancer cell MDA-MB-231 expressing B7-H4, but do not recognize the human normal breast cell MCF 10A, the human immortalized normal liver cell line L-02 and the human liver cancer cell line HepG2 which do not express B7-H4.
In conclusion, the aptamers 4s-7 and 4s-12 can specifically bind to the human B7-H4 protein, so that the aptamers can identify various human tumor cells expressing B7-H4 and can not identify other human cells, and have wide clinical application and drug application prospects in detection or targeted expression of the human cancer cells expressing B7-H4.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Qingdao university
<120> aptamer binding to human B7-H4 protein, use thereof, and detection method using same
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> oligonucleotide ligand sequence obtained by SELEX screening targeting human B7-H4 protein
<400> 1
ttagcaaagt agcgtgcact tttgtggaaa ccaccctaag gtccaggagt gatttggcgg 60
aagtaccgta ccattgc 77
<210> 2
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> oligonucleotide ligand sequence obtained by SELEX screening targeting human B7-H4 protein
<400> 2
ttagcaaagt agcgtgcact tttgttgtag gataatcctt actccaccgg gcccgaactg 60
gaagtaccgt accattgc 78
Claims (4)
1. 2 nucleic acid aptamers capable of binding to human B7-H4 protein, wherein the nucleic acid aptamers are sequences shown in SEQ ID NO.1 s-7 and SEQ ID NO.2 s-12.
2. According to claim 1, the aptamer is labeled at the 5 'end and/or 3' end with a fluorescent label, biotin label, drug label or chemical group.
3. The use of the nucleic acid aptamer according to any one of claims 1-2 in the preparation of a kit for detecting tumor cells and a tumor-targeted drug.
4. The use according to claim 3, wherein the tumor is breast cancer, lung cancer, ovarian cancer, bladder cancer, stomach cancer or pancreatic cancer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2892490A1 (en) * | 2012-11-26 | 2014-05-30 | Caris Science, Inc. | Biomarker compositions and methods |
| WO2016179319A1 (en) * | 2015-05-04 | 2016-11-10 | Cellerant Therapeutics, Inc. | Chimeric antigen receptors with ctla4 signal transduction domains |
| CN110981961A (en) * | 2019-11-28 | 2020-04-10 | 青岛大学 | Sequence and application of nano antibody H6 specifically binding to cancer cell protein B7-H4 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2892490A1 (en) * | 2012-11-26 | 2014-05-30 | Caris Science, Inc. | Biomarker compositions and methods |
| WO2016179319A1 (en) * | 2015-05-04 | 2016-11-10 | Cellerant Therapeutics, Inc. | Chimeric antigen receptors with ctla4 signal transduction domains |
| CN110981961A (en) * | 2019-11-28 | 2020-04-10 | 青岛大学 | Sequence and application of nano antibody H6 specifically binding to cancer cell protein B7-H4 |
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