CN112379094B

CN112379094B - Application of CST1-CTSH compound as esophageal cancer diagnosis marker

Info

Publication number: CN112379094B
Application number: CN202011135629.9A
Authority: CN
Inventors: 王力军; 孙玉龙; 杨亚云; 王弢
Original assignee: Shanghai Liangrun Biomedical Technology Co ltd
Current assignee: Shanghai Liangrun Biomedical Technology Co ltd
Priority date: 2020-10-22
Filing date: 2020-10-22
Publication date: 2022-07-26
Anticipated expiration: 2040-10-22
Also published as: WO2022083580A1; CN112379094A

Abstract

The invention relates to the field of medical diagnostics, in particular to application of a CST1-CTSH compound as an esophageal cancer diagnosis marker. The tissue specificity of the marker can be effectively improved through the detection of the CST1-CTSH compound, and the detection rate of early esophageal cancer can be effectively improved.

Description

Application of CST1-CTSH compound as esophageal cancer diagnosis marker

Technical Field

The invention relates to the field of medical diagnostics, in particular to application of a CST1-CTSH compound as an esophageal cancer diagnosis marker.

Background

Esophageal cancer (Esophageal carcinoma) is a common tumor of the digestive tract and one of the ten most common malignant tumors in the world.

Researches show that during the invasion and metastasis process of tumors, tumor cells must cross surrounding matrixes, the degradation of the matrixes is mainly related to various tissue proteolytic enzymes, and the researches show that the invasion and metastasis capacity of the tumor cells is closely related to the capacity of the cathepsin to degrade extracellular matrixes.

Cathepsin H (CTSH) is a lysosomal cysteine protease that is widely found in human tissue cells, an endopeptidase but also has exonuclease activity, which is usually synthesized in the inactive precursor form, with endopeptidase activity being obtained by autolysis at the acidic PH of lysosomes, and exopeptidase activity being obtained by endopeptidase activity. CTSH expression is up-regulated in most malignant tumors, such as breast, lung, stomach, melanoma, glioma, and its expression level is correlated with tumor malignancy. Cystatins SN (CST 1) is a cathepsin inhibitor, protein with 141 amino acids encoded by CST1 gene, and has a molecular weight of 16.4 kDa. The two disulfide bonds contained in the molecule of CST1 are typical secreted proteins that inhibit the activity of intracellular and extracellular tissue proteases, play an important role in tumor growth, angiogenesis, infiltration and metastasis, and it has been shown that high expression of CST1 is associated with various cancers.

The current tumor marker analysis for esophageal cancer mainly comprises CA125, CEA, CA199, SCCA, SCC and the like, but the positive rate of clinical detection is not higher than 30%. CTSH has poor tissue specificity when used as a tumor diagnostic marker, and is difficult to localize to a specific tumor type, and is expressed not only in high levels in gastric cancer but also in patients with gastric cancer, glioma, melanoma, and the like. Although CST1 has a certain differentiation in esophageal cancer, CST1 has a certain limitation as the esophageal cancer target due to its high homology in sequence with cystatins S (CST 4).

Disclosure of Invention

The invention relates to application of a quantitative detection agent of a cystatin SN and a Cathepsin H compound (CST1-CTSH compound) in preparation of a kit for diagnosis, auxiliary diagnosis or prognosis analysis of esophageal cancer.

Optionally, the quantitative detection agent is the cystatin SN and an antibody specific for the Cathepsin H, which can be used to perform co-immunoprecipitation or an elisa to detect the CST1-CTSH complex.

Optionally, the quantitative detection agent is an antibody specific to the CST1-CTSH complex.

Optionally, the specific antibody is a monoclonal antibody or a polyclonal antibody.

Optionally, the specific antibody is obtained by immunizing the amino acid sequence shown in SEQ ID NO. 1.

Optionally, the specific antibody has a label for indicating signal intensity.

Optionally, the label for indicating signal intensity is selected from any one or more of chromophore, digoxigenin-labeled probe, electron dense substance, colloidal gold, or enzyme.

The invention also relates to an esophageal cancer diagnosis, auxiliary diagnosis or prognosis analysis kit, which comprises the specific antibody defined above.

Optionally, the kit further comprises at least one of a solid support, a blocking solution, a color developing agent, a calibrator for CST1-CTSH fusion antigens, and a wash buffer.

Optionally, the solid support is a chemiluminescent plate.

The tissue specificity of the marker can be effectively improved through the detection of the CST1-CTSH compound, and the detection rate of early esophageal cancer can be effectively improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an SDS-PAGE electrophoresis of purified recombinant CST1-CTSH protein according to an embodiment of the present invention;

FIG. 2 is a calibration curve of the CST1-CTSH assay kit according to an embodiment of the present invention;

FIG. 3 is a sample concentration scatter plot of CST1-CTSH versus esophageal cancer and normal human findings in an embodiment of the present invention;

FIG. 4 is a ROC curve showing the diagnosis of esophageal cancer by CST1-CTSH according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention relates to application of a quantitative detection agent of a cystatin SN and a Cathepsin H complex (CST1-CTSH complex) in preparation of a kit for diagnosis, auxiliary diagnosis or prognostic analysis of esophageal cancer.

The present invention provides a novel marker for diagnosis: CST1-CTSH Complex. CTSH is reported to be inhibited by CST1 to bind to form a complex, whereas CST4 does not have this ability. Therefore, the tissue specificity of the marker can be effectively improved through the detection of the CST1-CTSH compound, and the detection rate of early esophageal cancer can be effectively improved.

The term "marker" as used herein refers to a molecule to be used as a target for the analysis of 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. The immunologically detectable fragment preferably comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 consecutive amino acids of the marker polypeptide. One skilled in the art will recognize that proteins released by cells or present in the extracellular matrix may be damaged (e.g., during inflammation) and may be degraded or cleaved into such fragments. Certain markers are synthesized in an inactive form, which can be subsequently activated by proteolysis. As will be appreciated by the skilled artisan, proteins or fragments thereof may also be present as part of a complex. Such complexes may also be used as markers in the sense of the present invention. In addition, or in the alternative, the marker polypeptide or variant thereof may carry post-translational modifications. Non-limiting examples of post-translational modifications are glycosylation, acylation and/or phosphorylation. In particular, the marker should be located at the binding site of CST1 and CTSH in the CST1-CTSH complex, and this "binding" refers to the site where the amino acid sequences of CST1 and CTSH interact with each other, and may be a linear epitope or a steric epitope.

In some embodiments, the quantitative detector is an antibody specific for the cystatin SN and the Cathepsin H, which can be used to perform co-immunoprecipitation or enzyme-linked immunosorbent assay to detect the CST1-CTSH complex.

The quantitative detection agent is generally a reagent that specifically detects the CST1-CTSH complex, for example, a lectin that specifically binds to the CST1-CTSH complex, an aptamer that specifically binds to the CST1-CTSH complex, or an antibody and an antibody fragment that specifically binds to the CST1-CTSH complex. The specific binding agent has at least 10 for its corresponding target molecule ⁷ l/mol affinity. The specific binding agent preferably has a binding specificity of 10 for its target molecule ⁸ l/mol, or more preferably 10 ⁹ l/mol affinity. The skilled person will understand that the term "specific" is used to indicate that other biomolecules present in the sample do not bind significantly to the quantitative detector of the CST1-CTSH complex, such biomolecules being in particular CST1 and CTSH, which are free alone.

In some embodiments, the quantitative detection agent is an antibody specific for the CST1-CTSH complex.

In some embodiments, the specific antibody is a monoclonal antibody or a polyclonal antibody.

In some embodiments, the specific antibody is derived from immunization with the amino acid sequence shown in SEQ ID NO. 1.

The antibody immunization may be followed by a screening process, as will be readily appreciated by those skilled in the art, by screening the recombinant protein CST1-CTSH for antibodies that specifically bind to the CST1-CTSH complex, optionally further including selecting antibodies with high antibody titers.

In some embodiments, the specific antibody has a label for indicating signal intensity.

In some embodiments, the label for indicating signal intensity is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron dense substance, colloidal gold, or an enzyme.

The following non-limiting section lists these markers:

enzymes which produce a detectable signal, e.g.by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.

Chromophores such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds (such as acridinium esters or derivatives thereof), and dyes.

Groups with electron density that can be detected by electron microscopy or by its electrical properties, such as conductivity, amperometry, voltage measurement and resistance.

A detectable group, such as one whose molecular size is sufficient to induce a detectable modification in its physical and/or chemical properties; such detection can be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variations, and contact variation angles) or physical methods (e.g., atomic force spectroscopy and tunneling).

Electron-dense substances, e.g. radioactive molecules (e.g. of the type ³² P， ³⁵ S or ¹²⁵ I)。

According to a further aspect of the invention, the invention also relates to an esophageal cancer diagnostic kit comprising specific antibodies as defined above.

In some embodiments, the kit further comprises at least one of a solid support, a blocking solution, a color developer, a calibrator for CST1-CTSH fusion antigens, and a wash buffer.

The calibrator for the CST1-CTSH fusion antigen preferably has the amino acid sequence shown in SEQ ID NO. 1.

The blocking solution may be one or more of BSA, bovine serum, skimmed milk, TBST, etc.

The color developing solution can be determined according to the substance labeled on the antibody, for example, when the labeled substance is horseradish peroxidase, the color developing solution can be luminol.

The washing buffer may be PBS, TBS, or the like.

The blocking solution, the developing solution, and the washing buffer solution may be packaged in the kit in the form of working concentrations, or may be packaged in the form of concentrated mother solutions thereof (e.g., mother solutions concentrated 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 times).

The solid phase carrier is usually used for coating the antibody, the solid phase carrier used for coating the antibody can be polystyrene, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, agarose gel and other materials, and the form of the carrier can be a test tube, an EP tube, a multi-well plate (especially a chemiluminescence plate), a micro-reaction plate concave hole, a bead (especially a magnetic bead), a small disc and the like.

The preferred solid support is a chemiluminescent plate. It may contain 16, 32, 48, 64, 96 or more holes.

According to a further aspect of the present invention, there is also provided a method for diagnosis, co-diagnosis or prognostic analysis of esophageal cancer, the method comprising: the content of the CST1-CTSH complex was measured using the quantitative detector/kit as described above.

The sample to be tested can be at least one of blood, serum, cerebrospinal fluid, tissue or tissue lysate, semen, and saliva sample of the subject.

The subject is typically a mammal, preferably a primate, more preferably a human.

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

Example 1 expression and purification of CST1-CTSH recombinant protein

Protein expression: according to the sequence table SEQ ID NO:1 and optimized to mammalian expression codons. The gene was inserted into pcDNA3.1 vector containing 6 XHis tag to obtain pcDNA3.1-CST 1-CTSH. Then pcDNA3.1-CST1-CTSH is transformed into DH5 alpha, after positive clone is picked up and mass culture, the recombinant plasmid pcDNA3.1-CST1-CTSH is extracted by a high-purity plasmid extraction kit. The recombinant plasmid is transferred into 293t cell, and pcDNA3.1 empty vector is simultaneously transfected as negative control, respectively in DMEM culture medium containing 10% fetal calf serum at 37 deg.C and 5% CO ₂ Culturing for 72h under the conditions, collecting the supernatant, and filtering the supernatant with 0.22 μm filter membrane.

SEQ ID NO:1

MWATLPLLCAGAWLLGVPVCGAAELCVNSLEKFHFKSWMSKHRKTYSTEEYHHRLQTFASNWRKINAHNNGNHTFKMALNQFSDMSFAEIKHKYLWSEPQNCSATKSNYLRGTGPYPPSVDWRKKGNFVSPVKNQGACGSCWTFSTTGALESAIAIATGKMLSLAEQQLVDCAQDFNNHGCQGGLPSQAFEYILYNKGIMGEDTYPYQGKDGYCKFQPGKAIGFVKDVANITIYDEEAMVEAVALYNPVSFAFEVTQDFMMYRTGIYSSTSCHKTPDKVNHAVLAVGYGEKNGIPYWIVKNSWGPQWGMNGYFLIERGKNMCGLAACASYPIPLVGGGSGGGSGGGSGGGSGGGSWSPKEEDRIIPGGIYNADLNDEWVQRALHFAISEYNKATKDDYYRRPLRVLRARQQTVGGVNYFFDVEVGRTICTKSQPNLDTCAFHEQPELQKKQLCSFEIYEVPWENRRSLVKSRCQES

Protein purification: the resulting 500mL of filtrate was subjected to Ni-NTA affinity chromatography under non-denaturing conditions in 50mM PBS, 10mM imidazole, 150mM NaCl, pH7.6 in equilibration buffer. After the sample loading is finished, washing by 10 mL; the eluate was collected by eluting with 50mM PBS, 250mM imidazole, 150mM NaCl, pH 7.6. The protein solution was concentrated using a 3kD ultrafiltration tube and the protein was stored in PBS buffer at pH 7.450 mM and at-80 ℃. The purity of the purified protein is identified by SDS-PAGE electrophoresis, the molecular weight is about 52kD, and gray analysis shows that the purity of the protein reaches more than 95 percent, which is shown in figure 1.

Example 2 validation of the Activity of CST1-CTSH recombinant protein and antibody pairing

And (3) activity analysis: the chemiluminescence plate is coated with 1ug/ml carbonate buffer (pH9.5) of recombinant CST1-CTSH protein at 100ul volume and 4 ℃ overnight, the capture antibody and the enzyme-labeled antibody (concentration is 0-1 ug/ml) are diluted in a gradient manner, and goat anti-mouse IgG-HRP (100ng/ml) is added. The detection shows that the luminescence values of the capture antibody and the detection antibody are not less than 20 ten thousand at 100ng/ml, and the reaction curve R of the protein and the antibody ² >0.99, the reactivity of the protein meets the requirement.

Antibody pairing: the chemiluminescence plate is coated with 1ug/mL capture antibody, 100uL of CST1-CTSH calibrator with different concentrations (5-1000 pg/mL) is added, incubation is carried out for 60min at 37 ℃, 100uL of horseradish peroxidase labeled detection antibody with concentration of 100ng/mL is added after washing, incubation is carried out for 60min at 37 ℃, chemiluminescence substrate is added after washing, and luminescence intensity of each well is measured. From the result, the capture antibody and the detection antibody are well paired, and can be used for constructing a double-antibody sandwich system.

Example 3 CST1-CTSH calibration Curve preparation

And (3) drawing a calibration curve: the capture antibody was first coated overnight on a chemiluminescent plate at 4 ℃ at a concentration of 1. mu.g/mL, recombinant human CST1-CTSH calibrator protein was diluted with protein stabilizer to 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1500pg/mL, 100. mu.L per well, and horseradish peroxidase-labeled detection antibody was added at a concentration of 5ng/mL, 100. mu.L per well and incubated for 1 hour at 37 ℃. PBST was washed 3 times, a chemiluminescent substrate was added and the luminescence intensity of each well was measured. The CST1-CTSH content of the tested sample is calculated from the calibration curve. The linear range of the calibration curve is 10-1500 pg/mL, and the attached figure 2 is a calibration curve of the CST1-CTSH detection kit, wherein a Y axis represents a light-emitting value logarithm value, and an X axis represents a concentration logarithm value of a CST1-CTSH calibrator.

Example 4 clinical Performance validation of CST1-CTSH kit

The CST1-CTSH detection kit is used for diagnosing esophageal cancer: collecting 50 cases of serum of esophageal cancer patients before operation from a hospital; serum was collected from 50 healthy blood donors at the same time. The CST1-CTSH detection kit is used for detecting the concentration of CST1-CTSH in the serum of esophagus cancer and normal human. The sample concentration scatter plot shows that CST1-CTSH has statistical significance for distinguishing the detection results of esophageal cancer and normal people, and the result is shown in figure 3. The ROC curve statistical result shows that the area under the curve is 0.907, 86pg/mL is used as a detection reference value, the specificity of the CST1-CTSH detection kit is 90.2%, and the sensitivity is 83.7%, see figure 4.

In conclusion, the kit provided by the invention adopts the monoclonal antibody specifically aiming at CST1-CTSH as the capture and detection antibody, so that the kit also has the characteristics of high sensitivity, good specificity, low detection limit, good stability and the like. The linear range reaches 10-1500 pg/mL, and the minimum detection limit can reach 5 pg/mL. The CST1-CTSH compound has the tissue specificity of CST1 and CTSH protein, and when an esophageal cancer sample is detected, the specificity is 90.2%, and the sensitivity can reach 83.7%. The CST1-CTSH detection kit can be used for early diagnosis of esophageal cancer, curative effect evaluation in the treatment process and metastatic relapse monitoring after treatment.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Application of <120> CST1-CTSH compound as esophageal cancer diagnosis marker

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Claims

1. Use of a quantitative detection agent for a cystatin SN and Cathepsin H complex, namely CST1-CTSH complex, in the preparation of a kit for the diagnosis, auxiliary diagnosis or prognostic analysis of esophageal cancer, wherein the quantitative detection agent is an antibody specific for the CST1-CTSH complex.

2. The use according to claim 1, wherein the specific antibody is a monoclonal antibody or a polyclonal antibody.

3. The use according to claim 2, wherein the specific antibody is obtained by immunization with the amino acid sequence shown in SEQ ID NO. 1.

4. The use according to any one of claims 1 to 3, wherein the specific antibody has a label for indicating signal intensity.

5. The use according to claim 4, wherein the label for indicating signal intensity is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme.

6. An esophageal cancer diagnosis, auxiliary diagnosis or prognostic assay kit, characterized in that it comprises a specific antibody as defined in any one of claims 1 to 5.

7. The kit according to claim 6, further comprising at least one of a solid support, a blocking solution, a color developing agent, a calibrator for CST1-CTSH fusion antigen, and a washing buffer.

8. The kit of claim 7, wherein the solid support is a chemiluminescent plate.

9. The kit of claim 7, wherein the calibrator for the CST1-CTSH fusion antigen has an amino acid sequence shown in SEQ ID No. 1.

10. The kit of claim 7, wherein the blocking solution is one or more of BSA, bovine serum, skim milk and TBST.