A probe for detecting and quantifying membrane surface proteins and its use method and application

Abstract

The present invention discloses a probe for detecting and quantifying membrane surface proteins, which includes a detection probe and a counting probe; the detection probe targets membrane characteristic proteins and membrane surface proteins that play a role in the occurrence and development of tumors. The method of use is as follows: when the detection probe and the counting probe are simultaneously bound to the membrane, the complementary regions bind, and under the action of the enzyme, the two chains are extended and complemented into a double chain; the extended product is then amplified by PCR, and the target band is purified and recovered and sent to NGS sequencing; after obtaining the sequencing results, the data is subjected to bioinformatics analysis, and clean data is obtained by screening and filtering the original data; then it is compared with the reference sequence, the data on the comparison is captured to extract information, and a protein counting matrix is ​​generated to achieve accurate quantification of multiple membrane surface proteins. The present invention can detect multiple membrane surface proteins simultaneously in a one-pot method, without the need for a cleaning step in the middle. The most important thing is that the surface protein detection method designed by the present invention does not rely on antibodies and can achieve accurate quantification.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a probe for detecting and quantifying membrane surface proteins, and a use method and application thereof. Background Art

[0002] Living organisms are rich in membrane structures. Cells, organelles, extracellular vesicles secreted by cells, and apoptotic bodies all possess membrane structures. Membranes are composed of a lipid bilayer, with the phospholipids at the head facing the aqueous environment, while the hydrophobic substances at the tail and other parts of the membrane are close together to form the interior. Proteins and carbohydrates are both important components of biological membranes and play important roles. Membrane surface proteins have many important functions, including mediating cell-to-cell recognition, adhesion, and interaction; acting as receptors for extracellular signaling molecules to trigger specific signal transduction pathways; forming channels or carriers to regulate the transport of substances across the membrane; and interacting with the cytoskeleton or other membrane proteins to provide structural support and stability to the cell membrane. The composition and abundance of membrane surface proteins can reflect cell type and state, and therefore the detection and quantification of surface proteins can be used as a tool for disease diagnosis.

[0003] Currently, the main methods for detecting membrane surface proteins are mass spectrometry, fluorescence spectroscopy, flow cytometry, microfluidic chips, Raman spectroscopy, and sequencing based on antibody recognition, but these methods all have their limitations. Mass spectrometry requires the cleavage of the analyte, and both experimental operations and data analysis require professional personnel, and a mass spectrometer is also required; the signal obtained by fluorescence spectroscopy is relatively rough, and due to the limitation of fluorescence wavelength, cross-talk is prone to occur and it is impossible to detect multiple proteins simultaneously; flow cytometry requires complex pre-processing and is limited by the resolution of the instrument; the design and preparation of microfluidic chips usually require precise microfabrication technology, special operations and equipment, and the detection method is usually based on antibody-antigen recognition, with limited specificity and sensitivity; Raman spectroscopy is a spectroscopic technology based on molecular vibrations, which is prone to mixed signals. The interpretation and analysis of spectral data may require special technology and experience; sequencing technology based on antibody recognition requires modifying DNA chains on antibodies for subsequent PCR amplification sequencing. There are certain technical difficulties in controlling only one DNA chain to modify a metabolite, and it is expensive.

[0004] The above detection methods are unable to achieve accurate quantification. Therefore, it is necessary to design a probe for detecting and quantifying membrane surface proteins and its use method and application. Summary of the Invention

[0005] In order to overcome the defects in the prior art, a probe for detecting and quantifying membrane surface proteins and a method for using and applying the probe are provided.

[0006] The present invention is achieved through the following solutions:

[0007] A probe for detecting and quantifying membrane surface proteins comprises a detection probe and a counting probe; the detection probe targets membrane characteristic proteins and membrane surface proteins that play a role in the occurrence and development of tumors.

[0008] The detection probe consists of five parts. The first part is the nucleic acid aptamer, which is used to recognize the surface protein; the second part is the PCR primer binding domain, which is used for subsequent PCR amplification; the third part is the molecular encoding part of the detection probe, and one UMI represents one detection probe; the fourth part is the nucleic acid aptamer tag, which indicates the type of aptamer and the type of surface protein; the fifth part is a 5nt complementary region that can complementarily bind to the 3' end of the counting probe for subsequent extension.

[0009] The counting probe consists of three parts: the first part is 5' modified cholesterol, which is used to target the lipid bilayer of the membrane; the second part is the primer binding domain of the PCR reaction, which is also used for subsequent PCR amplification; and the third part is a complementary sequence that binds to the 3' end of the detection probe.

[0010] A method for using a probe for detecting and quantifying membrane surface proteins, the method comprising the following steps: the probe comprises a detection probe and a counting probe, when the detection probe and the counting probe are simultaneously bound to the membrane, the complementary regions bind, and under the action of an enzyme, the two chains are extended to complement each other into a double chain;

[0011] The extended product is then amplified by PCR, and the target band is purified and recovered for NGS sequencing;

[0012] After obtaining the sequencing results, the data is subjected to bioinformatics analysis, and clean data is obtained by filtering the raw data;

[0013] Then compare it with the reference sequence, extract information from the data in the comparison, generate a protein count matrix, and achieve accurate quantification of multiple membrane surface proteins.

[0014] The enzyme is T4 DNA polymerase.

[0015] The target band was recovered by PAGE gel purification.

[0016] The detection probe can target membrane characteristic proteins and membrane surface proteins that play a role in the occurrence and development of tumors.

[0017] The invention discloses an application of a probe for detecting and quantifying membrane surface proteins. The probe is used for detecting and quantifying membrane surface proteins.

[0018] The probe is used for in situ detection and quantification of membrane surface proteins.

[0019] The probe is used for in situ detection and quantification of membrane characteristic proteins and a variety of membrane surface proteins that play a role in the occurrence and development of tumors.

[0020] The beneficial effects of the present invention are:

[0021] The present invention can detect multiple membrane surface proteins simultaneously in one pot without the need for a washing step. Most importantly, the surface protein detection method designed by the present invention does not rely on antibodies and can achieve accurate quantification. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the use of a probe for detecting and quantifying membrane surface proteins.

[0023] Figure 2 For the practical application of the detection method. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention are further described below:

[0025] A probe for detecting and quantifying membrane surface proteins comprises a detection probe and a counting probe; the detection probe targets membrane characteristic proteins and membrane surface proteins that play a role in the occurrence and development of tumors.

[0026] The detection probe consists of five parts. The first part is the nucleic acid aptamer, which is used to recognize the surface protein; the second part is the PCR primer binding domain, which is used for subsequent PCR amplification; the third part is the molecular encoding part of the detection probe, and one UMI represents one detection probe; the fourth part is the nucleic acid aptamer tag, which indicates the type of aptamer and the type of surface protein; the fifth part is a 5nt complementary region that can complementarily bind to the 3' end of the counting probe for subsequent extension.

[0027] The counting probe consists of three parts: the first part is 5' modified cholesterol, which is used to target the lipid bilayer of the membrane; the second part is the primer binding domain of the PCR reaction, which is also used for subsequent PCR amplification; and the third part is a complementary sequence that binds to the 3' end of the detection probe.

[0028] A method for using a probe for detecting and quantifying membrane surface proteins, the method comprising the following steps: the probe comprises a detection probe and a counting probe, and due to the proximity effect, when the detection probe and the counting probe are simultaneously bound to the membrane, the complementary regions bind, and under the action of an enzyme, the two chains are extended to complement each other into a double chain;

[0029] The extended product is then amplified by PCR, and the target band is purified and recovered for NGS sequencing;

[0030] After obtaining the sequencing results, the data is subjected to bioinformatics analysis, and clean data is obtained by filtering the raw data;

[0031] Then compare it with the reference sequence, extract information from the data in the comparison, generate a protein count matrix, and achieve accurate quantification of multiple membrane surface proteins.

[0032] The enzyme is T4 DNA polymerase.

[0033] The target band was recovered by PAGE gel purification.

[0034] The detection probe can target membrane-characterized proteins and membrane surface proteins that play a role in the development and progression of tumors. (In this embodiment, the detection probe targets membrane-characterized proteins and six surface proteins that play a role in the development and progression of tumors.)

[0035] The invention discloses an application of a probe for detecting and quantifying membrane surface proteins. The probe is used for detecting and quantifying membrane surface proteins.

[0036] The probe is used for in situ detection and quantification of membrane surface proteins.

[0037] The probe is used for in situ detection and quantification of membrane characteristic proteins and a variety of membrane surface proteins that play a role in the occurrence and development of tumors.

[0038] The present invention can achieve in situ and simultaneous precise quantification of multiple membrane surface proteins without the need for cleaning and without relying on antigen-antibody reactions.

[0039] The present application will be further described below with reference to specific embodiments:

[0040] First, they selected one membrane-characterized protein and six membrane surface proteins that play important roles in tumorigenesis and progression, and designed detection probes targeting these seven proteins. The detection probes consist of five main components: a nucleic acid aptamer that recognizes the surface protein; a primer-binding domain for PCR amplification; a molecular identifier (UMI) representing each detection probe; a label for the aptamer, indicating the specific aptamer (and thus the specific surface protein); and a 5-nt complementary region that binds to the 3' end of the counting probe for subsequent extension. The counting probes consist of three main components: a 5'-modified cholesterol residue that targets the lipid bilayer of the membrane; a primer-binding domain for PCR amplification; and a complementary sequence that binds to the 3' end of the detection probe. Once the two probes bind to the membrane simultaneously, the complementary regions bind due to the proximity effect. Under the action of T4 DNA polymerase, the two strands are extended to form a double strand. The extended product is then amplified by PCR, and the target band is recovered by PAGE gel purification and sent for NGS sequencing. After obtaining the sequencing results, the data is subjected to bioinformatics analysis. After filtering the raw data, the clean data is obtained and then aligned with the reference sequence. The data on the alignment is captured and information is extracted to generate a protein count matrix. The specific results are as follows Figure 1 As shown. Figure 1 In the figure, a: Schematic diagram of the simultaneous binding of the detection probe and the counting probe to the membrane. The detection probe relies on specific recognition, while the counting probe inserts into the lipid bilayer through hydrophobic interactions. b: The two probes approach each other, and the complementary regions bind, leading to enzyme-mediated extension, forming a double strand. c: Electrophoresis gel image shows that when the two probes bind to the membrane, distinct extension products are produced. d: Quantitative polymerase chain reaction (qPCR) results show that the maximum extension product is achieved when both probes and EVs are present, consistent with the gel image.

[0041] The key technology of the present invention lies in the design of the detection and counting probes and the conversion of protein signals. When the detection and counting probes are simultaneously bound to the membrane, the membrane's fluidity forces the two probes into close proximity, leading to extension mediated by the proximity effect under the action of DNA polymerase, forming a double-stranded structure. Only when the two probes are simultaneously bound to the membrane does the proximity effect-mediated extension efficiency reach its highest level, yielding the largest number of extension products. Furthermore, the protein recognition signal is converted into a uniquely encoded DNA strand. The extension products are then amplified by PCR, sequenced, and decoded to produce a protein count matrix, enabling accurate quantification of multiple membrane surface proteins.

[0042] The present invention quantitatively encodes protein recognition events into DNA chains with unique sequences for amplification, sequencing and decoding. It can simultaneously detect multiple proteins in situ and obtain accurate protein counts with simple and convenient operation and low cost. Figure 2 In Figure 1, a. shows that this detection method has a sensitivity of 96% and a specificity of 88.9% for prostate cancer diagnosis. b. shows that this method achieves an overall accuracy of 90% when applied to multiple cancer classifications. Experimental results show that the cancer diagnosis model established based on the obtained protein count matrix has a sensitivity of 96% and a specificity of 86.9%, and can distinguish multiple cancers with an accuracy of 90%.

[0043] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A probe for detecting and quantifying membrane surface proteins, characterized in that: The probe consists of a detection probe and a counting probe; the detection probe targets membrane characteristic proteins and membrane surface proteins that play a role in the occurrence and development of tumors; the detection probe consists of five parts, the first part of which is a nucleic acid aptamer for identifying surface proteins; The second part is the PCR primer binding domain, which is used for subsequent PCR amplification; The third part is the molecular encoding part of the detection probe, which is the UMI, and one UMI represents one detection probe; the fourth part is the nucleic acid aptamer tag, which indicates the type of nucleic acid aptamer and also indicates the type of surface protein; the fifth part is a 5nt complementary region that can complementarily bind to the 3' end of the counting probe for subsequent extension; the counting probe consists of three parts, the first part is the 5' end modified with cholesterol, which is used to target the lipid bilayer of the membrane; the second part is the primer binding domain of the PCR reaction, which is also used for subsequent PCR amplification; the third part is the complementary sequence that binds to the 3' end of the detection probe.

2. A method for using the probe for detecting and quantifying membrane surface proteins according to claim 1 for non-diagnostic purposes, characterized in that: The method comprises the following steps: the probe comprises a detection probe and a counting probe, when the detection probe and the counting probe are simultaneously bound to the membrane, the 5 nt complementary region of the detection probe is complementary to the 3' end of the counting probe, and under the action of the enzyme, the two chains are extended and complemented into a double chain; The extended product is then amplified by PCR, and the target band is purified and recovered for NGS sequencing; After obtaining the sequencing results, the data is subjected to bioinformatics analysis, and clean data is obtained by filtering the raw data; Then compare it with the reference sequence, extract information from the data in the comparison, generate a protein count matrix, and achieve accurate quantification of multiple membrane surface proteins.

3. The method for using a probe for detecting and quantifying membrane surface proteins for non-diagnostic purposes according to claim 2, characterized in that: The enzyme is T4 DNA polymerase.

4. The method for using a probe for detecting and quantifying membrane surface proteins for non-diagnostic purposes according to claim 2, characterized in that: The target band was recovered by PAGE gel purification.

5. The method for using a probe for detecting and quantifying membrane surface proteins for non-diagnostic purposes according to claim 2, characterized in that: The detection probe can target membrane characteristic proteins and membrane surface proteins that play a role in the occurrence and development of tumors.

6. A non-diagnostic use of the probe for detecting and quantifying membrane surface proteins according to claim 1, characterized in that: The probe is used to detect and quantify membrane surface proteins.

7. The use according to claim 6, characterized in that: The probe is used for in situ detection and quantification of membrane surface proteins.

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