阅读说明：本技术 用于胃癌诊断的CD44v3-v10结构域特异性多肽配体、获得方法及应用 (CD44v3-v10 structural domain specific polypeptide ligand for gastric cancer diagnosis, obtaining method and application ) 是由 张丹 任牡丹 卢桂芳 卢新兰 赵艳 和水祥 于 2019-11-28 设计创作，主要内容包括：本发明属于药物开发技术领域,具体涉及一种用于胃癌诊断的CD44v3-v10结构域特异性多肽配体、获得方法及应用。所述用于胃癌诊断的CD44v3-v10结构域特异性多肽配体的氨基酸序列如SEQ ID NO.1所示,为CV-1多肽：THENWPA。所述CV-1多肽能够特异性结合于CD44的v3-v10变异型外显子编码结构域,并通过该肿瘤标志物结合于人胃癌细胞及组织,该CV-1多肽可用于胃癌的分子影像诊断。(The invention belongs to the technical field of drug development, and particularly relates to a CD44v3-v10 domain specific polypeptide ligand for gastric cancer diagnosis, an obtaining method and application. The amino acid sequence of the CD44v3-v10 structural domain specific polypeptide ligand for gastric cancer diagnosis is shown as SEQ ID NO.1, and is CV-1 polypeptide: THENFPA. The CV-1 polypeptide can be specifically combined with a v3-v10 variant exon coding structure domain of CD44, and is combined with human gastric cancer cells and tissues through the tumor marker, and the CV-1 polypeptide can be used for molecular imaging diagnosis of gastric cancer.)

1. The CD44v3-v10 domain-specific polypeptide ligand for gastric cancer diagnosis is characterized in that the amino acid sequence of the CD44v3-v10 domain-specific polypeptide ligand for gastric cancer diagnosis is shown as SEQ ID NO.1, and is CV-1 polypeptide: THENFPA.

2. The ligand of a polypeptide specific to the CD44v3-v10 domain for gastric cancer diagnosis according to claim 1, wherein the CV-1 polypeptide is capable of binding to the coding region of the CD44 variant exon of the target protein.

3. The CD44v3-v10 structure for gastric cancer diagnosis of claim 1The method for obtaining the domain-specific polypeptide ligand is characterized in that the high-throughput screening of a phage peptide library is carried out by using an improved phage display technology to obtain a CD44v3-v10 structural domain-specific polypeptide ligand sequence aiming at a target protein CD44 variant exon coding region; wherein the phage peptide library is derived from ph.d. ^TM-12Phage Display Peptide Library kit。

4. Use of the polypeptide ligand specific to CD44v3-v10 domain according to claim 1 in the preparation of a gastric cancer diagnostic reagent.

5. The use of a polypeptide ligand specific to CD44v3-v10 domain for the preparation of a gastric cancer diagnostic reagent according to claim 4, wherein said gastric cancer diagnostic reagent is a molecular imaging diagnostic reagent.

6. The use of the CD44v3-v10 domain-specific polypeptide ligand of claim 1 in the preparation of a medicament for the targeted treatment of gastric cancer cells.

7. The use according to any one of claims 4 to 6, wherein the gastric cancer is gastric cancer cell SGC-7901, gastric cancer orthotopic tissue, metastatic lymph node tissue, animal gastric cancer transplantation tumor model.

Technical Field

The invention belongs to the technical field of drug development, and particularly relates to a CD44v3-v10 domain specific polypeptide ligand for gastric cancer diagnosis, an obtaining method and application.

Background

Early diagnosis is an important means for improving the treatment level of gastric cancer and reducing the recurrence and fatality rate of diseases. The existing stomach cancer detection methods, including gastroscopy, digestive tract radiography and serum tumor markers, lack sufficient sensitivity and specificity for early-stage stomach cancer. Therefore, the development of an economic, convenient and easily popularized early gastric cancer screening means is a target and a direction for research of many scientists.

Traditional imaging, including endoscopy, relies on the determination of morphological abnormalities in the lesion. With the maturation of disease-related molecular mechanism research and the development of various imaging techniques, imaging techniques based on the level of disease-specific molecules have emerged. Molecular imaging is the qualitative and quantitative study of the biological behavior of related molecules in the image by using imaging-related means to display specific molecules at the tissue, cellular, and subcellular levels, reflecting changes at the molecular level. The core technology of the method lies in a molecular probe capable of being combined with a special pathological marker and an imaging platform capable of detecting the probe.

Molecular probes generally include three elements: groups that can generate signals that are detected by molecular imaging equipment, carriers that can optimize the pharmacokinetics of the entire molecule, and affinity ligands that can target binding targets for detection. Affinity ligands are currently the major bottleneck in the development of molecular imaging technology. Ligands that have been developed now include mainly antibodies, modified antibodies, polypeptides, aptamers, and some other small molecules. Among them, only small molecule ligands are widely used in clinical applications. However, the micromolecule ligand has simple structure and high false positive rate. The antibody probes become the most probes in clinical tests at present due to high specificity, but the probes are difficult to prepare and store, high in cost, high in immunogenicity and toxicity, and limited in use to a certain extent. The polypeptide ligand and the antibody have the same molecular basis and high diversity, and have the advantages of low toxicity, low immunogenicity, good tissue penetration, easy metabolism, low price and easy synthesis and labeling, thereby becoming an important direction for probe research.

CD44 is used as a macromolecular membrane protein, the structure of the macromolecular membrane protein is favorable for the binding of a ligand with cells in vivo, and the macromolecular membrane protein is also an important gastric cancer tumor marker, and the research on the correlation between the macromolecular membrane protein and tumor and gastric cancer is very mature. Full-length CD44 contains 10 common exons and 9 variant exons. Ordinary CD44 is composed of only ordinary exons, and is designated as CD44s, and can be expressed in normal human cells to complete the physiological functions of intercellular connection with stroma and promotion of cell movement (Goodison, S., V.Urquidi, and D.Tarin, CD44 cell adhesion molecules molecular Pathology,1999.52(4): p.189-96). Variant CD44 refers to a CD44 molecule with a variant exon, written as CD44v, which has higher specificity for expression in gastric cancer relative to CD44 s. The variants from v3 to v10 of CD44 all have higher expression rates in gastric cancer. Its expression is related to various clinical pathological features, prognosis and drug resistance of gastric cancer. Therefore, the CD44v3-v10 specific polypeptide ligand (probe) has excellent application prospect in clinic.

Disclosure of Invention

In order to solve the technical problems, the invention provides a CD44v3-v10 domain specific polypeptide ligand for gastric cancer diagnosis, an obtaining method and application.

The first purpose of the invention is to provide a CD44v3-v10 domain-specific polypeptide ligand for gastric cancer diagnosis, the amino acid sequence of the CD44v3-v10 domain-specific polypeptide ligand for gastric cancer diagnosis is shown as SEQ ID NO.1, and the ligand is a CV-1 polypeptide: THENFPA.

Preferably, the CV-1 polypeptide is capable of binding to an exon coding region of a CD44 variant of the target protein.

The second objective of the invention is to provide a method for obtaining the CD44v3-v10 domain specific polypeptide ligand for gastric cancer diagnosis, which uses the improved phage display technology to carry out high-throughput screening of phage peptide library, and obtains the CD44v3-v10 domain specific polypeptide ligand sequence aiming at the coding region of the CD44 variant exon of the target protein; wherein the phage peptide library is derived from ph.d. ^TM-12Phage Display Peptide Library kit。

The third purpose of the invention is to provide the application of the CD44v3-v10 domain specific polypeptide ligand in preparing gastric cancer diagnostic reagents.

Preferably, the gastric cancer diagnostic reagent is a molecular imaging diagnostic reagent.

The fourth purpose of the invention is to provide an application of the CD44v3-v10 domain specific polypeptide ligand in preparing a medicine for targeted therapy of gastric cancer cells.

Preferably, the gastric cancer refers to gastric cancer cell SGC-7901, gastric cancer in-situ tissue, metastatic lymph node tissue and animal gastric cancer transplantation tumor model.

Compared with the prior art, the CD44v3-v10 domain specific polypeptide ligand for gastric cancer diagnosis, the obtaining method and the application have at least the following beneficial effects:

1. the invention uses the improved phage display technology to obtain a specific polypeptide ligand sequence of a special structural domain CD44v3-v10 aiming at a target protein, and the synthesized specific CV-1 polypeptide of CD44v3-v10 excludes the non-specific binding of the common phage capsid protein CD 44.

2. The affinity and specificity of the CV-1 polypeptide, gastric cancer cells and normal tissue level target protein are determined by calculating the affinity of the CV-1 polypeptide ligand and the protein level competitive inhibition.

Drawings

FIG. 1 is a schematic diagram of a screening process using an improved phage display technique;

FIG. 2 is a test for verifying the binding of different polypeptide phages to a target protein;

FIG. 3 shows the results of molecular level binding force and specificity verification of different polypeptide ligands and target proteins;

FIG. 3A is the result of competitive inhibition of a polypeptide against binding of the corresponding phage to a target protein, FIG. 3B is the result of affinity validation (large panel in FIG. 3B) and dissociation constant calculation (small panel in FIG. 3B) of the polypeptide with the target protein, FIG. 3C is the result of competitive inhibition of the anti-CD 44v3-v10 antibody against binding of the polypeptide to CD44v3-v10, and FIG. 3D is the result of competitive inhibition of the anti-CD 44 antibody against binding of the polypeptide to CD44v3-v 10;

FIG. 4 shows Western blot to verify the expression of CD44v3-v10 in gastric cancer cell SGC-7901 and human embryonic kidney cell HEK-293 cell lines;

FIG. 5 is the results of FITC and DAPI immunofluorescence verifying binding of the polypeptide ligands to the cells;

FIG. 5A is the result of FITC-labeled CV-1 polypeptide binding to gastric cancer cell SGC-7901, FIG. 5B is the result of FITC-labeled CV-1 polypeptide binding to human embryonic kidney HEK-293 cell line, FIG. 5C is the result of FITC-labeled control peptide binding to gastric cancer cell SGC-7901, FIG. 5D is the result of DAPI-labeled CV-1 polypeptide binding to gastric cancer cell SGC-7901, FIG. 5E is the result of DAPI-labeled CV-1 polypeptide binding to human embryonic kidney HEK-293 cell line, and FIG. 5F is the result of DAPI-labeled control peptide binding to gastric cancer cell SGC-7901;

FIG. 6 shows the result of staining the in situ tissue and the metastatic lymph node tissue of human gastric cancer;

FIGS. 6A-B are the results of staining cancer tissues and surrounding non-cancer tissues in situ human gastric cancer tissues with CV-1 polypeptide, and FIGS. 6C-D are the results of staining cancer tissues and surrounding non-cancer tissues in metastatic lymph node tissues with CV-1 polypeptide, respectively;

FIG. 7 is a statistical result of the grouping scores corresponding to the staining results in FIG. 6;

FIG. 7A is a cancer tissue score statistic and FIG. 7B is a non-cancer tissue score statistic;

FIG. 8 shows the in vivo imaging results of 2h animals after injection of CV-1 polypeptide and control peptide;

FIG. 8A is CV-1 polypeptide and FIG. 8B is control peptide.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention to be implemented, the present invention will be further described with reference to the following specific embodiments and accompanying drawings.

The following examples and test methods not specifically described in the summary of the invention were carried out according to conventional methods and conditions in the art, and materials, reagents and the like used in the following examples were commercially available unless otherwise specified.

The following examples 1-2 are based on ph.d. ^TM12Phage Display Peptide Library kit (New England BioLabs, Beverly, MA, USA) and its operating manual.