CN112924683B - Application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer - Google Patents
Application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer Download PDFInfo
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- CN112924683B CN112924683B CN201911235717.3A CN201911235717A CN112924683B CN 112924683 B CN112924683 B CN 112924683B CN 201911235717 A CN201911235717 A CN 201911235717A CN 112924683 B CN112924683 B CN 112924683B
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides application of urine exosome CD40 (also called tumor necrosis factor receptor superfamily member 5) protein and polypeptide fragments thereof, in particular application of urine exosome CD40 protein and polypeptide fragments thereof in preparation of a preparation for detecting and assisting in diagnosing bladder cancer. The research proves that compared with the normal control, the CD40 protein and the polypeptide fragment thereof are expressed in the urine exosome of the bladder cancer patient, and the expression is higher than that of the normal group, so that the invention can be used for detecting and assisting diagnosis of the bladder cancer. The invention plays the advantages of noninvasive acquisition of urine specimen, large-scale repeated sampling and convenient preservation, and utilizes the urine specimen to detect the urine exosome CD40 protein and the polypeptide fragment thereof.
Description
Technical Field
The invention relates to novel application of urine CD40 protein and polypeptide fragments thereof, in particular to application of urine exosome CD40 protein and polypeptide fragments thereof in diagnosis and treatment of bladder cancer.
Background
Bladder cancer is one of the most common malignant tumors of the urinary system, and cystoscopy and cytology are the main means of diagnosing bladder cancer at present. Among them, cystoscopy is an effective but invasive means of diagnosing bladder cancer. While its sensitivity to carcinoma in situ (tis) is low and the detection effect depends on the operator, urocytology is a non-invasive diagnostic method, but low sensitivity, cytologically negative does not preclude the presence of tumors. 20% of patients with bladder cancer will progress to invasive bladder cancer, and 50% of patients with invasive bladder cancer will progress to incurable metastatic disease. Therefore, there is an urgent need to find means for rapidly and effectively monitoring bladder cancer, and monitoring tumor progression in patients.
Urine exosome proteins have a higher value than urine soluble proteins because proteins secreted into the extracellular body are more directly involved in tumor development. Meanwhile, compared with urine soluble protein, the exosome is not easy to decompose, so that the false negative rate of protein detection is greatly reduced. During protein analysis, it has been found that exosomes exist in close to hundreds of proteins associated with diseases such as tumors. Therefore, the exosomes have higher research value in the research of bladder cancer.
CD40 is an important marker existing on the surface of tumor cells, and has been demonstrated to have the effect of predicting disease progression in various tumors such as ovarian cancer, hepatocellular carcinoma and the like. CD40 has the characteristic of differential expression in the occurrence and development of tumors, and has higher diagnostic and therapeutic values. CD40 has antiproliferative and pro-apoptotic effects. Research results show that the CD40 pathway has various anti-tumor activities in tumors, and provides possibility for future clinical application. In animal experiments, the recombinant CD40 ligand therapy of mice has remarkable anti-tumor effect on CD40 positive ovarian tumors, and the effect of cisplatin can be enhanced by influencing the expression of CD40, so that the experiments prove that the CD40 is a valuable marker in the tumors. However, in bladder cancer, the expression and predictive value of CD40 are not verified, and the invention provides the application of CD40 protein and polypeptide fragments thereof in human urine exosomes in bladder cancer.
Disclosure of Invention
The invention aims to provide an application of urine exosome CD40 protein and polypeptide fragments thereof in preparing a preparation for detecting or assisting in diagnosing bladder cancer.
Preferably, the amino acid sequence of the urine exosome CD40 protein is shown in SEQ ID NO.1 (MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ); or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function as the amino acid sequence shown in SEQ ID NO. 1.
Preferably, the CD40 protein and polypeptide fragments thereof are derived from urine exosomes.
Preferably, the urine exosome CD40 protein and polypeptide fragments thereof are highly expressed in bladder cancer patients.
Preferably, the preparation is a detection kit for urine exosome CD40 protein and polypeptide fragments thereof of a bladder cancer patient.
Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to CD40 protein and polypeptide fragments thereof.
Preferably, the kit further comprises a component selected from the group consisting of: solid phase carrier, diluent, reference substance, standard substance, quality control substance, detection antibody, secondary antibody diluent, luminous reagent, washing liquid, color developing liquid and stop solution.
Preferably, the standard comprises a CD40 protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: CD40 protein control and humanized tag antibody control; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates or affinity membranes.
Preferably, the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.
The inventor firstly collects urine samples of normal people and bladder cancer patients in the morning, takes supernatant after centrifugation, extracts urine exosomes, the collected protein is cracked and extracted by adopting a RIPA and heating method, and the CD40 in exosomes is verified by adopting a western blot method, and the result is combined with clinical progress of the patients to verify the correlation between the CD40 and disease progress in the urine exosomes.
Compared with a normal control group, the invention proves that the CD40 protein and the polypeptide fragment thereof are expressed in urine of patients with bladder cancer, are higher than the normal control group, and have correlation with disease progress of the patients with bladder cancer, thereby providing that the detection of the CD40 protein and the polypeptide fragment thereof in urine exosomes can be used for diagnosing and treating bladder cancer.
The invention plays the advantages of noninvasive acquisition of urine specimen, large-scale repeated sampling and convenient preservation, and utilizes the urine specimen to detect the urine exosome CD40 protein and the polypeptide fragment thereof.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments, as illustrated in the accompanying drawings.
Drawings
FIG. 1 is a graph showing differential expression of CD40 protein in urine exosomes in normal control and bladder cancer groups.
Detailed Description
Example 1Urine exosome extraction
Fresh morning urine from normal and bladder cancer patients was collected and placed on ice and centrifuged at 3000g for 10 minutes at 4 ℃ to remove cells or cell debris, after which the urine supernatant was transferred to a new tube. 5.0ml of the treated urine was placed in a 15ml centrifuge tube, 0.5ml of binding buffer was added, the lid was closed, and the mixture was inverted and mixed. Absorbing 300ul of binding resin, adding into a centrifuge tube, reversing at room temperature, uniformly mixing for 15min, centrifuging at room temperature for 2min, discarding 5.2ml of supernatant, suspending the rest supernatant liquid and the binding resin, transferring the liquid and the binding resin into a purification column, centrifuging at room temperature for 2min, discarding the collected liquid in a collecting pipe, adding 0.5ml of washing liquid into the centrifugal column, fully mixing the washing liquid and the binding resin by a pipettor, standing at room temperature for 1min, centrifuging at room temperature for 2min, discarding the collecting pipe and the collected liquid. And then placing the purification column into a 1.5ml centrifuge tube with low protein adsorption, adding 200ul of eluent, uniformly mixing and reacting for 10min, and centrifuging for 2min at room temperature with 3000g, wherein the liquid obtained in the centrifuge tube is the concentrated exosome solution.
Example 2Detection of urinary exosome proteins
And verifying the protein by adopting western blot. Adding RIPA protein lysate (1:4) into the extracted solution to extract total protein, performing 12% SDS-PAGE electrophoresis, transferring membrane at 12V for 60 min, sealing 2 h with 5% skimmed milk powder at room temperature, adding 1: primary anti-CD 40, beta-actin was incubated overnight at 4 ℃. PBST was rinsed 3 times for 20min each, 1 was added: 1000 secondary antibody, incubation for 90 min at room temperature, PBST rinsing, ECL color development. As shown in FIG. 1, the exocrine experience-evidence proteins CD63, CD9 and TSG101 are all expressed by using western blot verification, which indicates that urine exosomes are successfully extracted. Meanwhile, CD40 protein in urine exosomes of patients with bladder cancer is obviously increased compared with normal controls.
Although the invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and the invention is therefore to be limited only by the appended claims.
Sequence listing
<110> sheet, manchurian
<120> application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer
<130> 19PCD40-CN
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 277
<212> PRT
<213> urinary exosomes CD40 protein
<400> 1
Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr
1 5 10 15
Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu
20 25 30
Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val
35 40 45
Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu
50 55 60
Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His
65 70 75 80
Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr
85 90 95
Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr
100 105 110
Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly
115 120 125
Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu
130 135 140
Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys
145 150 155 160
Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln
165 170 175
Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu
180 185 190
Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile
195 200 205
Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn
210 215 220
Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp
225 230 235 240
Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His
245 250 255
Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser
260 265 270
Val Gln Glu Arg Gln
275
Claims (7)
1. The application of the urine exosome CD40 protein in preparing the preparation for detecting and assisting in diagnosing human bladder cancer is disclosed, wherein the amino acid sequence of the urine exosome CD40 protein is shown as SEQ ID NO. 1.
2. The use of claim 1, wherein the urinary exosome CD40 protein is highly expressed in bladder cancer patients.
3. The use according to claim 1, wherein the formulation is a urinary exosome CD40 protein detection kit for patients with bladder cancer.
4. The use according to claim 3, wherein the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to CD40 protein.
5. The use according to claim 3, wherein the kit further comprises a component selected from the group consisting of: solid phase carrier, diluent, reference substance, standard substance, quality control substance, detection antibody, secondary antibody diluent, luminous reagent, washing liquid, color developing liquid and stop solution.
6. The use of claim 5, wherein the standard comprises a CD40 protein standard, a humanized tag antibody standard; the quality control article comprises: CD40 protein control, humanized tag antibody control; the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates or affinity membranes.
7. The use according to claim 6, wherein the solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911235717.3A CN112924683B (en) | 2019-12-05 | 2019-12-05 | Application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911235717.3A CN112924683B (en) | 2019-12-05 | 2019-12-05 | Application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer |
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| CN112924683A CN112924683A (en) | 2021-06-08 |
| CN112924683B true CN112924683B (en) | 2023-08-29 |
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Citations (7)
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|---|---|---|---|---|
| CN1900113A (en) * | 2006-07-19 | 2007-01-24 | 张曼 | Tumor relative polypeptide and its coding gene sequence |
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| CN107177683A (en) * | 2017-06-13 | 2017-09-19 | 杨昭 | A kind of carcinoma of urinary bladder selective mechanisms kit |
| CN107573414A (en) * | 2017-08-14 | 2018-01-12 | 李翀 | Human bladder cancer mark AG CD71 and its antibody A BC71 and application |
| CN108289942A (en) * | 2015-09-25 | 2018-07-17 | 迈斯免疫公司 | With the cell inoculation vaccine of immune-separation of production immunomodulator |
| CN108414755A (en) * | 2017-10-25 | 2018-08-17 | 深圳市罗湖区人民医院 | Protein chip that is a kind of while detecting four bladder carcinoma markers in urine |
| CN109912717A (en) * | 2019-03-04 | 2019-06-21 | 北京天广实生物技术股份有限公司 | In conjunction with the antibody and application thereof of CD40 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050281815A1 (en) * | 2004-02-26 | 2005-12-22 | Dani Eshel | CD40 splice variants and their uses |
-
2019
- 2019-12-05 CN CN201911235717.3A patent/CN112924683B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101325970A (en) * | 2005-11-01 | 2008-12-17 | 诺华有限公司 | Uses of anti-CD40 antibodies |
| CN1900113A (en) * | 2006-07-19 | 2007-01-24 | 张曼 | Tumor relative polypeptide and its coding gene sequence |
| CN108289942A (en) * | 2015-09-25 | 2018-07-17 | 迈斯免疫公司 | With the cell inoculation vaccine of immune-separation of production immunomodulator |
| CN107177683A (en) * | 2017-06-13 | 2017-09-19 | 杨昭 | A kind of carcinoma of urinary bladder selective mechanisms kit |
| CN107573414A (en) * | 2017-08-14 | 2018-01-12 | 李翀 | Human bladder cancer mark AG CD71 and its antibody A BC71 and application |
| CN108414755A (en) * | 2017-10-25 | 2018-08-17 | 深圳市罗湖区人民医院 | Protein chip that is a kind of while detecting four bladder carcinoma markers in urine |
| CN109912717A (en) * | 2019-03-04 | 2019-06-21 | 北京天广实生物技术股份有限公司 | In conjunction with the antibody and application thereof of CD40 |
Non-Patent Citations (1)
| Title |
|---|
| 膀胱癌尿液差异蛋白鉴定及生物信息分析;雷婷 等;《标记免疫分析与临床》;20190531;第26卷(第5期);第815-821页 * |
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| CN112924683A (en) | 2021-06-08 |
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