阅读说明：本技术 特异性识别md-2的紫杉醇结合域的dna适配子及其应用 (DNA aptamer for specifically recognizing MD-2 paclitaxel binding domain and application thereof ) 是由 徐发良 郑晓东 李卫 蒋又新 魏珂 于 2019-04-22 设计创作，主要内容包括：本发明公开了特异性识别MD-2的紫杉醇结合域的DNA适配子及其应用。为解决现有技术中存在的问题,本发明确定一种紫杉醇结合域,即髓样分化蛋白-2(myeloid differential protein,MD-2)中的一段特殊氨基酸序列。通过人工合成的模板肽的氨基酸序列为SEQ ID NO.1。上述SEQ ID NO.1作为提高紫杉醇抗肿瘤敏感性的靶点。通过实验证明,本发明中的适配子具有潜在的促进紫杉醇导致乳腺癌细胞生长抑制的抗癌作用,得到的适配子在药物中得到应用后,可有效的增强紫杉醇敏感性的潜在作用。(The invention discloses a DNA aptamer specifically recognizing a paclitaxel binding domain of MD-2 and application thereof. In order to solve the problems in the prior art, the invention determines a paclitaxel binding domain, namely a section of special amino acid sequence in myeloid differentiation protein-2 (MD-2). The amino acid sequence of the artificially synthesized template peptide is SEQ ID NO. 1. The SEQ ID NO.1 is used as a target for improving the antitumor sensitivity of the paclitaxel. Experiments prove that the aptamer has a potential anticancer effect of promoting growth inhibition of breast cancer cells caused by paclitaxel, and the potential effect of effectively enhancing paclitaxel sensitivity can be achieved after the obtained aptamer is applied to a medicament.)

1. A paclitaxel binding domain for specifically recognizing MD-2, which is characterized in that the amino acid sequence of the binding domain is SEQ ID NO. 1;

The amino acid sequence of SEQ ID NO.1 is as follows:

The amino acid sequence of the 12 peptide derived from the structure of the MD-2 protein is NH 3-IKFSKGKYKCVV-COOH.

2. The paclitaxel-binding domain that specifically recognizes MD-2 according to claim 1, wherein: the SEQ ID NO.1 is used as a target for improving the antitumor sensitivity of the paclitaxel.

3. A set of aptamers, characterized in that: the aptamer is obtained based on the SEQ ID NO. 1.

4. The set of DNA aptamers according to claim 3, characterized in that: the nucleic acid sequence is SEQ ID NO.2, 3 and 4;

SEQ ID NO.2：

AACCGCCCAAATCCCTAAGAGTCTTTTTTAATGTTTTTTTTTAAGTTTTTTATTTTGTTTTTCCACAGACACACTACACACGCACA

SEQ ID NO.3：

AACCGCCCAAATCCCTAAGAGTCTTAATGTTGTTTATGATTAATCTCTGTTTATTTGTGAGGTCACAGACACACTACACACGCACA

SEQ ID NO.4：

AACCGCCCAAATCCCTAAGAGTCTTTTTTTTCTTTGTTGTTTCCTTTGTTTTTAATAGCTCTTCACAGACACACTACACACGCACA。

5. The aptamer according to claim 3 or 4, wherein: the application of the aptamer in preparing medicaments. The medicine is used for improving the antitumor sensitivity of the paclitaxel.

6. The aptamer according to claim 3 or 4, wherein: the application of the aptamer in preparing medicaments. The drug is a drug that delays or reverses paclitaxel resistance.

Technical Field

The invention relates to the field of biochemistry, in particular to application of a reagent for targeted recognition of MD-2 protein in preparation of a preparation for improving the sensitivity of an anti-tumor drug.

Background

Paclitaxel is extracted from the bark of the Pacific yew tree by the national cancer research center of America, and has been found to have anticancer effect, and through a series of clinical tests, the paclitaxel has been prepared to be used for treating ovarian cancer and breast cancer, and has excellent treatment effect.

according to the latest statistics from the united states, in 2006, the total international market sales of paclitaxel formulations including natural raw material processed paclitaxel injection and semi-synthetic paclitaxel injection has reached $ 37 billion, which is the top of the world anticancer drugs.

To date, paclitaxel is the first choice of antineoplastic drugs in hospitals in various countries throughout the world. The incidence of tumors in various countries in the world is improved by nearly one time in recent years compared with that before 10 years, and malignant tumors such as lung cancer, breast cancer, ovarian cancer and the like are also in a frequent trend, and all cancer patients are main users of paclitaxel. In general, the sales of paclitaxel only increased and did not decrease until no new plant anticancer drug could replace its site.

Paclitaxel plays an important role in tumor chemotherapy, but paclitaxel resistance can cause chemotherapy failure and tumor progression. The great consequences of paclitaxel resistance have been a great problem in cancer therapy, and this problem has not been solved so far.

Disclosure of Invention

The invention aims to solve the problems in the prior art and discloses the following technical scheme:

A paclitaxel binding domain, a specific amino acid sequence in myeloid differentiation protein-2 (MD-2), is identified. The amino acid sequence of the artificially synthesized template peptide is SEQ ID NO. 1.

the SEQ ID NO.1 is used as a target for improving the antitumor sensitivity of the paclitaxel.

And (3) taking the SEQ ID NO.1 sequence as a template to obtain a plurality of DNA aptamers. The existing SELEX technology can be used as the acquisition method. The nucleic acid sequences of the three aptamers are SEQ ID NO.2(M-42), SEQ ID NO.3(M-427) and SEQ ID NO.4 (M-430).

The aptamer can be applied to preparation of medicines. The medicine is a medicine (sensitizer) for improving the antitumor sensitivity of the paclitaxel and delaying or reversing the drug resistance of the paclitaxel.

The technical effects of the present invention are undoubted, and the present invention has the following advantages: experiments prove that the aptamer has a potential anticancer effect of promoting growth inhibition of breast cancer cells caused by paclitaxel, and the potential effect of enhancing the sensitivity of paclitaxel can be effectively achieved after the obtained aptamer is applied to a medicament.

Drawings

FIG. 1 binding of aptamers diluted in multiple ratios to template peptides (fluorescence colorimetry)

FIG. 2 shows the binding of aptamers to breast cancer cells (confocal laser)

FIG. 3 Effect of doublefold dilution of aptamer in combination with paclitaxel on growth inhibition of Breast cancer cells (MTT)

FIG. 4 Effect of multiple dilution of aptamer M42 in combination with paclitaxel on growth inhibition of Breast cancer cells (MTT)

Detailed Description

The present invention is further illustrated by the following examples, but it should not be construed that the scope of the above-described subject matter is limited to the following examples. Various alterations and modifications can be made without departing from the technical idea of the invention and according to the common technical knowledge and conventional means in the field, and all such alterations and modifications are intended to be included in the scope of the invention.