CN104277102B - Amino acid sequence for detecting breast cancer marker Annexin Al antigen epitope and application of amino acid sequence - Google Patents
Amino acid sequence for detecting breast cancer marker Annexin Al antigen epitope and application of amino acid sequence Download PDFInfo
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- CN104277102B CN104277102B CN201410293597.3A CN201410293597A CN104277102B CN 104277102 B CN104277102 B CN 104277102B CN 201410293597 A CN201410293597 A CN 201410293597A CN 104277102 B CN104277102 B CN 104277102B
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- breast cancer
- annexin
- amino acid
- acid sequence
- antigen
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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
-
- 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
- G01N33/57415—Specifically defined cancers of breast
-
- 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
-
- 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
Abstract
The invention relates to an amino acid sequence for detecting a breast cancer marker Annexin Al antigen epitope and application of the amino acid sequence, and belongs to the technical field of immunology. The invention provides the Annexin Al antigen amino acid sequence of a related breast cancer gene. An Annexin Al polypeptide antigen is applied to detect the corresponding specific autoantibody contained in the blood of a breast cancer patient, and the autoantibody can be used as the breast cancer marker for evaluating the risk degree of breast cancer occurrence; and the antigen polypeptide and the antibody thereof can be used for preparing a breast cancer early-diagnosis reagent and developing a targeted drug for treating breast cancer.
Description
Technical field
The invention belongs to immunological technique field, is that one kind treats tumour for preparing early diagnosis of tumor reagent and exploitation
Targeted drug.
Background technology
Numerous studies show that the tumor associated antigen in serum or blood plasma can induce body to produce autoantibody, in tumour
Both there is tumour antigen in patients serum, there is also the autoantibody for the tumour antigen.Therefore, both can be examined using antibody
Survey tumour antigen, it is also possible to detect the autoantibody of tumour antigen using antigen, but tumour is detected using tumour autoantibody
Specificity and the equal Billy of sensitiveness are much higher with tumour antigen detection tumour.Many tumor associated antigens are not only in tumor patient
Exist in vivo, there is also in normal human, therefore detect that tumor associated antigen is credible poor as diagnosis basis.And tumour is certainly
Body antibody be can't detect or not existed normal person's in-vivo content is very low, if in-vivo tumour autoantibody substantially increases
Height, then show there is abnormal immune situation in vivo, shows that internal related antigen level fluctuates, and indicates presence or the original of disease
There is disease aggravation.
Recent studies indicate that, the 3-5 before malignant tumour volume develops into available modern imaging technology detection,
The tumor associated antigen autoantibody of high concentration is may occur in which in patient's blood.Therefore, tumor associated antigen autoantibody in blood is detected
Important value with predicting tumors onset risk and early diagnosis tumour.It is the prior development direction of clinical tumor diagnostic field
One of.Diagnosing is had abroad and the early diagnosis kit of breast cancer is commercially available.However, the autoantibody reported at present
Detection method susceptibility is low, poor specificity, and false negative ratio may be up to more than 50%.It is primarily due to each tumour
Positive detection rate of the related antigen autoantibody in cancer patient is averagely 10% or so.How diagnostic reagent susceptibility is improved
It is to be currently needed for key issue urgently to be resolved hurrily.The effective method of comparison be find it is new may act as tumor markers from
Body antibody, is then combined into the diagnosis of and high specificity high with susceptibility with existing known tumor associated antigen autoantibody
Kit.
Breast cancer related gene Annexin A1 are positioned at human chromosome 9q12～q21.2, by 13 extrons and 12
Introne is constituted, and the amino acids of N-terminal 2～12 of Annexin A1 can be with S100C protein-interactings.With other annexins
(ANX) difference of topmost structure be its IIIth repetitive sequence with the hydrophobic domains at N- ends be not bound with calcium ion when
Wait, the helical structure is made up of 12 residues before N- ends and replaces D- spirals to be embedded in the core site of the IIIth repetitive sequence, and
D- spirals are then hung on outside the spiral of N- ends.Research shows, Annexin A1 participate in the Physiological effect process of cell, including inflammation is anti-
Should, the regulation of cell proliferation and differentiation apoptosis, phagocytosis secretion process and cell death signal, and play a great role, and with
The generation development of tumour is closely related.Experiment in vitro shows that Annexin A1C terminal domains are by hydrophobic effect with cPLA2's
Specific site is combined, and so as to disturb cPLA2 to be combined with plasma membrane, is suppressed the activity of cPLA2 and then is checked the proto-oncogenes such as c-fos
Expression.In scholars tissue microarray analysis normal breast tissue, breast cancer tissue and metastatic lymph node tissue
The expression of AnnexinA1, as a result finds：Annexin A1 express strong positive, breast cancer tissue in normal breast tissue
Middle expression is lowered, and expression strengthens in axillary gland, shows the expression of Annexin A1 with the generation development of breast cancer
Change.Recent studies have shown that：Annexin A1 high expression in the high transfer of breast cancer, hormone-sensitive clone, disturbs this cell
Cell migration and invasive ability is caused to decline after the expression of middle Annexin A1.Demonstrating Annexin A1 in cellular level can promote
Enter the invasion and attack transfer of breast cancer.The specificity that Annexin A1 are expressed in breast neoplasm, point out we its in breast cancer early stage
The using value of diagnosis is preferable, and as the possibility of potential source biomolecule mark.
The content of the invention
The technical problem to be solved in the present invention is to disclose a kind of detection markers for breast cancer Annexin A1 autoantibodies
Epitope sequence.
The present invention discloses the purposes of Annexin A1 epitopes.
A kind of epitope amino acid sequence of detection markers for breast cancer Annexin A1 autoantibodies that the present invention is provided
It is classified as：
H-DITSDTSGDFRNALLSEATIIDILTKRNNAQDC-OH
Its purity>95%, pH>7.0.
Application of the Annexin A1 antigen epitope polypeptides of the present invention in early diagnosing mammary cancer kit is prepared.
The present invention is suffered from using the linear polypeptide of the Annexin A1 albumen of designed, designed using ELISA method detection breast cancer
The specific Autologous IgG antibody of Annexin A1 albumen in person's serum and blood plasma.Autologous IgG antibody horizontal is raised and shows that tumour is suffered from
The expression of Annexin A1 albumen increases in person's body, and indication patient is likely to occur primary or secondary carcinoma of mammary gland, can be pre-
The danger of mammary gland carcinogenesis and recurrence is surveyed, early diagnosis of the clinician to breast cancer is instructed.
Tab.1Annexin A1 antigen epitope polypeptide sequence tables
The combination of antigen-antibody is actually only occurred between antigenic determinant and the antigen binding site of antibody, Liang Zhe
Complete complementary on space structure and steric configuration.Therefore antigenic determinant can just represent the state that whole albumen is combined with antibody
With affinity characteristic.In addition, with recombinant protein as antigen, be through the loaded down with trivial details mistake such as vector construction, transfection, expression, screening, purifying
Journey, protein steric structural is complicated, and epitope is difficult exposure, therefore the poor specificity that antigen-antibody is combined.Additionally, ELISA method
Stability requirement of the high sensitivity to purification technique it is high, cost intensive.
Inventor follows following principle and designs linear polypeptide antigen：1. epicyte protein surface region is selected；2. select not
Form the sequence of a-helix；3. the peptide fragment at two ends is more reasonable than middle arrangement；4. active site of protein is avoided to repeat；5. avoid homologous
The strong peptide fragment of property；6. Cys and Glu is avoided as far as possible in sequence, it is not possible to have too many Pro, but have 1-2 Pro to chain beneficial to peptide
Structure is stablized, beneficial to producing specific antibody.Additionally, the polypeptide antigen must contain class antigen (HLA) system of human leucocyte two
The restricted epitope of system, including the restricted epitope of HLA-DR, HLA-DP and HLA-DQ.These epi-positions can be by more than 90%
The class antigen systems of HLA bis- of Chinese colony are recognized.Based on above ANTIGEN DESIGNThe principle and the biology of Annexin A1 albumen
Characteristic, the present invention is analyzed and antigenicity associated parameter using bioinformatics and multiple Antigen Epitope Prediction simulation softwards, is devised
Linear amino acid sequence.Annexin A1 linear polypeptides antigens are made up of 33 amino acid residues, altogether containing 11 overlapping epitopes,
Can detect at least 11 kinds monoclonal antibodies, the specificity with height.
ELISA method detection epitope
We adopt ELISA method, and the blood to collecting is detected, and is obtained each sample OD values and be analyzed.
Quality Control
Each sample sets duplicate hole, takes average OD values.OD values dispersion judges：Dispersion=OD1-OD2/OD1+OD2, it is discrete
Degree≤0.1, is effective result；Dispersion>0.1, it is null result.Take 100 parts of Healthy Human Serum equal-volumes to mix as Quality Control
Blood (Quality control, QC), represents the common situation of crowd, 2 QC blood plasma holes is all provided with per plate, with the OD in QC blood plasma hole
The stability of value Deflection level result of determination, all batch QC hole OD averages of all batch QC hole SD/ of batch variation CV=<
20%.The daily each plate QC holes averages of the daily each plate QC holes SD/ of variation within batch CV=<10%.
Data analysis
Statistical analysis are carried out using SPSS17.0for windows.Using specific bond index (Specific
Binding index, SBI) judging the combination degree of Annexin A1 antigen polypeptides and blood plasma autoantibody, SBI=
Annexin A1 OD value-NC OD values/QC OD value-NC OD values, NC is the negative control of each sample.Compared respectively using t inspections
Compared with the difference between malignant breast carcinomas group and normal healthy controls between SBI values, a=0.05.
ROC curve be according to a series of different two mode classifications (cut off value determines threshold), it is (sensitive with True Positive Rate
Degree) it is ordinate, false positive rate (1- specificities) is the curve that abscissa is drawn.Area value under ROC curve is 1.0 and 0.5
Between.Area under the curve>In the case of 0.5, TG-AUC is closer to 1, illustrates that diagnosis accuracy is better.ROC curve
Sensitivity is combined together with specificity with graphic technique, the pass of certain analysis method specificity and sensitiveness can be accurately reflected
System, is the aggregate surrogates for testing accuracy.The invention adopts Analyse-it for Microsoft Excel Software on Drawing ROC
Curve, area (AU) under calculated curve judges sensitivity and specificity.
The present invention detects blood serum of patients with human breast carcinoma and the Annexin in blood plasma using Annexin A1 antigen epitope polypeptides
A1 specificity Autologous IgG antibody, and the reaction has high specific and high sensitivity.
Annexin A1 antigen epitope polypeptides can be used to prepare early diagnosing mammary cancer kit.
Description of the drawings
Accompanying drawing is the ROC curve analysis chart of the anti-Annexin A1 Autologous IgG antibody horizontals of patient with breast cancer's body.
Wherein, abscissa is 1- specificities, and ordinate is susceptibility.
Specific embodiment
With reference to example is embodied as, the present invention is expanded on further.It should be understood that these examples of implementation are merely to illustrate this
Invention rather than restriction the scope of the present invention.
Embodiment 1
It is prepared by kit
1 reagent kit is prepared and sees Tab.2～7.
2 operations
(1) it is coated with：ELISA Plate application lavation buffer solution is cleaned 3 times, and work antigen coating buffer is diluted to working concentration, wraps
By in ELISA Plate, 4 DEG C overnight.
(2) glutamic acid is added：Lavation buffer solution is cleaned 3 times, glutamic acid is diluted to the μ g/ml of concentration 100 with coating buffer, per hole
200 μ l, 37 DEG C or incubation at room temperature 1h；
(3) add blood plasma and Quality Control control (one resists):ELISA Plate application lavation buffer solution is cleaned 3 times, using coating buffer by blood
Starch and be diluted to suitable concn, generally 1:100～1:500, per the μ l of hole 100,37 DEG C or incubation at room temperature 1h；
(4) two anti-incubations：Lavation buffer solution is cleaned 3 times, and using coating buffer two anti-titer IgG, working concentration 1 are diluted:
20000, add 100 μ l per hole, 37 DEG C or incubation at room temperature 1h；
(5) develop the color：Lavation buffer solution is cleaned 3 times, and per hole 100 μ l substrate nitrite ions, room temperature 30～45min of lucifuge are added.
(6) detect：Add detection in 50 μ l terminate liquids, 10min per hole, Detection wavelength is 450nm, and reference wavelength is 630nm.
Embodiment 2
The AnnexinA1 Autologous IgG antibody tests of patient with breast cancer
1 sample collection：This research chooses Jing radiological examinations and histology from The Second Hospital of Jilin Universtiy, tumour hospital of province
311, breast cancer sample is made a definite diagnosis in inspection.Without any anticancer therapy before all serum sample collections, and with comprehensive clinic
Data and information.303, healthy control group sample is recruited simultaneously.It is ill that clinical interview and imaging examination exclude breast cancer
May.Health is organized with breast cancer group in sex, age-matched, with comparativity (P>0.05)
2 testing results：The autoantibodies level (Tab.8) of Annexin A1：Breast cancer group and healthy control group phase
Than there is significant difference (t=-7.163, P<0.001).
ROC curve is analyzed：Patient with breast cancer's Annexin A1 Autologous IgGs antibody tests area under ROC curve is 0.723
(standard error=0.028,95% fiducial limit：0.668-0.777) (Fig. 1 and Tab.9).
Data above fully shows, the patient with breast cancer obtained using the antigen epitope polypeptide detection designed by the present invention from
Body IgG antibody level compares with normal health group notable statistics difference.
Expression of the anti-Annexin A1 Autologous IgGs antibody of Tab.8 in patient with breast cancer and normal healthy controls sample
The ROC curve analysis of anti-Annexin A1 Autologous IgG antibody in Tab.9 breast cancer
H-DITSDTSGDFRNALLSEATIIDILTKRNNAQDC -OH
Its purity>95%, pH>7.0.
Claims (2)
- It is 1. a kind of to detect markers for breast cancer Annexin A1 antigen epitope polypeptides, it is characterised in that：Amino acid sequence isH-DITSDTSGDFRNALLSEATIIDILTKRNNAQDC -OHIts purity>95%, pH>7.0.
- 2. detection markers for breast cancer Annexin A1 antigen epitope polypeptides according to claim 1 are preparing breast cancer morning Application in phase diagnostic kit.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105111299B (en) * | 2015-09-24 | 2017-09-22 | 北华大学 | One kind detection markers for breast cancer TGF beta 1-6 antigens epitope amino acid sequence and application |
| CN106526187B (en) * | 2016-09-13 | 2018-04-13 | 浙江理工大学 | A kind of leukaemia detection kit based on Annexin A1 albumen |
| GB201813137D0 (en) * | 2018-08-10 | 2018-09-26 | Medannex Ltd | Cancer treatment with an antibody |
| CN116539885B (en) * | 2023-07-06 | 2023-09-29 | 上海秤信生物科技有限公司 | Tumor autoantigen/antibody combination for early detection of breast cancer and application thereof |
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2014
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| CN1378552A (en) * | 1999-08-13 | 2002-11-06 | 乔治华盛顿大学 | Novel transcription factor BP1 |
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| WO2009126966A2 (en) * | 2008-04-11 | 2009-10-15 | The Regents Of The University Of California | Genetic markers indicating biological response to a plk1 kinase inhibitor |
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