CN112430569B

CN112430569B - Application of protein SFTPC as lung cancer diagnosis marker and kit

Info

Publication number: CN112430569B
Application number: CN202110100174.5A
Authority: CN
Inventors: 张庆华; 宋凯; 匡鑫
Original assignee: Shanghai Wayen Biomedical Technology Co ltd
Current assignee: Shanghai Wayen Biomedical Technology Co ltd
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-05-11
Anticipated expiration: 2041-01-26
Also published as: CN112430569A

Abstract

The invention provides application of a quantitative detection agent of protein SFTPC in preparation of a kit for lung cancer diagnosis, auxiliary diagnosis or prognosis analysis and application in preparation of a kit for enriching lung-derived exosomes, which not only can achieve the effect of enriching lung tissue specific exosomes, but also can play a role in auxiliary lung cancer diagnosis, and directly uses the enriched specific exosomes in lung-related disease marker detection based on miRNA, protein and the like.

Description

Application of protein SFTPC as lung cancer diagnosis marker and kit

Technical Field

The invention relates to the field of medical diagnosis, in particular to application of protein SFTPC as a lung cancer diagnosis marker and a kit.

Background

The prevention and treatment of lung cancer is a common challenge in China and the world, and screening and early diagnosis and early treatment are one of the most effective means for reducing lung cancer death.

Extracellular Vesicles (EV) are membrane vesicles that are released from different cell types to the extracellular matrix and function to participate in the transport of molecules between cells, being present in almost all major body fluids. The EV contains different types of molecules, including protein, lipid, DNA, mRNA, miRNA and the like, and the content substances are changed along with the state of the parent cell secreting the EV and the change of the environment, namely the content substance information carried by the EV represents the cell state of the parent cell secreting the EV. EV's vary in size from 30 nm to 1000 nm and can be further classified according to their biogenesis, size and biophysical properties. The classical description of EV is based on vesicle size, and EV can be defined as exosome when the diameter is < 150 nm.

Exosomes are widely found in cell culture supernatants and in various body fluids (blood, lymph, saliva, urine, semen, milk) and also from various different tissues, such as lung tissue. Exosomes from different histiocytes have different compositions and functions due to different components such as carried proteins, and can exert different biological functions. At the same time, the difference is dynamically regulated by extracellular matrix and microenvironment, which also suggests that exosomes may have different effects in various lung diseases, and can be used as biomarkers of lung-related diseases, can provide rich, stable, specific biological information, and is expected to play a role in early diagnosis of diseases.

Actually, the separation and purification of exosomes are always the concerns of researchers, and obtaining high-purity exosomes is crucial to the subsequent research. At present, researchers mostly adopt methods such as ultracentrifugation, ultrafiltration, precipitation or a kit to realize the extraction and separation of exosomes. The magnetic bead immunocapture method is relatively convenient to operate, but at present, the method is generally based on exosome markers (CD 9, CD 63) and other exosome membrane proteins, is generally suitable for overall capture of exosomes, and cannot reflect the specificity of exosomes from specific sources. Due to the complex sources of exosomes in the body fluid sample, the exosomes from other cell sources interfere in the disease-related research by using the body fluid exosomes, for example, the exosomes from other tissues, organs, red blood cells and the like interfere in the lung cancer exosome-related research in blood, and the exosomes greatly obstruct the research process. The conventional exosome extraction technology cannot effectively enrich exosomes from specific tissues or disease sources, and how to obtain high-specificity exosomes from the tissues or the disease sources is very important for promoting research and application of the exosomes.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of protein SFTPC, which can realize rapid separation and extraction of exosomes from lung sources, does not need to adopt traditional physical methods such as separation, extraction and the like, saves operation time, can assist in the function of lung cancer diagnosis through research and discovery, and directly uses the enriched specific exosomes for detection on lung related disease markers such as miRNA, protein and the like.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention relates to application of a quantitative detection agent of a protein SFTPC in preparation of a kit for lung cancer diagnosis, auxiliary diagnosis or prognosis analysis and application in preparation of a kit for enriching lung-derived exosomes.

The protein SFTPC is identified in lung tissue exosomes through the first discovery in mass spectrum experimental data, and the SFTPC is not identified in liver tissue, stomach tissue, intestinal tissue, brain tissue and breast tissue exosomes. Pulmonary surfactant associated protein c (sftpc) is a protein specifically expressed in lung cells and is critical for postnatal lung function and homeostasis. Furthermore, in combination with the Human Protein Atlas database (http:// www.proteinatlas.org /), SFTPC was suggested to be specifically highly expressed only in lung tissue and not in other organ systems, e.g., brain, liver, bladder, platelets, etc. The GTEx database (https:// www.gtexportal.org/home /) also suggests that the expression of SFTPC is specific in lung tissue. In view of the specific expression and abundant quantity of the SFTPC in lung tissue cells, the protein SFTPC can be used as a surface marker of lung tissue specific exosomes by combining with subsequent specific experimental data and database information, so that the exosomes derived from lung tissue can be enriched, and the lung cancer diagnosis and detection effects can be realized.

It is to be understood that the term "marker", as used herein, refers to a molecule to be used as a target for analyzing a patient test sample. Examples of such molecular targets are proteins or polypeptides. Proteins or polypeptides for use as markers in the present invention are intended to include naturally occurring variants of said proteins as well as fragments, in particular immunologically detectable fragments, of said proteins or of said variants.

Optionally, the quantitative detection agent is a specific antibody of the protein SFTPC, and the specific antibody is used for performing a co-immunoprecipitation method or an enzyme-linked immunosorbent assay to detect the protein SFTPC.

The invention adopts an immunization and enrichment method to separate exosomes with SFTPC on the surface of a membrane from organism fluid, and can enrich lung tissue specific exosomes derived from organism fluid without non-specifically enriching exosomes derived from other tissue sources. The detection of the protein SFTPC by the quantitative detection agent can be carried out by methods known in the art. The method mainly comprises the following steps:

(1) preparing a biological fluid sample or extracting total exosomes in the biological fluid;

(2) incubating a sample with an antibody specific for SFTPC immobilized on a carrier, and adsorbing lung tissue specific exosomes to form a complex;

(3) the bound exosomes were isolated and purified to enrich lung tissue specific exosomes.

Optionally, the specific antibody is a monoclonal antibody.

Optionally, the specific antibody is a polyclonal antibody.

Of course, the recognition of the antigen SFTPC is not limited to the form of a specific antibody using SFTPC, and may include binding forms such as aptamers, receptors, and ligands.

The antibody of the present invention is preferably an antibody of the IgG type, and may be a polyclonal antibody or a monoclonal antibody. But also antibody fragments, synthetic antibodies, single chain antibodies, and the like. The source of the antibody is not particularly limited in the present invention, and commercially available products such as Thermo Scientific, cat # PA5-76631 can be used.

Preferably, the specific antibody of SFTPC is labeled with biotin, and based on the binding principle of biotin-streptavidin, the specific antibody of SFTPC may be immobilized on an agarose bead whose surface is modified with streptavidin. When incubated with a sample, antibodies specific for SFTPC can specifically bind to lung tissue specific exosomes, which can be further used and/or measured after elution of the exosomes.

The source of the agarose beads, which itself has the effect of binding to antibodies, is not so limited, as long as it is a commercially available product, such as that available from Thermo Scientific, cat # 20353.

The invention also relates to a kit for the diagnosis, auxiliary diagnosis or prognostic analysis of lung cancer, comprising a specific antibody as defined above.

The kit can obtain the lung tissue exosome with high specificity from a body fluid sample of a subject, and can be directly used for subsequent detection application, wherein the body fluid can comprise serum, plasma, urine, sputum, ascites, saliva, pleural effusion, bile and the like, and the preferable body fluid is the serum and the plasma.

Optionally, the kit further comprises at least one of a solid support, a blocking solution, a visualization reagent, a fusion antigen calibrator, and a wash buffer.

Optionally, the solid phase carrier comprises any one of agar balls, magnetic beads, particles and solid phase chips.

In practical application, the kit mainly comprises: reagent A, reagent B, agarose beads, biotin labeling reagents, antibodies specific for SFTPC, Glycine-HCL, buffers, and the like. Reagent a, reagent B used alone or in combination) are suitable for extracting total exosomes in serum, plasma and other common biological fluids. Reagent a is specifically adapted for serum samples and reagent B is specifically adapted for plasma samples.

The inventors have found that known lung-related disease biomarkers can be detected and/or quantified from lung tissue-specific exosomes enriched in biological fluids, and that the results can be used for the differential diagnosis of lung-related diseases.

The inventors have found that the lung-related disease biomarker may be a protein or a nucleic acid such as miRNA. For example, miRNA-31 is a novel biomarker of lung cancer metastasis; miRNA-4508 of peripheral blood lymphocytes can be used as a potential biomarker for diagnosing silicosis pulmonary fibrosis; neuron-specific enolase NSE was considered as the first marker for monitoring small cell lung cancer; annexin a2 is a biomarker for lung cancer diagnosis; CLM-1, IL12RB1, CD 83, FAM3B, IGFR1R and OPTC can predict the severity of neocoronary pneumonia; the combination of CC16, sRAGE, fibrinogen, CRP, and SP-D as biomarkers had some predictive effect on airflow limitation.

Protein biomarkers can be detected in enriched lung tissue specific exosomes, and protein markers exposed on the surface of exosomes can be detected directly without lysis. For a protein marker contained in an exosome, the exosome is cleaved and then detected. Detection methods such as Western Blot, ELISA, Luminex, flow cytometry, etc. are all preferred methods for protein biomarker detection.

The detection method can detect the nucleic acid biomarker in the enriched lung tissue specificity exosome, the detection is carried out after the exosome is cracked and the nucleic acid is extracted, and the detection methods such as qPCR, ddPCR, PCR Array and the like are all the preferable methods for detecting the nucleic acid biomarker.

The lung tissue specific exosome extracted from the biological fluid can be used for screening lung related disease markers. For example, protein markers of lung-related diseases can be specifically found by protein mass spectrometry technology, and nucleic acid markers of lung-related diseases can be specifically found by gene chip and sequencing technology.

The subject of the present embodiment is a mammalian subject, preferably a primate, more preferably a human.

In summary, lung tissue specific exosomes in biological fluids obtained by the method of the invention can be identified by Transmission Electron Microscopy (TEM), Nanoparticle Tracking Assay (NTA) and Western immunoblot (Western Blot) experiments. The results show that lung tissue specific exosomes obtained by the method of the invention are of similar quality to total exosomes obtained by other exosome separation techniques (e.g. ultracentrifugation). At present, specific exosomes derived from lung tissues cannot be obtained by traditional physical methods (such as ultracentrifugation and ultrafiltration), but the method disclosed by the invention does not relate to ultracentrifugation and has good practicability.

Compared with the prior art, the invention has the beneficial effects that:

(1) the method for extracting the lung tissue specific exosome mainly applies the lung tissue specific exosome surface marker SFTPC to the extraction of the lung tissue specific exosome, has the advantages of strong specificity, convenience and easiness, direct application to subsequent detection and good clinical application prospect;

(2) the exosome enrichment method is simple to operate, does not relate to expensive precise instruments, has stronger universality, can meet the condition in a general detection laboratory, and is more suitable for popularization in clinical application;

(3) compared with the existing magnetic bead affinity capture total exosome, the method has the advantages that the incubation combination of the SFTPC antibody and the exosome has lung tissue specificity, and the targeted capture of high-purity specific exosome can be realized;

(4) the exosome obtained after the elution is captured by the method has a complete structure, and is beneficial to downstream experiments;

(5) the method can simply and effectively realize the separation of the lung tissue specific exosome from the organism liquid, can effectively improve the accuracy of subsequent clinical diagnosis application and research, enables the subsequent mechanism research and immunotherapy application to become possible, and has great theoretical value and clinical application value.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

figure 1 is a representation of SFTPC results in different types of tissue exosomes and serum exosome profiling data;

FIG. 2 shows the identification results of Western blot on conventional exosome markers CD9, CD63 and target protein SFTPC;

in FIG. 2, panel A is the Western blot identification results of the conventional exosome markers CD9 (molecular weight of target protein 25 kDa) and CD63 (molecular weight of target protein 53 kDa), and panel B is the Western blot identification result of the target protein SFTPC (molecular weight of target protein 21 kDa);

FIG. 3 shows the result of identifying SFTPC of supernatant exosomes of different tissue-derived cell lines by Western blot;

FIG. 4 is a schematic illustration of the capture and isolation of lung tissue specific exosomes using immunoaffinity;

FIG. 5 shows the NTA detection result of a serum sample after the specific exosome is extracted by using the kit of the present invention, the abscissa is the size of the particle size, and the ordinate is the number of the particles;

FIG. 6 shows The Electron Microscope (TEM) detection result of exosomes after specific exosomes are extracted from serum samples by using the kit of the present invention;

FIG. 7 shows the Western blot identification results of the exosome marker CD9 (target protein molecular weight 25 kDa) and the target protein SFTPC (target protein molecular weight 21 kDa) after a serum sample is subjected to specific exosome extraction by using the kit of the invention;

FIG. 8 shows the results of PCR detection of Lunx in serum total exosomes and lung tissue specific exosomes;

FIG. 9 shows the PCR detection of miR-31 in lung tissue-specific exosomes in healthy subjects and lung cancer patients;

FIG. 10 shows ELISA detection of NSE in lung tissue-specific exosomes of healthy subjects and lung cancer patients;

FIG. 11 shows the secretion of exosomes with SFTPC from lung tissue and a schematic diagram of SFTPC-based enrichment.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In some embodiments, the isolated exosomes are predominantly exosomes, but may also include small amounts of other types of extracellular vesicles, such as microvesicles. The total exosomes described in the examples are almost exclusively exosomes, but may or may not necessarily include very small quantities of vesicles of other sizes, such as microvesicles.

In some embodiments, the discovery and validation of SFTPC indices in different tissues and serum exosomes is illustrated.

In some embodiments, results display details of SFTPC in different public databases are provided to demonstrate that SFTPC expression is tissue specific and demonstrated by another example, suggesting that SFTPC may be used as a lung tissue specific exosome surface marker and subsequently applied.

In some embodiments, the art recognizes binders to SFTPC including, but not limited to, DNA, RNA, antibodies, antibody fragments, lectins, chemical compounds, ligands, and the like, and combinations thereof.

In some embodiments, the antibodies of the invention can be anti-animal, particularly primate, SFTPC antibodies can be prepared by methods known in the art or obtained commercially.

It is also noted that common exosome biomarkers include: CD9, CD63, and the like.

One or more lung cancer specific biomarkers, e.g., miR-31, NSE, etc., are known in the art.

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

Materials, reagents and the like used in the following examples were purchased from commercial reagents unless otherwise specified. The corresponding reagent is not limited to the following companies and models, and can be made by using corresponding models of other companies or synthesized by themselves.

In the examples of the present invention, the buffer solution is a PBS buffer solution and a DPBS balanced salt solution, unless otherwise specified. The elution buffer was glycine-HCl (pH 3.0). The enrichment carrier is streptavidin modified agar ball.

Embodiments of the present invention will be described in detail with reference to examples.

Example 1: discovery of lung tissue exosome surface marker SFTPC

The method comprises the following steps:

the proteomics analysis is carried out on exosomes and serum exosomes of different types of tissue sources (lung tissue, liver tissue, stomach tissue, intestinal tissue, brain tissue and mammary tissue) by applying a mass spectrometry technology. The results are shown in FIG. 1, and the specific steps are as follows:

1. preparation of tissue exosomes

(1) Washing the tissue sample with PBS, washing to remove blood stain, and removing redundant tissues such as adipose tissue, connective tissue, necrotic tissue, etc. and original preservation medium;

(2) transferring the tissue sample to a culture dish, adding a proper amount of PBS, and shearing the tissue sample by using an ophthalmic scissors;

(3) adding collagenase to make the concentration of the working solution be 1 mg/mL, placing the mixture in a constant temperature shaking table at 37 ℃ and 90rpm, and incubating for about 60min until the tissue block has good light transmittance and is flocculent;

(4) collecting the supernatant collected in the experiment in a 50mL centrifuge tube, centrifuging at 4 ℃, collecting the supernatant after 300g and 5 min;

(5) centrifuging the supernatant sample at 4 deg.C for 10min at 2000g, and collecting the supernatant;

(6) centrifuging the supernatant at 4 deg.C for 30min at 10,000g, and collecting the supernatant;

(7) transferring the supernatant sample to an ultra-high-speed centrifuge tube, centrifuging at 4 ℃ for 75min at 110000g, and discarding the supernatant;

(8) resuspend the pellet with 1mL of 1xPBS, dilute to 15mL with 1XPBS after resuspension, filter with 0.22 μm filter membrane;

(9) transferring the sample to an ultra-high-speed centrifuge tube, centrifuging at 4 ℃, 110000g for 75min, and discarding the supernatant;

(10) the pellet was resuspended in the corresponding 1mL of 1XPBS, aliquoted and stored at-80 ℃.

2. Preparation of serum exosomes

(1) Taking out the sample at-80 ℃, and unfreezing the sample in water bath at 25 ℃;

(2) centrifuging at 4 deg.C and 1500 Xg for 10min, and transferring supernatant;

(3) centrifuging at 4 deg.C and 10000 Xg for 10min, and transferring supernatant;

(4) removing the buffer solution on the top of the sieve plate with a pipette before the column removes the bottom sliding cover;

(5) adding 500 mu L of sample (1 ml of sample needs to be added in two times, namely, the subsequent steps are repeated);

(6) immediately removing the bottom sliding cover;

(7) immediately begin collecting 0.5 mL fractions (the first 3mL is void volume, contains no vesicles);

(8) when the last sample has just entered (is flat with) the top plate of the column, more buffer is added;

(9) collecting 1.5mL of liquid with the void volume as the exosome solution with higher purity;

(10) the combined collected exosome solutions were concentrated to a final volume of 200 μ L of exosome suspension by ultrafiltration tube.

3. Analysis of exosome body mass spectrum

(1) Proteome sample pretreatment

a) Adding a protein lysate (7M urea, 2% SDS, 1 Xprotease Inhibitor Cocktail before use) with the same volume as the sample;

b) performing ultrasonic treatment on ice for 2s and stopping for 5s for 1min by using an ultrasonic cell disruptor, and performing cracking on ice for 2 h after all the ultrasonic treatment is finished;

c) the lysate was centrifuged at 13,000 rpm for 20min at 4 ℃ and the supernatant was pipetted into a new 1.5mL EP tube;

d) adding 6 times of 100% acetone by volume, and precipitating at-20 ℃ overnight;

e) the next day, the pellet was centrifuged and 500 μ L precooled (ethanol: acetone: acetic acid =50:50: 0.1) washing the pellet 2 times, centrifuging at 13,000 rpm for 15 min at 4 ℃, (6M guanidine hydrochloride, 300 mM TEAB) redissolving the pellet and determining the sample concentration again;

f) the sample was kept in a refrigerator at 4 ℃ and a portion of the sample was diluted and the concentration was measured by BCA method.

(2) Enzymatic hydrolysis of FASP

a) Taking 20 mu g of protein solution samples from each sample, and using 25 mM ammonium bicarbonate to fix the volume to 100 mu L;

b) reductive alkylation: to the protein solution, 1M DTT (added at a ratio of 2. mu.L of 1M DTT to 100. mu.L of protein, final concentration: 20 mM) was added, mixed well, and incubated at 57 ℃ for 1 h. Adding 10 μ L of 1M Iodoacetimide/100 μ L solution (final concentration 90 mM, solid powder, 25 mM ammonium bicarbonate dissolved, ready for use), mixing, and standing at room temperature in dark for 40 min;

c) adding the protein subjected to reductive alkylation into a 10K ultrafiltration tube, centrifuging at 12,000 rpm, and discarding the solution at the bottom of the collection tube;

d) adding ammonium bicarbonate (precipitation Buffer) into the ultrafiltration tube, and washing for 4 times;

e) adding Trypsin prepared by resolution Buffer, and performing enzymolysis at 37 ℃ overnight;

f) the next day, the peptide fragments after enzymolysis and digestion are collected by centrifugation, concentrated and dried.

(3) Desalination

The dried polypeptide was concentrated by centrifugation and desalted using a Ziptip C18 column, dried and ready for mass spectrometric analysis. The desalting method comprises the following steps:

a) dissolving the dried mixed peptide fragment by using 0.1% Formic Acid (FA) solution;

b) activating the desalting column with 100% acetonitrile;

c) equilibrating the desalting column with 0.1% FA solution;

d) adding the re-dissolved sample into a desalting column, enabling the sample to slowly flow through the desalting column, trapping the peptide segment by the desalting column, and discarding other non-hydrophobic small molecules such as salt and the like;

e) then 0.1% FA solution is added to clean the desalting column and remove the residual salt;

f) adding 0.1% FA and 80% acetonitrile solution, allowing the liquid to slowly flow through a desalting column, eluting the peptide fragment, and collecting the eluate with a new EP tube;

g) the eluted solution is centrifugally concentrated and dried to remove the acetonitrile.

(4) LC-MS analysis

Adding 0.1% FA into the sample after vacuum drying for redissolving, and taking 1-2 μ g of the sample to be loaded on a machine. Separation was performed using EASY-nLC 1200 (Thermo Scientific, USA) using an analytical column (C18, 1.9 μm, 75 μm 20 cm) at a flow rate of 200 nl/min. The mass spectrometer was an Orbitrap Fusion Lumos (Thermo Scientific, USA). Tandem mass spectrometry detection uses a Data Dependent scanning (DDA) mode. The full scan resolution was 60,000 (FWHM), the mass-to-charge ratio range was 1600, and the collision energy was set at 30% in the HCD fragmentation mode.

FIG. 1 results:

the results of the different tissue exosome and serum exosome body profile data show that: the index SFTPC was identified in lung tissue exosomes, while no SFTPC was identified in other tissue exosomes and serum exosome samples.

As can be seen from fig. 11, exosomes secreted from lung tissues are widely distributed in body fluids such as serum, urine, and the like, and circulate throughout the body through the blood. Exosomes isolated from serum, urine and the like by conventional means such as superionization comprise exosomes derived from various organs of a human body, and the exosomes derived from each organ account for a small part of the exosomes, are low in abundance, and carry some markers which are not easy to detect. The invention discovers that the content of a lung cell specific surface marker (SFTPC) in exosomes is very high, invents the method and the kit for enriching exosomes from lung tissue sources based on the SFTPC, can directly enrich the exosomes from the lung tissue sources from biological body fluid or total exosomes in the biological body fluid, and lays a foundation for later application.

Example 2: validation of lung tissue exosome surface marker SFTPC

The method comprises the following steps:

and (3) taking PBS resuspension of tissue exosomes and serum exosomes, adding equal-volume RIPA (strong) lysate for cracking and protein extraction, and detecting an exosome marker and SFTPC by Western blot. The results are shown in FIG. 2, the specific steps are as follows:

(1) preparing glue: preparing a 10% separation gel and a 5% concentration gel by using a 1.5 mm glass plate and a 15-hole sample comb according to the molecular weight of the target protein;

(2) sample loading

(3) Electrophoresis: carrying out electrophoresis at a stable voltage of 80 v until the Loading Buffer (indicator) enters the separation gel, changing the electrophoresis into a stable voltage of 120 v, and continuing the electrophoresis until the Loading Buffer (indicator) reaches the bottommost part of the gel, thus terminating the electrophoresis;

(4) film transfer: and selecting a PVDF membrane with the aperture of 0.22um, keeping the constant current at 200 mA, and rotating the membrane for 90 min.

(5) And (3) sealing: PBST diluted 5% skimmed milk powder, sealed for 1h, washed 3 times with PBST, each time for 10 min;

(6) incubating the primary antibody: putting the membrane into a hybridization box, adding a corresponding antibody, and putting the membrane in a decoloring shaker at 4 ℃ overnight;

(7) incubation of secondary antibody: the hybridization box was placed on a shaker and shaken slowly to return to room temperature. Removing the primary antibody, washing the membrane for 3 times by using PBST (Poly-p-Phenylene Benzobisoxazole) (PBST), 10min each time, putting the secondary antibody and the membrane into a hybridization box again, slowly shaking the hybridization box on a shaking table, and incubating the hybridization box for 1h at room temperature;

(8) remove secondary antibody, wash membrane 3 times with PBST for 10min each;

(9) exposure: an appropriate amount of ECL luminescence was added and the film was photographed using a digital imager.

FIG. 2 results:

panel A shows the Western blot identification results of the conventional exosome markers CD9 (molecular weight of target protein: 25 kDa) and CD63 (molecular weight of target protein: 53 kDa), and panel B shows the Western blot identification result of the target protein SFTPC (molecular weight of target protein: 21 kDa). The results show that: the conventional markers of exosomes, CD9 and CD63, were detectable in different types of tissue exosomes and serum exosomes, and the target protein SFTPC was detected only in lung tissue exosomes.

Example 3: specificity verification of lung tissue exosome surface marker SFTPC

The method comprises the following steps:

the expression conditions of the mass spectrum screening index SFTPC in different tissues are inquired by utilizing a public database Human Protein Atlas (http:// www.proteinatlas.org /) and a GTEx database (https:// www.gtexportal.org/home /), and the inquiry result shows that: SFTPC is specifically highly expressed only in lung tissue.

Example 4: specificity confirmation of lung tissue exosome surface marker SFTPC

The method comprises the following steps:

and extracting cell lines (lung cancer cell lines, liver cancer cell lines, colorectal cancer cell lines, breast cancer cell lines, stomach cancer cell lines and glioblastoma cell lines) of different types of tissue sources by using a super-dissociation technology to culture supernatant exosomes, and detecting target protein SFTPC by using a Western blot technology. The results are shown in FIG. 3, the specific steps are as follows:

1. preparation of culture supernatant exosomes of different tissue-derived cell lines

(1) Taking out the sample, thawing in a water bath at 25 ℃, and placing on ice;

(2) centrifuging at 4 deg.C for 10min at 2,000 Xg, and collecting supernatant;

(3) centrifuging at 4 deg.C for 30min at 10,000 Xg, and collecting supernatant;

(4) transferring the sample into a super-high-speed centrifuge tube, centrifuging at 4 ℃ for 75min at 110,000 Xg, and discarding the supernatant;

(5) resuspend the pellet with 1mL of 1XPBS, dilute with 1XPBS after resuspension, filter with 0.22um membrane;

(6) transferring the sample to an ultra-high-speed centrifuge tube, centrifuging at 4 ℃ for 75min at 110,000 Xg, and discarding the supernatant;

(7) the pellet was resuspended in the corresponding 1XPBS, aliquoted and stored at-80 ℃.

2. Exosome SFTPC detection

Western blot detects the exosome specific marker, and the experimental method is the same as that described above.

FIG. 3 results:

the Western blot identification result of the supernatant exosome target protein SFTPC (target protein molecular weight of 21 kDa) cultured by different tissue-derived cell lines shows that: SFTPC was detected only in lung cancer cell line supernatant exosome samples.

Example 5: lung tissue specificity exosome extraction kit

The kit comprises the following specific components: reagent A, reagent B, DPBS balanced salt solution, 3% BSA, streptavidin-agarose resin, EZ-Link sulfo-NHS-biotin, SFTPC-antibody, 0.05M glycine-HCl (pH 3.0), 1M Tris-HCl (pH = 8.6).

Example 6: extraction of lung tissue specific exosomes

The method comprises the following steps:

the total exosomes in the serum sample are separated by using the extraction reagent in the kit in example 5, and then the lung tissue specific exosomes are extracted according to the kit using instructions and step operation. A schematic diagram of the use of the kit for isolating lung tissue specific exosomes is shown in FIG. 4, and the specific steps are as follows:

1. enrichment of total exosomes in serum

(1) Sample preparation

a) The serum sample needs to be placed on ice, if the initial serum sample is a frozen sample, the sample needs to be thawed in a water bath at 25 ℃ until the sample is completely thawed, and then the sample is placed on ice;

b) serum samples were centrifuged at 4 ℃ for 10min at 3,000 Xg.

(2) Exosome extraction

a) Taking the centrifuged serum sample, adding the extraction reagent A with the corresponding volume, and repeatedly blowing and beating the serum sample uniformly or uniformly mixing the serum sample by using a vortex mixer;

b) standing the mixed solution at 4 ℃, and incubating for 30 min;

c) after the incubation is finished, centrifuging the mixed solution at room temperature for 3,000 Xg for 10min, removing supernatant, and allowing a precipitate to appear at the bottom of the tube;

d) centrifuging the obtained precipitate at room temperature for 5min at 3,000 Xg, removing residual supernatant, and avoiding contacting the precipitate at the bottom of the tube;

e) resuspending the pellet with 1 × PBS, and repeatedly and uniformly blowing (the volume of resuspension is equal to that of the initial serum);

f) adding a reagent B with a corresponding volume into the heavy suspension, and repeatedly blowing and uniformly stirring or uniformly mixing by using a vortex mixer;

g) incubating the mixed solution for 30min at 4 ℃;

h) after the incubation is finished, centrifuging the mixed solution at room temperature for 3,000 Xg for 10min, and removing the supernatant;

i) centrifuging the obtained precipitate again at room temperature for 3,000 Xg for 5min, and removing the residual supernatant;

j) resuspending the precipitate with 1 × PBS, repeatedly beating, and filtering with 0.22 um;

k) subpackaging, storing at-80 ℃ for downstream analysis.

2. Immunoaffinity enrichment purification of lung tissue specific exosomes

(1) Biotinylated modification of antibodies

a) The vial of biotin reagent was removed from the refrigerator and equilibrated to room temperature;

b) preparing a 10mM biotin labeling reagent solution;

c) adding a volume of 10mM biotin reagent solution to the antibody solution (so that the molar ratio of the labeled reagent to the antibody is 20: 1);

d) incubate the reaction on ice for 2 hours or at room temperature for 30 minutes;

e) the labeled antibody was purified through an ultrafiltration tube.

(2) Application of modified antibody in capturing lung tissue specific exosome

a) Adding 100 mu L of 3% BSA to each exosome suspension, and incubating for 1h at 4 ℃ with 2 mu g of SFTPC biotinylated antibody;

b) adding 25 μ L of streptavidin agarose resin and 50 μ L of 3% BSA, and incubating at 4 deg.C for 0.5 h;

c) centrifuge at 200 x g for 10min at 4 ℃ and remove the supernatant to a new centrifuge tube.

(3) Elution of Lung tissue specific exosomes

a) The pellet was resuspended in 250. mu.L of 0.05M glycine-HCl (pH 3.0) and vortexed for 10 s;

b) centrifuging at 200 x g for 10min at 4 deg.C, removing supernatant, transferring to a new centrifuge tube, and adjusting pH to 7 with 1M Tris-HCl (pH = 8.6);

c) the samples were placed in a-80 ℃ freezer for subsequent experiments.

FIG. 4 results:

the serum sample lung tissue specific exosomes were isolated using the example 5 kit and further characterization of the enriched exosomes was confirmed in connection with the following examples.

Example 7: characterization after exosome enrichment by using the kit of the invention

The method comprises the following steps:

the lung tissue specific exosomes extracted in example 6 were characterized by NTA, TEM, Western Blot experiments. The results are shown in fig. 5-7, and the specific steps are as follows:

1. exosome NTA validation

And determining the dispersibility, particle size and distribution of the exosomes by adopting a nanoparticle tracking analysis technology.

(1) Taking a frozen sample, thawing in a water bath at 25 ℃, and placing on ice;

(2) opening a switch at the back of the host, and clicking to open ZetaView connected with the host and a computer;

(3) initializing software;

(4) injecting 10mL of ultrapure water into the one-way valve on the right side of the main machine by using a disposable needle tube injector, and automatically detecting the cleanliness of the sample pool by software;

(5) preparing a PS100 standard substance suspension, slowly injecting PS100 into the sample pool, and starting laser and camera focusing after particles are stable;

(6) determining a blank control group;

(7) setting camera parameters;

(8) after the particles are stabilized, clicking a button Run Video Acquisition;

(9) entering a sample measurement interface, setting parameters and selecting proper SOP;

(10) clicking on the expert Parameters, and starting the test after all the Parameters are confirmed;

(11) injecting the diluted sample suspension into 3-5mL from a one-way valve by using a needle tube;

(12) setting corresponding parameters when the particles are stable, and starting testing after all the particles are confirmed;

(13) after the software tests and analyzes the samples, the software automatically obtains and stores the particle size distribution report.

2. Exosome electron microscopy (TEM) analysis

The size, shape and the like of the exosome are observed by adopting an exosome electron microscope detection technology.

(1) Dripping 5uL of the exosome sample on a copper mesh, and incubating for 5min at room temperature;

(2) after the incubation is finished, sucking off the redundant liquid on one side by using absorbent paper;

(3) dripping a drop of 2% uranyl acetate on the copper net, and incubating for 1min at room temperature;

(4) after the incubation is finished, sucking off the redundant liquid on one side by using absorbent paper;

(5) drying at room temperature for about 20min, and observing on a machine.

(6) Information on instruments used for testing

The instrument name: nano transmission electron microscope detector

Production company: FEI

The instrument model is as follows: tecnai G2 Spirit BioTwin

Setting an acceleration voltage: 80Kv

3. Exosome protein marker analysis (Western Blot)

Western blot was used to detect the exosome marker CD9 (exosome) and the target protein SFTPC (lung). The experimental procedure was the same as described above.

FIG. 5-FIG. 7 results:

FIG. 5 is the result of detecting the exosome NTA, FIG. 6 is the result of detecting the exosome electron microscope (TEM), and FIG. 7 is the result of identifying the Western blot of the exosome marker CD9 (target protein molecular weight 25 kDa) and the target protein SFTPC (target protein molecular weight 21 kDa). The results show that: the lung tissue specific exosomes isolated in example 6 have particle size distribution mainly around 100nm, and the particle size range meets the literature report. Under a microscope, the exosome presents 50-100nm size double molecular layer membrane structure vesicles, and the morphology is consistent with literature reports. Western blot also identified CD9, SFTPC target protein.

Example 8: extraction effect detection of lung tissue specificity exosome extraction kit

The method comprises the following steps:

the kit of example 5 was used to extract lung tissue-specific exosomes from 5 serum samples, wherein total exosomes in the serum samples were used as control samples, and the mRNA expression level of lung tissue-specific gene (lung-specific X protein gene) Lunx was detected by PCR assay. The results are shown in FIG. 8, with the following specific steps:

1. extracting total exosomes and lung tissue specific exosomes from serum samples

The experimental procedure was the same as described above.

2. Extraction of purified exosome total RNA by Qiagen kit

(1) Collecting an exosome sample;

(2) add appropriate volume of QIAzol Lysis Reagent to the exosome sample, mix immediately;

(3) disrupting the exosome sample by vortexing for 1 minute;

(4) the lysed exosome sample was left at room temperature (15-25 ℃) for 10 minutes;

(5) adding a suitable volume of chloroform to the tube containing the homogenate and shaking the tube vigorously for 15 seconds;

(6) the treated exosome samples were left at room temperature (15-25 ℃) for 3 minutes;

(7) centrifugation at 12,000 Xg for 15 minutes at 4 ℃;

(8) the samples were removed slowly from the centrifuge without disturbing the middle layer and the upper aqueous phase was transferred to a fresh self-contained RNase-free collection tube. Adding 100% ethanol 1.5 times volume, and mixing thoroughly;

(9) not more than 700. mu.L of sample was pipetted into an RNeasy MinElute spin column in a2 mL collection tube. Lightly covering the cover, centrifuging at room temperature (15-25 deg.C) and 12,000 Xg for 15 s, pouring off waste liquid in the collecting tube, and placing the adsorption column back into the collecting tube;

(10) if the sample still remains, repeating the experimental steps, pouring the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe;

(11) mu.l Buffer RPE was applied to RNeasy MinElute spin columns. Gently cover the cover, washing the column at room temperature (15-25 ℃) at 12,000 Xg, centrifuging for 15 seconds, pouring off the waste liquid in the collection tube, and putting the adsorption column back into the collection tube;

(12) 500 μ L of 80% ethanol was applied to RNeasy MinElute spin column. Capping, washing the membrane of the column at room temperature at 12,000 Xg, centrifuging for 2 minutes, discarding the collection tube and the liquid removed;

(13) RNeasy MinElute spin columns were placed into new 2 mL collection tubes. Opening the cap of the spin column, centrifuging at room temperature at 12,000 Xg for 5 minutes at full speed to dry the membranes of the spin column, discarding the process collection tube and the liquid removed from the tube;

(14) RNeasy MinElute spin columns were placed into a new 1.5mL collection tube. Add 14. mu.L of RNase-free water to the center of the spin column membrane. The cap was gently closed, and the RNA was eluted at room temperature at 12,000 Xg by centrifugation at full speed for 1 minute.

3. PCR detection of exosome mRNA expression level

(1) Reverse transcribing all RNAs to form cDNA according to the concentration determined by Nanodrop;

(2) the primer sequence is synthesized by biological engineering company,

lunx sense primer 5'-AGGTCTTCTGGACAGCCTCAC-3'

Antisense primer 5'-CAGGGTGATGTCCAAGCCTCT-3'

GAPDH sense primer 5'-AGAAAAACCTGCCAAATATGATGAC-3'

An antisense primer 5'-GCCCAGGATGCCCTTGA-3';

(3) reaction system (ABI SYBR)

(4) Reaction conditions

(5) Data were collected and quantitatively analyzed using software.

FIG. 8 results:

the PCR results show that the lung tissue specific exosomes isolated using the kit of example 5 of the present invention have higher Lunx mRNA levels relative to the total serum exosome samples.

Example 9: application of lung tissue specific exosome in lung cancer marker detection

The method comprises the following steps:

after the kit in the embodiment 5 is used for extracting lung tissue specific exosomes in serum samples of 35 lung cancer patients and 35 healthy people, a marker miR-31 in the enriched exosomes is detected by combining a PCR experiment, and a marker NSE in the enriched exosomes is detected by combining an ELISA experiment. The results are shown in fig. 9 and 10, and the specific steps are as follows:

1. lung cancer miRNA marker detection

(1) Immunoaffinity enrichment purification of lung tissue specific exosomes

The experimental procedure was the same as described above.

(2) Extraction of purified exosome total RNA by Qiagen kit

The experimental procedure was the same as described above.

(3) qPCR (quantitative polymerase chain reaction) detection of exosome miRNA expression level

The experimental procedure was the same as described above.

Wherein, the miR-31 sequence: 5'-AGGCAAGATGCTGGCATAGCT-3'

RNU6B sequence: 5'-ACGCAAATTCGTGAAGCGTT-3' are provided.

FIG. 9 results:

the lung tissue specific exosome miR-31 levels extracted from serum samples of healthy people and lung cancer patients are obviously distinguished, and the expression level of the marker miR-31 in the lung cancer patient sample is higher than that of the healthy people.

2. Lung cancer NSE protein marker detection

(1) Immunoaffinity enrichment purification of lung tissue specific exosomes

The experimental procedure was the same as described above.

(2) Extracting and purifying total protein of exosome

The experimental procedure was the same as described above.

(3) ELISA (enzyme-Linked immuno sorbent assay) for detecting expression quantity of specific exosome protein

The enriched lung tissue specific exosomes were tested for NSE index according to the instructions.

a) Adding 100 mu L of standard working solution or sample into the corresponding plate hole, and incubating for 90 minutes at 37 ℃;

b) after the liquid in the plate is discarded, 100 mu L of biotinylated antibody working solution is immediately added, and incubation is carried out for 60 minutes at 37 ℃;

c) discarding the liquid in the plate, and washing the plate for 3 times;

d) adding 100 mu L of HRP enzyme conjugate working solution into each hole, incubating for 30 minutes at 37 ℃, discarding the liquid in the plate, and washing the plate for 5 times;

e) adding 90 mu L of substrate solution into each hole, and incubating for about 15 minutes at 37 ℃;

f) adding 50 mu L of stop solution into each hole;

g) the data were processed by reading immediately at a wavelength of 450 nm.

FIG. 10 results:

the NSE levels in lung tissue specific exosomes extracted from serum samples of healthy people and lung cancer patients are obviously distinguished, and the NSE expression level of a marker in the lung cancer patient sample is higher than that of the healthy people.

Claims

1. Application of a quantitative detection agent of protein SFTPC in preparation of a kit for enriching lung-derived exosomes.

2. The use of claim 1, wherein the quantitative detection agent is an antibody specific to the protein SFTPC, and the antibody is used for performing co-immunoprecipitation or enzyme-linked immunosorbent assay to detect the protein SFTPC.

3. The use according to claim 2, wherein the specific antibody is a monoclonal antibody.

4. The use according to claim 2, wherein the specific antibody is a polyclonal antibody.