A short peptide specifically binding to cytokeratin 19 fragment and its use

Abstract

The present invention belongs to the technical field of protein short peptides, and discloses a short peptide specifically binding to a cytokeratin 19 fragment and its use. The cytokeratin 19 fragment is a recognized tumor marker with high specificity and sensitivity, and plays an important role in the early diagnosis, efficacy monitoring and prognosis judgment of non-small cell lung cancer. The present invention relates to a method for obtaining a cytokeratin 19 fragment short peptide Y10 by phage display technology. The amino acid sequence of the short peptide Y10 is shown in SEQ ID NO.1. The specific short peptide screened by the present invention has the characteristics of small relative molecular mass and high affinity, so that a probe can be designed for detecting the cytokeratin 19 fragment, which provides a new direction for the early diagnosis of tumors and the research and development of targeted drugs in clinical practice.

Description

Technical Field

[0001] The invention belongs to the technical field of proteins and relates to a cytokeratin 19 fragment-specific binding short peptide and a use thereof. Background Art

[0002] Cytokeratin 19 fragment is a soluble protein. When tumor cells are destroyed or apoptotic, it will be released into human body fluids. Therefore, its content is directly related to the level of tumor load in the body. It mainly comes from the cytoplasm of tumor cells of epithelial cell origin such as lung cancer and esophageal cancer. Cytokeratin 19 fragment is the preferred marker for non-small cell lung cancer, especially the preferred tumor marker for squamous cell carcinoma, with a sensitivity of 60% and a specificity of 95%.

[0003] Cytokeratin 19 fragments are recognized as tumor markers with high specificity and sensitivity, and play an important role in the early diagnosis, efficacy monitoring and prognosis judgment of non-small cell lung cancer. The current technical means for detecting cytokeratin 19 fragments mainly rely on enzyme-linked immunosorbent assay, chemiluminescence and radioimmunoassay detection methods. These detection methods generally have the disadvantages of poor specificity, false positives, complex operations, and long detection time. Therefore, it is necessary to design a highly sensitive and specific method to detect cytokeratin 19 fragments. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a short peptide that can specifically recognize a cytokeratin 19 fragment, that is, a short peptide sequence that can specifically bind to a cytokeratin 19 fragment.

[0005] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0006] A carcinoembryonic antigen-specific binding short peptide is named Y10, and its amino acid sequence is shown in SEQ ID NO.1: QSYFNACWSCNH.

[0007] The present invention uses phage display technology to target cytokeratin 19 fragment protein and performs "elution-binding-amplification" to obtain a short peptide Y10 that can specifically bind to the cytokeratin 19 fragment. The molecular interaction instrument (Fortebio) technology can be used to quantitatively verify the binding force between the short peptide Y10 and the cytokeratin 19 fragment.

[0008] The cytokeratin 19 fragment-specific binding short peptide can be used for designing probes for detecting cytokeratin 19 fragments and developing targeted therapeutic drugs.

[0009] Beneficial effects of the present invention:

[0010] The present invention utilizes phage display technology to screen short peptides that specifically bind to cytokeratin 19 fragments, and identifies that the short peptides have high specificity and affinity. At the same time, the short peptides also have the advantages of small relative molecular mass, high affinity and specificity, and play an important role in the early diagnosis, screening and targeted treatment of tumor marker cytokeratin 19 fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the experimental flow chart for screening cytokeratin 19 fragments using the M13 phage random short peptide library.

[0012] Figure 2 This is the ELISA test result of phage binding clone and keratin fragment 19.

[0013] Figure 3 This is a schematic diagram of the binding activity of short peptides with cytokeratin 19 fragments. DETAILED DESCRIPTION

[0014] The present invention is further illustrated by examples below in conjunction with the accompanying drawings, but is not intended to limit the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, and materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0015] Example 1: Three rounds of panning of cytokeratin 19 fragments using phage display technology

[0016] Bio-panning of binding peptides, flow chart see Figure 1 .

[0017] Target molecule fixation: Coat the 96-well ELISA plate with cytokeratin 19 fragment protein solution (100 μg / mL) at 4°C overnight, add 200 μl blocking solution, incubate at 4°C for 3-4 hours, and then wash the plate 6 times with TBST (0.1% Tween-20) buffer;

[0018] Binding of target molecules to peptide library: dilute phages with TBST buffer, 150 μl / well (containing 2×10 8 pfu phage), gently shake at room temperature for 60 minutes, pour out to remove unbound phage, wash the plate 6 times with TBST (0.1% Tween-20) buffer, add 100 μl of elution buffer, gently shake at room temperature for 20 minutes, then add 15 μl of neutralization solution and add glycerol to store at -20°C.

[0019] Phage amplification and purification: 50 μl of eluted and neutralized phage was added to 20 mL of ER2738 in logarithmic growth period, cultured at 37°C 270 rpm for 4.5 hours, and centrifuged to keep the supernatant. 1 / 6 volume of PEG / NaCl was added to the supernatant, precipitated at 4°C overnight, centrifuged, and the precipitate was resuspended in 200 μl TBS, 0.02% NaN3 in TBS, which was the eluate after amplification.

[0020] The first step of bio-panning of binders, the second step of binding of target molecules to peptide library and the last step of amplification and purification of phages were repeated for the next two rounds of panning.

[0021] Determination of phage single-stranded DNA sequence: After three rounds of panning, the phage eluted in the last round was infected with the host bacteria and then spread on the LB / IPTG / Xgal plate and cultured overnight. The phage blue plaques grew, picked the blue plaques and amplified the monoclonal phage. Take 500μl of the above-amplified phage, centrifuge, add 200μl PEG / NaCl to the supernatant, leave it at room temperature for 10 minutes, and centrifuge. The precipitate was thoroughly resuspended in 100μl iodide buffer, 250μl ethanol was added, and incubated at room temperature for 10 minutes. The precipitate was washed with 70% ethanol, air-dried at room temperature, and the precipitate was resuspended in 30μl TE buffer, which is the phage single-stranded DNA. Using the specific downstream primer M13-96 gⅢ primer provided by the kit, the short peptide Y10 was obtained, and its amino acid sequence is as shown in SEQ ID NO.1.

[0022] Example 2 ELISA method to detect the specific binding of short peptides to cytokeratin 19 fragments

[0023] After coating with cytokeratin 19 fragment protein solution (100 μg / mL) at 4°C overnight, add 200 μL blocking solution, let it bind at room temperature for 1 hour, and wash it 3 times with TBST (0.5% Tween-20). Add 100 μL phage monoclonal amplification product (Y10), let it bind at room temperature for 1 hour, and then wash it 3 times with TBST (0.5% Tween-20). Add 100 μL / well of M13 monoclonal antibody (1:5,000 dilution) and let it bind at room temperature for 1 hour. Wash it 3 times with TBST (0.5% Tween-20). Add 100 μL / well of HRP-labeled goat anti-rabbit IgG (1:5,000 dilution), let it bind at room temperature for 1 hour, and wash it 3 times with TBST (0.5% Tween-20). Add 100 μL / well of colorimetric solution with TMB as substrate, react for 5-10 min in the dark, and add 50 μL / well of stop solution (2 mol / L H2SO4).

[0024] Determine the OD value, the result is as follows Figure 2 As shown, add phage monoclonal amplification product (Y10) OD 450 Compared with the control group without phage addition, OD450 The results show that the short peptide Y10 can effectively bind to the cytokeratin 19 fragment specifically. In order to further quantitatively study the binding strength of the short peptide Y10 and the cytokeratin 19 fragment, the Fortebio bio-layer interferometry technology was used in Example 3 to detect the binding force between the short peptide Y10 and the cytokeratin 19 fragment.

[0025] Example 3: Determination of the binding force between short peptide Y10 and cytokeratin 19 fragment using Fortebio biolayer interferometry

[0026] (1) Pretreatment of AR2G sensor: unused AR2G sensor was placed in 200 μL mixed solution (20 mM EDC and 10 mM sulfo-NHS) for 1 h;

[0027] (2) Immobilization of target protein: The cytokeratin 19 fragment is immobilized on the sensor surface in the form of a covalent bond. Through the EDC / NHS esterification reaction, the amino group on the protein and the carboxyl group on the sensor surface form an amide bond;

[0028] (3) Equilibration: The sensor with the immobilized target protein is equilibrated in water;

[0029] (4) Quenching: The sensor with the target protein equilibrated in water is activated in ethanolamine;

[0030] (5) Binding: Immersing the sensor with the target protein into the analyte;

[0031] (6) Dissociation: Immerse the sensor in water to dissociate the small molecule analyte bound to the target protein;

[0032] (7) Data collection: ForteBio Data Acquisition software is used to collect molecular interaction data in real time;

[0033] (8) Data analysis: The real-time collected data were analyzed using ForteBio Data Analysis software to calculate the phase shift intensity (Response, nm). The response values ​​corresponding to different concentrations in Table 1 were plotted as follows: Figure 3 , the response value of the short peptide Y10 to the cytokeratin 19 fragment at a concentration of 500-2000 nM, and the K D =1.60E±6.1E-08M(R 2 =0.999493949114857). The results showed that the short peptide Y10 interacted with the cytokeratin 19 fragment and the binding ability was strong.

[0034] Table 1

[0035]

Claims

1. A cytokeratin 19 fragment-specific binding short peptide, characterized in that: The amino acid sequence of the cytokeratin 19 fragment-specific binding short peptide is shown in SEQ ID NO.

1.

2. Use of a cytokeratin 19 fragment-specific binding short peptide as claimed in claim 1 in preparing a cytokeratin 19 fragment detection probe.

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