CN105218656A - A kind of detection cervical cancer mark Survivin epitope aminoacid sequence and application - Google Patents
A kind of detection cervical cancer mark Survivin epitope aminoacid sequence and application Download PDFInfo
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- CN105218656A CN105218656A CN201510555205.0A CN201510555205A CN105218656A CN 105218656 A CN105218656 A CN 105218656A CN 201510555205 A CN201510555205 A CN 201510555205A CN 105218656 A CN105218656 A CN 105218656A
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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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
- G01N33/57411—Specifically defined cancers of cervix
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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
Abstract
Detect aminoacid sequence and the application of cervical cancer mark Survivin epitope, belong to immunological technique field, the invention provides the antigen aminoacid sequence of a kind of cervical cancer-related genes Survivin.Application Survivin polypeptide antigen detects corresponding specific autoantibody in cervical cancer patient blood, and this autoantibody can be used as risk level and the prognosis curative effect of the generation of cervical cancer mark assessment cervical cancer.This antigenic peptide and antibody thereof can be used for preparing the targeted drug that cervical cancer is treated in cervical cancer early diagnosis, prognosis prediction reagent and exploitation.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind ofly detect cervical cancer mark Survivin polypeptide fragment by having of information biology virtual sifting, population epidemiology detection validation its detection sensitivity and specific degree, for being further used for preparation cervical cancer early diagnosis and prognosis prediction test kit lays the foundation.
Background technology
Cervical cancer is one of common cancer of global women, and its sickness rate is only second to mammary cancer.Present the phenomenon that area increases and age of onset shifts to an earlier date in recent years.The histological type of cervical cancer, clinical phase not and prognosis closely related.Cervical cancer early diagnosis is conducive to patient and obtains treating in time, effectively, controls disease progression, improves the healthy and quality of life of women.Hematological examination is the simplest Wicresoft detection methods, and sensitivity, targetedly tumor markers contribute to the auxiliary diagnosis of clinician, relieve patient ' s burden for early diagnosis cervical cancer, improves early diagnostic rate.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.
Mankind survivin gene is found the earliest by Ambrosini for 1997, be positioned at karyomit(e) 17q25, total length 14.7kb, be made up of 3 introns and 4 exons, the albumen that its coding produces is made up of 142 amino acid, and relative molecular weight is 16.5kD, is the IAP (inhibitorofapoptosisprotein of discovered in recent years, IAP) newcomer in family is also one of significant survivin found at present.Survivin, with inhibited apoptosis and the dual-use function maintaining cell mitogen, generally expresses in the normal tissue hardly.But under pathological state, Survivin can wide expression in various malignant tumor tissue.The process LAN meeting inhibition tumor cell apoptosis of Survivin, promote tumour cell malignant proliferation, Survivin protein overexpression and course advancement are obviously relevant, suppress Survivin to express the means being considered to a kind of effective tumor remission.In the research of cervical cancer, researchist finds the phenomenon that Survivin raises successively in uterine neck chronic inflammatory diseases, Cervical intraepitheliaI neoplasia and cervical cancer.Also have report to show that Survivin is the index judging cervical cancer early prognosis, in early cervical carcinoma, Survivin expresses and significantly raises, and prompting Survivin has important value at the diagnosis and prognosis of cervical cancer.
The greatest differences of expressing between tumor tissues and healthy tissues based on Survivin and its vital role in tumor development, Survivin has attracted increasing concern as prediction, prognostic indicator and an anticancer therapy target spot.Point out our its using value in cervical cancer early diagnosis better simultaneously, and as potential source biomolecule mark may.The present invention is by the Survivin antigen epitope polypeptide of designed, designed, detect autoantibody in Serum of Cancer Patients and blood plasma and develop corresponding reagent, the danger that prediction cervical cancer occurs and prognosis prediction, and provide reliable data for cervical cancer new drug research.
The present invention is by the Survivin antigen epitope polypeptide of designed, designed, detect autoantibodies level in cervical cancer patient serum and blood plasma and develop corresponding reagent, the danger that prediction cervical cancer occurs, and be that preparation cervical cancer early diagnosis and prognosis prediction test kit lay the foundation.
Summary of the invention
The technical problem to be solved in the present invention is open a kind of epitope sequence detecting cervical cancer mark Survivin autoantibody.
The present invention discloses the purposes of Survivin epitope.
A kind of epitope aminoacid sequence detecting cervical cancer mark Survivin autoantibody provided by the invention is:
H-DACTPERMAEAGFIHCPAGFIHCPTENEPD-OH
Its purity >95%, pH>7.0.
The application of Survivin antigen epitope polypeptide of the present invention in preparation cervical cancer early diagnosis kit.
The present invention utilizes the linear polypeptide of the Survivin albumen of designed, designed, adopts ELISA method to detect the specificity Autologous IgG antibody of anti-Survivin albumen in cervical cancer patient serum and blood plasma.Autologous IgG antibody horizontal raises and shows that the expression amount of Survivin albumen in tumour patient body increases, primary or Secondary cases cervical cancer may be there is in indication patient, can predict that cervical cancer occurs, with the danger of recurrence, to instruct clinician to the early diagnosis of cervical cancer and prognosis prediction.
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-DPandHLA-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 Survivin albumen, the present invention utilizes information biology and multiple Antigen Epitope Prediction simulation software, analyzes and antigenicity associated parameter, designed linear amino acid sequence.Survivin linear polypeptide antigen is made up of 30 amino-acid residues, altogether containing 8 overlapping epitope, can detect at least 8 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 Controlduplicate 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 (Qualitycontrol, 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 analysissPSS17.0forwindows is adopted to carry out statistical analysis.Adopt specific combination index (Specificbindingindex, SBI) to judge the combination degree of SURVIVIN antigenic peptide and blood plasma autoantibody, SBI=SURVIVINOD value – NCOD value/QCOD value – NCOD value, NC is the negative control of each sample.Utilize
tthe difference of inspection respectively between more pernicious cervical cancer 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-itforMicrosoftExcel Software on Drawing ROC curve, and area (AU) under calculated curve, judges sensitivity and specific degree.
The present invention's application Survivin antigen epitope polypeptide detects the Survivin specificity Autologous IgG antibody in cervical cancer patient serum and blood plasma, and this reaction has high specific and high sensitivity.
Survivin antigen epitope polypeptide can be used for preparation cervical cancer early diagnosis and prognosis prediction test kit.
Accompanying drawing explanation
Accompanying drawing is the ROC tracing analysis figure of cervical cancer patient body anti-Survivin Autologous IgG antibody horizontal.
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
cervical cancer patientsurvivin
autologous IgG antibody test
1 sample collection:this research is chosen and is made a definite diagnosis cervical cancer sample 111 example from hospital of Jilin University the 3rd, 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 147 example simultaneously.Clinical interview and imaging examination all get rid of the ill possibility of cervical cancer.Healthy group with cervical cancer group in sex, age-matched, have comparability (
p>0.05)
2 detected results: the autoantibodies level (Tab.8) of Survivin: cervical cancer group exist compared with normal healthy controls group significant difference (
t=3.841,
p<0.001).
ROC tracing analysis: the antibody test of cervical cancer patient Survivin Autologous IgG is 0.723(SE=0.034,95%CI:0.593-0.728 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 cervical cancer patient autoantibody IgG level obtained and compare with normal health group and have notable statistics difference.
tab.8the expression level of anti-Survivin Autologous IgG antibody in cervical cancer patient and normal healthy controls sample
| Antibody a | Mean±SD（n） | t | P b |
| control | 0.613±0.147 (147) | ||
| malignant | 0.689±0.174 (111) | 3.841 | 0.000 |
aStandarderror
tab.9the ROC tracing analysis of anti-Survivin Autologous IgG antibody in cervical cancer
| Antibody | AUC | SE a | 95%CI | Sensitivity (%) | Specificity (%) |
| malignant | 0.660 | 0.034 | 0.593-0.728 | 22.5 | 90 |
aStandarderror。
Aminoacid sequence is: H-DACTPERMAEAGFIHCPAGFIHCPTENEPD-OH, its purity >95%, pH>7.0.
Claims (2)
1. detect a cervical cancer mark Survivin antigen epitope polypeptide, it is characterized in that: aminoacid sequence is: H-DACTPERMAEAGFIHCPAGFIHCPTENEPD-OH, its purity >95%, pH>7.0.
2. detection cervical cancer mark Survivin antigen epitope polypeptide according to claim 1 is preparing the application of cervical cancer early diagnosis and prognosis prediction test kit.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112229996A (en) * | 2020-08-25 | 2021-01-15 | 深圳市第二人民医院(深圳市转化医学研究院) | Application of protease activated receptor 2 as cervical cancer metastasis detection marker |
-
2015
- 2015-09-02 CN CN201510555205.0A patent/CN105218656A/en active Pending
Non-Patent Citations (4)
| Title |
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| 惠燕等: "survivin的研究进展", 《肿瘤研究与临床》 * |
| 陈明水等: "Survivin HLA-A2+高亲和性CTL表位的预测及鉴定", 《中国肿瘤生物治疗杂志》 * |
| 陈明水等: "Survivin表位肽诱导CTL免疫学效应及杀瘤效应研究", 《中国免疫学杂志》 * |
| 陶谦等: "人Survivin蛋白B细胞表位的初步预测", 《中华肿瘤防治杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112229996A (en) * | 2020-08-25 | 2021-01-15 | 深圳市第二人民医院(深圳市转化医学研究院) | Application of protease activated receptor 2 as cervical cancer metastasis detection marker |
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