CN109468326A - A kind of CD4+T nucleic acid aptamer and its application - Google Patents

Abstract

The invention discloses a CD4⁺T nucleic acid aptamer and an application thereof, belonging to the technical fields of genetic engineering, immunology and oncology. The aptamer is any one or several sequences of the nucleotide sequences shown in SEQ ID NOs: 1-5. The nucleic acid aptamer of the present invention is screened by using the Cell-SELEX technology to reduce the living cells, has a small molecular weight, is easy to synthesize and modify, can recognize with high specificity and bind to CD4⁺T with high affinity, does not bind to other cells in a characteristic way, and has no immunity. Original, stable and easy to modify, easy to synthesize and preserve. The CD4⁺T detection kit and reagent paper prepared with the aptamer have high sensitivity for CD4⁺T detection and are easy to optimize. Application in tumor diagnosis, evaluation of tumor prognosis and monitoring, and tumor diagnosis.

Description

A kind of CD4+T nucleic acid aptamer and its application

technical field

The invention belongs to the technical fields of genetic engineering, immunology and oncology, and particularly relates to a CD4⁺T nucleic acid aptamer and its application.

Background technique

Malignant tumor has become a major disease endangering the world, seriously endangering human health. Hepatocellular carcinoma (HCC) is one of the common primary malignant tumors, and its mortality rate ranks third in the global malignant tumor mortality rate. At present, most patients are treated with chemotherapy, radiotherapy and surgery, but the effect still needs to be improved. As a new treatment mode, tumor immune targeted therapy is gradually being accepted by people. In recent years, more and more attention has been paid to the role of activated CD4⁺T cells in the antitumor immune response. It not only has the functions of immune memory and direct killing of tumor cells, but also can assist CD8 ⁺ T cells in killing tumor cells. However, its poor activity and lack of targeting limit its clinical application. Therefore, the use of bispecific aptamers to effectively mediate the targeting of activated CD4⁺T cells to local tumors and improve the anti-tumor effect of cellular immunotherapy will provide a new therapeutic strategy for the clinical application of cellular immunotherapy .

Nucleic acid aptamers are truncated single-stranded DNA(single-stranded DNA, ssDNA) or RNA molecules that have been screened for binding to specific targets. They are easy to synthesize, have low immunogenicity and are small in molecular weight (10-15kDa). ) and other advantages. Because it has a similar effect to antibodies, it is also called "chemical antibody". The emergence of nucleic acid aptamers provides a new identification tool for the chemical biology and biomedical fields, especially in the early diagnosis of liver cancer, showing a good application prospect. At present, there is no report on the screening of CD4⁺T aptamers. If the aptamer that can specifically bind to CD4⁺T can be screened, it will undoubtedly provide a broader platform for the detection of CD4⁺T.

As helper T cells, CD4⁺T cells can assist the anti-tumor effects of various immune cells in the body. Its helper effect is necessary for the activation phase of CD8 ⁺ T cells. CD4⁺T cells can secrete cytokines such as IL-2 and promote the expression of co-stimulatory molecules such as B7 in APCs, assist in the activation of CD8 ⁺ T cells, and are of great significance to the activation, proliferation and differentiation of CD8 ⁺ T cells. CD4⁺T cells can also help activate B cells and produce antibodies, stimulate CD8 ⁺ T cells to secrete IFN-γ, activate macrophages, and enhance the activity of NK cells to improve the anti-tumor ability of the immune system. CD4⁺T cells can promote the generation of memory cytotoxic cells (CTL) and maintain long-term memory CTL responses, and CD4⁺T cells also play an important role in maintaining and promoting memory immune responses of CD8 ⁺ T cells .

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a CD4⁺T nucleic acid aptamer and its application.

The technical scheme used for realizing the purpose of the present invention is:

A CD4⁺T nucleic acid aptamer, wherein the aptamer is any one or several sequences of the nucleotide sequences shown in SEQ ID NOs: 1 to 5.

Preferably, the aptamer has a sequence that has a homology of more than 60% with any one or several sequences of the nucleotide sequences shown in SEQ ID NOs: 1 to 5.

Preferably, the aptamer has a sequence that hybridizes with any one or several sequences of the nucleotide sequences shown in SEQ ID NOs: 1 to 5.

Preferably, the aptamer has a sequence that is transcribed with any one or several of the nucleotide sequences shown in SEQ ID NOs: 1 to 5.

Preferably, several positions on the aptamer sequence are phosphorylated, methylated, aminated, thiolated or isotopolated.

Preferably, the aptamer sequence is conjugated with biotin, digoxigenin, fluorescent substance, nanoluminescent material, polyethylene glycol, peptide segment, protein, folic acid or enzyme label.

Preferably, the CD4⁺T nucleic acid aptamer has a corresponding peptide nucleic acid transformed from any one or several sequences of the nucleotide sequences shown in SEQ ID NOs: 1 to 5.

The present invention also provides an application of the CD4⁺T nucleic acid aptamer in preparing a CD4⁺T detection kit or detection reagent paper.

The outstanding substantive features and remarkable progress of the present invention are:

The nucleic acid aptamer of the present invention is screened by whole cell subtraction Cell-SELEX technology, has a small molecular weight, is easy to synthesize and modify, can recognize with high specificity and bind to CD4⁺T with high affinity, does not bind to other bacteria, and has no immunity. Original, stable and easy to modify, easy to synthesize and preserve, can be used to detect CD4 ⁺ T, and effectively prevent and control CD4⁺T, which is of great significance. The CD4⁺T detection kit and reagent paper prepared with the aptamer have high sensitivity for CD4⁺T detection and are easy to optimize.

Description of drawings

Figure 1 shows the results of flow cytometry of the activation degree of Naive CD4⁺T cells before and after stimulation with ConA.

Figure 2 is the flow cytometer monitoring the binding ability of the enriched library to activated CD4⁺T cells and Naive CD4⁺T cells; A. The flow cytometry results of the binding of the enriched library to activated CD4⁺T cells in each round of screening; B . Flow cytometry results of the binding of enriched pools to Naive CD4⁺T cells in each round.

Figure 3 shows the flow cytometry results of the binding of different cells to candidate aptamers. The detected cells are activated CD4⁺T cells (A), Naive CD4⁺T cells (B), BNL.CL2 cells (C), and BSR cells. (D), DC2.4 cells (E) and 3T3 cells (F).

Figure 4 shows the binding of Act-12a, Act-12b and Act-12c to activated CD4⁺T cells (A) and Naive CD4⁺T cells (B).

Figure 5 shows the fluorescence imaging results (1000 x) of Act-12c binding to activated CD4⁺T cells and Naive CD4⁺T cells.

Detailed ways

The solution of the present invention will be further described in detail below with reference to the embodiments. The following description is only for explaining the present invention, and does not limit its content.

Example

1 ConA stimulates Naive CD4⁺T to obtain activated CD4⁺T cells

The present invention uses activated CD4⁺T cells obtained by stimulating Naive CD4⁺T cells with ConA as positive sieve cells, and the activation degree before and after stimulation is shown in Figure 1. CD4⁺T cells (i.e. CD4 ⁺ CD69 ⁺ T cells) were 3.8 ± 1.3%, and among Naive CD4⁺T cells stimulated with ConA for 48 h, activated CD4⁺T cells were 84.9 ± 5.2%. This result demonstrated that the activated CD4⁺T cells obtained by stimulation with ConA were feasible as positive sieve cells.

2 Screening of activated CD4⁺T cell aptamers using Cell-SELEX technology

First, the initial ssDNA library was subjected to a counter-screening operation with Naive CD4⁺T cells (anti-screening cells), and the ssDNA that could be combined with the counter-screening cells was discarded, and the non-target sequences were removed. Then, collect the ssDNA that does not bind to the reverse screening cells, perform positive screening with activated CD4⁺T cells (positive screening cells), remove the non-specifically bound sequences by washing, collect the ssDNA sequences that can specifically bind to the target cells and use the It is amplified by PCR, and the secondary library formed after single-stranded treatment is put into the next round of screening. Screening is repeated according to the above process, and when the enrichment of the library reaches saturation, the enriched library is cloned and sequenced. Finally, by analyzing the primary mechanism and secondary structure of the sequence, several candidate aptamers were selected for functional identification.

3 Flow cytometry to monitor the degree of enrichment of the screening library

To judge the screening progress of the present invention, the saturation state of the enriched library is judged. The initial library and the 5th, 7th, 10th, and 11th rounds of screening products were labeled with FITC fluorescence, incubated with positive screening cells and negative screening cells, respectively, and then detected by flow cytometry. The results are shown in Figure 2. With the increase of the number of screening rounds, the fluorescence curve of the enriched library binding to activated CD4⁺T cells gradually shifted to the right, and the fluorescence intensity gradually increased, and the fluorescence curve of the 11th round was higher than that of the 10th round. The fluorescence curve of the round shifts very slightly to the right, indicating that the enriched library has tended to be saturated by the 11th round of screening. However, the fluorescence curve of each round of screening library binding to Naive CD4⁺T cells did not shift to the right obviously. It indicates that the screening has reached the end point, and the enriched library in the 11th round can bind to the target cells but not to the control cells.

4 Secondary structure analysis of sequencing results

Since the binding of an aptamer to a target cell is based on the spatial binding of the special three-dimensional structure of the aptamer sequence to the target cell, its secondary structure largely determines the binding ability of the aptamer to the target. The present invention selects a candidate aptamer Act-4, Act-5, Act-12, Act-15 and Act-20 with high repetition rate from each of the five families. The secondary structures of candidate aptamers were simulated using NUPACK (http://www.nupack.org). The candidate aptamer sequences are shown in Table 1.

Table 1 Candidate aptamer sequences name Sequence (5'-3') Act-4 <u>ATACCAGCTTATTCAATT</u>CATCCGCAACTTTGCCCCTCCACTCCCCTATCTCCTTTCGCCATC<u>AGATAGTAAGTGCAATCT</u> Act-5 <u>ATACCAGCTTATTCAATT</u>CGCCAAACCCGGATTTTTTGTTTTCCCCTTTATAGTACCGTCACA<u>AGATAGTAAGTGCAATCT</u> Act-12 <u>ATACCAGCTTATTCAATT</u>CGGGGAAAGTCACGGGGGGTTTCAGATGTTCTGATCGGTGTGGAG<u>AGATAGTAAGTGCAATCT</u> Act-15 <u>ATACCAGCTTATTCAATT</u>CGGGGAAAGTCACGGGGGGTTTCAGATGTTCTGATCGGTGTGGAG<u>AGATAGTAAGTGCAATCT</u> Act-20 <u>ATACCAGCTTATTCAATT</u>CGGCGGCCGTGCTATAGCGGAGTCCCTTTTCTTCCCCCTATGTACAG<u>AGATAGTAAGTGCAATCT</u>

5 Cell-specific analysis of candidate aptamers

In order to investigate the specific binding ability of the five candidate aptamers to target cells, the present invention uses flow cytometry to detect the binding conditions of the five candidate aptamers to six kinds of cells respectively. The results are shown in Fig. 3. All five candidate aptamers can bind to activated CD4⁺T cells (target cells), but bind to control cells Naive CD4⁺T cells, BNL.CL2 cells, BSR cells, DC2.4 cells and control cells. 3T3 cells do not bind.

6Act-12 optimized sequence design

According to the design of the initial library of the present invention, the aptamer sequence Act-12 obtained by screening is 81bp, and not all nucleotides are involved in the recognition of target cells, and the excess nucleotides may accumulate by themselves to form steric hindrance or Hybridizes with key functional nucleotides, thereby reducing the binding ability of nucleic acid aptamers. Therefore, it is necessary to optimize the sequence truncation of Act-12. By analyzing the primary and secondary structures of the Act-12 sequence, the sequence was truncated, and the optimized truncated sequence is shown in Table 2.

Table 2 Act-12 optimized truncated sequences name Sequence (5'-3') Act-12 <u>ATACCAGCTTATTCAATT</u>CGGGGAAAGTCACGGGGGGTTTCAGATGTTCTGATCGGTGTGGAG<u>AGATAGTAAGTGCAATCT</u> Act-12a CGGGGAAAGTCACGGGGGGTTTCAGATGTTCTGATCGGTGTGGAG<u>AGATAGTAAGTGCAATCT</u> Act-12b <u>ATACCAGCTTATTCAATT</u>CGGGGAAAGTCACGGGGGTTTCAGATGTTCTGATCGGTGTGGAG Act-12c CGGGGAAAGTCACGGGGGTTTCAGATGTTCTGATCGGTGTGGAG

According to the original sequence Act-12, three truncated sequences were designed. Act-12a deletes the upstream primer sequence on the basis of the original sequence, and the length of the truncated sequence is 63 bp; Act-12b deletes the downstream primer sequence on the basis of the original sequence, and the length of the truncated sequence is 63 bp; and Act-12c is The upstream and downstream primer sequences at both ends were deleted, and the length of the truncated sequence was 45 bp.

7 The optimized sequences of Act-12 can bind to activated CD4⁺T cells

In order to further investigate the binding ability of the truncated and optimized three aptamer sequences to target cells, the present invention used flow cytometry to measure the binding of FITC-labeled Act-12a, Act-12b and Act-12c to target cells. detection. The results are shown in Figure 4. The three truncated and optimized aptamer sequences Act-12a, Act-12b and Act-12c can bind well to target cells. The sequence Act-12 was not significantly changed. And Act-12a, Act-12b and Act-12c did not bind to the reverse screening cells.

8 Act-12c can specifically bind to activated CD4⁺T cells

The FITC-labeled Act-12c was incubated with target cells and reverse screening cells, respectively, and its binding was observed by confocal fluorescence microscopy. The results are shown in Figure 5. After Act-12c was incubated with activated CD4⁺T cells, there was strong green fluorescence expression on the cell membrane surface, while after incubation with Naive CD4⁺T cells, there was no green fluorescence expression on the cell membrane surface. After the initial library and the control aptamer TLS9a were incubated with activated CD4⁺T cells and Naive CD4⁺T cells, respectively, there was no green fluorescence expression on the cell membrane surface.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included within the scope of the present invention.

sequence listing

<110> Guangxi Medical University

<120> A kind of CD4⁺ T nucleic acid aptamer and its application

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Claims (9)

1. A CD4⁺T nucleic acid aptamer, wherein the aptamer is any one or several sequences of the nucleotide sequences shown in SEQ ID NOs: 1 to 5. 2. The CD4⁺T nucleic acid aptamer according to claim 1, wherein the aptamer has any one or several sequences with the nucleotide sequences shown in SEQ ID NOs: 1 to 5 The sequence homology is more than 60%. 3. The CD4⁺T nucleic acid aptamer according to claim 1, wherein the aptamer has any one or several sequences with the nucleotide sequences shown in SEQ ID NOs: 1 to 5 Sequences to hybridize. 4. The CD4⁺T nucleic acid aptamer according to claim 1, wherein the aptamer has any one or several sequences with the nucleotide sequences shown in SEQ ID NOs: 1 to 5 Sequence to be transcribed. 5. The CD4⁺T nucleic acid aptamer according to claim 1, wherein the aptamer has any one or several sequences with the nucleotide sequences shown in SEQ ID NOs: 1 to 5 Perform optimization truncated sequences. 6 . The CD4⁺T nucleic acid aptamer according to claim 1 , wherein several positions on the aptamer sequence are phosphorylated, methylated, aminated, thiolated or isotopic. 7 . 7. The CD4⁺T nucleic acid aptamer according to claim 1, wherein the aptamer sequence is combined with biotin, digoxigenin, fluorescent substance, nanoluminescent material, polyethylene glycol, Peptide, protein, folic acid or enzyme tags. 8. The CD4⁺T nucleic acid aptamer according to claim 1, wherein the CD4⁺T nucleic acid aptamer has any of the nucleotide sequences shown in SEQ ID NOs: 1 to 5. One or more sequences have been engineered into the corresponding peptide nucleic acid. 9. The application of the CD4⁺T nucleic acid aptamer according to any one of claims 1-7 in the preparation of a CD4⁺T detection kit or detection reagent paper.