CN115856300A - Lung cancer-related plasma exosome protein marker, antibody and application thereof - Google Patents
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Abstract
The invention belongs to the field of molecular biology, and discloses a plasma exosome marker related to lung cancer and application thereof. The marker is plasma exosome protein TLR7. The plasma exosome protein TLR7 screened by the invention has higher specificity and sensitivity on the detection of lung cancer patients, and can greatly improve the accuracy of lung cancer diagnosis. The marker can be used for preparing a diagnostic kit and used for the auxiliary early diagnosis of lung cancer.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to a plasma exosome protein marker related to lung cancer, two antibodies and application thereof.
Background
Lung cancer is the most common malignant tumor with the highest incidence and mortality worldwide, wherein non-small cell lung cancer (NSCLC) accounts for more than 85% of all lung cancer types, and the mortality rate is as high as 80% -90%. Due to the lack of effective early diagnosis and screening methods, 70% of lung cancer patients are diagnosed in the late stage, and the 5-year survival rate is only 16% -18%. Early diagnosis of NSCLC is therefore particularly important. Currently, clinical early screening of NSCLC is mainly based on examination of Carcinoembryonic antigen (CEA), low-dose computed tomography (LDCT), and fibrobronchoscopy, which are classical blood tumor markers. However, these detection approaches have limitations such as low sensitivity or invasiveness, and therefore, development of noninvasive and sensitive screening based on liquid biopsy is a significant challenge for early diagnosis of NSCLC.
Exosomes (Exosomes) are nanoscale extracellular vesicles with a diameter between 50-150 nm, originating from the endosomal system, present in almost all body fluids including blood. It carries DNA, mrna, non-coding RNA, proteins and lipids, and it is able to protect its contents from degradation by the lipid bilayer membrane structure. All cell types, including tumor cells, are capable of releasing exosomes into the extracellular space, and cancer patients have significantly higher numbers of exosomes than healthy people. Tumor-derived exosomes can act as a sentinel once released, target cells through fusion with a plasma membrane or interaction with protein receptors, and further participate in processes such as proliferation, metastasis, escape and the like of tumors widely. The exosome can provide important information for early diagnosis of lung cancer, and has the potential of becoming a lung cancer tumor marker. Therefore, exosomes are one of the most promising liquid biopsy markers in the cancer field.
The research shows that partial exosome protein is abnormally expressed in a tumor patient body and has the potential of becoming a tumor diagnosis marker. Compared with the conventional tumor diagnosis method, the method has the advantages of simple and convenient sampling, low detection cost and the like, and has wide development and application prospects in the aspect of tumor diagnosis biomarkers.
However, the application prospect of the plasma exosome protein in noninvasive lung cancer diagnosis still needs to be discovered, and if the plasma exosome protein abnormally expressed in the lung cancer can be screened as a biomarker and a corresponding diagnosis kit is developed, the current lung cancer diagnosis situation can be greatly promoted.
Disclosure of Invention
The invention mainly aims to provide a plasma exosome protein marker related to lung cancer and application of the plasma exosome protein marker in preparation of a lung cancer diagnosis kit aiming at the problem in clinical noninvasive diagnosis of the lung cancer.
In order to achieve the purpose, the invention adopts the following technical scheme:
a plasma exosome protein marker related to lung cancer is a plasma exosome protein TLR7.
In a further technical scheme, the plasma exosome protein marker is a combination of plasma exosome proteins CD47, CD63 and TLR7.
CD47 is a widely expressed transmembrane glycoprotein, also known as integrin-associated protein (IAP), with a molecular weight of 52 kDa. The protein has variable N-terminal structural domain of immunoglobulin, 5 transmembrane structural domains and a short C-terminal intracellular tail, and the intracellular tail has four alternative different splicing isomers, thereby forming 4 subtypes. Subtype 2 CD47 is expressed mainly in hematopoietic, vascular endothelial and epithelial cells, subtype 1 in keratinocytes and subtypes 3 and 4 in neuronal, intestinal mucosal and testicular cells.
CD63 is a protein antigen, encoded by the CD63 gene in humans, and CD63 is found primarily on the surface of extracellular vesicles, as well as on the surface of common cell membranes.
Toll-like receptor (TLR) family members are named for the closely related Toll-like receptors in drosophila and play an important role in the innate immune response. TLRs recognize conserved motifs present in various pathogens and mediate defense responses. Triggering the TLR pathway leads to activation of NF- κ B and subsequent regulation of immune and inflammatory genes.
The invention provides an antibody capable of specifically capturing the plasma exosome protein marker related to the lung cancer. The antibody is a monoclonal antibody against TLR7 protein and a biotinylated anti-TLR 7 protein antibody which are custom-synthesized by Abcam company; further, the antibody is a monoclonal antibody against CD47, CD63, TLR7 protein custom-synthesized by Abcam corporation and a biotinylated antibody against CD47, CD63, TLR7 protein.
The invention provides application of the plasma exosome protein marker and the antibody in preparation of a lung cancer diagnosis kit.
The invention provides a lung cancer diagnosis kit, which is used for detecting the expression quantity of the protein marker in blood plasma.
In a further technical scheme, the kit comprises the antibody.
In a further technical scheme, the kit also comprises a reagent and an enzyme which are commonly used for enzyme-linked immunity.
According to a further technical scheme, the kit comprises a monoclonal antibody for resisting TLR7 protein, a TLR7 standard substance, a biotinylated anti-TLR 7 protein antibody, horseradish peroxidase-labeled avidin and biotin.
In a further technical scheme, the kit comprises monoclonal antibodies for resisting CD47, CD63 and TLR7 proteins, CD47, CD63 and TLR7 standards, biotinylated antibodies for resisting CD47, CD63 and TLR7 proteins, horseradish peroxidase-labeled avidin and biotin.
The invention has the beneficial effects that:
the plasma exosome protein marker provided by the invention as a marker for lung cancer diagnosis has the advantages that:
(1) The traditional plasma marker of lung cancer is mainly a protein biomarker represented by carcinoembryonic antigen (CEA), has the defects of low sensitivity and specificity, and plasma exosome TLR7 protein is a novel biomarker, is specifically expressed in the plasma of lung cancer patients, is stably expressed, and is easy to detect and quantify. The TLR7 protein or the combination of CD47, CD63 and TLR7 protein provided by the invention is used for noninvasive diagnosis of lung cancer plasma, which is beneficial to the auxiliary diagnosis of the current lung cancer and can greatly improve the sensitivity and specificity of noninvasive diagnosis of the lung cancer.
(2) The differential expression of the plasma exosome protein in the plasma exosome of the lung cancer patient is researched by the system through the exosome protein mass spectrometry and the Western blot method, and the application value of the plasma exosome protein in the lung cancer diagnosis is further verified.
(3) The invention carries out multi-stage verification on a large sample by ELISA, and adopts absolute quantification for verification: the sensitivity and specificity are obviously improved when the plasma exosome TLR7, CD47 and CD63 proteins are used in a combined manner and compared with the single use of the plasma exosome TLR 7; ensures the application of the lung cancer plasma exosomes CD47, CD63 and TLR7 protein biomarkers and a diagnostic kit, and also provides a method and a reference on strategies for the development of other disease biomarkers.
Drawings
FIG. 1 (a) is a plasma exosome morphogram;
FIG. 1 (b) is a graph of plasma exosome particle size;
FIG. 1 (c) is a drawing of plasma exosome surface marker proteins;
FIG. 2 is a thermal map of mass spectrometry analysis of 21 proteins whose expression is most significantly different in plasma of lung cancer patients and healthy controls.
FIG. 3 is a Western blot result chart of the expression difference of 3 selected significant difference proteins (CD 63, PRSS1, CD47, TLR 7) in plasma exosomes of lung cancer patient groups and healthy control groups.
FIG. 4 is a graph showing the ELISA results of differential expression of proteins CD47, CD63, TLR7 in plasma exosomes of lung cancer patient group and healthy control group.
FIG. 5 is a ROC graph of the effect of TLR7 on the diagnosis of lung cancer in patients;
FIG. 6 is a ROC graph showing the effect of CEA protein on the diagnosis of lung cancer in patients using CD47, CD63, TLR7 alone, in combination with TLR7.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Examples
A plasma exosome protein marker related to lung cancer is a plasma exosome protein TLR7, preferably a combination of plasma exosome proteins CD47, CD63 and TLR7.
The invention provides an antibody capable of specifically capturing the plasma exosome protein marker related to the lung cancer. The antibody is a monoclonal antibody against TLR7 protein and a biotinylated anti-TLR 7 protein antibody which are custom-synthesized by Abcam company; preferably, the antibodies are monoclonal antibodies against CD47, CD63, TLR7 proteins and biotinylated anti-CD 47, CD63, TLR7 proteins custom-synthesized by Abcam.
The invention provides application of the plasma exosome protein marker and the antibody in preparation of a lung cancer diagnosis kit.
The invention provides a lung cancer diagnosis kit, which is used for detecting the expression quantity of the protein marker in blood plasma.
The kit comprises the antibody. The kit also comprises reagents and enzymes commonly used by enzyme-linked immunization.
The kit comprises a monoclonal antibody for resisting TLR7 protein, a TLR7 standard substance, a biotinylated anti-TLR 7 protein antibody, horseradish peroxidase-labeled avidin and biotin.
The kit comprises monoclonal antibodies for resisting CD47, CD63 and TLR7 proteins, CD47, CD63 and TLR7 standards, biotinylated antibodies for resisting CD47, CD63 and TLR7 proteins, horseradish peroxidase-labeled avidin and biotin.
According to the invention, firstly, a lung cancer patient and a healthy control plasma sample matched with information such as age, sex and the like of the patient are separated, exosomes are extracted for protein mass spectrometry, a group of plasma exosome markers which are highly related to lung cancer and have higher sensitivity and specificity are screened out through sample verification after primary screening, and a kit applicable to clinical diagnosis of lung cancer is developed on the basis of the plasma exosome markers, so that a basis is provided for screening and early diagnosis of lung cancer.
The technical scheme for solving the problems comprises the following steps:
(1) Blood samples meeting the standard are collected by a Standard Operating Procedure (SOP), and complete clinical case information data are collected by the system.
(2) Screening and analyzing differential expression profiles of plasma exosome proteins: screening lung cancer patients and healthy people matched with the age and sex of the lung cancer patients, separating plasma exosomes, obtaining a plasma exosome protein expression profile through protein mass spectrometry, screening differentially expressed proteins and verifying the differentially expressed proteins through a sample.
(3) Quantitative analysis is carried out on the plasma exosome protein which is screened and differentially expressed, and the plasma exosome protein related to the lung cancer onset is determined.
(4) Based on the plasma exosome proteins screened above, diagnostic kits were developed.
Specifically, the method comprises the following parts:
(1) Collecting a patient sample: (1) cases of lung cancer diagnosed by imaging and confirmed by pathology; (2) all samples were preoperative, without radiotherapy, chemotherapy and neoadjuvant treatment; (3) healthy controls are normal persons matched to the age and sex of the patient. In the study, 38 standard lung cancer patients with plasma samples are used for the study.
(2) Plasma exosomes were extracted using the Total Exosome Isolation Kit (from plasma) (Invitrogen thermoses Fisher) Kit.
(3) Detecting the quality of the exosome: identifying the plasma exosomes through a transmission electron microscope, nanosight and Western blot.
(4) Protein mass spectrometry analysis:
(1) extracting total protein of plasma exosome, and performing protein mass spectrometry;
(5) Western blot method
(1) Extracting plasma exosomes;
(2) western blot analysis was performed using a protein antibody produced by Abcam corporation;
(4) detecting and comparing the expression change of the exosome protein in the plasma sample of the lung cancer patient with that of a healthy normal control.
(6) Diagnostic kit for preparing lung cancer plasma exosome protein
(1) And (3) detecting proteins with type difference and expression difference between the lung cancer patient and the normal control by protein mass spectrometry at the early stage, and further detecting plasma exosome proteins with more obvious difference in the lung cancer patient and the healthy control by Western blot technology to serve as an index for assisting lung cancer diagnosis. Finally, plasma exosome proteins associated with lung cancer were screened: CD47, CD63, TLR7 protein as diagnostic markers. A lung cancer diagnosis kit is developed on the basis, and comprises monoclonal antibodies resisting CD47, CD63 and TLR7 proteins, CD47, CD63 and TLR7 standard substances, biotinylated anti-CD 47, CD63 and TLR7 protein antibodies, horseradish peroxidase-labeled avidin, biotin and the like.
(7) Data analysis
All statistical tests were performed using GraphPad Prism software 7. Data are presented as mean ± SEMs. P < 0.05 is statistically significant for the differences. The normality and the equal variance of samples between groups were evaluated using the Shapiro-Wilk test and the Brown-Forsythe test, respectively. When the normality and variance equality between the sample groups was achieved, one-way analysis of variance (followed by Bonferroni multiple comparison test), two-way analysis of variance (followed by Bonferroni multiple comparison test) or t-test was used.
The following is a further description of the invention:
firstly, identifying the extracted plasma exosomes, and observing the exosome morphology by observing the morphology through a transmission electron microscope, as shown in fig. 1 (a); nanoparticle size analysis of exosomes, as shown in figure 1 (b); western Blot detection of exosome surface marker proteins, as shown in FIG. 1 (c); it can be seen that the form, size and biochemical indexes of the exosome all meet the test and research standards.
In the exploration stage, the inventor finds that 21 proteins have significant changes (fold change > 2) in lung cancer plasma exosome samples by screening 4 cases of lung cancer plasma exosomes and 4 cases of healthy control plasma exosomes respectively through protein mass spectrometry, and the result is shown in fig. 2.
According to the sequencing result, the inventor selects exosome proteins with remarkably increased expression in lung cancer patients and the increased levels in the first 4 positions to analyze (the increase multiple is greater than 2), the corresponding protein antibodies are customized and synthesized by Abcam company to detect, the proteins with statistically significant difference are further verified in 4 lung cancer cases and 4 controls one by one, and then the stability degree of the research result is observed, and the result shows that the increase of CD47, CD63 and TLR7 proteins in exosomes of a lung cancer case sample is most significant, and the result is shown in FIG. 3.
According to the above results, the above CD47, CD63, TLR7 proteins were further validated by ELISA in the plasma of 4 additional healthy controls and 4 lung cancer patients, and the results show that: CD47, CD63, TLR7 proteins were significantly elevated in lung cancer samples, and the results are shown in figure 4.
Further analyzing the effect of the protein on lung cancer diagnosis, using 30 patient samples to make ROC curve (Receiver operating characterization curve) analysis, finding that the TLR7 protein has better diagnosis effect on lung cancer, the result is shown in FIG. 5, the sensitivity reaches 83.33%, and the specificity reaches 80%.
The comparison of the combined use of CD47, CD63 and TLR7 proteins with the use of TLR7 protein alone and the diagnosis effect of the traditional carcinoembryonic protein CEA-based lung cancer is further analyzed, and the result is shown in FIG. 6. The combined use has better diagnosis effect on lung cancer, the area under the curve reaches 0.9328, the sensitivity reaches 93.33 percent, the specificity reaches 80 percent, and the sensitivity (66.67 percent) and the specificity (76.67 percent) of the traditional detection mode based on oncofetal protein CEA or the sensitivity (83.33 percent) and the specificity (80 percent) of a TLR7 protein single detection mode are obviously improved.
Claims (10)
1. A plasma exosome protein marker associated with lung cancer, characterized by: the plasma exosome protein marker is plasma exosome protein TLR7.
2. The plasma exosome protein marker associated with lung cancer according to claim 1, characterized in that: the plasma exosome protein marker is a combination of plasma exosome proteins CD47, CD63 and TLR7.
3. An antibody capable of specifically capturing a plasma exosome protein marker comprising a lung cancer according to claim 1 or claim 2.
4. Use of the plasma exosome protein marker according to claim 1 or claim 2 in the preparation of a lung cancer diagnostic kit.
5. Use of the antibody of claim 3 in the preparation of a lung cancer diagnostic kit.
6. An ELISA kit for lung cancer diagnosis, characterized in that the kit is used for detecting the amount of the exosome protein marker of claim 1 or claim 2 in plasma.
7. The diagnostic kit of claim 6, wherein said kit comprises the antibody of claim 3.
8. The diagnostic kit of claim 6 or 7, wherein said kit further comprises conventional reagents and enzymes.
9. The diagnostic kit of claim 8, wherein the kit comprises a monoclonal antibody against TLR7 protein, a TLR7 standard, a biotinylated anti-TLR 7 protein antibody, horseradish peroxidase-labeled avidin, biotin.
10. The diagnostic kit of claim 8, wherein the kit comprises monoclonal antibodies against CD47, CD63, TLR7 proteins, CD47, CD63, TLR7 standards, biotinylated antibodies against CD47, CD63, TLR7 proteins, horseradish peroxidase-labeled avidin, biotin.
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