CN108872599A - PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker - Google Patents
PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker Download PDFInfo
- Publication number
- CN108872599A CN108872599A CN201810747903.4A CN201810747903A CN108872599A CN 108872599 A CN108872599 A CN 108872599A CN 201810747903 A CN201810747903 A CN 201810747903A CN 108872599 A CN108872599 A CN 108872599A
- Authority
- CN
- China
- Prior art keywords
- pdlim3
- gastric cancer
- expression
- antibody
- individual
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Application the invention discloses PDLIM3 as diagnosing gastric cancer marker.The research of the invention finds that the expression of patients with gastric cancer PDLIM3 gene or albumen is compared with Healthy People or cancer beside organism, expression is significantly increased, therefore can be used as diagnosing gastric cancer marker, is used to prepare diagnosing gastric cancer preparation and reagent kit product.The present invention provides a kind of gastric cancer biomarker PDLIM3 of new specificity, and give the detection method of the PDLIM3 with reference value.Gastric cancer screening/diagnosis effect of the gastric cancer biomarker PDLIM3 for gastric cancer is good, and accuracy is high, has a good application prospect, and is worth a wide range of and promotes.
Description
Technical field
The present invention relates to biotechnologys and field of immunology, and in particular to a kind of PDLIM3 is used as the molecule mark of detection gastric cancer
The application of note.
Background technique
Gastric cancer ranks first in the various malignant tumours in China, and incidence gastric cancer has apparent region gender gap, in the west in China
North is evident as with coastal region in east China incidence gastric cancer rate than southern area high.The hair age is at 50 years old or more well, men and women's disease incidence it
Than being 2: 1.The prognosis of gastric cancer is related with the pathological staging of gastric cancer, position, organization type, biological behaviour and remedy measures.
Relatives its incidence gastric cancer rate that heredity shows that Patients with Gastric Cancer has relationship by blood with molecular biology research is compared with control group
It is 4 times high.The canceration of gastric cancer is multifactor, a multi-step, Multi stage development process, is related to oncogene, tumor suppressor gene, apoptosis phase
The change of correlation gene and metastasis related gene etc., and the form of gene alteration is also diversified.
Early carcinoma of stomach majority patient's non-evident sympton, a few peoples have the upper digestive tract of Nausea and vomiting or similar canker
Symptom.Pain and weight loss are the most common clinical symptoms of advanced gastric carcinoma.The more specific upper digestive tract disease of patient Chang You
Shape, it is loss of appetite, out of strength as disease progression Upper abdominal pain aggravates as glutted after epigastric discomfort, feed.According to the portion of tumour
Position is different, also there is its Special Manifestations.Cardia stomach bottom cancer can have retrosternal pain and progressive dysphagia；Gastric cancer near pylorus
There is pyloric stenosis performance；There can be the hemorrhages of digestive tract symptom such as spitting blood, melena after tumor destruction blood vessel.Abdomen constant pain often prompts
Expanding tumor exceeds recessed before stomach wall, such as supraclavicular lymph nodes enlargement, ascites, jaundice, abdominal mass, rectum lay one's hand on and lump.Evening
Phase Patients with Gastric Cancer often may occur in which anaemia, syntexis, malnutritive even cachexia etc. performance.
Current diagnostic method has x-ray barium meal examination, fibergastroscopy, abdominal ultrasonic, spiral CT and positron emission
Imaging inspection etc..All there is respective defect in these methods, if Imaging Technology is difficult to find the lesser tumour of knurl, early stage is general
The omission factor looked into is higher.Lack a species specific gastric cancer biomarker, the phase of specific gastric cancer biomarker at present
Research is closed to have great importance.
(HGNC accession number is 20767 to PDLIM3；Entrez Gene accession number is 27295；EnsembI accession number is
ENSG00000154553；OMIM accession number is 605889；UniProtKB accession number is Q53GG5) the main table in muscle of albumen
It reaches, belongs to FOZ/LIM protein family, directly combined at skeletal muscle Z-line with actin, in mechanics of muscle perception and signal
Key effect is played in conduction.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide a species specific gastric cancer biomarker.
Application the first purpose of the invention is to provide PDLIM3 as diagnosing gastric cancer marker.
A second object of the present invention is to provide application of the PDLIM3 protein fragments in the detection of PDLIM3 protein quantification.
Third object of the present invention is to provide PDLIM3 albumen to prepare answering in gastric cancer screening/diagnosis kit
With.
Fourth object of the present invention is to provide PDLIM3 albumen and is preparing the application in gastric cancer screening/diagnosis reagent.
Fifth object of the present invention is to provide PDLIM3 protein fragments in preparing gastric cancer screening/diagnosis kit
Using.
Sixth object of the present invention is to provide PDLIM3 protein antibodies in preparing gastric cancer screening/diagnosis kit
Using.
7th purpose of the invention is to provide a kind of for gastric cancer screening/diagnosis kit.
To achieve the goals above, the present invention is achieved by the following technical programs：By screening in mankind's stomach
The protein of differential expression in cancerous tissue and corresponding cancer beside organism, present inventor have found it is a kind of in stomach organization and
The protein (up-regulated expression in cancerous tissue) that expression is had differences in corresponding cancer beside organism is PDLIM3 through Mass Spectrometric Identification.
Immunoblot experiment confirms that PDLIM3 has differences expression (in cancerous tissue in stomach organization and corresponding cancer beside organism really
Middle expression up-regulation).It is further demonstrate,proved by the immunohistochemical assay that the cancerous tissue to 55 pairs of human gastric cancers is carried out with cancer beside organism
Real PDLIM3 has differences expression (expression is raised in cancerous tissue) in the cancerous tissue of Human Gastric carcinoma and cancer beside organism, and
The immunological experiment of serum also shows PDLIM3 high expression in Human Gastric carcinoma patient.
This correlation based on PDLIM3 with gastric cancer, PDLIM3 can be used as a kind of protein molecular marker object to it
Expression quantity, which carries out detection, can be used for detecting gastric cancer.
Therefore, primary and foremost purpose of the invention is to be to provide the PDLIM3 application for being used as detection gastric cancer.
The application of PDLIM3 provided by the invention is, as the protein molecular marker object for detecting gastric cancer.
PDLIM3 provided by the invention is used to detect the application of the protein molecular marker object of gastric cancer to prepare Quantitative Western
The kit of matter group technology.
It is another object of the present invention to provide the applications of the antibody of PDLIM3.
The application of the antibody of PDLIM3 provided by the invention is to be used to prepare the preparation of detection gastric cancer and/or be used to prepare
Detect the kit of gastric cancer.
According to the present invention, the antibody of anti-PDLIM3 includes monoclonal antibody and polyclonal antibody.
Yet another object of the invention is that whether the expression that provides PDLIM3 in a kind of vitro detection gastric tissue is abnormal
Method.
Method provided by the invention is：Detect the amount of PDLIM3 in gastric tissue/serum to be measured, and with normal gastric mucosa/blood
The amount of PDLIM3 in clear is compared.
In view of up to the present, there are no the correlation reports of PDLIM3 and gastric cancer.Therefore, this discovery of the invention will
A completely new approach is provided for diagnosing and/or treating for gastric cancer.
Moreover, it relates to following embodiments：
1. the method for diagnosis of gastric cancer, the method includes：
A) the doubtful cancerous tissue of individual is obtained,
B) expression of PDLIM3 therein is measured,
C) the same tissue of normal individual or the normal tissue of same individual are obtained, and
D) expression of PDLIM3 therein is measured as control expression level,
E) by the b) expression of the middle PDLlM3 obtained and d), the control expression level of the middle PDLIM3 obtained compares
Compared with when the expression of the PDLIM3 obtained in b) improves compared with the control expression level of the PDLIM3 obtained in d), really
The fixed individual suffers from gastric cancer.
2. according to method described in embodiment 1, wherein the tissue is gastric tissue.
3. the method according to embodiment 1 or 2, wherein the expression is examined by either method selected from the following
It surveys：
(a) mRNA of detection coding PDLIM3, and
(b) protein of PDLIM3 is detected.
4. the method according to any one of embodiment 1-3, wherein the amount of the PDLIM3 is by using anti-
The antibody of PDLIM3 is measured.
5. the method according to any one of embodiment 1-4, wherein the antibody of the anti-PDLIM3 is that monoclonal is anti-
Body or polyclonal antibody.
6. the method for diagnosis of gastric cancer, the method includes：
A) serum of individual is obtained,
B) expression of PDLIM3 therein is measured,
C) serum of normal individual is obtained,
D) expression of PDLIM3 therein is measured as control expression level, and
E) by the b) expression of the middle PDLIM3 obtained and d), the control expression level of the middle PDLIM3 obtained compares
Compared with when the expression of the PDLIM3 obtained in b) improves compared with the control expression level of the PDLIM3 obtained in d), really
The fixed individual suffers from gastric cancer.
7. according to method described in embodiment 6, wherein the expression of the PDLIM3 is by using anti-PDLIM3's
Antibody is measured.
8. the method according to embodiment 6 or 7, wherein the antibody of the anti-PDLIM3 is monoclonal antibody or more grams
Grand antibody.
9. purposes of the antibody of anti-PDLIM3 in reagent preparation box, the kit is used to diagnose the gastric cancer of individual.
10. the kit for the gastric cancer for diagnosing individual, the kit include
A) it is used to measure the reagent of the expression of PDLIM3, and
B) operation instructions.
Other aspects and advantages of the present invention can be known by the description refering to embodiment in detail below and in conjunction with attached drawing
Dawn.
Detailed description of the invention
Fig. 1 carries out data to the immunoblotting map of PDLIM3 and analyzes the protein expression quantity Relative distribution figure obtained.
Fig. 2 carries out data to PDLIM3 immunohistochemical assay result and analyzes the protein expression quantity Relative distribution obtained
Figure.
Fig. 3 carries out data to PDLIM3 Proteins in Serum content experimental result and analyzes Relative distribution figure.
Specific embodiment
In a preferred embodiment, the segment of the albumen is preferably used.The segment should be with the egg in sample
White or its segment obtained after digesting (such as trypsin digestion) sequence is completely the same.For convenience of operation, the segment
Length be preferably 5-50 amino acid, preferably 6-30 amino acid, more preferably 6-25 amino acid.In addition, the segment
Best Transition should have preferable signal-to-noise ratio.It is described preferably pass through heavy isotope (such as 13C, 14N) label, so as to by its with
Un-marked sample is mutually distinguished.
Table 1.PDLIM3 peptide section sequence.
It include by a certain amount of segment by heavy label to the step of carrying out early detection with patients with gastric cancer
It is added in the patient serum sample digested through enzyme (such as trypsase), in the technology detection patients serum of quantitative proteomics
The level of the segment, so that it is determined that PDLIM3 is horizontal in serum；Measured level is determined with according to the level of normal person
Critical value compare, if measured level be higher than critical value, then it represents that gastric cancer illness probability is big.
The critical value can be determined by those skilled in the art according to conventional means.For example, those skilled in the art
Receiver operating curve can be drawn according to the serum protein content data of the normal person's (group) measured, and (ROC is bent
Line), it is later determined that critical value (cut-off).
In a more preferred embodiment, the quantitative proteomics technology can with it is exhausted based on section of synthesized peptide
Quantitative technique (absolute quantification analysis, AQUA) is combined, it thus can be directly to multiple
PDLIM3 in sample directly carries out the detection of absolute content.By measured level compared with the level of normal control serum,
If measured level is higher than the level of normal control serum, then it represents that the object suffers from gastric cancer.
In another preferred embodiment, the substance in conjunction with people's PDLIM3 protein-specific is antibody.This
The term " antibody " at place has most broadly meaning, specifically includes：Monoclonal antibody (monoclonal antibody including overall length),
Polyclonal antibody, multi-specific antibody (such as bispecific antibody).Term " antibody " further includes the segment of entire molecule, such as
It is capable of the Fab and F (ab ') 2 of combining response antigen.Fab and 2 segment of F (ab ') lack the Fe segment of complete antibody, can be more fast
Fast ground is removed from the circulatory system, and with lower non-specific tissue binding compared with complete antibody.In the present invention
Useful Fab and F (ab ') 2 and other antibody fragments can also be used for detecting and quantitative determining people PDLIM3, as long as they are showed
The activity of required specific binding out.The usually available papain (generating Fab segment) of these segments or pepsin (generate F
(ab ') 2 segment) it is generated by protease hydrolytic cutting.
It can be made with conventional method for polyclonal antibody and monoclonal antibody of the invention.In general, first with albumen come
Suitable animal is immunized, it is preferred that mouse, rat, rabbit or goat.Since obtainable serum volume is more, can be marked
Anti- rabbit and anti-goat antibody, therefore for preparing for polyclonal antiserum, rabbit and goat are preferable.It is immune usual
It carries out in this way：Albumen is mixed or emulsified with salt water (preferably with adjuvant such as Freund's complete adjuvant), then parenteral is (usually
Subcutaneously or intramuscularly) inject the mixture or emulsion.The egg matched after 2-6 weeks with salt water (preferably not exclusively helping neat U with Freund)
White matter injection is one or many with reinforced immunological.Animal blood after will be immune is drawn into glass or plastic containers, 25 DEG C of trainings
It educates the blood I hours, then 4 DEG C cultivation 2-18 hours, obtains polyclonal antiserum.Monoclonal antibody can with Kohler and
Standard method [the Nature (1975) 256 of Milstein：495-96] or its improved method be made.In general, as described above to small
Mouse or rat immunity.However, being not to take blood then to extract serum to animal, but take out spleen and (and optionally take out several
A big lymph node), it is dispersed into unicellular.If desired, can be by cell suspension (in the cell for removing non-specific adhesion
It is added and has been coated in the plate or hole of proteantigen afterwards), splenocyte is screened.The film for expressing antigentic specificity, which combines, to be immunized
The B cell of globulin is integrated on plate, is washed away not as suspension other materials.Then to gained B cell or all dissociation
Splenocyte induced, make it merge to form hybridoma with myeloma cell, cultivate it is (as follows yellow fast in selective medium
Purine, aminopterin, thymidine medium, " HAT ") in.The hybridoma as obtained by limiting dilution inoculation, and measure specific binding and exempt from
The generation of the antibody of epidemic disease antigen (and not combining irrelevant antigen).Then, external (such as reacted in tissue culture flasks or doughnut
In device) or in vivo (such as in mouse ascites) the selected secrete monoclonal antibody of culture hybridoma.
Immunofluorescence technique can be used with antigen in antibody determination serum, using the antibody of fluorescent marker and in conjunction with optical microphotograph
Art, flow cytometry or fluorescence metering art are detected and are realized.Test method is for example including making biological sample (such as biofluid
Such as serum) it is cultivated in the presence of can identify detectable label antibody, then in numerous methods well known in the art
Any method detects antibody.
Biological sample can with solid support or carrier (such as nitrocellulose) or be capable of fixing cell, cell granulations or
Other solid supports or carrier of soluble protein are handled.Then support or carrier, then root are washed with suitable buffer
It is handled like that with the antibody of detectable label according to the present invention is above-mentioned.Solid support or load are washed with second of buffer again
Body removes unbonded antibody.Then conventional method can be used to detect the label combined on the solid support or carrier
Amount.Well known support or carrier include：It is glass, polystyrene, polypropylene, polyethylene, glucan, nylon amylases, natural
And modified cellulose, polyacrylamide, gabbro and magnetite.The structure of support or carrier can be spherical (such as exist
In bead), cylindrical (inner surface of such as test tube or the outer surface of stick).In addition, surface can be flat, such as plate, test
Band etc..Preferable support or carrier include polystyrene bead.Skilled person will appreciate that road is for binding antibody or resists
Other former many suitable carriers, or these carriers can be determined by routine experiment.
In the present invention, antibody can be connected with enzyme, is subsequently used for EIA enzyme immunoassay.Then, when the enzyme and suitable bottom
When object contacts, enzyme meeting and substrate reactions, to generate and can be detected (such as pass through spectrophotometric analysis, fluorescence analysis or visually
Observation) chemicals.The enzyme that can be used for detectably labelled antibody includes but is not limited to：Malic dehydrogenase, grape ball
Bacterium nuclease, S-5- steroid isomeras, yeast alcohol dehydrogenase, a- glycerolphos phate dehydrogenase, phosphotriose isomerase, horseradish
Peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta galactosidase, ribalgilase, urase, mistake
Hydrogen oxide enzyme, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.Using chromophore's substrate of enzyme, utilize
Colorimetric method can be completed to detect.Detection can also by the degree of reacting enzyme-to-substrate and the reference substance of similar preparation into
Row naked eyes are relatively realized.
Detection can be realized with other various immunity tests.For example, passing through radiolabelled antibody, so that it may by using putting
Radioimmunoassay (RIA) detects.Radioactive isotope can with the equipment such as scintillation counter or by radioactive automatic developing come into
Row detection.
Antibody of the invention can also be marked with fluorescent chemicals.When the antibody of fluorescent marker is exposed to appropriate wavelength
Light when, its presence can be detected because of fluorescence.Most common fluorescent labelling compound has fluorescein isothiocynate, Luo Dan
Bright, phycoerythrin, phycocyanin, allophycocyanin, phthalic aldehyde and fluorescamine.The metal of sending fluorescence also can be used (such as in antibody
152E or other lanthanide series metals) detectably marked.These metals can pass through chelation group (such as diethyl three of these metals
Triamine pentaacetic acid (ETPA)) it is connect with antibody.Antibody coupling chemiluminescence compound can also be subjected to detectable mark to antibody
Note.It is then detected that the presence to shine in chemical reaction process detects the presence of the antibody with chemiluminescent labeling.Especially
The example of useful chemiluminescent labeling compound is luminol, different luminol, hot A Ding Gun ester, imidazoles, A Ding Gun salt and grass
Acid esters.Same way, it is also possible to mark antibody of the present invention with bioluminescent compound.Bioluminescence is found in biosystem
A kind of chemiluminescence, wherein catalytic protein improves the efficiency of chemiluminescence reaction.The presence of bioluminescent protein can pass through
Detection shines to determine.Important bioluminescent compound for labeling purposes is luciferin, luciferase and aequorin
Albumen.
Antibody of the invention can also be used for immunoassay test, such as sandwich assay.It, will in typical immunoassay test
A certain amount of unmarked antibody (or antibody fragment) is incorporated on solid support or carrier, and the detectable mark of a certain amount of band is added
The solvable antibody of note, so as to the ternary detected and/or quantitative analysis is formed between solid phase antibody, antigen and labelled antibody
Compound.Typical and preferable immunometric assays are tested including " forward direction ", first with the antibody of solid phase binding in this experiment
It contacts with sample to be tested, is extracted antigen from sample by forming binary solid matrix antibody-antigen compound.It is training
After educating the suitable time, wash solid support or carrier with remove fluid sample residue (including unreacted antigen, if
If having), then contacted again with the solution of the labelled antibody containing unknown quantity.Cultivating at second makes labelled antibody by not marking
After note antibody and the antigen being incorporated on solid support or carrier are compound, second of washing solid support or carrier are removed
Remove unreacted labelled antibody.
Third aspect present invention provides a kind of kit that gastric cancer whether is suffered from for test object, the kit packet
Containing amino acid sequence shown in table 1.
In a preferred embodiment, the amino acid sequence can also carry label.The preferred isotope of label
Label.The kit also may include specification, and the specification provides instruct user to apply aforementioned polypeptides sequence as described above
Column carry out the explanation whether test object suffers from gastric cancer.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this
Invention is not for limiting the scope of the invention.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as Sambrook et al.,
Molecular cloning：Laboratory manual (New York：Cold Spring Harbor Laboratory Press, 1989) described in
Condition, or according to the normal condition proposed by manufacturer.
In following embodiments of the invention, urea, 3- [(3_ gallbladder amido propyl) _ diethyl ammonium] -1- propane sulfonic acid
(CHAPS), dodecyl sodium sulfate (SDS), dithiothreitol (DTT) (DTT), three (methylol) aminoethanes (Tris), iodoacetamido
Amine (IAA) is purchased from Bio-Rad company；The chemical reagent such as beta -mercaptoethanol are purchased from Sigma company；Trypsase (Trypsin,
Sequencing grade) it is purchased from Promega company；iTRAQ(isobaric tags for relative and
Absolute quantitation) reagent be purchased from AB SCIEX；SCX column and SAX column and pH buffer kit are purchased from
Waters company.
In following embodiments of the invention, the solution formula used is as follows：
Lysate：8mol/L urea, 4%CHAPS, 40mmol/L Tris and 65mmol/L DTT.
TBST：Tris 2.42g/L, sodium chloride 8g/L, Tween_201ml/L adjust pH to 7.6 with HCl.
The preparation of embodiment 1, the cancerous tissue of gastric cancer and cancer beside organism's protein example.The present inventor is prepared with enzymolysis sample
The cancerous tissue and cancer beside organism's protein example of the gastric cancer of method (enzymatic sample preparation, ESP) preparation, with
ITRAQ (isobaric tags for relative and absolute quantitation) Different Proteomics mass spectrum
It is quantitative.As a result, it has been found that PDLIM3 high expression in gastric cancer cancerous tissue.Immunoblot experiment and immunohistochemical assay are further
Confirm that PDLIM3 has differences expression in the cancerous tissue of gastric cancer and cancer beside organism really.The present invention is completed on this basis.
Therefore, detection is carried out using PDLIM3 as expression quantity of the protein molecular marker object to it can be used for detecting
Gastric cancer, i.e. PDLIM3 may be used as the protein molecule of detection gastric cancer.
The cancerous tissue of 4 patients with gastric cancer and cancer beside organism, 4 patients are made a definite diagnosis by 2 Pathologis, suffer from stomach
The pathological data of cancer, 4 patients is as shown in table 2.
Table 2：The pathological data of 4 patients with gastric cancer.
The cancerous tissue and cancer beside organism's protein sample of the gastric cancer of above-mentioned 4 patients are prepared with enzymolysis sample the preparation method in solution
Product (cancerous tissue used and cancer beside organism's sample be the paired samples for being derived from same patients with gastric cancer), detailed process is as follows：
Operation excision flesh tissue block be immediately placed on ice, be quickly cut into several naked eyes it is visible, without the small of necrotic zone
Block.After washing tissue fritter for several times with the PBS solution of pre-cooling, it is quickly ground into cell precipitation in liquid nitrogen, then cell sinks
Shallow lake is dissolved in lysate respectively, then under condition of ice bath, uses ultrasonic cell disintegration instrument (Soniprep 150, Britain, MSE
Company) after interval ultrasonication 2min, in 15000g/min, 4 DEG C of centrifugation 1h, take supernatant, with the Bradford method of improvement (see
Bio-Rad Products specification) to carry out gross protein quantitative.
Embodiment 2：The screening of cancerous tissue and the differentially expressed protein of cancer beside organism
Using iTRAQ technology (referring to Pichler P et al.Anal Chem.2010Aug 1；82(15)：6549-
58.) zymolysis technique (reference William M.Old et al.Mol Cell Proteomics.2005 in binding soln；4：
1487-1502) with yin-yang multidimensional Liquid Chromatography-Tandem Mass Spectrometry technology (referring to Jie Dai et al.J
ProteomeRes.2007；6(1)：The screening of differentially expressed protein 250-262) is carried out, detailed process is as follows：
The 4 pairs of stomach organizations and each 200ug of cancer beside organism's protein example obtained in Example 1, with enzyme in solution
Solution technology is digested into peptide fragment, is then replaced as iTRAQ reaction reagent.100ug is respectively taken to mix respectively with iTRAQ reagent, room
Temperature stands 1hr.8uL azanol is added, is stored at room temperature 15min, terminates reaction.Merge 8 samples, concussion makes to be sufficiently mixed, and removes
The impurity such as the salt in sample.
Peptide fragment mixture is analyzed with SCX/SAX column first, then with the multidimensional liquid chromatography tandem being fully automated
Mass spectrograph LTQTMQ-ExactiveTM(being purchased from Thermo Fisher company) is analyzed, and obtained initial data uses
Maxquant software (German Ma Pu research institute) carries out database search, and carries out quantitative analysis to the protein identified, fixed
The results are shown in Table 2 for amount.
Table 3：The quantitative result of PDLIM3 in stomach organization and cancer beside organism：
According to the result of table 3, expression contents of the PDLIM3 in stomach organization are compared with cancer beside organism, in stomach organization
2 times or so of height expression, expression in gastric carcinoma obviously raises.
Embodiment 3：The verifying of PDLIM3
The protein example of 6 pairs of stomach organizations and corresponding cancer beside organism is taken, pathological data is as shown in table 4.
Table 4：The pathological data of 6 gastric cancer samples
Using purchase anti-PDLIM3 antibody to the protein example of above-mentioned 6 pairs of stomach organizations and corresponding cancer beside organism into
Row immunoblotting assay, process are as follows：
Each sample takes 20ug protein example to be separated with 12%SDS-PAGE, is transferred to pvdf membrane (purchased from GE
Healthcare company) on；
Using rabbit-anti people PDLIM3 polyclonal antibody (being purchased from Abcam company, 1: 1000 dilution), 4 DEG C are incubated overnight primary antibody,
It is washed three times with TBST, 5 minutes every time；
Secondary antibody is anti-rabbit antibody (being purchased from Santa Cruz company, I: 10000 dilutions), is incubated at room temperature I hours, then use TBST
It washs three times, 10 minutes every time；
ECL plus reagent (being purchased from GE Healthcare company) reaction is added after five minutes, with X- mating plate exposure tests.
To immunoblotting map, using Gel-Pro Analyzer quantitative gel analysis software, (Media Cybernetic is public
Department) data analysis is carried out, obtain the Relative distribution figure of protein expression quantity, as a result as shown in Figure 1.
The results show that the expression quantity in stomach organization is apparently higher than corresponding cancer beside organism, mean ratio is Fig. 1
2.10, P values (paired t-test) are 0.004.
According to Fig. 1's as a result, PDLIM3 has high expression in stomach organization, the result is consistent with mass spectral results.
Embodiment 4.It is random to select in order to further confirm differential expression of the PDLIM3 between stomach organization and cancer beside organism
The cancerous tissue and corresponding cancer beside organism's sample for taking 55 pairs of gastric cancers carry out immunohistochemistry research using stomach organization chip,
Wherein, the physical resource difference of the sample of selection is as shown in table 5.
Table 5：Stomach organization chip data.
The experimentation of immunohistochemistry research is as follows：
Histotomy is placed in 60 DEG C of insulating boxs and toasts about 3 hours, dimethylbenzene, dimethylbenzene/second are successively used after taking-up
Alcohol (I: I), 100% ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol, 70% ethyl alcohol, 50% second alcohol and water, carry out dewaxing hydration process；
PBS is washed 3 times, every time 5 minutes；0.3%H2O2(methanol dilution) impregnates half an hour, and PBS is washed 3 times, every time 5 minutes；
Pressure method antigen, distilled water are washed 2 times, every time 5 minutes；PBS is washed 2 times, every time 5 minutes；10% serum room temperature envelope
It closes 20 minutes；
Rabbit-anti people PDLIM3 polyclonal antibody (being purchased from Abcam company, I: 50 dilutions) is added, 4 DEG C are overnight, and PBST is washed 3 times,
5 minutes every time；
The goat anti-rabbit antibody (coming from ABC kit, be purchased from VECTOR company) of biotin labeling is added, is incubated at room temperature 30 points
Clock, PBST are washed 3 times, every time 5 minutes；PBS is washed 2 times, every time 5 minutes；
ABC solution (come from ABC kit, be purchased from VECTOR company) is added, is incubated at room temperature 30 minutes, PBS washes 3 times, often
Secondary 5 minutes；
DAB solution (being purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) colour developing；Hematoxylin is (public purchased from VECTOR
Department, H3404) dyeing 20 seconds；
Histotomy successively uses：50% ethyl alcohol, 70% ethyl alcohol, 80% ethyl alcohol, 90% ethyl alcohol, 100% ethyl alcohol, diformazan
Benzene/ethyl alcohol (I: I), dimethylbenzene, carry out dehydration transparent processing.
Then resinene mounting is used, in micro- sem observation.Organization chip is assessed, as the result is shown：Organize core
Totally 55 pairs of effective samples in piece, and being given a mark according to staining power and positive rate, wherein staining power scoring criterion is：Feminine gender is O
Divide, weakly positive is I points, moderate positive is 2 points, strong positive is 3 points；Positive rate scoring criterion is：It is O points lower than 5%, 5%~
30% is I point, and 31%~60% is 2 points, and 60% the above are 3 points.The score of staining power is added with the score of positive rate, is obtained
The comprehensive score of histotomy is obtained, scoring event is as shown in table 6.
Table 6：Stomach organization chip integration score.
Fig. 2 is calculated according to the result of table 6：The average aggregate of stomach cancer cell cancerous tissue is scored at 3.09, cancer beside organism
Average aggregate be scored at 2.27, the two has extremely significant difference, and P value is 0.0006.Statistics discovery, more than 64% (35 pairs)
Sample centering, PDLIM3 high expression in stomach cancer cell cancer.Mass spectral results of the result with front, immunoblot results phase one
It causes.
Embodiment 5.In order to be further discovered that differential expression of the PDLIM3 between gastric cancer serum and normal human serum, at random
The serum and 30 normal human serum samples of 30 patients with gastric cancer are chosen, carries out enzyme linked immunosorbent assay (ELISA), wherein the sample of selection
This physical resource difference is as shown in table 7.
Table 7：Gastric cancer serum sample information.
Table 7：Normal serum sample information.
Fig. 3 is obtained according to the result of enzyme linked immunosorbent assay (ELISA)：Statistics discovery, PDLIM3 are high in the patients serum of gastric cancer
Expression.The result is consistent with the mass spectral results of front, immunoblot results and ImmunohistochemistryResults Results.
In conclusion there are apparent differential expressions in the cancerous tissue of gastric cancer and cancer beside organism by PDLIM3, and in stomach
It expresses and increases in the serum of cancer patient, it is clear that the occurrence and development with gastric cancer have close correlation, therefore its expression quantity can
For detecting gastric cancer.Correspondingly, the antibody of specificity PDLIM3, monoclonal antibody and Anti-TNF-α including various PDLIM3
Body due to it can be used to detect the expression quantity of PDLIM3, thus can be used for detecting gastric cancer, or be used to prepare detection gastric cancer
Preparation or kit, this will be apparent to the person skilled in the art.
Although the dynamic biological function of related PDLIM3 and tumour related mechanism need further to study, by it
Marker as detection gastric cancer is affirmative.PDLIM3 can be used as the potential mark of gastric cancer, and it is in biology intracellular
Function prompt PDLIM3 may be marked as the prognosis molecule of gastric cancer and the target molecule of clinical treatment.
Claims (10)
1. the method for diagnosis of gastric cancer, the method includes：
A) the doubtful cancerous tissue of individual is obtained,
B) expression of PDLIM3 therein is measured,
C) the same tissue of normal individual or the normal tissue of same individual are obtained, and
D) expression of PDLIM3 therein is measured as control expression level,
E) b) expression of the middle PDLIM3 obtained is compared with the control expression level of d) the middle PDLIM3 obtained, when
B) when the control expression level for the PDLIM3 that the expression of the PDLIM3 obtained in obtains in d) is compared to improving, it is determining described in
Individual suffers from gastric cancer.
2. according to the method described in claim 1, wherein the tissue is gastric tissue.
3. method according to claim 1 or 2, wherein the expression is detected by either method selected from the following：
(a) mRNA of detection coding PDLIM3, and
(b) protein of PDLIM3 is detected.
4. method according to any one of claim 1-3, wherein the amount of the PDLIM3 is by using anti-PDLIM3's
Antibody is measured.
5. method according to any of claims 1-4, wherein the antibody of the anti-PDLIM3 be monoclonal antibody or
Polyclonal antibody.
6. the method for diagnosis of gastric cancer, the method includes：
A) serum of individual is obtained,
B) expression of PDLIM3 therein is measured,
C) serum of normal individual is obtained,
D) expression of PDLIM3 therein is measured as control expression level, and
E) b) expression of the middle PDLIM3 obtained is compared with the control expression level of d) the middle PDLIM3 obtained, when
B) when the control expression level for the PDLIM3 that the expression of the PDLIM3 obtained in obtains in d) is compared to improving, it is determining described in
Individual suffers from gastric cancer.
7. according to the method described in claim 6, wherein the expression of the PDLIM3 by using anti-PDLIM3 antibody
It is measured.
8. method according to claim 6 or 7, wherein the antibody of the anti-PDLIM3 is monoclonal antibody or Anti-TNF-α
Body.
9. purposes of the antibody of anti-PDLIM3 in reagent preparation box, the kit is used to diagnose the gastric cancer of individual.
10. the kit for the gastric cancer for diagnosing individual, the kit include
A) it is used to measure the reagent of the expression of PDLIM3, and
B) operation instructions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810747903.4A CN108872599A (en) | 2018-07-09 | 2018-07-09 | PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810747903.4A CN108872599A (en) | 2018-07-09 | 2018-07-09 | PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108872599A true CN108872599A (en) | 2018-11-23 |
Family
ID=64300226
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810747903.4A Pending CN108872599A (en) | 2018-07-09 | 2018-07-09 | PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108872599A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109613268A (en) * | 2019-01-09 | 2019-04-12 | 新疆医科大学 | Differential protein system and its screening technique and application in a kind of Erectile Dysfunction patients serum |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103608683A (en) * | 2011-06-02 | 2014-02-26 | 诺华股份有限公司 | Biomarkers for HEDGEHOG inhibitor therapy |
| CN103627785A (en) * | 2011-12-16 | 2014-03-12 | 香港中文大学 | Biomarker DACT1 for gastric cancer |
| CN107267580A (en) * | 2017-06-22 | 2017-10-20 | 西北工业大学 | It is a kind of to suppress the purification process that mechanics perceives albumen PDLIM3 degradeds |
| US20180188256A1 (en) * | 2011-09-08 | 2018-07-05 | The Regents Of The University Of California | Salivary Biomarkers for Gastric Cancer Detection |
-
2018
- 2018-07-09 CN CN201810747903.4A patent/CN108872599A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103608683A (en) * | 2011-06-02 | 2014-02-26 | 诺华股份有限公司 | Biomarkers for HEDGEHOG inhibitor therapy |
| US20180188256A1 (en) * | 2011-09-08 | 2018-07-05 | The Regents Of The University Of California | Salivary Biomarkers for Gastric Cancer Detection |
| CN103627785A (en) * | 2011-12-16 | 2014-03-12 | 香港中文大学 | Biomarker DACT1 for gastric cancer |
| CN107267580A (en) * | 2017-06-22 | 2017-10-20 | 西北工业大学 | It is a kind of to suppress the purification process that mechanics perceives albumen PDLIM3 degradeds |
Non-Patent Citations (1)
| Title |
|---|
| WEI ZENG 等: "Genome-wide Analyses on Single Disease Samples for Potential Biomarkers and Biological Features of Molecular Subtypes: A Case Study in Gastric Cancer", 《INT. J. BIOL. SCI.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109613268A (en) * | 2019-01-09 | 2019-04-12 | 新疆医科大学 | Differential protein system and its screening technique and application in a kind of Erectile Dysfunction patients serum |
| CN109613268B (en) * | 2019-01-09 | 2021-10-22 | 新疆医科大学 | Differential protein system in serum of patient with penile erectile dysfunction and screening method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200200753A1 (en) | Methods and kits for detecting prostate cancer biomarkers | |
| EP3545309B1 (en) | Antibody assay | |
| Sun et al. | CPA4 is a novel diagnostic and prognostic marker for human non-small-cell lung cancer | |
| JP7492287B2 (en) | MMP9 as a marker for endometrial cancer | |
| CN102432683B (en) | Preparation of multi-epitope TK1 antibody and application of multi-epitope TK1 antibody to evaluation on recurrence risk and prognosis of tumor patient at early stage | |
| CN110187109A (en) | A kind of autoantibody joint-detection ELISA kit for Grades of Gastric Cardia Adenocarcinoma early screening | |
| US20140066325A1 (en) | Protein Biomarkers for the Diagnosis of Prostate Cancer | |
| KR102208140B1 (en) | Methods and arrays for use in biomarker detection for prostate cancer | |
| CN109517062B (en) | Antibodies capable of binding to LINC00266-1 polypeptides | |
| EP3655778B1 (en) | Agrin as a marker for endometrial cancer | |
| CN108872599A (en) | PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker | |
| US20200018758A1 (en) | Methods for differentiating benign prostatic hyperplasia from prostate cancer | |
| CN108872600A (en) | PDLIM4 is used as the application of stomach cancer marker | |
| CA2799359C (en) | Method for the diagnosis/prognosis of colorectal cancer | |
| KR20130005087A (en) | Protein marker melanotransferrin for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same | |
| CN108330192A (en) | Smell mediates application of the element -4 as non-small cell lung carcinoma marker | |
| EP3698138A1 (en) | Assay and kit for the diagnosis of ovarian carcinoma | |
| CN115385990B (en) | Isolated polypeptides and their use in diagnosis or prognosis of ovarian cancer and detection devices | |
| CN108490179A (en) | Applications of the Metaxin-2 as stomach cancer marker | |
| CN116413430A (en) | Autoantibody/antigen combination and detection kit for early prediction of liver cancer | |
| GB2541712A (en) | Autoantibody biomarkers for gastric cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181123 |
|
| RJ01 | Rejection of invention patent application after publication |