CN101921335B - Hybrid tumor generating anti-AMP-18 (Antrum Mucosalprotein-18) monoclonal antibody as well as anti-AMP-18 monoclonal antibody and application thereof in gastric carcinoma detection - Google Patents
Hybrid tumor generating anti-AMP-18 (Antrum Mucosalprotein-18) monoclonal antibody as well as anti-AMP-18 monoclonal antibody and application thereof in gastric carcinoma detection Download PDFInfo
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- CN101921335B CN101921335B CN2009100863522A CN200910086352A CN101921335B CN 101921335 B CN101921335 B CN 101921335B CN 2009100863522 A CN2009100863522 A CN 2009100863522A CN 200910086352 A CN200910086352 A CN 200910086352A CN 101921335 B CN101921335 B CN 101921335B
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Abstract
The invention aims at preparing a hybrid tumor of an anti-antrum mucosalprotein-18 (AMP-18) monoclonal antibody and relates to application thereof in preparing a tumor clinical detection reagent. Particularly, in the invention, a hybrid tumor cell (70110-4) capable of secreting the monoclonal antibody specifically recognizing the AMP-18 is screened through a molecular cloning technology and a monoclonal antibody technology. The monoclonal antibody secreted by the 70110-4 can be used for the clinical detection of carcinoma lesion in gastric tissues of human bodies.
Description
Technical field
The present invention relates to immunochemistry, oncobiology and cytobiology field.Particularly, the objective of the invention is to prepare a kind of and the secreted natural antrum secretory protein-AMP-18 (Antrum mucosal protein-18) of human stomach normal mucosa, the hybridoma of the monoclonal antibody that combines; Diagnostic reagent or association area that anti-AMP-18 monoclonal antibody that is produced by this hybridoma and corresponding product thereof can be applicable to people's cancer of the stomach.
Technical background
Cancer of the stomach is one of serious harm population health most common tumor.Its sickness rate and lethality rate are just listed after lung cancer, occupy time position of the cancer cause of the death.The main method of at present, treating tumour in the world is four kinds of operation, radiotherapy, chemotherapy and biotherapies etc.See that from medical angle tumor treatment does not have a kind of means in full force and effect so far, the tumour patient that has especially shifted, the whole body diffusion of the uncontrollable tumour of traditional treatment.Research confirms that back 5 years survival rates of diameter of tumor and operation are closely related, diameter of tumor＜2cm, and survival rate 100% in 5 years, the every increase of diameter of tumor 1cm, survival rate descended 20% in 5 years.So early diagnosis, even the diagnosis of cancer stage has significance for improving the cancer curative ratio in position.
Along with development of molecular biology, tumor markers has obtained paying attention to widely and using.Its appearance makes people express great hope to the early diagnosis of tumour.Tumor markers (tumor marker; TM) be meant in the generation and evolution of tumour; By tumour cell itself or by body tumour cell reaction is produced with the remarkable one type of different material of normal body; They both possibly be protein or nucleic acid molecule, also can be carbohydrate or lipid molecule.The detection of tumor markers in tumour patient blood or the body fluid has higher using value to auxiliary diagnosis, differential diagnosis, observation of curative effect, state of illness monitoring and the evaluating prognosis of tumour.
AMP-18 be one find recently by gastric gland body epithelial cell synthetic autocrine protein, molecular weight is about 18kDa.This albumen is expressed in stomach mucous membrane single-mindedly, and in its hetero-organization of human body, does not have or extremely low the expression.According to this breadboard early-stage Study and other bibliographical informations, we find that the generation of AMP-18 and cancer of the stomach and development have substantial connection, and its principal character does; 1) the expression degree of AMP-18 and gastric epithelial cell differentiation positive correlation, i.e. when differentiation was low, the expression amount of AMP-18 was very low; Even can not examine; Otherwise when cell differentiation was higher, the expression level of AMP-18 approached normally; 2) AMP-18 down-regulated expression or disappearance in tumour cell or tumor tissues, and in healthy tissues, be high expression level; 3) the stomach Helicobacter pylori infection is one of major reason that causes cancer of the stomach, and the intravital Hp amount of the expression amount of AMP-18 and patient is proportionate; 4) the prognosis positive correlation of the expression of AMP-18 and Patients with Gastric Cancer.In sum, AMP-18 can be used as molecular marker and is applied to the clinical diagnosis of cancer of the stomach and the assessment of prognosis curative effect.Because the height of AMP-18 secretion characteristic can detect its existence at normal stomach-tissue mucous layer; But in the zone that cancer of the stomach takes place, AMP-18 significantly descends along with the process of cancer, and therefore, the AMP-18 antibody of a kind of high specific and sensitivity is that the production reagent for clinical diagnosis is necessary.
The present invention is through this albumen of prokaryotic expression, and immune Balb/c mouse and process fusion, colony screening, preparation ascites, affinity chromatography technology purifying ascites have obtained the mouse resource monoclonal antibody.Immunohistochemical experiment detects the visible AMP-18 high expression level in healthy tissues of experiment of the expression in the cancer of the stomach pairing tissue, expresses or does not express but in stomach organization, hang down.Immunoblot experiment detect in eight pairs of stomach organizations and six kinds of stomach cancer cells systems in the experiment of expression of AMP-18 show: in all eight pairs of cancer of the stomach and cancer beside organism, AMP-18 is whole high expression levels in healthy tissues, in tumor tissues, do not express basically.In addition, we only can detect the expression of AMP-18 in normal gastric mucous membrane clone GES, and the expression of MP-18 is all negative in 6 kinds of stomach cancer cell systems.The experiment of immunofluorescence location external source AMP-18 shows: AMP-18 is an autocrine protein of cell, and this just means that AMP-18 can be secreted into blood, and possibly in stomach-tissue mucous membrane or serum, detect the existence of AMP-18.
Summary of the invention
The objective of the invention is to, provide a kind of and can specificity be directed against monoclonal antibody hybridoma of AMP-18 and preparation method thereof, and the purposes of this anti-AMP-18 monoclonal antibody hybridoma excretory antibody in lesion detection; Provide a specific specificity good monoclonal antibody method, the subclass of this antibody is IgG2a, ability specific combination AMP-18 recombinant antigen and natural antigen; A kind of immune diagnostic reagent or detection reagent that can be used for laboratory or clinical diagnosis tumor tissues AMP-18 expression level is provided.
Be the technique means that realizes that above goal of the invention adopts, the inventor is through this albumen of prokaryotic expression, and immune Balb/c mouse and process fusion, colony screening, preparation ascites, affinity chromatography technology purifying ascites have obtained the mouse resource monoclonal antibody.Immunohistochemical experiment detects the visible AMP-18 high expression level in healthy tissues of experiment of the expression in the cancer of the stomach pairing tissue, expresses or does not express but in stomach organization, hang down.Immunoblot experiment detect in eight pairs of stomach organizations and six kinds of stomach cancer cells systems in the experiment of expression of AMP-18 show: in all eight pairs of cancer of the stomach and cancer beside organism, AMP-18 is whole high expression levels in healthy tissues, in tumor tissues, do not express basically.And the positive expression that AMP-18 is arranged in normal gastric mucous membrane clone GES, and the expression of MP-18 is all negative in 6 kinds of stomach cancer cell systems.The experiment of immunofluorescence location external source AMP-18 shows: AMP-18 is an autocrine protein of cell, and this just means that AMP-18 can be secreted into blood, and possibly in stomach-tissue mucous membrane or serum, detect the existence of AMP-18.
Main contents of the present invention are following:
One) monoclonal antibody of hybridoma of the present invention (70110-4, preserving number are CGMCC 2839) secretion generation is mouse IgG2a hypotype monoclonal antibody.This antibody not only has specificity and susceptibility with the AMP-18 antigen of reorganization, and with AMP-18 in stomach-tissue and the cell extremely strong specificity and susceptibility is arranged.
Two) monoclonal antibody that hybridoma (70110-4, preserving number are CGMCC 2839) produces among the present invention can be applicable to immunoblotting, in the immunological experiment technology such as immunofluorescence and immunohistochemical methods.And, be that the cancer of the stomach mark diagnostic reagent of body making will have application promise in clinical practice to the monitoring of the early diagnosis and therapy of cancer of the stomach with the 70110-4 monoclonal antibody among the present invention.
Three) hybridoma (70110-4, preserving number are CGMCC 2839) excretory monoclonal antibody can be applicable to study Subcellular Localization among the present invention, in the scientific research of signal paths such as protein-interacting.
Description of drawings
Fig. 1 representes to adopt monoclonal antibody of the present invention to carry out the experimental result that immunohistochemical experiment detects the expression in the cancer of the stomach pairing tissue.Concrete visible AMP-18 high expression level in healthy tissues is expressed or is not expressed but in stomach organization, hang down.
Fig. 2 represent that monoclonal antibody of the present invention carries out that immunoblot experiment detects in eight pairs of stomach organizations and six kinds of stomach cancer cells systems in the experimental result of expression of AMP-18.The visible AMP-18 of experimental result is high expression level in the healthy tissues of all eight pairs of cancer of the stomach and cancer beside organism, in tumor tissues, does not express basically.In normal gastric mucous membrane clone GES, detect the expression of AMP-18, and the expression of MP-18 is all negative in 6 kinds of stomach cancer cell systems.
Fig. 3 representes that monoclonal antibody is in the experimental result of carrying out immunofluorescence location external source AMP-18 among the present invention.By the removal of expressing in the visible e. coli bl21 (DE3) of experimental result the AMP-18 protein-t-AMP-18 of signal peptide just be incorporated on the cytolemma, AMP-18 can combine with gastric epithelial cell, is an autocrine protein of this cell.
Embodiment
The antigenic preparation of embodiment 1:AMP-18
AMP-18 antigen is to be template with normal gastric mucosa cDNA, and according to AMP-18 sequence (SEQ ID No.1) design special primer, the primer two ends are connected into BamHI and SalI restriction enzyme site
Upstream primer: 5 '-TGAAGGATCCAACTATAATATCAACGTC-3 ' (SEQ ID NO.2)
Downstream primer: 5 '-AAATGTCGACGTTCTCCACCGTGTCTCC-3 ' (SEQ ID NO.3)
Pcr amplification is gone out except that the gene of AMP-18 signal peptide sequence (PCR parameter: 95 ℃ 5 minutes; 72 ℃ of 60s of 55 ℃ of 30s of 95 ℃ of 30s, totally 40 circulations; Extended after 72 ℃ 10 minutes) behind BamHI and SalI double digestion, is connected with pQE30a, chemical conversion competent cell JM109, picking mono-clonal extraction plasmid checks order, and verifies that sequence is errorless.Select the correct positive bacteria of order-checking, 1mmol/L IPTG abduction delivering 4 hours.After inducing, collect thalline, centrifugal after ultrasonication, go up cleer and peaceful precipitation and do not carry out the SDS-PAGE electrophoresis, the result shows that expressed products is a non-solubility albumen.
Carry out abduction delivering behind a large amount of culturing bacterium, extract AMP-18 albumen and carry out purifying through the Ni-NAT affinity column, 50mM imidazoles wash-out AMP-18 albumen, the SDS-PAGE electrophoresis cuts band and carries out the MALDI-TOF/TOF evaluation, and the confirmation expression product is AMP-18.
Embodiment 2: the preparation of anti-AMP-18 hybridoma
1) immunity
The AMP-18 albumen that will obtain through the Ni-NAT affinitive layer purification, subcutaneous abdomen immunity (eye socket is got 20 μ L serum and done negative control before the immunity) 4-6 age in week female Balb/c mouse; Dosage is every mouse 60 μ g albumen+saline water to 200 μ L+CFA200 μ L.Per 14 days subcutaneous booster immunizations once, dosage is every mouse 30 μ g albumen+saline water to 200 μ L+IFA200 μ L.7 days eye sockets are got blood and are surveyed and tire behind the 3rd booster immunization, reach the demander and impact immunity, 50 μ g albumen+saline water to 100 μ L; Tail vein injection is treated to merge after 3 days.
2) merge
The mouse spleen of fresh cutting-out is put on the cell sieve pulverizes filtration, with the mixed that the sp2/0 cell is pressed 1:5,1500 rev/mins centrifugal 5 minutes.Put into 37 ℃ of warm water baths with centrifugal good and centrifuge tube cell mixing is housed, the limit is stirred the cell limit and is slowly added 1mL PEG1500.In water-bath, left standstill 1 minute.Slowly add the serum-free of 10mL IMDM (Sigma, H0262-10VL) 1000 rev/mins centrifugal 5 minutes.Abandon supernatant, add careful cell is blown and beaten of serum of 10mL, and add thymocyte and 25mL semisolid medium, fully mixing is evenly poured in 20 Tissue Culture Dishs.Tissue Culture Dish is put in the wet box, put into 37 ℃, 5%CO
2Cultivate in the incubator.
3) choose the clone
Merged the back 7-10 days, observing clone cell, to roll into a ball big or small density moderate.Under anatomical lens, draw circle, real, big cloning cluster in the substratum that is prepared in advance 96 orifice plates of (containing feeder cell and 1%HT liquid).Put into 37 ℃, 5%CO
2Cell culture incubator.
4) screening
A. one sieve: chose the clone back about 3 days, and when the observation of cell amount accounts for floorage 2/3 greatly, got 100 μ L supernatant ELISA screening.Positive colony changes liquid, adds 200 μ L perfect mediums (containing 1%HT liquid).
B. two sieve: repeating step a carries out postsearch screening after 2 days.Positive strain changes the substratum that is prepared in advance 24 orifice plates of (containing feeder cell and 1%HT liquid) over to.
C. three sieve: after 5 days,, get 100 μ L supernatant ELISA screening with other recombinant protein wrapper sheet that contains label H is.Satisfactory clone changes 6 orifice plates or Tissue Culture Flask enlarged culturing over to.
5) cell cryopreservation
The logarithmic phase cell takes floorage about 80% can be frozen.Collect impurity such as supernatant and removal dead cell, supernatant is stored in-20 ℃.Directly freeze-stored cell is put 4 ℃ of half a hour, put then-20 ℃ two hours, change-80 ℃ of frozen spending the night, put liquid nitrogen container next day.
Embodiment 3: by preserving number is that the hybridoma cell line of CGMCC 2839 prepares anti-AMP-18
Monoclonal antibody
The contriver is with hybridoma (70110-4; December 30 2008 preservation day; Preserving number is CGMCC 2839, and preservation ground: the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms) be used to prepare mouse-anti AMP-18 monoclonal antibody, the concrete operations step is following:
1) cell recovery
From-80 ℃ of refrigerators or liquid nitrogen container, take out frozen pipe rapidly; Put into 37 ℃ of water-baths rapidly and stir fast, make frozen storing liquid in 2 minutes, all be melted into liquid.In the 15mL centrifuge tube, add the 3mL blood serum medium, frozen storing liquid is sucked centrifuge tube, 1500 rev/mins, centrifugal 5 minutes.Abandon supernatant, hanged cell, be incubated in 6 orifice plates (3mL) or the bottle (5mL) with perfect medium.
2) ascites preparation
The logarithmic phase cell washs with serum free medium and hangs; Count about 5 * 10
5/ 1mL.The cell intraperitoneal injection of mice that suspends.Generally just can collect ascites after 7-10 days.Can collect about 3mL for the first time, whenever can repeat to get, only finally can collect 8mL/ at a distance from 2,3 days.4000 rev/mins of each ascites of taking out, centrifugal 10 minutes; The centre is an ascites.Careful sucking-off ascites is collected in the centrifuge tube 4 ℃ of preservations.
3) Purification of Monoclonal Antibodies
With HiTrap rProtein A FF (GE Healthcare, 17-5079-01) affinity column monoclonal antibody purification.
With ascites under 4 ℃ of conditions, 10000 rev/mins centrifugal 10 minutes, remove lipid material.Supernatant is drawn in centrifugal back, and with coupling buffer with 1: 3 (ascites: coupling liquid) dilute, with 0.45 μ m membrane filtration.The chromatography column that appearance is crossed to the coupling buffer balance on the filtrating.After washing pillar with coupling buffer,, be collected in the collection tube that neutralizer is housed in advance with elution buffer wash-out antibody.
Embodiment 4:ELISA method is identified the monoclonal antibody subclass
1) experimental implementation
Encapsulate sheep anti-mouse igg to 0.5 μ g/mL with 100mM PBS (pH7.4) dilution, every hole adds 100 μ L, 4 ℃, spends the night.The sky liquid that inclines is washed 3 times with the PBS (PBST) that contains 0.05%Tween, and every hole adds 200 μ L confining liquids, hatches 1 hour for 37 ℃.The sky liquid that inclines cleans 3 times with PBST.Every hole adds 0.1mL hybridoma supernatant, hatches 1 hour for 37 ℃.The sky liquid that inclines cleans 3 times with PBST.With the sheep anti mouse of confining liquid 1: 1000 dilution HRP mark (κ, λ) sheep anti mouse of antibody or 1: 2000 dilution HRP mark (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) the every hole of antibody 0.1mL adds respectively in the suitable hole, 37 ℃ with hatching 1 hour.The sky liquid that inclines cleans 3 times with PBS-T.Every hole adds 50 μ L substrate solutions, surveys the OD value under the 405nm wavelength in 10-20 minute.
2) experimental result
Experimental result shows that monoclonal antibody of the present invention is an IgG2a type mouse resource monoclonal antibody.
Embodiment 5:ELISA method is measured the monoclonal antibody affinity costant
1) experimental procedure:
Encapsulate immunity with reorganization AMP-18, encapsulating concentration is 2 μ g/mL, 100 μ L/ holes, and 4 ℃ encapsulate and spend the night, and 1 * PBST washes 3 times.Every hole adds 37 ℃ of sealings of 200 μ L confining liquids 2 hours, and 1 * PBST washes 3 times.Anti-AMP-18 monoclonal antibody, since 1: 200 2 times of gradient dilution, the contrast that blanks of last 1 hole was hatched 1 hour for 37 ℃, and 1 * PBST washes 3 times.Sheep anti mouse two dilutions in anti-1: 20000 of HRP mark, every hole 100 μ L were hatched 1 hour for 37 ℃, and 1 * PBST washes 3 times.Colour developing liquid 100 μ L/ holes colour developing 10 minutes, 50 μ L/ hole stop buffer termination reactions.Measure light absorption value with ELIASA.
2) data analysis: find out >=the corresponding extension rate A of 1/2 " platform OD value ".
3) result: the affinity costant of monoclonal antibody of the present invention is 1.08 * 10
9
Embodiment 6: the purposes checking-immunohistochemical methods (IHC) of anti-AMP-18 monoclonal antibody of the present invention
1) material source:
The used stomach organization sample of the specific embodiment of the invention derives from School of Clinical Oncology, Peking University tissue sample storehouse, comprises cancer of the stomach and the corresponding other healthy tissues sample of cancer altogether.Sample draw materials be the fresh stomach organization of the underwent operative excision cancer beside organism corresponding with it after the saline water washing, put into the liquid nitrogen preservation.Sample all confirms through pathology, and patient's Informed Consent Form is arranged.
2) experimental procedure:
Roasting sheet, YLENE dewaxing, gradient ethanol aquation.0.3%H
2O
2Methyl alcohol room temperature 10 minutes, PBS washed 3 times * 5 minutes then; 5% skim-milk, 37 ℃ were sealed 2 hours; Anti-AMP-18 monoclonal antibody (dilution in 1: 2000), wet box is hatched 4 ℃ and is spent the night; Drip sheep anti mouse two anti-(middle China fir Golden Bridge, PV-6002), incubated at room 30 minutes; DAB-H
2O
2(middle China fir Golden Bridge, ZLI-9031) colour developing, mirror control 3-5 minute down, PBS rinsing, color development stopping; (middle China fir Golden Bridge ZLI-9040) redyes 45 seconds to Hematorylin, 1% hydrochloride alcohol differentiation then; The tap water flushing is anti-blue; 95% and 100% ethanol dehydration each 5 minutes, YLENE is transparent, the neutral gum mounting.
3) experimental result (Fig. 1):
The immunohistochemical experiment result is carried out statistical results show, AMP-18 all strong positive expression in 36 routine normal gastric mucosas (+++), and positive expression (+) a little less than only in 9 routine stomach organizations, whole negative results (-) in the remaining 27 example tissues.The p value is less than 0.01.Therefore, the experimental result of organization chip is also supported the conclusion of Proteomic analysis fully, and promptly AMP-18 high expression level in healthy tissues is expressed or do not expressed but in stomach organization, hang down.
Embodiment 7: the purposes checking-immunoblotting (WB) of anti-AMP-18 monoclonal antibody of the present invention
The inventor further uses immunoblotting WB and measures the protein expression of AMP-18 in stomach organization and 6 kinds of stomach cancer cells systems.
1) experimental procedure:
Albumen 30 μ g samples in stomach organization and the 6 kinds of stomach cancer cells system are splined on 12%SDSPAGE glue; After electrophoresis finishes, glue was soaked 10 minutes in 1 * transfer liquid; 350mA after the pvdf membrane methanol treatment changeed film 70 minutes; Place the TBST confining liquid that contains 5% skim-milk to seal 1 hour for 37 ℃ film; Anti-AMP-18 monoclonal antibody (dilution in 1: 1000) was hatched 1 hour or 4 ℃ of incubated overnight for 37 ℃; Corresponding sheep anti mouse two anti-(middle China fir Golden Bridge, dilution in 1: 2500) incubated at room 1 hour; Make public with ImageQuant ECL instrument.
2) experimental result (Fig. 2):
In all eight pairs of cancer of the stomach and cancer beside organism, whole high expression levels in the AMP-18 healthy tissues are not expressed in tumor tissues basically.When the expression of AMP-18 is as positive control in healthy tissues, all there is not the expression of AMP-18 in 6 kinds of stomach cancer cells systems.
Embodiment 8: the checking-immunofluorescence of the purposes of anti-AMP-18 monoclonal antibody of the present invention (IF)
For state after the secretion of simulating AMP-18, the inventor has made up escherichia expression system, in e. coli bl21 (DE3), has expressed AMP-18 protein--the t-AMP-18 that removes signal peptide.The inventor adopts immunofluorescence technique to detect the cellular localization of t-AMP-18.
1) experimental procedure:
T-AMP-18 and BGC-823 are cultivated altogether; 3.7% formaldehyde fixed 15 minutes; 0.2%TritonX-100 punching 5 minutes; The PBS solution that contains 1%BSA sealed 1 hour; Add in special anti-AMP-18 monoclonal antibody (dilution in 1: the 100) magazine and hatched 2 hours; Hatched 1 hour in the sheep anti mouse two of FITC mark anti-(middle China fir Golden Bridge, dilution in 1: the 200) magazine; 0.25 μ g/ μ LDAPI solution transfect cell 4 minutes; Deckglass taken out to cover dripping to be had on the slide glass of the anti-fluorescence decay mountant of 10 μ L, after the nail varnish mounting, and observation under laser scanning co-focusing microscope.
2) experimental result (Fig. 3):
T-AMP-18 just is incorporated on the cytolemma.Utilization obtains conclusion like this, and AMP-18 can combine with gastric epithelial cell, is an autocrine protein of this cell.This just means that AMP-18 can be secreted into blood, therefore can in serum, detect the existence of AMP-18, and this provides convenience for the detection of AMP-18.Specifically, because AMP-18 is a good tumor markers, it has the advantage that other tumor markerses do not have, and the method for available ELISA detects the content of AMP-18 in the serum clinically.Therefore, monoclonal antibody provided by the invention is used for preparing the diagnostic reagent and the test kit that detect serum AMP-18 tumor markers clinically has high application prospect.
Sequence table
< 110>BeiJing HuaDa protein Research Center Co., Ltd
< 120>hybridoma, anti-AMP-18 monoclonal antibody and the application in lesion detection thereof of the anti-AMP-18 monoclonal antibody of generation
<130>
<160>3
<170>PatentIn version 3.5
<210>1
<211>165
<212>PRT
< 213>AMP-18 protein sequence
<400>1
Asn Tyr Asn Ile Asn Val Asn Asp Asp Asn Asn Asn Ala Gly Ser Gly
1 5 10 15
Gln Gln Ser Val Ser Val Asn Asn Glu His Asn Val Ala Asn Val Asp
20 25 30
Asn Asn Asn Gly Trp Asp Ser Trp Asn Ser Ile Trp Asp Tyr Gly Asn
35 40 45
Gly Phe Ala Ala Thr Arg Leu Phe Gln Lys Lys Thr Cys Ile Val His
50 55 60
Lys Met Asn Lys Glu Val Met Pro Ser Ile Gln Ser Leu Asp Ala Leu
65 70 75 80
Val Lys Glu Lys Lys Leu Gln Gly Lys Gly Pro Gly Gly Pro Pro Pro
85 90 95
Lys Gly Leu Met Tyr Ser Val Asn Pro Asn Lys Val Asp Asp Leu Ser
100 105 110
Lys Phe Gly Lys Asn Ile Ala Asn Met Cys Arg Gly Ile Pro Thr Tyr
115 120 125
Met Ala Glu Glu Met Gln Glu Ala Ser Leu Phe Phe Tyr Ser Gly Thr
130 135 140
Cys Tyr Thr Thr SerVal Leu Trp Ile Val Asp Ile Ser Phe Cys Gly
145 150 155 160
Asp Thr Val Glu Asn
165
<210>2
<211>28
<212>DNA
< 213>upstream primer
<400>2
tgaaggatcc aactataata tcaacgtc 28
<210>3
<211>28
<212>DNA
< 213>downstream primer
<400>3
aaatgtcgac gttctccacc gtgtctcc 28
Claims (9)
1. a monoclonal antibody is characterized in that said monoclonal antibody is on December 30th, 2008 by preservation day, and preserving number is that the mouse hybridoma cell of CGMCC 2839 is that 70110-4 produces.
2. with the immunohistochemical methods reagent of the described Monoclonal Antibody of claim 1, it is used for detecting the differential expression of people's stomach organization and the other tissue of cancer of the stomach AMP-18.
3. with the immunofluorescence reagent of the described Monoclonal Antibody of claim 1, it is used for detecting the differential expression of people's stomach organization and the other tissue of cancer of the stomach AMP-18.
4. with the western blot reagent of the described Monoclonal Antibody of claim 1, it is used for detecting the differential expression of people's stomach organization and the other tissue of cancer of the stomach AMP-18.
5. the purposes of the described monoclonal antibody of claim 1, it is used for preparing the purposes of the immunohistochemical methods reagent that detects people's stomach organization and the AMP-18 of cancer beside organism differential expression.
6. the purposes of the described monoclonal antibody of claim 1, it is used for preparing and detects the purposes of immunofluorescence reagent that stomach cancer cell system and normal cell are the differential expression of AMP-18.
7. the purposes of the described monoclonal antibody of claim 1, it is used for preparing and detects the purposes of western blot reagent that SGC-7901 and normal cell are the differential expression of AMP-18.
8. the purposes of the described monoclonal antibody of claim 1, it is used for preparing the purposes of the detection reagent that detects stomach-tissue mucous layer AMP-18 differential expression.
9. a mouse hybridoma cell is 70110-4, and preserving number is CGMCC 2839, it is characterized in that said mouse hybridoma cell strain stably secretes the said monoclonal antibody of claim 1.
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| CN103130896B (en) * | 2013-03-20 | 2014-05-07 | 上海麦柏星生物科技有限公司 | Monoclonal antibody for resisting cell surface ectopic expression, and preparation method and application thereof |
| KR101988120B1 (en) * | 2017-08-21 | 2019-09-27 | 가톨릭대학교 산학협력단 | Diagnosis of gastric cancer using gastrokine 1 protein within blood |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1595547A2 (en) * | 2004-05-11 | 2005-11-16 | University of Chicago, UC-Tech Office of Technology & Intellectual Property | Use of the growth factor AMP for the treatment of gastrointestinal injuries and acute renal failure |
| EP1885390A2 (en) * | 2005-05-10 | 2008-02-13 | The University of Chicago | Use of peptides derived from the growth factor amp-18 for the treatment of mucositis |
-
2009
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1595547A2 (en) * | 2004-05-11 | 2005-11-16 | University of Chicago, UC-Tech Office of Technology & Intellectual Property | Use of the growth factor AMP for the treatment of gastrointestinal injuries and acute renal failure |
| EP1885390A2 (en) * | 2005-05-10 | 2008-02-13 | The University of Chicago | Use of peptides derived from the growth factor amp-18 for the treatment of mucositis |
Non-Patent Citations (1)
| Title |
|---|
| 陈美娅.AMP-18，一种新发现的胃黏膜保护因子.《世界华人消化杂志》.2006,第14卷(第8期),805-809. * |
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