CN117777232A

Movatterモバイル変換

Info

Publication number: CN117777232A
Application number: CN202211197570.5A
Authority: CN
Inventors: 赵睿; 徐坤; 黄嫣嫣; 金钰龙
Original assignee: Institute of Chemistry CAS
Current assignee: Institute of Chemistry CAS
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2024-03-29

Abstract

本发明公开了一种靶向人CD81蛋白的亲和肽及其应用。本发明提供的亲和肽的氨基酸序列如SEQ ID NO：7所示。本发明提供的亲和肽与CD81蛋白胞外小环片段具有高亲和力、高选择性，能够识别并结合游离以及活细胞中的CD81蛋白，可用于CD81蛋白的高灵敏检测和原位成像，并进一步用作表达CD81蛋白的肿瘤细胞迁移过程的选择性抑制剂。本发明提供的亲和肽可以特异性靶向识别CD81蛋白，是CD81蛋白分析检测和功能研究的特异性探针，在恶性肿瘤等疾病的检测和靶向药物开发中具有应用前景。本发明具有重要的应用价值。The invention discloses an affinity peptide targeting human CD81 protein and its application. The amino acid sequence of the affinity peptide provided by the present invention is shown in SEQ ID NO: 7. The affinity peptide provided by the invention has high affinity and selectivity with the extracellular small ring fragment of CD81 protein, can recognize and bind to CD81 protein in free and living cells, and can be used for highly sensitive detection and in-situ imaging of CD81 protein, and It is further used as a selective inhibitor of the migration process of tumor cells expressing CD81 protein. The affinity peptide provided by the invention can specifically target and recognize CD81 protein, is a specific probe for CD81 protein analysis, detection and functional research, and has application prospects in the detection of malignant tumors and other diseases and the development of targeted drugs. The invention has important application value.

Description

Translated fromChinese

一种靶向人CD81蛋白的亲和肽及其应用An affinity peptide targeting human CD81 protein and its application

技术领域Technical field

本发明属于生物分析技术领域，具体涉及一种靶向人CD81蛋白的亲和肽及其应用。The invention belongs to the field of biological analysis technology, and specifically relates to an affinity peptide targeting human CD81 protein and its application.

背景技术Background technique

四次跨膜蛋白CD81在人体内参与蛋白质转运、细胞融合、信号转导等重要生理过程，与多种疾病的发生、发展密切相关。研究表明，CD81通过与其他蛋白质相互作用形成跨膜蛋白相互作用网络，调节肿瘤细胞的粘附、增殖、迁移与侵袭等过程，是癌症发展、转移的关键信号蛋白。此外，作为丙型肝炎病毒(HCV)包膜糖蛋白的受体，CD81能够介导病毒与细胞的融合，在病毒侵入、感染宿主细胞的过程中发挥着不可或缺的作用。因此，CD81是肿瘤转移和丙肝病毒感染检测和干预的理想靶点。以CD81为靶标，筛选新型亲和识别分子，是疾病检测和新靶向药物开发的关键。然而，CD81缺少天然结合配体，其人工亲和配体的研究也鲜有报道，严重阻碍其发展成为新型生物标志物和药物靶点。高亲和力、高特异性的CD81配体分子的发现有望为转移性肿瘤等重大疾病的早期检测和靶向药物的开发带来新机遇。The tetraspanning protein CD81 participates in important physiological processes such as protein transport, cell fusion, and signal transduction in the human body, and is closely related to the occurrence and development of various diseases. Studies have shown that CD81 forms a transmembrane protein interaction network by interacting with other proteins, regulating the adhesion, proliferation, migration and invasion of tumor cells, and is a key signaling protein for cancer development and metastasis. In addition, as a receptor for the hepatitis C virus (HCV) envelope glycoprotein, CD81 can mediate the fusion of viruses and cells and plays an indispensable role in the process of virus invasion and infection of host cells. Therefore, CD81 is an ideal target for detection and intervention of tumor metastasis and HCV infection. Using CD81 as a target to screen new affinity recognition molecules is the key to disease detection and the development of new targeted drugs. However, CD81 lacks natural binding ligands, and there are few reports on its artificial affinity ligands, which seriously hinders its development into new biomarkers and drug targets. The discovery of CD81 ligand molecules with high affinity and specificity is expected to bring new opportunities for the early detection of major diseases such as metastatic tumors and the development of targeted drugs.

多肽作为内源性生理活性物质，可参与生命体内众多的分子事件，发挥重要的生理功能。以多肽作为识别单元的分子探针不仅生物相容性好、免疫原性低，而且可设计性强、易于合成与修饰，被广泛应用于生理活性物质的分析检测。同时，基于靶向多肽与目标蛋白分子的特异性结合，发展出了越来越多的新型抗癌、抗病毒等靶向药物和疫苗。因此，针对CD81蛋白的关键结构片段，利用化学方法设计、构建新型人工多肽识别分子，通过特异性结合CD81蛋白，既可以发展高选择性、高灵敏度的疾病分析检测新方法，又能够为调控和干预CD81功能、开发新型靶向药物提供新的、有价值的思路和手段。As endogenous physiologically active substances, peptides can participate in numerous molecular events in life and play important physiological functions. Molecular probes using peptides as recognition units not only have good biocompatibility and low immunogenicity, but are also highly designable and easy to synthesize and modify. They are widely used in the analysis and detection of physiologically active substances. At the same time, more and more new anti-cancer, anti-viral and other targeted drugs and vaccines have been developed based on the specific combination of targeted peptides and target protein molecules. Therefore, by targeting the key structural fragments of the CD81 protein and using chemical methods to design and construct new artificial peptide recognition molecules, by specifically binding to the CD81 protein, we can not only develop new methods for disease analysis and detection with high selectivity and sensitivity, but also provide new methods for regulation and control. Intervening CD81 function and developing new targeted drugs provide new and valuable ideas and means.

发明内容Contents of the invention

本发明的目的是提供一种靶向人CD81蛋白的亲和肽。The purpose of the present invention is to provide an affinity peptide targeting human CD81 protein.

本发明首先保护一种多肽，其氨基酸序列如SEQ ID NO：7所示。The present invention firstly protects a polypeptide, whose amino acid sequence is shown in SEQ ID NO:7.

所述多肽既可以利用固相肽合成方法(BOC方法或FMOC方法)合成，也可以利用多肽自动合成仪进行合成，亦可将编码所述多肽的基因克隆到表达载体中进行生物合成。The polypeptide can be synthesized using a solid-phase peptide synthesis method (BOC method or FMOC method), an automatic polypeptide synthesizer can be used, or the gene encoding the polypeptide can be cloned into an expression vector for biosynthesis.

本发明还保护所述多肽的应用，可为如下(c1)-(c12)中至少一种：The invention also protects the application of the polypeptide, which can be at least one of the following (c1)-(c12):

(c1)制备用于检测CD81蛋白的试剂盒；(c1) Prepare a kit for detecting CD81 protein;

(c2)制备用于检测多肽EL1的试剂盒；(c2) preparing a kit for detecting polypeptide EL1;

(c3)制备用于检测表达CD81蛋白的细胞的试剂盒；(c3) Preparing a kit for detecting cells expressing CD81 protein;

(c4)制备用于抑制表达CD81蛋白的细胞的迁移和/或侵袭的试剂盒；(c4) Preparing a kit for inhibiting the migration and/or invasion of cells expressing CD81 protein;

(c5)制备用于抑制表达CD81蛋白的癌组织的迁移和/或侵袭的试剂盒；(c5) Preparing a kit for inhibiting the migration and/or invasion of cancer tissue expressing CD81 protein;

(c6)制备用于抑制CD81蛋白功能的试剂盒；(c6) preparing a kit for inhibiting the function of CD81 protein;

(c7)检测CD81蛋白；(c7) Detection of CD81 protein;

(c8)检测多肽EL1；(c8) Detection of polypeptide EL1;

(c9)检测表达CD81蛋白的细胞；(c9) Detect cells expressing CD81 protein;

(c10)抑制表达CD81蛋白的细胞的迁移和/或侵袭；(c10) Inhibit the migration and/or invasion of cells expressing CD81 protein;

(c11)抑制表达CD81蛋白的癌组织的迁移和/或侵袭；(c11) inhibiting the migration and/or invasion of cancer tissues expressing CD81 protein;

(c12)作为CD81蛋白功能抑制剂；(c12) as an inhibitor of CD81 protein function;

多肽EL1的氨基酸序列如SEQ ID NO：10所示。The amino acid sequence of polypeptide EL1 is shown in SEQ ID NO: 10.

上述应用中，所述表达CD81蛋白的细胞可为人乳腺癌细胞MCF-7、人肝癌细胞Huh-7或人胶质瘤细胞U251。In the above application, the cells expressing CD81 protein can be human breast cancer cells MCF-7, human liver cancer cells Huh-7 or human glioma cells U251.

本发明还保护一种偶联物，由所述多肽和物质偶联获得。The present invention also protects a conjugate obtained by coupling the polypeptide and the substance.

所述偶联物中，所述物质可为药物。所述药物的功能可为如下(1)、(2)、(3)和/或(4)：(1)抑制癌细胞增殖、迁移和/或侵袭；(2)杀伤癌细胞；(3)抑制癌组织生长和/或转移；(4)治疗癌症。该偶联物中，所述多肽作为将所述药物靶向CD81蛋白的载体。In the conjugate, the substance may be a drug. The functions of the drug may be as follows (1), (2), (3) and/or (4): (1) inhibit cancer cell proliferation, migration and/or invasion; (2) kill cancer cells; (3) Inhibit cancer tissue growth and/or metastasis; (4) Treat cancer. In this conjugate, the polypeptide serves as a carrier to target the drug to the CD81 protein.

所述药物可为化合物或蛋白质。所述药物可为毒素、细胞因子、酶或凝集素。The drug may be a compound or protein. The drug may be a toxin, cytokine, enzyme or lectin.

所述偶联物中，所述物质可为标记基团，即所述偶联物由所述多肽与所述标记基团偶联而成。该偶联物可作为检测探针，检测CD81蛋白。In the conjugate, the substance may be a labeling group, that is, the conjugate is formed by coupling the polypeptide with the labeling group. The conjugate can be used as a detection probe to detect CD81 protein.

所述标记基团可为放射性元素、荧光基团、量子点或高吸光系数发色团。所述荧光基团具体可为硫氰酸荧光素荧光基团(FITC)或四苯乙烯荧光基团(TPE)。The labeling group may be a radioactive element, a fluorescent group, a quantum dot or a high absorption coefficient chromophore. The fluorescent group may specifically be a fluorescein thiocyanate fluorescent group (FITC) or a tetraphenylethylene fluorescent group (TPE).

所述偶联物具体可为由所述多肽与FITC或TPE偶联获得的偶联物。The conjugate may specifically be a conjugate obtained by conjugating the polypeptide with FITC or TPE.

上述任一所述FITC的结构式如式(Ⅰ)所示；The structural formula of any of the above-mentioned FITC is shown in formula (I);

上述任一所述TPE的结构式如式(Ⅱ)所示；The structural formula of any of the above-mentioned TPEs is shown in formula (II);

本发明还保护上述任一所述偶联物的应用，可为如下(c1)-(c12)中至少一种：The present invention also protects the application of any of the above-mentioned conjugates, which can be at least one of the following (c1)-(c12):

(c2)制备用于检测多肽EL1的试剂盒；(c2) Prepare a kit for detecting polypeptide EL1;

(c6)制备用于抑制CD81蛋白功能的试剂盒；(c6) Prepare a kit for inhibiting CD81 protein function;

(c7)检测CD81蛋白；(c7) Detection of CD81 protein;

(c8)检测多肽EL1；(c8) Detection of polypeptide EL1;

(c11)抑制表达CD81蛋白的癌组织的迁移和/或侵袭；(c11) Inhibit the migration and/or invasion of cancer tissues expressing CD81 protein;

本发明还保护试剂盒甲，其含有上述任一所述多肽；所述试剂盒甲的用途可为(c7)-(c12)中至少一种：The present invention also protects a kit A, which contains any of the above-mentioned polypeptides; the purpose of the kit A can be at least one of (c7)-(c12):

(c7)检测CD81蛋白；(c7) Detection of CD81 protein;

(c8)检测多肽EL1；(c8) detecting polypeptide EL1;

(c12)作为CD81蛋白功能抑制剂；(c12) As a CD81 protein function inhibitor;

多肽EL1的氨基酸序列如SEQ ID NO：10所示。The amino acid sequence of polypeptide EL1 is shown in SEQ ID NO:10.

所述试剂盒甲中，表达CD81蛋白的细胞可为人乳腺癌细胞MCF-7、人肝癌细胞Huh-7或人胶质瘤细胞U251。In the kit A, the cells expressing CD81 protein can be human breast cancer cells MCF-7, human liver cancer cells Huh-7 or human glioma cells U251.

本发明还保护试剂盒乙，其含有上述任一所述偶联物；所述试剂盒乙的用途可为(c7)-(c12)中至少一种：The present invention also protects kit B, which contains any of the conjugates described above; the use of kit B can be at least one of (c7)-(c12):

(c7)检测CD81蛋白；(c7) Detection of CD81 protein;

(c8)检测多肽EL1；(c8) detecting polypeptide EL1;

所述试剂盒乙中，表达CD81蛋白的细胞可为人乳腺癌细胞MCF-7、人肝癌细胞Huh-7或人胶质瘤细胞U251。In the kit B, the cells expressing CD81 protein can be human breast cancer cells MCF-7, human liver cancer cells Huh-7 or human glioma cells U251.

CD81蛋白是一个25kD的4次跨膜蛋白，其存在4个疏水跨膜区和2个胞外区。与胞外大环相比，胞外小环SEL(N端34-63位氨基酸)的构象更为延展，适合于配体的对接。CD81蛋白的胞外小环含有亲水性较好的多肽EL1(N端44-54位氨基酸)，可作为识别CD81蛋白的靶点。CD81 protein is a 25kD 4-transmembrane protein with 4 hydrophobic transmembrane regions and 2 extracellular regions. Compared with the extracellular macrocycle, the conformation of the small extracellular ring SEL (amino acids 34-63 at the N terminus) is more extended and is suitable for docking of ligands. The extracellular small loop of the CD81 protein contains the hydrophilic peptide EL1 (N-terminal amino acids 44-54), which can be used as a target to identify the CD81 protein.

实验证明，本发明提供的多肽及多肽偶联物除了能高选择性、高亲和力地结合细胞表面的CD81蛋白，实现CD81的高选择性分析检测外，还能够通过靶向结合CD81蛋白，选择性抑制细胞的迁移，可作为人CD81蛋白的新型功能抑制剂。Experiments have proven that the polypeptides and polypeptide conjugates provided by the present invention can not only bind to the CD81 protein on the cell surface with high selectivity and high affinity to achieve highly selective analysis and detection of CD81, but can also selectively bind to the CD81 protein through targeting. Inhibits cell migration and can be used as a new functional inhibitor of human CD81 protein.

由此可见，本发明提供的多肽(即亲和肽)与CD81蛋白胞外小环片段具有高亲和力、高选择性，能够识别并结合游离以及活细胞中的CD81蛋白，可用于CD81蛋白的高灵敏检测和原位成像，并进一步用作表达CD81蛋白的肿瘤细胞迁移过程的选择性抑制剂。同时本发明提供的多肽的氨基酸序列较短，易于实现大规模生产，并且对细胞无毒性，弥补了抗体等生物制剂制备繁琐、稳定性较差、造价昂贵、易引起免疫反应、穿透力弱等缺点，可作为一种靶向CD81蛋白的新型小分子识别工具。本发明提供的亲和肽可以特异性靶向识别CD81蛋白，是CD81蛋白分析检测和功能研究的特异性探针，在恶性肿瘤等疾病的检测和靶向药物开发中具有应用前景。本发明具有重要的应用价值。It can be seen that the polypeptide (i.e., affinity peptide) provided by the present invention has high affinity and selectivity with the extracellular small ring fragment of the CD81 protein, can recognize and bind to the CD81 protein in free and living cells, and can be used for high-sensitivity detection of the CD81 protein. Sensitive detection and in situ imaging, and further used as a selective inhibitor of the migration process of tumor cells expressing CD81 protein. At the same time, the polypeptide provided by the invention has a short amino acid sequence, is easy to realize large-scale production, and is non-toxic to cells, which makes up for the tedious preparation, poor stability, expensive production, easy induction of immune reactions and weak penetrating power of biological preparations such as antibodies. and other shortcomings, it can be used as a new small molecule recognition tool targeting CD81 protein. The affinity peptide provided by the invention can specifically target and recognize CD81 protein, is a specific probe for CD81 protein analysis, detection and functional research, and has application prospects in the detection of malignant tumors and other diseases and the development of targeted drugs. The invention has important application value.

附图说明Description of drawings

图1为筛选探针TPE-EL1的化学结构示意图。FIG1 is a schematic diagram of the chemical structure of the screening probe TPE-EL1.

图2为9条多肽与TPE-EL1孵育后相对荧光强度统计结果。Figure 2 shows the statistical results of relative fluorescence intensity after incubation of 9 polypeptides with TPE-EL1.

图3为多肽APQQ与CD81蛋白及其他多肽、蛋白孵育后相对荧光强度统计结果。Figure 3 shows the statistical results of relative fluorescence intensity after incubation of polypeptide APQQ with CD81 protein and other polypeptides and proteins.

图4为多肽APQQ对CD81蛋白亲和力及检出限的测定结果。Figure 4 shows the measurement results of the affinity and detection limit of polypeptide APQQ for CD81 protein.

图5为人肝癌细胞Hep3B、人肝癌细胞Huh-7、人乳腺癌细胞MCF-7和人胶质瘤细胞U251细胞中CD81表达量的免疫荧光分析及FITC-APQQ染色后细胞成像分析。Figure 5 shows the immunofluorescence analysis and cell imaging analysis of CD81 expression in human liver cancer cells Hep3B, human liver cancer cells Huh-7, human breast cancer cells MCF-7 and human glioma cells U251 cells after FITC-APQQ staining.

图6为多肽APQQ通过靶向结合CD81蛋白抑制细胞迁移。FIG. 6 shows that the peptide APQQ inhibits cell migration by targeting and binding to CD81 protein.

具体实施方式Detailed ways

下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

以下实施例中的定量实验，均设置三次重复实验，结果取平均值。The quantitative experiments in the following examples were repeated three times, and the results were averaged.

实施例1、靶向人CD81蛋白的亲和肽的设计与筛选Example 1. Design and screening of affinity peptides targeting human CD81 protein

一、靶向人CD81蛋白的亲和肽的设计1. Design of affinity peptide targeting human CD81 protein

以人CD81蛋白胞外小环中十一肽(简称多肽EL1或EL1)为靶点，基于正-反义肽相互作用原理和亮氨酸遗传密码子简并性，设计了特异结合EL1的一组功能导向候选肽，分别命名为多肽APEQ(简称APEQ)、多肽APEK(简称APEK)、多肽APEE(简称APEE)、多肽APKQ(简称APKQ)、多肽APKK(简称APKK)、多肽APKE(简称APKE)、多肽APQQ(简称APQQ)、多肽APQK(简称APQK)和多肽APQE(简称APQE)。Taking the undecapeptide in the extracellular small loop of human CD81 protein (referred to as polypeptide EL1 or EL1) as the target, based on the principle of sense-antisense peptide interaction and the degeneracy of the leucine genetic code, a peptide that specifically binds to EL1 was designed. The group of function-oriented candidate peptides are named peptide APEQ (APEQ for short), APEK for short (APEK for short), APEE for short (APEE for short), APKQ for short (APKQ for short), APKK for short (APKK for short), and APKE for short (APKE for short). , peptide APQQ (APQQ for short), peptide APQK (APQK for short) and peptide APQE (APQE for short).

EL1和上述9条候选肽的氨基酸序列见表1；其中各字母代表氨基酸如下：A为丙氨酸，D为天冬氨酸，P为脯氨酸，Q为谷氨酰胺，Y为酪氨酸，S为丝氨酸，E为谷氨酸，L为亮氨酸，G为甘氨酸，I为异亮氨酸，R为精氨酸，V为缬氨酸，K为赖氨酸。The amino acid sequences of EL1 and the above nine candidate peptides are shown in Table 1; the amino acids represented by each letter are as follows: A is alanine, D is aspartic acid, P is proline, Q is glutamine, and Y is tyrosine. Acid, S is serine, E is glutamic acid, L is leucine, G is glycine, I is isoleucine, R is arginine, V is valine, and K is lysine.

表1Table 1

二、亲和肽的筛选2. Screening of affinity peptides

聚集诱导发光荧光团由于其在聚集状态或分子内旋转受限时荧光打开的特性，在分子间相互作用研究中得到了越来越多的应用。TPE是一种典型的聚集诱导发光荧光团，当TPE与多肽偶联之后，亲水性多肽的存在使得其在生理溶液中荧光明显减弱。Aggregation-induced emission fluorophores have been increasingly used in the study of molecular interactions due to their characteristic of fluorescence turning on in an aggregated state or when the intramolecular rotation is restricted. TPE is a typical aggregation-induced emission fluorophore. When TPE is coupled to a peptide, the presence of the hydrophilic peptide significantly weakens its fluorescence in physiological solutions.

在多肽与其他分子产生相互作用时，TPE呈现荧光打开的特性，从而指示体系中相互作用的强弱。基于这一原理，可以通过筛选探针TPE-EL1的荧光强度来考察9条候选肽与EL1之间特异性相互作用的强弱，从而获得与EL1相互作用最强的优选肽。When the peptide interacts with other molecules, TPE exhibits the characteristic of fluorescence, thus indicating the strength of the interaction in the system. Based on this principle, the strength of the specific interaction between the 9 candidate peptides and EL1 can be examined by screening the fluorescence intensity of the probe TPE-EL1, thereby obtaining the preferred peptide with the strongest interaction with EL1.

1、多肽筛选荧光探针的制备1. Preparation of fluorescent probes for peptide screening

用FMOC固相多肽合成法在Wang树脂上由C端开始合成多肽筛选探针(简称TPE-EL1)。具体方法为：将多肽EL1的氨基酸逐个偶联到固相树脂上，在EL1的N端连接一个甘氨酸作为间隔手臂，最后将多肽的N端氨基与具有羧基的荧光打开型分子TPE偶联，然后使用裂解液(由95％(v/v)三氟乙酸、2.5％(v/v)三异丙基硅烷和2.5％(v/v)水组成)将肽链从树脂上裂解，同时去除侧链保护基团。The FMOC solid-phase peptide synthesis method was used to synthesize the peptide screening probe (referred to as TPE-EL1) on Wang resin starting from the C-terminus. The specific method is: couple the amino acids of the polypeptide EL1 to the solid-phase resin one by one, connect a glycine to the N-terminal of EL1 as a spacer arm, and finally couple the N-terminal amino group of the polypeptide to a fluorescent open molecule TPE with a carboxyl group, and then Use cleavage solution (composed of 95% (v/v) trifluoroacetic acid, 2.5% (v/v) triisopropylsilane and 2.5% (v/v) water) to cleave the peptide chain from the resin while removing the side chains. chain protecting group.

TPE-EL1的化学结构示意图如图1所示。The schematic diagram of the chemical structure of TPE-EL1 is shown in Figure 1.

2、亲和肽的筛选2. Screening of affinity peptides

为了考察TPE-EL1与候选肽(APEQ、APEK、APEE、APKQ、APKK、APKE、APQQ、APQK或APQE)的相互作用强弱，进行如下实验：In order to examine the strength of the interaction between TPE-EL1 and candidate peptides (APEQ, APEK, APEE, APKQ, APKK, APKE, APQQ, APQK or APQE), the following experiments were performed:

(1)使用二甲基亚砜(DMSO)作为溶剂，配制TPE-EL1储备液和候选肽储备液。(1) Use dimethyl sulfoxide (DMSO) as the solvent to prepare TPE-EL1 stock solution and candidate peptide stock solution.

(2)将含有0.05％Tween-20的10mmol/L磷酸盐缓冲液(pH7.4)、TPE-EL1储备液和候选肽储备液混合，得到混合体系1；混合体系1中，TPE-EL1的浓度为10μmol/L，候选肽的浓度为20μmol/L，DMSO的含量为0.3％。(2) 10 mmol/L phosphate buffer (pH 7.4) containing 0.05% Tween-20, TPE-EL1 stock solution and candidate peptide stock solution were mixed to obtain mixed system 1; in mixed system 1, the concentration of TPE-EL1 was 10 μmol/L, the concentration of the candidate peptide was 20 μmol/L, and the content of DMSO was 0.3%.

(3)取步骤(2)得到的混合体系1，避光孵育1h。(3) Take the mixed system 1 obtained in step (2) and incubate it in the dark for 1 hour.

(4)完成步骤(3)后，使用酶标仪(Microplate reader，美国)对溶液进行快速扫描，激发波长为320nm，记录发射波长457nm的荧光强度数据，即候选肽的荧光强度。(4) After completing step (3), use a microplate reader (USA) to quickly scan the solution with an excitation wavelength of 320 nm and record the fluorescence intensity data at an emission wavelength of 457 nm, which is the fluorescence intensity of the candidate peptide.

(5)按照上述步骤(2)-(4)，将混合体系1替换为混合体系2，其它步骤均不变，得到空白对照的荧光强度。混合体系2由含有0.05％Tween-20的10mmol/L磷酸盐缓冲液(pH7.4)和TPE-EL1储备液组成；混合体系2中，TPE-EL1的浓度为10μmol/L，DMSO的含量为0.3％。(5) Follow the above steps (2)-(4), replace mixed system 1 with mixed system 2, keep other steps unchanged, and obtain the fluorescence intensity of the blank control. Mixed system 2 consists of 10 mmol/L phosphate buffer (pH 7.4) containing 0.05% Tween-20 and TPE-EL1 stock solution; in mixed system 2, the concentration of TPE-EL1 is 10 μmol/L, and the content of DMSO is 0.3%.

(6)计算候选肽的相对荧光强度。候选肽的相对荧光强度＝(候选肽的荧光强度-空白对照的荧光强度)/空白对照的荧光强度(6) Calculate the relative fluorescence intensity of the candidate peptide. Relative fluorescence intensity of candidate peptide = (fluorescence intensity of candidate peptide - fluorescence intensity of blank control) / fluorescence intensity of blank control

结果如图2所示。结果表明，9条候选肽与EL1的结合能力不同，在pH 7.4、10mmol/L的磷酸盐缓冲液中，多肽APQQ与EL1的靶向结合能力最强，在457nm处的荧光强度提高了2.98倍。将多肽APQQ作为优选肽，进一步考察其对CD81蛋白的特异性识别性能。The results are shown in Figure 2. The results show that the nine candidate peptides have different binding abilities to EL1. In the phosphate buffer solution with pH 7.4 and 10mmol/L, the peptide APQQ has the strongest target binding ability with EL1, and the fluorescence intensity at 457nm increases by 2.98 times. . Polypeptide APQQ was selected as the preferred peptide to further investigate its specific recognition performance for CD81 protein.

实施例2、多肽APQQ特异性识别与高灵敏检测CD81蛋白Example 2. Specific recognition and highly sensitive detection of CD81 protein by polypeptide APQQ

以筛选所得到的多肽APQQ为识别分子，在其N端修饰TPE分子，考察多肽APQQ与CD81蛋白的特异性识别。当多肽APQQ与CD81蛋白产生相互作用时，TPE荧光打开，从而指示相互作用强弱。The polypeptide APQQ obtained by screening was used as the recognition molecule, and the TPE molecule was modified at its N-terminus to investigate the specific recognition of the polypeptide APQQ and the CD81 protein. When polypeptide APQQ interacts with CD81 protein, TPE fluorescence turns on, indicating the strength of the interaction.

1、制备TPE标记的多肽荧光探针TPE-APQQ1. Preparation of TPE-labeled peptide fluorescent probe TPE-APQQ

用FMOC固相多肽合成法在Wang树脂上由C端开始直接合成多肽，并在其N端偶联Fmoc-Lys(Dde)-OH，保留N端FMOC保护基，加入Dde脱保护液(Dde脱保护液由咪唑、盐酸羟胺、5mLN-甲基吡咯烷酮和1mL二氯甲烷组成；Dde脱保护液中，咪唑的物质的量为1.35mmol，盐酸羟胺的物质的量为1.80mmol)，室温脱除3h。称取一定量上述树脂，将3倍量TPE-COOH、3倍量HATU和3倍量HOBt溶于添加有0.4mol/LN-甲基吗啡啉的DMF溶液中，室温下避光偶联2.5h，DMF清洗后加入含有20％哌啶的DMF溶液，脱除N端FMOC保护基，之后使用裂解液(由95％(v/v)三氟乙酸、2.5％(v/v)三异丙基硅烷和2.5％(v/v)水组成)将修饰有TPE的肽链从树脂上裂解，同时去除所有侧链保护基，得到裂解产物。Use FMOC solid-phase peptide synthesis method to directly synthesize peptides starting from the C-terminus on Wang resin, and couple Fmoc-Lys(Dde)-OH to its N-terminus, retain the N-terminal FMOC protective group, and add Dde deprotection solution (Dde deprotection solution). The protective solution consists of imidazole, hydroxylamine hydrochloride, 5mL N-methylpyrrolidone and 1mL dichloromethane; in the Dde deprotection solution, the amount of imidazole is 1.35mmol, and the amount of hydroxylamine hydrochloride is 1.80mmol), and the removal is done at room temperature for 3 hours . Weigh a certain amount of the above resin, dissolve 3 times the amount of TPE-COOH, 3 times the amount of HATU, and 3 times the amount of HOBt in a DMF solution with 0.4 mol/L N-methylmorpholine added, and couple in the dark at room temperature for 2.5 hours. , after washing with DMF, add a DMF solution containing 20% piperidine to remove the N-terminal FMOC protecting group, and then use a lysis solution (composed of 95% (v/v) trifluoroacetic acid, 2.5% (v/v) triisopropyl Composed of silane and 2.5% (v/v) water), the peptide chain modified with TPE is cleaved from the resin, and all side chain protecting groups are removed to obtain the cleaved product.

将裂解产物用HPLC纯化，所用HPLC纯化的参数如下：色谱柱为Diamonsil C18(2)250×4.6mm；流动相A为添加有0.1％(v/v)三氟乙酸的水溶液，流动相B为添加有0.1％(v/v)三氟乙酸的乙腈溶液，在25min内，流动相B百分比从47％上升至67％进行洗脱。收集对应目标物馏分，冻干后得到纯度高且结构正确的TPE-APQQ。The cleavage product was purified by HPLC. The parameters used for HPLC purification were as follows: the chromatographic column was Diamonsil C18(2) 250×4.6mm; the mobile phase A was an aqueous solution with 0.1% (v/v) trifluoroacetic acid added, and the mobile phase B was A solution of acetonitrile with 0.1% (v/v) trifluoroacetic acid was added, and the percentage of mobile phase B increased from 47% to 67% within 25 min for elution. Collect the fractions corresponding to the target substance and obtain TPE-APQQ with high purity and correct structure after freeze-drying.

2、多肽APQQ与CD81蛋白的识别选择性2. Recognition selectivity of polypeptide APQQ and CD81 protein

(1)将含有0.05％Tween-20的10mmol/L磷酸盐缓冲液(pH7.4)、TPE-APQQ和待测物质混合，得到混合体系甲；混合体系甲中，TPE-APQQ的浓度为1μmol/L，待测物质的浓度为2μmol/L。(1) Mix 10 mmol/L phosphate buffer (pH 7.4) containing 0.05% Tween-20, TPE-APQQ and the substance to be tested to obtain mixed system A; in mixed system A, the concentration of TPE-APQQ is 1 μmol /L, the concentration of the substance to be tested is 2μmol/L.

待测物质为CD81蛋白、转铁蛋白(Transferrin)、人血清白蛋白(HSA)、细胞色素C(Cytochrome-C)、胰蛋白酶(Trypsin)、辣根过氧化酶(HRP)、溶菌酶(Lysozyme)、催产素(Oxytocin)、C型心房钠尿肽(CNP)、血管加压素(Vasopressin)、物质P(Substance P)或L-谷胱甘肽(L-GSH)。The substances to be tested are CD81 protein, transferrin, human serum albumin (HSA), cytochrome C (Cytochrome-C), trypsin (Trypsin), horseradish peroxidase (HRP), lysozyme (Lysozyme), oxytocin (Oxytocin), C-type atrial natriuretic peptide (CNP), vasopressin (Vasopressin), substance P (Substance P) or L-glutathione (L-GSH).

(2)取步骤(1)得到的混合体系，避光孵育1h。(2) Take the mixed system obtained in step (1) and incubate it in the dark for 1 hour.

(3)使用荧光仪(Hitachi F-4600，日本)对完成步骤(2)的溶液进行荧光扫描，激发波长为320nm，记录发射波长457nm的荧光强度数据，即待测物质的荧光强度。(3) Use a fluorometer (Hitachi F-4600, Japan) to perform a fluorescence scan on the solution that has completed step (2). The excitation wavelength is 320 nm, and the fluorescence intensity data at the emission wavelength of 457 nm is recorded, which is the fluorescence intensity of the substance to be tested.

(4)按照上述步骤(1)-(3)，将混合体系甲替换为混合体系乙，其它步骤均不变，得到空白对照的荧光强度。混合体系乙由含有0.05％Tween-20的10mmol/L磷酸盐缓冲液(pH7.4)和TPE-APQQ组成；混合体系乙中，TPE-APQQ的浓度为1μmol/L。(4) According to the above steps (1)-(3), the mixed system A was replaced by the mixed system B, and the other steps remained unchanged to obtain the fluorescence intensity of the blank control. The mixed system B consisted of 10 mmol/L phosphate buffer (pH 7.4) containing 0.05% Tween-20 and TPE-APQQ; in the mixed system B, the concentration of TPE-APQQ was 1 μmol/L.

(5)计算待测物质的相对荧光强度。待测物质的相对荧光强度＝(待测物质的荧光强度-空白对照的荧光强度)/空白对照的荧光强度。(5) Calculate the relative fluorescence intensity of the substance to be tested. The relative fluorescence intensity of the substance to be tested = (fluorescence intensity of the substance to be tested - fluorescence intensity of the blank control)/fluorescence intensity of the blank control.

结果如图3所示。结果表明，除CD81蛋白之外，其余多肽或蛋白与APQQ均无特异性结合，荧光信号明显弱于CD81蛋白，说明多肽APQQ与CD81蛋白的结合具有高选择性。The results are shown in Figure 3. The results showed that, except for the CD81 protein, the other polypeptides or proteins had no specific binding to APQQ, and the fluorescence signal was significantly weaker than that of the CD81 protein, indicating that the binding of the polypeptide APQQ to the CD81 protein is highly selective.

3、多肽APQQ与CD81蛋白的亲和力与检出限3. Affinity and detection limit of peptide APQQ and CD81 protein

(1)将含有0.05％Tween-20的10mmol/L磷酸盐缓冲液(pH7.4)、TPE-APQQ和CD81蛋白混合，得到混合体系A；混合体系A中，TPE-APQQ的浓度为1μmol/L，CD81蛋白的浓度为0.2μmol/L、0.4μmol/L、0.6μmol/L、0.8μmol/L、1μmol/L、1.5μmol/L或2.0μmol/L。(1) Mix 10mmol/L phosphate buffer (pH7.4) containing 0.05% Tween-20, TPE-APQQ and CD81 protein to obtain mixed system A; in mixed system A, the concentration of TPE-APQQ is 1 μmol/ L, the concentration of CD81 protein is 0.2μmol/L, 0.4μmol/L, 0.6μmol/L, 0.8μmol/L, 1μmol/L, 1.5μmol/L or 2.0μmol/L.

(3)使用荧光仪对完成步骤(2)的溶液进行荧光扫描，激发波长为320nm，记录发射波长457nm的荧光强度数据，即实验组的荧光强度。(3) Use a fluorometer to perform a fluorescence scan on the solution that has completed step (2). The excitation wavelength is 320nm, and the fluorescence intensity data at the emission wavelength of 457nm is recorded, which is the fluorescence intensity of the experimental group.

(4)按照上述步骤(1)-(3)，将混合体系A替换为混合体系B，其它步骤均不变，得到空白对照的荧光强度。混合体系B由含有0.05％Tween-20的10mmol/L磷酸盐缓冲液(pH7.4)和TPE-APQQ组成；混合体系B中，TPE-APQQ的浓度为1μmol/L。(4) Follow the above steps (1)-(3), replace mixed system A with mixed system B, keep other steps unchanged, and obtain the fluorescence intensity of the blank control. Mixed system B consists of 10 mmol/L phosphate buffer (pH 7.4) containing 0.05% Tween-20 and TPE-APQQ; in mixed system B, the concentration of TPE-APQQ is 1 μmol/L.

(5)计算实验组的相对荧光强度(实验组的相对荧光强度＝(实验组的荧光强度-空白对照的荧光强度)/空白对照的荧光强度)，使用SigmaPlot软件计算平衡解离常数K_D。以相对荧光强度对CD81蛋白浓度进行线性拟合，根据检出限公式LOD＝3N/S(N为空白标准偏差，S为标准曲线斜率)，计算检出限。(5) Calculate the relative fluorescence intensity of the experimental group (relative fluorescence intensity of the experimental group = (fluorescence intensity of the experimental group - fluorescence intensity of the blank control)/fluorescence intensity of the blank control), and use SigmaPlot software to calculate the equilibrium dissociation constant K_D . The relative fluorescence intensity was used to linearly fit the CD81 protein concentration, and the detection limit was calculated according to the detection limit formula LOD=3N/S (N is the blank standard deviation, S is the slope of the standard curve).

结果如图4所示。结果表明，多肽APQQ与CD81蛋白的K_D为514.1±81.0nM，显示出APQQ对CD81蛋白的高亲和力，其检出限为61.1nM，说明多肽APQQ可作为检测CD81蛋白的识别探针。The results are shown in Figure 4. The results show that the K_D of polypeptide APQQ and CD81 protein is 514.1±81.0nM, showing the high affinity of APQQ for CD81 protein, and its detection limit is 61.1nM, indicating that polypeptide APQQ can be used as a recognition probe for detecting CD81 protein.

实施例3、多肽APQQ在活细胞中的CD81蛋白成像分析应用Example 3: Application of peptide APQQ in CD81 protein imaging analysis in living cells

1、FTIC-APQQ偶联物的制备1. Preparation of FTIC-APQQ conjugate

用FMOC固相多肽合成法在Wang树脂上由C端开始直接合成多肽，并在其N端偶联Fmoc-Lys(Dde)-OH，保留N端FMOC保护基，加入Dde脱保护液(Dde脱保护液由咪唑、盐酸羟胺、5mLN-甲基吡咯烷酮和1mL二氯甲烷组成；Dde脱保护液中，咪唑的物质的量为1.35mmol，盐酸羟胺的物质的量为1.80mmol)，室温脱除3h。称取一定量上述树脂，将2倍量FITC和4倍量N,N-二异丙基乙胺溶于DMF溶液中，加入溶胀后的树脂中，室温下避光偶联3h，DMF清洗后加入含有20％哌啶的DMF溶液，脱除N端FMOC保护基，之后使用裂解液(由95％(v/v)三氟乙酸、2.5％(v/v)三异丙基硅烷和2.5％(v/v)水组成)将修饰有FITC的肽链从树脂上裂解，同时去除所有侧链保护基，得到裂解产物。Use FMOC solid-phase peptide synthesis method to directly synthesize peptides starting from the C-terminus on Wang resin, and couple Fmoc-Lys(Dde)-OH to its N-terminus, retain the N-terminal FMOC protective group, and add Dde deprotection solution (Dde deprotection solution). The protective solution consists of imidazole, hydroxylamine hydrochloride, 5mL N-methylpyrrolidone and 1mL dichloromethane; in the Dde deprotection solution, the amount of imidazole is 1.35mmol, and the amount of hydroxylamine hydrochloride is 1.80mmol), and the removal is done at room temperature for 3 hours . Weigh a certain amount of the above resin, dissolve 2 times the amount of FITC and 4 times the amount of N,N-diisopropylethylamine in the DMF solution, add it to the swollen resin, and couple in the dark at room temperature for 3 hours. After cleaning with DMF A DMF solution containing 20% piperidine was added to remove the N-terminal FMOC protecting group, and then a lysis solution (composed of 95% (v/v) trifluoroacetic acid, 2.5% (v/v) triisopropylsilane and 2.5% (v/v) water composition) cleave the peptide chain modified with FITC from the resin and remove all side chain protecting groups to obtain the cleavage product.

将裂解产物用HPLC纯化，所用HPLC纯化的参数如下：色谱柱为Diamonsil C18(2)250×4.6mm；流动相A为添加有0.1％(v/v)三氟乙酸的水溶液，流动相B为添加有0.1％(v/v)三氟乙酸的乙腈溶液，在25min内，流动相B百分比从28％上升至35％进行洗脱。收集对应目标物馏分，冻干后得到纯度高且结构正确的FITC-APQQ。The cleavage product was purified by HPLC. The parameters used for HPLC purification were as follows: the chromatographic column was Diamonsil C18(2) 250×4.6mm; the mobile phase A was an aqueous solution with 0.1% (v/v) trifluoroacetic acid added, and the mobile phase B was A solution of acetonitrile with 0.1% (v/v) trifluoroacetic acid was added, and the percentage of mobile phase B increased from 28% to 35% for elution within 25 min. Collect the fractions corresponding to the target substance and obtain FITC-APQQ with high purity and correct structure after freeze-drying.

2、免疫荧光染色结合流式细胞术分析细胞中CD81蛋白表达量2. Immunofluorescence staining combined with flow cytometry to analyze CD81 protein expression in cells

(1)分别将人乳腺癌细胞MCF-7、人肝癌细胞Huh-7和人胶质瘤细胞U251用含10％胎牛血清和1％双抗(由10000Units/mL的青霉素和10000μg/mL的链霉素组成)的DMEM培养基培养，达到所需密度后，使用0.25％胰蛋白酶溶液消化，依次得到MCF-7悬浮细胞、Huh-7悬浮细胞和U251悬浮细胞。将人肝癌细胞Hep3B用含10％胎牛血清和1％双抗(由10000Units/mL的青霉素和10000μg/mL的链霉素组成)的RPMI 1640培养基培养，达到所需密度后，使用0.25％胰蛋白酶溶液消化，得到Hep3B悬浮细胞。(1) Human breast cancer cell MCF-7, human liver cancer cell Huh-7 and human glioma cell U251 were treated with 10% fetal calf serum and 1% double antibody (composed of 10000 Units/mL penicillin and 10000 μg/mL After reaching the required density, it was digested with 0.25% trypsin solution to obtain MCF-7 suspension cells, Huh-7 suspension cells and U251 suspension cells in sequence. Human liver cancer cell Hep3B was cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% double antibody (composed of 10000 Units/mL penicillin and 10000 μg/mL streptomycin). After reaching the required density, use 0.25% Digest with trypsin solution to obtain Hep3B suspension cells.

(2)将1×10⁶个悬浮细胞(MCF-7悬浮细胞、Huh-7悬浮细胞、U251悬浮细胞或Hep3B悬浮细胞)置于离心管中，加入封闭液(由牛血清白蛋白和PBS组成，牛血清白蛋白的质量浓度为5％)，室温下封闭1h。(2) Place 1×10⁶ suspension cells (MCF-7 suspension cells, Huh-7 suspension cells, U251 suspension cells or Hep3B suspension cells) in a centrifuge tube, and add blocking solution (composed of bovine serum albumin and PBS , the mass concentration of bovine serum albumin is 5%), and blocked for 1 h at room temperature.

(3)完成步骤(2)后，弃溶液，加入PBS清洗一次后，加入CD81抗体溶液，室温下孵育1h；CD81抗体溶液由CD81抗体(Abcam,货号ab219209，英国)、牛血清白蛋白和PBS组成；CD81抗体稀释比例为1:500，牛血清白蛋白的质量浓度为0.5％；(3) After completing step (2), discard the solution, add PBS to wash once, add CD81 antibody solution, and incubate at room temperature for 1 hour; the CD81 antibody solution is composed of CD81 antibody (Abcam, Cat. No. ab219209, UK), bovine serum albumin and PBS Composition; CD81 antibody dilution ratio is 1:500, and the mass concentration of bovine serum albumin is 0.5%;

(4)完成步骤(3)后，弃溶液，加入PBS清洗一次，加入二抗溶液，室温下孵育1h；二抗溶液由Alexa Fluor 488标记山羊抗兔IgG抗体(Abcam，货号ab150077，英国)、牛血清白蛋白和PBS组成；二抗稀释比例为1:2000，牛血清白蛋白的质量浓度为0.5％。(4) After completing step (3), discard the solution, add PBS to wash once, add secondary antibody solution, and incubate at room temperature for 1 hour; the secondary antibody solution consists of Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (Abcam, Cat. No. ab150077, UK), Composed of bovine serum albumin and PBS; the secondary antibody dilution ratio is 1:2000, and the mass concentration of bovine serum albumin is 0.5%.

(5)完成步骤(4)后，弃溶液，加入PBS清洗，将细胞重新分散于PBS中，使用流式细胞仪(BD，美国)进行分析，使用FlowJo软件进行数据处理。(5) After completing step (4), discard the solution, add PBS for washing, redisperse the cells in PBS, analyze using a flow cytometer (BD, USA), and use FlowJo software for data processing.

结果如图5中a)所示。结果表明，人乳腺癌细胞MCF-7、人肝癌细胞Huh-7和人胶质瘤细胞U251中均有CD81蛋白表达，为CD81阳性细胞；人肝癌细胞Hep3B中没有CD81蛋白表达，为CD81阴性细胞。The results are shown in Figure 5a). The results showed that CD81 protein was expressed in human breast cancer cells MCF-7, human liver cancer cells Huh-7 and human glioma cells U251, which were CD81 positive cells; CD81 protein was not expressed in human liver cancer cells Hep3B, which were CD81 negative cells.

3、活细胞中CD81蛋白的原位成像分析3. In situ imaging analysis of CD81 protein in living cells

(1)以约1×10⁶个/皿的密度将癌细胞(人乳腺癌细胞MCF-7、人肝癌细胞Huh-7、人胶质瘤细胞U251或人肝癌细胞Hep3B)种于共聚焦皿(Φ＝15mm)中，37℃、5％CO₂条件下培养过夜，使其贴壁。(1) Seed cancer cells (human breast cancer cell MCF-7, human liver cancer cell Huh-7, human glioma cell U251 or human liver cancer cell Hep3B) on the confocal dish at a density of approximately 1×10⁶ cells/dish. (Φ=15mm), culture overnight at 37°C and 5%_CO2 to allow it to adhere to the wall.

(2)完成步骤(1)后，取装有癌细胞的共聚焦皿，分为组Ⅰ(FITC-APQQ组)和组Ⅱ(EL1+FITC-APQQ组)，进行如下操作：(2) After completing step (1), take the confocal dishes containing cancer cells and divide them into group I (FITC-APQQ group) and group II (EL1+FITC-APQQ group), and perform the following operations:

组Ⅰ：弃去共聚焦皿原本的培养液，然后加入FITC-APQQ溶液(用DMEM培养基稀释)并使FITC-APQQ在体系中的浓度为25μmol/L，之后37℃、5％CO₂避光孵育1h，弃溶液，用PBS清洗一次。Group I: Discard the original culture medium of the confocal dish, then add FITC-APQQ solution (diluted with DMEM culture medium) to make the concentration of FITC-APQQ in the system 25 μmol/L, and then incubate at 37°C and 5%_CO2. Incubate with light for 1 hour, discard the solution, and wash once with PBS.

组Ⅱ：弃去共聚焦皿原本的培养液，然后加入FITC-APQQ+EL1混合液(由FITC-APQQ溶液(用DMEM培养基稀释)和EL1溶液(用DMEM培养基稀释)混合而成)并使FITC-APQQ和EL1在体系中的浓度均为25μmol/L，之后37℃、5％CO₂避光孵育1h，弃溶液，用PBS清洗一次。Group II: Discard the original culture medium of the confocal dish, then add FITC-APQQ+EL1 mixture (mixed from FITC-APQQ solution (diluted with DMEM culture medium) and EL1 solution (diluted with DMEM culture medium)) and The concentrations of FITC-APQQ and EL1 in the system were both 25 μmol/L, and then incubated at 37°C and 5%_CO2 in the dark for 1 hour. The solution was discarded and washed once with PBS.

(3)用激光扫描共聚焦显微镜(Olympus FV1000-IX81，日本)检测细胞中的荧光分布。(3) Use a laser scanning confocal microscope (Olympus FV1000-IX81, Japan) to detect the fluorescence distribution in cells.

结果如图5中b)所示。对于CD81阳性的人乳腺癌细胞MCF-7、人肝癌细胞Huh-7和人胶质瘤细胞U251，加入FITC-APQQ后，细胞膜上存在着明显的绿色荧光，与CD81蛋白的膜定位分布一致。对于CD81阴性的人肝癌细胞Hep3B，视野中无明显绿色荧光存在。由此可见，FITC-APQQ与CD81蛋白具有特异性识别，且信号响应强度与CD81蛋白表达量正相关。而将FITC-APQQ与EL1预先混合后，加入各组细胞中孵育1h，细胞中几乎观察不到绿色荧光。这是由于FITC-APQQ优先结合溶液中游离的EL1片段，阻断了与细胞中CD81蛋白上的EL1位点的结合。FITC-APQQ与EL1之间的这种竞争性关系也验证了APQQ对细胞中EL1位点的靶向识别。The results are shown in b) in Figure 5. For CD81-positive human breast cancer cells MCF-7, human liver cancer cells Huh-7 and human glioma cells U251, after adding FITC-APQQ, there was obvious green fluorescence on the cell membrane, which was consistent with the membrane localization distribution of CD81 protein. For the CD81-negative human liver cancer cell Hep3B, there is no obvious green fluorescence in the field of view. It can be seen that FITC-APQQ specifically recognizes CD81 protein, and the signal response intensity is positively correlated with the expression level of CD81 protein. However, after premixing FITC-APQQ and EL1, they were added to cells in each group and incubated for 1 hour, and almost no green fluorescence was observed in the cells. This is because FITC-APQQ preferentially binds to free EL1 fragments in the solution, blocking the binding to the EL1 site on the CD81 protein in cells. This competitive relationship between FITC-APQQ and EL1 also verifies APQQ's targeted recognition of the EL1 site in cells.

实施例4、多肽APQQ通过靶向结合CD81蛋白抑制细胞迁移Example 4. Polypeptide APQQ inhibits cell migration through targeted binding to CD81 protein.

1、伤口愈合实验1. Wound healing experiment

(1)在六孔板中以5×10⁴个/孔的密度将癌细胞(人乳腺癌细胞MCF-7、人肝癌细胞Huh-7、人胶质瘤细胞U251或人肝癌细胞Hep3B)植入伤口愈合插件(Ibidi，德国)中，在37℃、5％CO₂条件下培养至100％汇合度。(1) Plant cancer cells (human breast cancer cell MCF-7, human liver cancer cell Huh-7, human glioma cell U251 or human liver cancer cell Hep3B) in a six-well plate at a density of 5×10⁴ cells/well. into wound healing inserts (Ibidi, Germany) and cultured to 100% confluence at 37°C, 5%_CO2 .

(2)完成步骤(1)后，用镊子将插件小心移除，留下宽度为500μm的划痕区域。(2) After completing step (1), carefully remove the insert with tweezers, leaving a scratch area with a width of 500 μm.

(3)完成步骤(2)后，弃去培养液，用PBS缓冲液小心清洗掉落细胞，之后加入待测溶液至总体积为1mL。(3) After completing step (2), discard the culture medium, carefully wash the fallen cells with PBS buffer, and then add the test solution to a total volume of 1 mL.

待测溶液为浓度为10μmol/L的APQQ溶液(用DMEM培养基稀释)(作为实验组)、浓度为10μmol/L的蜂毒明肽(Apamin)溶液(用DMEM培养基稀释)(作为阳性组)或DMEM培养基(作为空白组)。The solutions to be tested are APQQ solution (diluted with DMEM medium) with a concentration of 10 μmol/L (as the experimental group), and Apamin solution (diluted with DMEM medium) with a concentration of 10 μmol/L (as the positive group). ) or DMEM medium (as a blank group).

(4)完成步骤(3)后，在37℃、5％CO₂条件下继续孵育24h，分别在0h和24h时使用相机拍摄划痕区域，通过image J软件统计划痕区愈合面积。以实验组愈合面积与空白组愈合面积之比衡量细胞的迁移能力。(4) After completing step (3), continue to incubate for 24 hours at 37°C and 5%_CO2 . Use a camera to take pictures of the scratched area at 0h and 24h respectively, and use image J software to calculate the healing area of the scratched area. The migration ability of cells was measured by the ratio of the healing area of the experimental group to the healing area of the blank group.

实验结果见图6中a)和b)(Control为空白组)。结果表明，APQQ通过靶向结合CD81蛋白，能够选择性抑制人乳腺癌细胞MCF-7、人肝癌细胞Huh-7和人胶质瘤细胞U251的迁移，三种细胞的迁移率分别降至47.4％,31.1％和37.1％，与细胞迁移抑制剂蜂毒明肽的抑制水平相当。而对于CD81阴性的人肝癌细胞Hep3B，APQQ难以抑制其迁移过程。The experimental results are shown in a) and b) in Figure 6 (Control is the blank group). The results show that APQQ can selectively inhibit the migration of human breast cancer cells MCF-7, human liver cancer cells Huh-7 and human glioma cells U251 by targeting the CD81 protein. The migration rates of the three cells were reduced to 47.4% respectively. , 31.1% and 37.1%, which are comparable to the inhibition levels of melittin, a cell migration inhibitor. For CD81-negative human liver cancer cell Hep3B, APQQ is difficult to inhibit its migration process.

2、竞争性结合实验2. Competitive binding experiment

(1)在六孔板中以5×10⁴个/孔的密度将癌细胞(人乳腺癌细胞MCF-7、人肝癌细胞Huh-7或人胶质瘤细胞U251)植入伤口愈合插件(Ibidi，德国)中，在37℃、5％CO₂条件下培养至100％汇合度。(1)^Implant cancer cells (human breast cancer cells MCF-7, human liver cancer cells Huh-7 or human glioma cells U251) into a wound healing plug-in ( Ibidi, Germany) and cultured to 100% confluence at 37°C, 5%_CO2 .

(3)完成步骤(2)后，弃去培养液，用PBS缓冲液小心清洗掉落细胞，之后加入待测溶液至总体积为1mL。(3) After completing step (2), discard the culture medium, carefully wash the fallen cells with PBS buffer, and then add the solution to be tested until the total volume is 1 mL.

待测溶液为浓度为10μmol/L的APQQ溶液(用DMEM培养基稀释)(作为APQQ实验组)、混合溶液(作为APQQ+EL1实验组)或DMEM培养基(作为空白组)。混合溶液由APQQ、EL1和DMEM培养基组成；混合溶液中APQQ的浓度为10μmol/L，EL1的浓度为20μmol/L。The solution to be tested was an APQQ solution (diluted with DMEM culture medium) with a concentration of 10 μmol/L (as the APQQ experimental group), a mixed solution (as the APQQ+EL1 experimental group) or DMEM culture medium (as the blank group). The mixed solution consists of APQQ, EL1 and DMEM medium; the concentration of APQQ in the mixed solution is 10 μmol/L, and the concentration of EL1 is 20 μmol/L.

实验结果见图6中c)和d)(Control为空白组)。结果表明，当加入靶点EL1后，APQQ优先结合溶液中的EL1，阻断了与细胞中CD81蛋白上EL1位点的结合，细胞迁移能力得以恢复。The experimental results are shown in c) and d) in Figure 6 (Control is the blank group). The results show that when the target EL1 is added, APQQ preferentially binds to EL1 in the solution, blocking the binding to the EL1 site on the CD81 protein in cells, and the cell migration ability is restored.

由此可见，APQQ可作为CD81蛋白的抑制剂，其通过靶向结合CD81蛋白的胞外小环片段EL1，从而选择性抑制其在细胞迁移过程中的作用。It can be seen that APQQ can be used as an inhibitor of CD81 protein by targeting the extracellular small ring fragment EL1 that binds to CD81 protein, thereby selectively inhibiting its role in the cell migration process.

以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.