CN104277102A - Amino Acid Sequence and Application of Detecting Breast Cancer Marker Annexin A1 Antigen Epitope - Google Patents
Amino Acid Sequence and Application of Detecting Breast Cancer Marker Annexin A1 Antigen Epitope Download PDFInfo
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- 239000000439 tumor marker Substances 0.000 title claims abstract 5
- 125000003275 alpha amino acid group Chemical group 0.000 title claims abstract 4
- 239000000427 antigen Substances 0.000 title abstract description 34
- 102000036639 antigens Human genes 0.000 title abstract description 34
- 108091007433 antigens Proteins 0.000 title abstract description 34
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- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 12
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Classifications
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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
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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
- G01N33/57415—Specifically defined cancers of breast
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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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- 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
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Abstract
一种检测乳腺癌标志物Annexin A1抗原表位的氨基酸序列及应用，属于免疫学技术领域，本发明提供了一种乳腺癌相关基因Annexin A1的抗原氨基酸序列。应用Annexin A1多肽抗原检测乳腺癌患者血中相应的特异性自身抗体，这种自身抗体可作为乳腺癌标志物评估乳腺癌发生的危险度。这种抗原多肽及其抗体可用于制备乳腺癌早期诊断试剂和开发治疗乳腺癌的靶向药物。
An amino acid sequence and application for detecting the antigen epitope of Annexin A1, a breast cancer marker, belongs to the field of immunology technology. The present invention provides an antigen amino acid sequence of Annexin A1, a breast cancer-related gene. Annexin A1 polypeptide antigen is used to detect the corresponding specific autoantibodies in the blood of breast cancer patients. This autoantibody can be used as a breast cancer marker to assess the risk of breast cancer. This antigen polypeptide and its antibody can be used to prepare early diagnosis reagents for breast cancer and develop targeted drugs for treating breast cancer.
Description
Technical field
The invention belongs to immunological technique field, is a kind of targeted drug for the treatment of tumour for the preparation of early diagnosis of tumor reagent and exploitation.
Background technology
Large quantity research shows, the tumor associated antigen in serum or blood plasma can induce body to produce autoantibody, both there is tumour antigen, also there is the autoantibody for this tumour antigen in Serum of Cancer Patients.Therefore, both can utilize antibody test tumour antigen, and also can utilize the autoantibody of Detection of antigen tumour antigen, but it is much higher to utilize the specificity of tumour autoantibody detection tumour and the equal Billy of susceptibility to detect tumour with tumour antigen.A lot of tumor associated antigen not only exists in tumour patient body, also exists in normal human, therefore detects tumor associated antigen credible poor as diagnosis basis.And tumour autoantibody can't detect or not exist normal human's intensive amount is very low, if in-vivo tumour autoantibody obviously increases, then show to there is abnormal immune situation in body, show that in body, related antigen level fluctuates, the existence of indication disease or original disease aggravation.
Research in recent years shows, develops into 3-5 before available modern imaging technology detects at malignant tumour volume, can occur the tumor associated antigen autoantibody of high density in patient's blood.Therefore, detect tumor associated antigen autoantibody in blood and there is the important value of predicting tumors onset risk and early diagnosis tumour.It is one of prior development direction of clinical tumor diagnostic field.The early diagnosis kit of existing diagnosing and mammary cancer is commercially available abroad.But current reported autoantibody detection method susceptibility is low, and poor specificity, false negative ratio can up to more than 50%.Its major cause is because the positive detection rate of each tumor associated antigen autoantibody in cancer patients is on average about 10%.How improving diagnostic reagent susceptibility is current needs key issues urgently to be resolved hurrily.More effective method finds the autoantibody of new served as tumor markers, is then combined into the diagnostic kit with susceptibility height and high specificity with existing known tumor associated antigen autoantibody.
Breast cancer related gene Annexin A1 is positioned human chromosome 9q12 ~ q21.2, is made up of 13 exons and 12 introns, the N of Annexin A1 hold 2 ~ 12 amino acids can with S100C protein-interacting.Distinguish with other annexins (ANX) topmost structure and be that its IIIth tumor-necrosis factor glycoproteins is with the hydrophobic domains that N-holds is not in conjunction with calcium ion time, this spirane structure holds front 12 residues form and replace D-spiral to be embedded in the core site of the IIIth tumor-necrosis factor glycoproteins by N-, and D-spiral then hangs on N-and holds outside spiral.Research shows, Annexin A1 participates in the physiological regulation process of cell, comprises inflammatory reaction, cell proliferation and differentiation apoptosis, engulfs the adjustment of secretion process and cell death signal, and play a great role, and develop closely related with the generation of tumour.Experiment in vitro shows, Annexin A1 C terminal domains is combined with the specific site of cPLA2 by hydrophobic interaction, thus interference cPLA2 is combined with plasma membrane, suppresses the activity of cPLA2 and then checks the expression of the proto-oncogenes such as c-fos.The expression of AnnexinA1 in scholars' tissue microarray analysis normal breast tissue, breast cancer tissue and metastatic lymph node tissue, found that: Annexin A1 expresses strong positive in normal breast tissue, expression in breast is lowered, and in axillary gland, express enhancing, the expression of display Annexin A1 changes with the generation development of mammary cancer.Recent studies have shown that: Annexin A1 is high expression level in the transfer of mammary cancer height, hormone-sensitive clone, after disturbing the expression of Annexin A1 in this cell, cause cell migration and invasive ability to decline.The Invasion and Metastasis that Annexin A1 can promote mammary cancer is demonstrated at cell levels.The specificity that Annexin A1 expresses in breast neoplasm, points out our its using value at early diagnosing mammary cancer better, and as potential source biomolecule mark may.
Summary of the invention
The technical problem to be solved in the present invention is open a kind of epitope sequence detecting markers for breast cancer Annexin A1 autoantibody.
The present invention discloses the purposes of Annexin A1 epitope.
A kind of epitope aminoacid sequence detecting markers for breast cancer Annexin A1 autoantibody provided by the invention is:
H-DITSDTSGDFRNALLSEATIIDILTKRNNAQDC -OH
Its purity >95%, pH>7.0.
Annexin A1 antigen epitope polypeptide of the present invention is preparing the application in early diagnosing mammary cancer test kit.
The present invention utilizes the linear polypeptide of the Annexin A1 albumen of designed, designed, adopts ELISA method to detect the specificity Autologous IgG antibody of Annexin A1 albumen in blood serum of patients with human breast carcinoma and blood plasma.Autologous IgG antibody horizontal raises and shows that the expression amount of Annexin A1 albumen in tumour patient body increases, primary or secondary carcinoma of mammary gland may be there is in indication patient, can predict that mammary cancer occurs, with the danger of recurrence, to instruct clinician to the early diagnosis of mammary cancer.
In fact the combination of antigen-antibody only occurs between antigenic determinant and the antigen binding site of antibody, and both are complete complementary on space structure and sterie configuration.Therefore antigenic determinant just can represent state and the affinity characteristic of whole albumen and antibodies.In addition, take recombinant protein as antigen, through the loaded down with trivial details process such as vector construction, transfection, expression, screening, purifying, protein steric structural is complicated, and epitope not easily exposes, therefore the poor specificity that combines of antigen-antibody.In addition, the stability requirement of high sensitivity to purification technique of ELISA method is high, cost intensive.
Contriver follows following principle and designs linear polypeptide antigen: 1. select epicyte protein surf zone; 2. the sequence not forming a-helix is selected; 3. the peptide section at two ends is more reasonable than middle arrangement; 4. active site of protein is avoided to repeat; 5. the peptide section that homology is strong is avoided; 6. avoid Cys and Glu in sequence as far as possible, too many Pro cannot be had, but there have 1-2 Pro to be beneficial to peptide chain structure to be stable, useful to generation specific antibody.In addition, this polypeptide antigen must contain the restricted epitope of human leucocyte two class antigen (HLA) system, comprises HLA-DR, the restricted epitope of HLA-DP and HLA-DQ.These epi-positions can identify by the HLA bis-class antigen systems of more than 90% Chinese colony.Based on the biological characteristics of above ANTIGEN DESIGNThe principle and Annexin A1 albumen, the present invention utilizes information biology and multiple Antigen Epitope Prediction simulation software, analyzes and antigenicity associated parameter, designed linear amino acid sequence.Annexin A1 linear polypeptide antigen is made up of 33 amino-acid residues, altogether containing 11 overlapping epitope, can detect at least 11 kinds of monoclonal antibodies, has the specificity of height.
method detectable antigens epi-position
we adopt ELISA method, detect, and obtain each sample OD value and analyze the blood collected.
quality Control
Duplicate hole established by each sample, is averaged OD value.OD value plastisied dispersion judges: plastisied dispersion=OD1-OD2/OD1+OD2, and plastisied dispersion≤0.1 is effective result; Plastisied dispersion >0.1 is null result.Get 100 parts of Healthy Human Serum equal-volume mixing as Quality Control blood (Quality control, QC), represent the common situation of crowd, 2 QC blood plasma holes all established by every plate, with the stability of the OD value Deflection level result of determination in QC blood plasma hole, batch variation CV=all batches of QC hole SD/ all batches of QC hole OD average <20%.Variation within batch CV=each plate QC every day hole each plate QC hole average <10% SD/ every day.
data analysis
SPSS17.0 for windows is adopted to carry out statistical analysis.Adopt specific combination index (Specific binding index, SBI) combination degree of Annexin A1 antigenic peptide and blood plasma autoantibody is judged, SBI=Annexin A1 OD value – NC OD value/QC OD value – NC OD value, NC is the negative control of each sample.Utilize
tcheck the difference compared respectively between malignant breast carcinomas group and normal healthy controls between SBI value, a=0.05.
ROC curve is according to a series of two different mode classifications (cut off value or decision threshold), with True Positive Rate (sensitivity) for ordinate zou, and the curve that false positive rate (1-specific degree) is drawn for X-coordinate.ROC area under a curve value is between 1.0 and 0.5.When AU>0.5, AU, more close to 1, illustrates that diagnosis accuracy is better.Sensitivity and specificity combine with graphic technique by ROC curve, accurately can reflect the relation of certain analytical procedure specificity and susceptibility, are the aggregate surrogates of test accuracy.This invention adopts Analyse-it for Microsoft Excel Software on Drawing ROC curve, and area (AU) under calculated curve, judges sensitivity and specific degree.
The present invention's application Annexin A1 antigen epitope polypeptide detects the Annexin A1 specificity Autologous IgG antibody in blood serum of patients with human breast carcinoma and blood plasma, and this reaction has high specific and high sensitivity.
Annexin A1 antigen epitope polypeptide can be used for preparing early diagnosing mammary cancer test kit.
Accompanying drawing explanation
Accompanying drawing is the ROC tracing analysis figure of patient with breast cancer's body anti-Annexin A1 Autologous IgG antibody horizontal.Wherein, X-coordinate is 1-specific degree, and ordinate zou is susceptibility.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these examples of implementation are only not used in for illustration of the present invention to limit the scope of the invention.
embodiment 1
prepared by test kit
Tab.2 ~ 7 are shown in 1 reagent kit preparation.
2 operations
(1) bag quilt: enzyme plate application lavation buffer solution cleans 3 times, work antigen coating buffer is diluted to working concentration, and be coated in enzyme plate, 4 DEG C are spent the night.
(2) L-glutamic acid is added: lavation buffer solution cleans 3 times, with coating buffer dilution L-glutamic acid to concentration 100 μ g/ml, every hole 200 μ l, 37 DEG C or incubated at room 1h;
(3) add blood plasma and Quality Control contrast (primary antibodie): enzyme plate application lavation buffer solution cleans 3 times, utilize coating buffer by diluted plasma to suitable concn, be generally 1:100 ~ 1:500, every hole 100 μ l, 37 DEG C or incubated at room 1h;
(4) two anti-hatch: lavation buffer solution cleans 3 times, and utilize coating buffer to dilute two anti-reference liquid IgG, working concentration 1:20000, every hole adds 100 μ l, 37 DEG C or incubated at room 1h;
(5) develop the color: lavation buffer solution cleans 3 times, and every hole adds 100 μ l substrate nitrite ions, room temperature lucifuge 30 ~ 45min.
(6) detect: every hole adds 50 μ l stop buffers, and detect in 10min, determined wavelength is 450nm, and reference wavelength is 630nm.
embodiment 2
patient with breast cancer'sannexin A1
autologous IgG antibody test
1 sample collection:this research is chosen and is made a definite diagnosis mammary cancer sample 311 example from The Second Hospital of Jilin Universtiy, tumour hospital of province through radiological examination and histological examination.Without any anticancer therapy before all serum sample collection, and there is comprehensive clinical data and information.Recruited normal healthy controls group sample 303 example simultaneously.Clinical interview and imaging examination all get rid of the ill possibility of mammary cancer.Healthy group with mammary cancer group in sex, age-matched, have comparability (
p>0.05)
2 detected results: the autoantibodies level (Tab.8) of Annexin A1: mammary cancer group exist compared with normal healthy controls group significant difference (
t=-7.163,
p<0.001).
ROC tracing analysis: the antibody test of patient with breast cancer Annexin A1 Autologous IgG is 0.723(SE=0.028,95%CI:0.668-0.777 in ROC area under curve) (Fig. 1 and Tab.9).
Above data fully show, utilize the antigen epitope polypeptide designed by the present invention to detect the patient with breast cancer's autoantibody IgG level obtained and compare with normal health group and have notable statistics difference.
tab.8the expression level of anti-Annexin A1 Autologous IgG antibody in patient with breast cancer and normal healthy controls sample
a Standard error
tab.9the ROC tracing analysis of anti-Annexin A1 Autologous IgG antibody in mammary cancer
| Antibody | AUC | SE a | 95%CI | Sensitivity (%) | Specificity (%) |
| malignant | 0.732 | 0.028 | 0.668-0.777 | 20.4 | 90.6 |
a Standard error
H-DITSDTSGDFRNALLSEATIIDILTKRNNAQDC -OH
Its purity >95%, pH>7.0.
Claims (2)
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| CN105111299A (en) * | 2015-09-24 | 2015-12-02 | 北华大学 | Epitode amino acid sequence of TFG-beta1 (transforming growth factor beta-1) as breast cancer detecting marker and application of epitode amino acid sequence |
| CN106526187A (en) * | 2016-09-13 | 2017-03-22 | 浙江理工大学 | Leukemia detection kit based on AnnexinA1 protein |
| CN112770786A (en) * | 2018-08-10 | 2021-05-07 | 米达内克斯股份有限公司 | Cancer treatment using antibodies |
| CN116539885A (en) * | 2023-07-06 | 2023-08-04 | 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 |
| JP7630676B2 (en) | 2015-02-06 | 2025-02-17 | セル アイディーエックス， インコーポレイテッド | Antigen-coupled immunoreagents |
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