Specific binding of a gastrin releasing peptide precursor to a short peptide and uses thereof

Abstract

The present application belongs to the technical field of protein short peptide, and discloses a gastrin releasing peptide precursor specific binding short peptide and application. The gastrin releasing peptide precursor is a tumor marker with high specificity and sensitivity, and plays an important role in early diagnosis, curative effect monitoring and prognosis judgment of non-small cell lung cancer. The present application obtains a gastrin releasing peptide precursor short peptide P2 through phage display technology, and the amino acid sequence is shown as SEQ ID NO. 1. The specific short peptide screened by the present application has the characteristics of small relative molecular mass and high affinity, and thus a probe can be designed for detecting the gastrin releasing peptide precursor, thereby providing a new direction for early diagnosis of tumors and research and development of targeted drugs in clinic.

Description

Technical Field

[0001] The invention belongs to the technical field of proteins and relates to a gastrin-releasing peptide precursor-specific binding short peptide and its application. Background Art

[0002] Progastrin-releasing peptide (PGRP) is an important regulatory molecule involved in many physiological functions and pathological conditions in the human body. It can promote the growth of small cell lung cancer (SCLC) and indirectly reflect SCLC tumor burden. The rate of change in serum PGRP levels before and after chemotherapy in patients with different stages of SCLC has value in evaluating the efficacy of tumor control and predicting prognosis and survival. PGRP is a recognized tumor marker with high specificity and sensitivity, playing a crucial role in the early diagnosis, efficacy monitoring, and prognosis of non-small cell lung cancer (NSCLC).

[0003] Progastrin-releasing peptide (PGP) is easily detected in peripheral blood, and its levels indirectly reflect its expression. It is considered a good biomarker for the diagnosis, treatment response monitoring, and prognosis of small cell lung cancer. Current methods for detecting PGP rely primarily on enzyme-linked immunosorbent assays (ELISAs), chemiluminescence, and radioimmunoassays (RIAs). These methods suffer from poor specificity, false positives, and complex procedures, resulting in lengthy testing times. Therefore, a highly sensitive and specific method for detecting PGP is needed. 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 gastrin-releasing peptide precursor, which is a short peptide sequence that can specifically bind to gastrin-releasing peptide precursor.

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

[0006] A gastrin-releasing peptide precursor-specific binding short peptide is named P2, and its amino acid sequence is shown in SEQ ID NO. 1: KIWLIPGGDVLA.

[0007] The above-mentioned method for screening short peptides that specifically bind to gastrin-releasing peptide precursor uses phage display technology to target gastrin-releasing peptide precursor protein and performs "elution-binding-amplification" to obtain short peptide P2 that can specifically bind to gastrin-releasing peptide precursor.

[0008] The gastrin-releasing peptide precursor-specific binding short peptide is used in preparing a probe for detecting the gastrin-releasing peptide precursor and detecting a gastrin-releasing peptide precursor targeted therapeutic drug.

[0009] Beneficial effects of the present invention:

[0010] The present invention utilizes phage display technology to screen short peptides that specifically bind to gastrin-releasing peptide precursor, and identifies them as having high specificity and affinity. At the same time, the short peptide also has the advantages of small relative molecular mass, high affinity and specificity, and plays an important role in the early diagnosis, screening and targeted treatment of the tumor marker gastrin-releasing peptide precursor. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Experimental workflow for screening gastrin-releasing peptide precursors from an M13 phage random short peptide library.

[0012] Figure 2 This is the ELISA test result of phage binding clone and gastrin-releasing peptide precursor.

[0013] Figure 3 Schematic diagram of the binding activity of short peptides with gastrin-releasing peptide precursor. 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. 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 be obtained from commercial sources.

[0015] Example 1 Three rounds of panning of gastrin-releasing peptide precursor using phage display technology

[0016] Biopanning of binding peptides, see the flow chart Figure 1 , as follows:

[0017] Target molecule immobilization: Gastrin-releasing peptide precursor protein solution (100 μg / mL) was coated on a 96-well enzyme-linked microplate. After overnight coating at 4°C, 200 μl of blocking solution was added and incubated at 4°C for 3-4 hours. The plate was then washed six times with TBST (0.1% Tween-20) buffer.

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

[0019] Phage amplification and purification: Add 50 μl of eluted, neutralized phage to 20 mL of ER2738 in logarithmic growth. Incubate at 37°C, 270 rpm, and retain the supernatant. Add 1 / 6 volume of PEG / NaCl to the supernatant, precipitate overnight at 4°C, and centrifuge. Resuspend the pellet in 200 μl of TBS containing 0.02% NaN₃ (TBS) to obtain the amplified eluate.

[0020] Repeat 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 for the next two rounds of panning.

[0021] Determination of phage single-stranded DNA sequence: After three rounds of panning, phage eluted from the final round were infecting host bacteria and plated onto LB / IPTG / Xgal plates for overnight incubation. Blue plaques of phage were grown, picked, and monoclonal phage amplification was performed. 500 μl of the amplified phage was centrifuged, and 200 μl of PEG / NaCl was added to the supernatant. The supernatant was allowed to stand at room temperature for 10 minutes and then centrifuged. The precipitate was thoroughly resuspended in 100 μl of iodide buffer, 250 μl of ethanol was added, and the precipitate was incubated at room temperature for 10 minutes. The precipitate was washed with 70% ethanol, air-dried at room temperature, and resuspended in 30 μl of TE buffer, which was the phage single-stranded DNA. Using the specific downstream primer M13-96 gⅢ provided in the kit, the short peptide P2 was obtained, whose amino acid sequence is shown in SEQ ID NO. 1.

[0022] Example 2 ELISA method to detect the specific binding of short peptides to gastrin-releasing peptide precursor

[0023] After coating overnight at 4°C with a 100 μg / mL solution of gastrin-releasing peptide precursor protein, 200 μL of blocking buffer was added and allowed to bind at room temperature for 1 hour. The cells were then washed three times with TBST (0.5% Tween-20). Then, 100 μL of phage monoclonal amplicon (P2) was added and allowed to bind at room temperature for 1 hour. The cells were then washed three times with TBST (0.5% Tween-20). Then, 100 μL / well of M13 monoclonal antibody (1:5,000 dilution) was added and allowed to bind at room temperature for 1 hour. The cells were then washed three times with TBST (0.5% Tween-20). Then, 100 μL / well of HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution) was added and allowed to bind at room temperature for 1 hour. The cells were then washed three times with TBST (0.5% Tween-20). Add 100 μL / well of color development solution with TMB as substrate, react for 5-10 minutes 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 (P2) OD 450 Compared with the control group without phage OD450 Compared with the results of the previous study, the peptide P2 was significantly enhanced. This preliminary study shows that the short peptide P2 can specifically and effectively bind to the pro-gastrin-releasing peptide. To further quantitatively study the binding strength of the short peptide P2 and the pro-gastrin-releasing peptide, the Fortebio (molecular interaction instrument) bio-layer interferometry technique was used to detect the binding force between the short peptide P2 and the pro-gastrin-releasing peptide.

[0025] Example 3 Fortebio biolayer interferometry determination

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

[0027] (2) Immobilization of the target protein: The gastrin-releasing peptide precursor is covalently fixed to the sensor surface. Through the EDC / NHS esterification reaction, the amino groups on the protein and the carboxyl groups on the sensor surface form amide bonds;

[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 was 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 short peptide P2 to gastrin-releasing peptide precursor at concentrations of 781.5-3125nM, and the K D =9.10E-7±6.2E-09M(R 2 =0.999954487689176). The results showed that the short peptide P2 interacted with the gastrin-releasing peptide precursor and the binding ability was strong.

[0034] Table 1

[0035]

Claims

1. A gastrin-releasing peptide precursor-specific binding short peptide, characterized in that: The amino acid sequence of the gastrin-releasing peptide precursor specifically binding to the short peptide is shown in SEQ ID NO.

1.

2. Use of a gastrin-releasing peptide precursor-specific binding short peptide as claimed in claim 1 in preparing a probe for detecting gastrin-releasing peptide precursor.

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