CN101735317A - Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide - Google Patents
Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide Download PDFInfo
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- CN101735317A CN101735317A CN200810227252A CN200810227252A CN101735317A CN 101735317 A CN101735317 A CN 101735317A CN 200810227252 A CN200810227252 A CN 200810227252A CN 200810227252 A CN200810227252 A CN 200810227252A CN 101735317 A CN101735317 A CN 101735317A
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Abstract
The invention provides a tumor marker Cathepsin D antigen polypeptide which has an amino acid sequence shown as SEQ IDNo.1 in a sequence table. The invention also discloses a method for cloning and expressing the protein by a gene, preparing a specific antibody of the protein and judging gastric cancer biological behavior. The Cathepsin D antigen polypeptide and the antibody thereof prepared by the method can be used for preparing the tumor detecting kit and the research and development of a drug target.
Description
Technical field
The present invention relates to a kind of Cathepsin D antigenic peptide, a kind of tumor marker CathepsinD antigenic peptide of more specifically saying so, autoantibody belongs to bioengineering field preparing the detection kit that detects the application in the tumor reagent box and contain this polypeptide.
Background technology
In China, lung cancer is first cancer, is that sickness rate or mortality ratio all occupy first of the tumour.The diagnosis of lung cancer and treatment technology have had very much progress in recent years, but the five year survival rate of lung cancer still is lower than 15%.The human at present diagnosis to tumour mainly still relies on iconography and pathological technique means, and the patient is just diagnosed after obvious space occupying lesion and clinical symptom are arranged mostly, and most of patients has arrived middle and advanced stage, is difficult to obtain the effective treatment.And, lack clinically now the effective monitoring index of tumor development process, thereby cause the early diagnosis difficulty.Therefore, seek and set up the biomarker that lung cancer reaches precancerous lesion in early days, and prepare diagnostic kit with it, be the important subject of lung cancer prevention and early diagnosis always.
Along with the development of molecular cytobiology research and information biology, some new technique means continue to bring out, the proposition of the notion of more and more groups, and tumor research has also entered a new epoch.People begin the biological research of system tumor, wish to realize the unification of gene, albumen and biological metabolism research, and the lot of experiments data of acquisition will help human knowledge's oncobiology essence, and be converted into the effective technology means of treatment and prevention of tumour.
Proteomics be exactly protein group with biology be research object, by on a large scale, high-throughout analysis, attempt fundamentally cracks the mystery of life.In recent years, the fast development of proteomic techniques and application are for solid basis has been established in the screening and the early stage diagnosis of tumor markers.
Summary of the invention
An object of the present invention is to provide a kind of specific tumour mark Cathepsin D antigenic peptide and derivative thereof.
Second purpose of the present invention is to provide a kind of antibody of specific tumour mark Cathepsin D antigenic peptide preparation.
A further object of the present invention provides this in the application of Cathepsin D antigenic peptide in preparation detection tumor reagent box.
To achieve these goals, invention thinking of the present invention is:
The present invention utilizes proteomic techniques to identify and filtered out and the relevant protein C athepsin D of lung cancer generation, and it has been carried out clonal expression according to gene order, prepared antibody, further research can be applicable to the target of clinical diagnosis and research and development medicine.
By the method that two-dimensional electrophoresis and mass spectrum are identified, the contriver finds protein C athepsin D along with the increase for age in the secretory protein of three differences for the M-BE cell conditioned medium in age, and its content in conditioned medium also increases.The M-BE cell be the LT antigen of normal people's bronchial epithelial cell transfection SV40 and obtain with biochemical clone.It just aggravates with the increase for age, and the secretion characteristic that it showed may be consistent with the secretion characteristic of lung carcinoma cell, that is, Cathepsin D may secrete in a large number for lung carcinoma cell.Cathepsin D has been in the news and has participated in the development and the transfer of tumour.Therefore the difference of the secretory volume of this albumen in the tumour cell conditioned medium provides new tumor markers for further disclosing the tumor development rule, provides important test basis for being applied to clinical diagnosis from now on.
For further function and the effect of research Cathepsin D albumen in the tumour generating process, we are according to its gene order, clonal expression total length Cathepsin D albumen and multiple polypeptides fragment thereof, respectively through ni-sepharose purification.The hydrophobicity of Cathepsin D full-length proteins and each peptide species thereof is stronger, we utilize the Sarkosyl of different concns Cathepsin D full-length proteins and polypeptide thereof to be carried out the purifying of sex change and renaturation, finally obtain the higher Soluble Ca thepsin D antigen protein of purity and adopted highly purified antigen immune rabbit, obtained high tire how anti-, detect through ELISA and Western Blotting, confirm its specificity and susceptibility.
Concrete technical scheme of the present invention:
A kind of Cathepsin D antigenic peptide, it has aminoacid sequence shown in the SEQ ID No.1 in the sequence table.At first serum free medium is cultivated the M-BE cell, extracts protein from this serum free medium, behind the protein process Bradford standard measure of extraction, adopts two-dimensional electrophoresis to separate.Glue map analysis and mass spectrum identify and found 78 for relevant differential protein spot in age, and 44 protein spots wherein are along with for the painted intensification of the increase in age, and 34 protein sites show as with for painted the weakening of increase in age.These 78 protein sites are downcut, carry out enzyme and cut digestion, identify that through MALDI-TOF and LC-MS/MS mass spectrum one of them is Cathepsin D.Extract total mRNA of culturing cell, be that the template reverse transcription synthesizes cDNA first chain.According to Cathepsin D full length sequence design primer, the primer two ends are connected into EcoR I and Xho I restriction enzyme site:
Forward：5’CCGGAATTCATGCAGCCCTCCAGCCTTCTG 3’
Reverse：5’CCGCTCGAGGAGGCGGGCAGCCTCGGCGAA 3’
With cDNA first chain is that template is carried out the PCR reaction, and reaction conditions is 95 degree sex change, 56 degree annealing, and 72 degree extend, and MgCl2 concentration is 2mM.The PCR product reclaims in the glue, behind EcoR I and the Xho I double digestion after electrophoresis detection, be connected with the pET30a of same double digestion, chemical conversion competent cell DH5 α is coated with the Kana plate screening and transforms positive bacteria, choose mono-clonal and spend the night and shake bacterium, extract plasmid and check order, the checking sequence is errorless.
The derived sequence of above-mentioned a kind of Cathepsin D antigenic peptide, its be by aminoacid sequence shown in the SEQ ID No.1 through replacement, disappearance or the interpolation of amino-acid residue and the amino acid derived sequence that forms, its aminoacid sequence is shown in SEQ ID No.2.
For the further function of research Cathepsin D in the tumor development process, contriver's clonal expression Cathepsin D full-length proteins, behind ni-sepharose purification, obtained the CathepsinD antigen protein of purity very high (＞95%).Adopt highly purified antigen immune rabbit and mouse, obtained high tire how anti-, detect, confirmed its specificity and susceptibility through ELISA and Western Blotting.
A kind of can with above-mentioned Cathepsin D antigenic peptide specificity bonded antibody.
The application of Cathepsin D antigenic peptide in preparation detection tumor reagent box.
A kind of test kit that detects tumour, it contains described Cathepsin D antigenic peptide specificity bonded antibody.This test kit also comprises: (1) is the solid phase carrier (polystyrene) of envelope antigen or antibody; (2) substrate of enzyme: tetramethyl benzidine (3,3,5,5 ,-TMB); (3) negative control product and positive reference substance, reference standard product and control serum; (4) diluent of binding substances and sample: Tween-20, bovine serum albumin, phosphate buffered saline buffer, pH7.2 ± 0.2; (5) washings: contain 0.05% polysorbas20 phosphate-buffered saline; (6) enzyme reaction stop buffer: 2mol/L sulfuric acid.
Advantage of the present invention and beneficial effect:
(1) Cathepsin D antigenic peptide of the present invention has extremely strong specificity, can be used as tumor markers and prepares detection kit, has good application prospects and market outlook.
(2) the present invention has cloned its total length and has prepared corresponding antibody, has immunoreactive high specific and hypersensitivity concurrently; It can prepare the especially test kit of lung cancer of detection tumour, the detection efficiency height, and easy to use, this test kit has higher using value.
(3) in oncobiology research, the antigen that the present invention expresses can be applicable to study protein interactions etc., and the antibody of generation can be applicable to Western blotting, immunofluorescence and immunohistochemical methods etc.
(4) in addition, the present invention can also be as the target of new drug development, for the antitumor drug research and development provide new research material.
The present invention will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can clearerly learn technical scheme of the present invention, is not limitation of the present invention.
Description of drawings
Fig. 1 is for being in harmonious proportion the representative differential protein spot that raises under the increase in age with cell.
Fig. 2 immunohistochemical methods method detects the expression amount of Cathepsin D in the different lung tissues sample.A: lung phosphorus cancer, b: the lung phosphorus cancer of nodus lymphoideus transferring rate, c: normal segmental bronchus, d: normal alveolar.
Embodiment
Material and reagent source:
The used M-BE clone of this test is made up by Tumour Inst., Chinese Medical Academy journey book an ancient unit of weight academician laboratory.It is the non-tumour patient of China adult bronchial epithelial cell through former be commissioned to train support after, the LT antigen of transfection virus SV40 and the clone of the immortalization that obtains.Use MCDB 151 substratum (serum-free) that add somatomedin and hypophysis extract, in 37 ℃, 4% CO
2Incubator is cultivated.
Concrete steps:
The proteic preparation of embodiment 1Cathepsin D
1.M-BE the serum-free culture of cell
Three M-BE cells for age (P40, P140 and P200), with MCDB 151 substratum (serum-free) that are added with somatomedin and hypophysis extract, in 37 ℃, 4%CO
2Incubator is cultivated.Cell density reaches at 80% o'clock, behind PBS cleaning cell three times, changes MCDB 151 conditioned mediums of no somatomedin and hypophysis extract, continues at 37 ℃ 4%CO
2Incubator is cultivated.Behind the 48h, the collection condition substratum, and in 4 ℃, the centrifugal 10min of 1000g removes cell debris; Cell carries out proteinic extraction after PBS washing three times.
2. the proteins extraction in the serum free medium
The conditioned medium of collecting utilizes the dialysis membrane of 3500Da, dialyses in NaCl concentration gradient solution, and dialysate concentration and time are respectively: 100mM 2h; 50mM 4h; 25mM 8h; 10mM 10h; 0mM 24h.Cultivation after the dialysis based on-80 ℃ of refrigerators freezing after, with Maxi-dry Lyo concentration systems vacuum-freeze-dry.Dry powder is used lysate (8M urea, 4%CHAPS, 0.5%Ampholyte (pH 3.5-9.5), 2mM EDTA, 1mMPMSF, 10mM DTT) dissolving subsequently.
3. proteinic separation
Behind the protein process Bradford standard measure that extracts, adopt two-dimensional electrophoresis (2-DE) to separate.One what adopt mutually is 18cm PH 3-10NL adhesive tape, focus on to be set to the no-voltage bubble and to rise 4 hours, 50 volts 8 hours, 500 volts, 1000 volts and 8000 volts respectively one hour successively afterwards keep 8000 volts to reach 56,000 volts hours until total volt hours at last.Focus on the good balance liquid balance of a phase adhesive tape after 15 minutes, be transferred to two-phase 13%SDS-PAGE and separate through containing dithiothreitol (DTT) (DTT) and iodo-acetamide (IAA) respectively.Electrophoresis finishes the back and adopts silver to dye colour developing.
In order to obtain the further analysis that reliable 2-DE result is used for glue figure, we collect for the conditioned medium of the M-BE cell in age at twice to each, carry out proteinic extraction respectively; Protein for each extraction carries out the 2-DE separation with two parallel glue.Like this, each just has four corresponding glue figure for the protein in the age M-BE cell conditioned medium.
4. glue map analysis and mass spectrum are identified
The glue figure that silver dyes adopts ImageMaster 2D Platinum analysis software to analyze after scanner scanning, and the optical density(OD) difference of setting more than 3 times is threshold value.In order to obtain believable variance analysis result, at first relatively P40 and P200 glue figure find the point with significant difference, check the existence whether this point is arranged on the position corresponding on the P140 glue figure then.Fig. 1 has shown with cell for being in harmonious proportion the representative differential protein spot that raises under the increase in age.Shown in Figure 1A, on P40 glue figure, painted very dark spot a arranged, and there is this point in P200 glue figure this spot not on the P140 figure, illustrate that albumen that a orders is with cell secretory volume decline for the increase in age; And deepened gradually for painted on the glue figure in age at three in the b among Figure 1B o'clock, show that it increases with the increase secretory volume of cell for age.
Found 78 altogether for relevant differential protein spot in age, wherein 44 protein spots are along with for the painted intensification of the increase in age, and 34 protein sites show as with for increasing painted weakening age.
These 78 protein sites are downcut, carry out enzyme and cut digestion, identify that through MALDI-TOF/TOF and LC-MS/MS mass spectrum one of them is Cathepsin D.
5, the clone of Cathepsin D full-length proteins
Extract total mRNA of culturing cell, be that the template reverse transcription synthesizes cDNA first chain.According to Cathepsin D full length sequence design primer, the primer two ends are connected into EcoR I and Xho I restriction enzyme site:
Forward：5’CCGGAATTCATGCAGCCCTCCAGCCTTCTG 3’
Reverse：5’CCGCTCGAGGAGGCGGGCAGCCTCGGCGAA 3’
With cDNA first chain is that template is carried out the PCR reaction, and reaction conditions is 95 degree sex change, 56 degree annealing, and 72 degree extend, and MgCl2 concentration is 2mM.The PCR product reclaims in the glue, behind EcoR I and the Xho I double digestion after electrophoresis detection, be connected with the pET30a of same double digestion, chemical conversion competent cell DH5 α is coated with the Kana plate screening and transforms positive bacteria, choose mono-clonal and spend the night and shake bacterium, extract plasmid and check order, the checking sequence is errorless.
6.Cathepsin protein induced expression of D and purifying
From the correct positive bacteria that checks order, extract pET30a-Cathepsin D plasmid, transform the BL21 competent cell, be coated with the Kana plate screening and transform positive bacteria, choose mono-clonal and spend the night and shake bacterium, next day, enlarged culturing was when the OD reading is 0.6-0.8,1mmol/L IPTG abduction delivering.Induce later bacterial strain centrifugal after ultrasonic, go up cleer and peaceful precipitation and do not carry out the SDS-PAGE electrophoresis, the result shows that expressed products is an insoluble protein.
Carry out abduction delivering after shaking bacterium in a large number, the hydrophobicity of Cathepsin D full-length proteins and each peptide species thereof is stronger, the Sarkosyl that we utilize different concns carries out the purifying of sex change and renaturation to Cathepsin D full-length proteins and polypeptide thereof, has finally obtained the higher Soluble Ca thepsin D antigen protein of purity.Extract soluble albumen and cross the nickel post and carry out purifying, 100mM imidazoles wash-out, the SDS-PAGE electrophoresis cuts band and carries out MALDI-TOF and identify, confirms that expression product is Cathepsin D.
The preparation of embodiment 2Cathepsin D antigenic peptide polyclonal antibody
After the total length Cathepsin of ni-sepharose purification D antigen was fully dialysed by PBS, being concentrated into concentration was 1.0mg/ml.1.0mg antigen concentrated solution and isopyknic complete freund adjuvant are evenly mixed, fully emulsified back is in the subcutaneous multi-point injection in New Zealand white rabbit back.Get blood system from serum from rabbit leg vein before the initial immunity, contrast as preimmune serum; Carry out booster immunization first after two weeks, antigen concentrated solution and the isopyknic incomplete freund adjuvant of 500 μ g is evenly mixed, and fully emulsified back is in the subcutaneous multi-point injection in New Zealand white rabbit back.Afterwards, every 2 all booster immunizations once, and behind second time booster immunization, got blood from rabbit leg vein in 7 days, measure antibody titer with the ELISA method.Carotid artery bloodletting in the 7th day behind the 4th booster immunization, room temperature were placed after 4-5 hour, and centrifugal 5 minutes of 4 ℃ of 3000rpm collect supernatant, and are frozen stand-by in-80 ℃ after the packing.
Immunity back serum use protein A carries out the purifying of Ig G, has obtained the antibody of anti-Cathepsin D.We adopt ELISA and two kinds of methods of Western blotting to verify the specificity and the sensitivity of antibody.
ELISA experiment showed, that the titre of this antibody is 1: 10000, and background value is very low.Western blotting result shows, this antibody capable special and antigen-reactive, cross reaction is very weak.
Embodiment 3Cathepsin D specific antibody prepares the test kit composition
The preparation basic step of antibody kit is as follows:
(1) select suitable polystyrene elisa plate, the difference between the light absorption value of assurance single hole and the mean value of 96 hole light absorption values is in 10%;
(2) be labeled the antibody (100ng/ml) of the anti-Cathepsin D of horseradish peroxidase at every hole Zhong Bao with the micro-sampling instrument; Antibody (100ng/ml);
(3) seal with 0.5% bovine serum albumin;
(4) antibody to sheet material and bag quilt carries out drying treatment and vacuum packaging.
Embodiment 4Cathepsin D specific antibody is used for the judgement of lung cancer biological behaviour
We adopt the method for Tissue micro-array (TMA), analyze 346 routine paraffin-embedded tissue samples, comprised lung squamous cancer (SCC) sample of 178 routine nodus lymphoideus transferring rates (LNM), SCC sample, the normal alveolar of 40 examples and the segmental bronchus sample that 128 examples do not have LNM.As shown in Figure 2, utilize the immunohistochemical methods method to detect the expression amount of Cathepsin D in the different lung tissues sample.A: lung phosphorus cancer, B: the lung phosphorus cancer of nodus lymphoideus transferring rate, C: normal segmental bronchus, D: normal alveolar.Through statistical calculations, find that the positive tinctorial yield of Cathepsin D in 306 routine SCC samples is 87.9%, be higher than the positive tinctorial yield (being respectively 42.1% and 14.3%, p＜0.001) of normal segmental bronchus and alveolar significantly.And, the tinctorial yield of Cathepsin D in the lung squamous cancer sample and the positive correlation by stages of lung squamous cancer, Cathepsin D tinctorial yield is respectively 80.4%, 89.7%, 90.6% and 100% in I phase, II phase, III phase and the IV phase tumor tissues.
We select mono-clonal and the polyclonal antibody of Cathepsin D for use, 155 routine plasma samples have been carried out sandwich ELISA detected.In 104 routine SCC blood plasma, the middle site concentration of Cathepsin D is 93.12ng/ml, and the middle site concentration of Cathepsin D is respectively 63.87ng/ml and 79.46ng/ml in lung's illness blood plasma of 36 routine normal peoples, 15 routine non-tumours.By Kruskal-Walls test statistical analysis, the concentration of Cathepsin D is significantly higher than other two classes (p≤0.015) in the SCC blood plasma.Yet the concentration of Cathepsin D in lung's illness human plasma of normal people and non-tumour is comparable, does not have significant difference (p=0.812).If is two classes with the blood plasma of patients with lung cancer according to having or not LNM to shift non-, find that the concentration of Cathepsin D in 65 routine LNM patients' the blood plasma also is significantly higher than the patient (p≤0.038) of no LNM.
Show by this examination: the high-content of Cathepsin D in cancerous lung tissue and patients serum shows that all it can be used as the biomarker of lung cancer, can be used to indicate the generation of lung cancer; The more important thing is the high expression level of Cathepsin D in the lung squamous cancer of nodus lymphoideus transferring rate, show that it can be used to refer to the nodus lymphoideus transferring rate of lung cancer.Therefore, utilize the detection tumor reagent box of Cathepsin D antigenic peptide, have excellent specificity in preparation.
Sequence table
＜110〉Beijing Institute of Gene Science, Chinese Academy of Sciences
＜120〉Cathepsin D antigenic peptide, autoantibody are preparing the detection kit that detects the application in the tumor reagent box and contain this polypeptide
<130>
<160>2
<170>PatentIn?version?3.5
<210>1
<211>412
<212>PRT
＜213〉artificial sequence
<400>1
Met?Gln?Pro?Ser?Ser?Leu?Leu?Pro?Leu?Ala?Leu?Cys?Leu?Leu?Ala?Ala
1 5 10 15
Pro?Ala?Ser?Ala?Leu?Val?Arg?Ile?Pro?Leu?His?Lys?Phe?Thr?Ser?Ile
20 25 30
Arg?Arg?Thr?Met?Ser?Glu?Val?Gly?Gly?Ser?Val?Glu?Asp?Leu?Ile?Ala
35 40 45
Lys?Gly?Pro?Val?Ser?Lys?Tyr?Ser?Gln?Ala?Val?Pro?Ala?Val?Thr?Glu
50 55 60
Gly?Pro?Ile?Pro?Glu?Val?Leu?Lys?Asn?Tyr?Met?Asp?Ala?Gln?Tyr?Tyr
65 70 75 80
Gly?Glu?Ile?Gly?Ile?Gly?Thr?Pro?Pro?Gln?Cys?Phe?Thr?Val?Val?Phe
85 90 95
Asp?Thr?Gly?Ser?Ser?Asn?Leu?Trp?Val?Pro?Ser?Ile?His?Cys?Lys?Leu
100 105 110
Leu?Asp?Ile?Ala?Cys?Trp?Ile?His?His?Lys?Tyr?Asn?Ser?Asp?Lys?Ser
115 120 125
Ser?Thr?Tyr?Val?Lys?Asn?Gly?Thr?Ser?Phe?Asp?Ile?His?Tyr?Gly?Ser
130 135 140
Gly?Ser?Leu?Ser?Gly?Tyr?Leu?Ser?Gln?Asp?Thr?Val?Ser?Val?Pro?Cys
145 150 155 160
Gln?Ser?Ala?Ser?Ser?Ala?Ser?Ala?Leu?Gly?Gly?Val?Lys?Val?Glu?Arg
165 170 175
Gln?Val?Phe?Gly?Glu?Ala?Thr?Lys?Gln?Pro?Gly?Ile?Thr?Phe?Ile?Ala
180 185 190
Ala?Lys?Phe?Asp?Gly?Ile?Leu?Gly?Met?Ala?Tyr?Pro?Arg?Ile?Ser?Val
195 200 205
Asn?Asn?Val?Leu?Pro?Val?Phe?Asp?Asn?Leu?Met?Gln?Gln?Lys?Leu?Val
210 215 220
Asp?Gln?Asn?Ile?Phe?Ser?Phe?Tyr?Leu?Ser?Arg?Asp?Pro?Asp?Ala?Gln
225 230 235 240
Pro?Gly?Gly?Glu?Leu?Met?Leu?Gly?Gly?Thr?Asp?Ser?Lys?Tyr?Tyr?Lys
245 250 255
Gly?Ser?Leu?Ser?Tyr?Leu?Asn?Val?Thr?Arg?Lys?Ala?Tyr?Trp?Gln?Val
260 265 270
His?Leu?Asp?Gln?Val?Glu?Val?Ala?Ser?Gly?Leu?Thr?Leu?Cys?Lys?Glu
275 280 285
Gly?Cys?Glu?Ala?Ile?Val?Asp?Thr?Gly?Thr?Ser?Leu?Met?Val?Gly?Pro
290 295 300
Val?Asp?Glu?Val?Arg?Glu?Leu?Gln?Lys?Ala?Ile?Gly?Ala?Val?Pro?Leu
305 310 315 320
Ile?Gln?Gly?Glu?Tyr?Met?Ile?Pro?Cys?Glu?Lys?Val?Ser?Thr?Leu?Pro
325 330 335
Ala?Ile?Thr?Leu?Lys?Leu?Gly?Gly?Lys?Gly?Tyr?Lys?Leu?Ser?Pro?Glu
340 345 350
Asp?Tyr?Thr?Leu?Lys?Val?Ser?Gln?Ala?Gly?Lys?Thr?Leu?Cys?Leu?Ser
355 360 365
Gly?Phe?Met?Gly?Met?Asp?Ile?Pro?Pro?Pro?Ser?Gly?Pro?Leu?Trp?Ile
370 375 380
Leu?Gly?Asp?Val?Phe?Ile?Gly?Arg?Tyr?Tyr?Thr?Val?Phe?Asp?Arg?Asp
385 390 395 400
Asn?Asn?Arg?Val?Gly?Phe?Ala?Glu?Ala?Ala?Arg?Leu
405 410
<210>2
<211>348
<212>PRT
＜213〉artificial sequence
<400>2
Gly?Pro?Ile?Pro?Glu?Val?Leu?Lys?Asn?Tyr?Met?Asp?Ala?Gln?Tyr?Tyr
1 5 10 15
Gly?Glu?Ile?Gly?Ile?Gly?Thr?Pro?Pro?Gln?Cys?Phe?Thr?Val?Val?Phe
20 25 30
Asp?Thr?Gly?Ser?Ser?Asn?Leu?Trp?Val?Pro?Ser?Ile?His?Cys?Lys?Leu
35 40 45
Leu?Asp?Ile?Ala?Cys?Trp?Ile?His?His?Lys?Tyr?Asn?Ser?Asp?Lys?Ser
50 55 60
Ser?Thr?Tyr?Val?Lys?Asn?Gly?Thr?Ser?Phe?Asp?Ile?His?Tyr?Gly?Ser
65 70 75 80
Gly?Ser?Leu?Ser?Gly?Tyr?Leu?Ser?Gln?Asp?Thr?Val?Ser?Val?Pro?Cys
85 90 95
Gln?Ser?Ala?Ser?Ser?Ala?Ser?Ala?Leu?Gly?Gly?Val?Lys?Val?Glu?Arg
100 105 110
Gln?Val?Phe?Gly?Glu?Ala?Thr?Lys?Gln?Pro?Gly?Ile?Thr?Phe?Ile?Ala
115 120 125
Ala?Lys?Phe?Asp?Gly?Ile?Leu?Gly?Met?Ala?Tyr?Pro?Arg?Ile?Ser?Val
130 135 140
Asn?Asn?Val?Leu?Pro?Val?Phe?Asp?Asn?Leu?Met?Gln?Gln?Lys?Leu?Val
145 150 155 160
Asp?Gln?Asn?Ile?Phe?Ser?Phe?Tyr?Leu?Ser?Arg?Asp?Pro?Asp?Ala?Gln
165 170 175
Pro?Gly?Gly?Glu?Leu?Met?Leu?Gly?Gly?Thr?Asp?Ser?Lys?Tyr?Tyr?Lys
180 185 190
Gly?Ser?Leu?Ser?Tyr?Leu?Asn?Val?Thr?Arg?Lys?Ala?Tyr?Trp?Gln?Val
195 200 205
His?Leu?Asp?Gln?Val?Glu?Val?Ala?Ser?Gly?Leu?Thr?Leu?Cys?Lys?Glu
210 215 220
Gly?Cys?Glu?Ala?Ile?Val?Asp?Thr?Gly?Thr?Ser?Leu?Met?Val?Gly?Pro
225 230 235 240
Val?Asp?Glu?Val?Arg?Glu?Leu?Gln?Lys?Ala?Ile?Gly?Ala?Val?Pro?Leu
245 250 255
Ile?Gln?Gly?Glu?Tyr?Met?Ile?Pro?Cys?Glu?Lys?Val?Ser?Thr?Leu?Pro
260 265 270
Ala?Ile?Thr?Leu?Lys?Leu?Gly?Gly?Lys?Gly?Tyr?Lys?Leu?Ser?Pro?Glu
275 280 285
Asp?Tyr?Thr?Leu?Lys?Val?Ser?Gln?Ala?Gly?Lys?Thr?Leu?Cys?Leu?Ser
290 295 300
Gly?Phe?Met?Gly?Met?Asp?Ile?Pro?Pro?Pro?Ser?Gly?Pro?Leu?Trp?Ile
305 310 315 320
Leu?Gly?Asp?Val?Phe?Ile?Gly?Arg?Tyr?Tyr?Thr?Val?Phe?Asp?Arg?Asp
325 330 335
Asn?Asn?Arg?Val?Gly?Phe?Ala?Glu?Ala?Ala?Arg?Leu
340 345
Claims (10)
1. a Cathepsin D antigenic peptide is characterized in that, it has the aminoacid sequence shown in the SEQ ID No.1 in the sequence table.
2. a kind of Cathepsin D antigenic peptide according to claim 1 is characterized in that, it is through replacement, disappearance or the interpolation of amino-acid residue and the amino acid derived sequence that forms by aminoacid sequence shown in the SEQID No.1.
3. a kind of Cathepsin D antigenic peptide according to claim 2 is characterized in that the aminoacid sequence of Cathepsin D antigenic peptide derived sequence is SEQ ID No.2.
4. any described Cathepsin D antigenic peptide specificity bonded antibody in an energy and the claim 1 to 3.
5.Cathepsin the application of D antigenic peptide in preparation detection tumor reagent box.
6. a test kit that detects tumour is characterized in that, it contains the described Cathepsin D of claim 4 antigenic peptide specificity bonded antibody.
7. a kind of test kit that detects tumour according to claim 6 is characterized in that this test kit comprises: has wrapped by the solid phase carrier of Cathepsin D antigen or antibody (1); (2) substrate of enzyme: tetramethyl benzidine (3,3,5,5 ,-TMB); (3) negative control product and positive reference substance, reference standard product and control serum; (4) diluent of binding substances and sample: Tween-20, bovine serum albumin, phosphate buffered saline buffer, pH 7.2 ± 0.2; (5) washings: contain 0.05% polysorbas20 phosphate-buffered saline; (6) enzyme reaction stop buffer: 2mol/L sulfuric acid.
8. according to claim 5,6 or 7 described detection tumor reagent boxes, it is characterized in that described tumour is a lung cancer.
9. purposes that contains the test kit of Cathepsin D antibody as the detection of lung cancer nodus lymphoideus transferring rate.
10.Cathepsin the D antigenic peptide is as the application of the target of medicine for treating tumor thing.
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| CN200810227252A CN101735317A (en) | 2008-11-25 | 2008-11-25 | Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide |
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| CN200810227252A CN101735317A (en) | 2008-11-25 | 2008-11-25 | Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide |
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| CN200810227252A Pending CN101735317A (en) | 2008-11-25 | 2008-11-25 | Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2605016A1 (en) * | 2011-12-14 | 2013-06-19 | Philip Morris Products S.A. | Biomarkers related to lung cancer |
| CN105137085A (en) * | 2010-06-23 | 2015-12-09 | 阿斯图特医药公司 | Methods and compositions for diagnosis and prognosis of renal injury and renal failure |
| WO2018220106A1 (en) * | 2017-05-31 | 2018-12-06 | Artialis Sa | Biomarker molecules for sarcopenia and uses thereof |
| CN114015709A (en) * | 2021-11-04 | 2022-02-08 | 塔里木大学 | Application of sheep CTSD gene in regulation and control of initiation of primitive phase |
| CN115806623A (en) * | 2023-01-05 | 2023-03-17 | 广州达魏科技有限公司 | Application of Cathepsin L inhibition composition in cell amplification |
-
2008
- 2008-11-25 CN CN200810227252A patent/CN101735317A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105137085A (en) * | 2010-06-23 | 2015-12-09 | 阿斯图特医药公司 | Methods and compositions for diagnosis and prognosis of renal injury and renal failure |
| EP2605016A1 (en) * | 2011-12-14 | 2013-06-19 | Philip Morris Products S.A. | Biomarkers related to lung cancer |
| WO2018220106A1 (en) * | 2017-05-31 | 2018-12-06 | Artialis Sa | Biomarker molecules for sarcopenia and uses thereof |
| CN114015709A (en) * | 2021-11-04 | 2022-02-08 | 塔里木大学 | Application of sheep CTSD gene in regulation and control of initiation of primitive phase |
| CN115806623A (en) * | 2023-01-05 | 2023-03-17 | 广州达魏科技有限公司 | Application of Cathepsin L inhibition composition in cell amplification |
| CN115806623B (en) * | 2023-01-05 | 2023-11-21 | 营龄(武汉)生物科技有限公司 | Application of Cathepsin L inhibition composition in cell expansion |
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Open date: 20100616 |