CN100569800C - The monoclonal antibody of resisting human myeloblast differentiation marker gene and application thereof - Google Patents
The monoclonal antibody of resisting human myeloblast differentiation marker gene and application thereof Download PDFInfo
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- CN100569800C CN100569800C CNB2006100496619A CN200610049661A CN100569800C CN 100569800 C CN100569800 C CN 100569800C CN B2006100496619 A CNB2006100496619 A CN B2006100496619A CN 200610049661 A CN200610049661 A CN 200610049661A CN 100569800 C CN100569800 C CN 100569800C
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Abstract
The invention provides the monoclonal antibody and the application thereof of resisting human myeloblast differentiation marker gene.Through the antigen peptide immune mouse, mouse bone-marrow-derived lymphocyte and the murine myeloma cell of collecting sensitization merge, utilize enzyme-linked immunologic adsorption test method to judge positive colony, set up the clone of the monoclonal antibody of secretion resisting human myeloblast differentiation marker gene, the positive cells amplification cultivation and be injected to the mouse peritoneal of strain and induce generation ascites, through collecting and purifying, obtain the monoclonal antibody of resisting human myeloblast differentiation marker gene.The monoclonal antibody that the invention provides resisting human myeloblast differentiation marker gene can be used for the evaluation of human myeloblast differentiation marker gene molecule and human myeloblast differentiation marker gene molecule quantitatively.
Description
Technical field
The invention belongs to bioengineering field, relate to the MONOCLONAL ANTIBODIES SPECIFIC FOR and the application of resisting human myeloblast differentiation marker gene (hMYADM), be to utilize cell engineering, antibody engineering technology, obtain the clone of the monoclonal antibody of the anti-hMYADM of secretion, induce ascites by mouse, prepare the monoclonal antibody of anti-hMYADM with strain; Again by the application of technology such as protein purification, electrophoresis, immunity realization to this antibody.
Background technology
The immune molecule participation comprises the proliferation and differentiation and the function adjusting of the various kinds of cell of immunocyte.A kind of important immune recruit's discovery, often to the essence of the great vital processes such as differentiation and development that disclose immunne response, inflammation and hematopoietic cell and regulate rule and exert far reaching influence, therefore immune recruit's discovery and research have important theory and explore meaning and practical value.Marrow stromal cell (Bone MarrowStroma Cells, BMSC) be the very important cell colony of a class that constitutes the marrow hemopoiesis microenvironment, the discovered in recent years hematopoieticmicroenviron-ment plays an important role to hyperplasia and the differentiation of regulating hematopoietic cell, stroma cell and the interdependence of hematopoiesis parenchyma cell, stroma cell has special cell phenotype, can produce and secrete numerous cytokines, and at many cytokine receptors of film surface expression and other active substance, and effect such as directly contact with intercellular by cell, settling down of hematopoiesis support stem cell, propagation, its growth is regulated in differentiation, self, transition process in intercellular adhesion and the marrow.Discover that in addition BMSC plays an important role to selection, propagation, differentiation, migration, the apoptosis of leukemia cell's malignant clone.And BSMC still is the target cell of a kind of comparatively ideal cell and gene therapy.The immune recruit in research BMSC source is significant to migration in the differentiation and development of deeply illustrating BMSC, hematopoietic cell, the body, biological action and molecule mechanism thereof, the mechanism that can be understanding disease (as leukemia) generation development provides new angle, also may provide new thinking and means for the control of disease.
The applicant has set up the extensive random sequencing system of human bone marrow substrate cell (BMSC) cDNA gene library, has been cloned into the immune recruit that new cytodifferentiation antigen, immunity receptor, Ig superfamily member, signal transduction molecule etc. have the critical function prompting from nearly ten thousand EST that obtain.Find an immune recruit who derives from BMSC highly significant by the research applicant, obtained its full-length cDNA, be 2080bp, comprised 169bp 5 ' non-coding region, 969bp coding region, 322 amino acid of encoding, and 942bp 3 ' non-coding region.The myeloid cell differentiation marker gene (MYADM) of its aminoacid sequence and mouse has 86% homology, we are with its called after human myeloblast differentiation marker gene hMYADM (human Myeloid-associated Differentiation Marker gene), and in GenBank, logined, accession number is AY037147, and applied for patent of invention (title " Novel Human myeloblast differentiation marker gene; its encoding sequence and uses thereof ", application number 02136523.7).
By discovering to this recruit, this gene Selection is expressed in the myeloid cell and the marrow series leukemia cell in peripheral blood source, induce marrow series leukemia cell differentiation and marrow CD34+ cell in grain, monocyte differentiation at PMA, the expression of hMYADM is obviously raised.Laser Scanning Confocal Microscope proof hMYADM is a kind of transmembrane protein, is expressed on the cytolemma.These results disclose this new gene more fully, might and induce in leukemic pathogenesis and play certain vital role in the differentiation.The monoclonal antibody of preparation hMYADM, significant to its function of further further investigation, can be applicable to the detection of this recruit in different cells and the tissue, might disclose the possible application prospect of this recruit in the clinical disease control, enrich the fundamental research of hematopoietic cell differentiation and development process, and evocation is played in the research of aspects such as disease pathogenesis, diagnosis, prognosis and efficacy determination.
The present invention uses hybridoma cell technology.This technology merges myeloma cell and bone-marrow-derived lymphocyte, to set up the hybridoma cell line that waits the secretion homogeneous antibody, is also referred to as monoclonal antibody technique.This technology relates to serial of methods such as animal immune, cell cultures, cytogamy, cell clone cultivation and immunoassay.
Summary of the invention
The monoclonal antibody that the purpose of this invention is to provide a kind of resisting human myeloblast differentiation marker gene (hMYADM), resisting human myeloblast differentiation marker gene (hMYADM) is logined in GenBank, accession number is AY037147, and resisting human myeloblast differentiation marker gene (hMYADM) monoclonal antibody hybridoma cell is by China's typical culture collection center preservation; Address: Chinese Wuhan Wuhan University; Preservation date: on February 23rd, 2006; Classification name and deposit number are respectively:
Resisting human myeloblast differentiation marker gene (hMYADM) monoclonal antibody hybridoma cell ZMA1, CCTCC NO:C200615,
Resisting human myeloblast differentiation marker gene (hMYADM) monoclonal antibody hybridoma cell ZMA6, CCTCC NO:C200616,
Resisting human myeloblast differentiation marker gene (hMYADM) monoclonal antibody hybridoma cell ZMB5, CCTCC NO:C200617.
The monoclonal antibody of anti-hMYADM obtains by following steps:
(1) immunity of animal: select of the right age mouse, mouse is carried out immunity with antigen peptide (the 266-283 amino acids of hMYADM, 18 peptide KYGGQPRRSRDVSCSRSH are referring to SEQ No 1).18 peptide KYGGQPRRSRDVSCSRSH are synthetic by solid phase method, carry out on the Peptide synthesizer (431A) of American AB I company, adopt Fmoc (9-fluorenylmethyloxycarbonyl) scheme, and synthesis step carries out according to the polypeptide synthetic operation handbook of ABI company.Through high-efficient liquid phase chromatogram purification, purity＞95%, Sequence Identification is by mass spectroscopy.
(2) cultivation of murine myeloma cell: cultivation murine myeloma cell SP2/0 and the growth conditions that makes it to keep good are used for cytogamy.
(3) cytogamy: as feeder cell, merging the day before yesterday with the BALB/C mice peritoneal macrophage, inoculation BALB/C mice peritoneal macrophage is in 96 well culture plates, contains the HAT culture medium culturing one day of 20%FCS.The mouse of getting ready in (1) is put to death, obtain spleen lymphocyte.Collect the murine myeloma cell in (2).Above-mentioned two kinds of cytomixis are centrifugal, merge with polyoxyethylene glycol (PEG) mediated cell then.Cell after the fusion suitably dilutes, and is seeded to culture plate, and felicity condition is cultivated.
(4) hybridoma screening: above-mentioned culture is cultivated in selective medium.When cell colony length arrives size to fit, draw the nutrient solution supernatant and do antibody evaluation, screening positive clone.
(5) hybridoma cloning: with limiting dilution assay clone hybridization oncocyte, will be diluted to cell inoculation to 96 orifice plate of certain density, make every hole have only a cell growth.The hole of formation cell colony is got supernatant and is done enzyme-linked immunosorbent assay (ELISA), identifies positive colony.Can the repeated cloning several times, reach 100% up to the positive porosity of hybridoma.Hybridoma enlarged culturing after the cloning is done antibody to be identified.
(6) inducing of mouse ascites: inoculating preceding 10 days of hybridoma, give every 0.5ml of BALB/C mice abdominal injection whiteruss, then every inoculation 1.0 * 10
6Positive hybridoma cell, centrifugal after the collection ascites after 10 days, measure antibody titer, and monoclonal antibody purification.
(7) Purification of Monoclonal Antibodies: utilize the monoclonal antibody in sad-ammonium sulfate precipitation method purifying ascites supernatant.
(8) the present invention obtains three hybridoma cell lines of producing anti-hMYADM monoclonal antibodies, i.e. ZMA1, ZMA6, ZMB5, ZMA1, ZMA6, ZMB5 cell strain be through 3 time cloningizations, and it is surplus to continue to cultivate June, and secretory antibody is stable.This cell strain is through liquid nitrogen cryopreservation, and recovery is well-grown afterwards, and antibody-secreting is not seen decline.The ELISA indirect method records ZMA1, ZMA6, the ZMB5 supernatant is tired and is respectively 1: 128,1: 4,1: 32, tire be respectively ZMA1 1: 16384, ZMB5 1: 16384 of ascites.Cell culture supernatant after concentrating, immune double diffusion test-results show that three strain of hybridoma produce the antibody type and are IgG.
The preserving number of the CCTCC of ZMA1, ZMA6, ZMB5 is respectively:
ZMA1 CCTCC-C200615
ZMA6 CCTCC-C200616
ZMB5 CCTCC-C200617
Another object of the present invention provides anti-hMYADM monoclonal antibody and uses in human myeloblast differentiation marker gene is identified, realizes by following approach:
1. use the SDS-PAGE electrophoresis, by western blotting method (Western Blot) method, pair cell is expressed the situation of hMYADM molecule and is identified.
2. come the expression of identification of cell surface hMYADM molecule with immunofluorescence experiment.
Another purpose of the present invention provides anti-hMYADM monoclonal antibody and uses in human myeloblast differentiation marker gene is quantitative, realizes by following approach:
Utilize anti-hMYADM monoclonal antibody in conjunction with cell, combined with fluorescent two is anti-again, and the expression of the hMYADM molecule by flow cytometer pair cell surface is carried out quantitatively.
The invention has the advantages that the monoclonal antibody that a kind of anti-new gene hMYADM is provided, do not see bibliographical information as yet.The preparation method is simple, the monoclonal antibody of this method preparation of what is more important can have multiple use, qualitative and quantitative etc. as hMYADM developed by molecule situation in pair cell and the tissue, can be applicable to detect this new gene at different tissues and cell, and, be applied to the biology and the immunologic function research of this new gene more easily at the expression characteristic of bone marrow stem cell to the medullary system different differentiation phases.
Figure of description
Fig. 1 is hMYADM monoclonal antibody (ZMB5) the immunofluorescence experiment result of THP-1 cell.
Fig. 2 is the expression of flow cytometer detection by quantitative leukemia cell HL-60 surface hMYADM.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
The MONOCLONAL ANTIBODIES SPECIFIC FOR method of embodiment 1. anti-hMYADM
(1) immunity of mouse: first immunisation, with Freund's complete adjuvant emulsification, the 2nd, 3 time with Freund emulsification with 18 peptides of the hMYADM of crosslinked BSA, every mouse 0.2ml (containing hMYADM peptide 100 μ g), back intracutaneous multi-point injection, weekly.Booster immunization once merged after 3 days after one month.
(2) cultivation of murine myeloma cell: the SP2/0 myeloma cell strain from BALB/C mice is gone down to posterity with the DMEM culture medium culturing, containing 5%CO
2Cultivate in 37 ℃ of incubators of saturated humidity.Merge and enter logarithmic phase when going down to posterity the day before yesterday with the assurance fusion.
(3) cytogamy: with the BALB/C mice peritoneal macrophage as feeder cell, merging the day before yesterday, inoculation BALB/C mice peritoneal macrophage is in 96 well culture plates, contain the HAT culture medium culturing one day of 20%FCS, get spleen next day, put in the 200 order stainless steel meshs, reject fat and reticular tissue, tear coating, resuspended behind the separating Morr. cell, centrifuge washing cell 1～2 time with nutrient solution.Collect mouse SP2/0 myeloma cell, centrifugal, wash 2 times after the usefulness nutrient solution resuspended, as SP2/0 cell to be merged.With 1.0 * 10
8Individual splenocyte and 1.0 * 10
7Individual murine myeloma cell SP2/0 mixes, and merges under the 50%PEG effect.The cytomixis after scouring once, the centrifugal supernatant of abandoning, outstanding open cell (not adding liquid) and in 60～90 seconds, dropwise add in the cell precipitation with 37 ℃ of 50%PEG (pH＞8.0) 0.8ml, jolting centrifuge tube gently therebetween, but not piping and druming, left standstill 1 minute, then by elder generation's fast principle in slow back, in 1 minute, add 1ml serum-free RPMI1640, the serum-free RPMI1640 substratum 30～40ml that in 5 minutes, adds 37 ℃ of pre-temperature gradually, 37 ℃ warm, and gently suct down agitated liquid with suction pipe, low-speed centrifugal 5～10 minutes.Add 20%FCS HAT substratum then, be inoculated into 96 well culture plates that are rich in feeder cell respectively, put 37 ℃, 5%CO
2Cultivate in the incubator.
(4) hybridoma screening: changed 1/2 nutrient solution (HAT) every 3 days once, use the HT nutrient solution instead after 12 days.This selectivity is cultivated approximately and was continued for two weeks.When cell colony length arrives suitable size, draw the nutrient solution supernatant, be ELISA, screening positive clone.Adopt ELISA indirect method screening positive hybridoma.Key step: 1. 0.01M pH9.6 carbonate buffer solution dilutes the hMYADM recombinant protein, 1 μ g/ml, and 0.1ml/ hole wrapper sheet, 4 ℃ are spent the night; 2. 0.01M pH7.4PBS-Tween 20 washes plate three times; 3. the PBS of 10%FCS 0.01M pH 7.2 seals 30s; 4. the same plate of washing; 5. add the hybridoma culture supernatant, the 0.1ml/ hole is established positive control, negative control and blank simultaneously, 37 ℃ * 1hr; 6. wash plate; 7. add goat anti-mouse igg/HRP, 0.1ml/ hole, 37 ℃ * 1hr; 8. wash plate; 9. add 37 ℃ * 30s of substrate (TMB); 10. 2M H
2The SO4 termination reaction; 490nm measures its OD value, and is positive with P/N 〉=2.1.
(5) hybridoma cloning: hybridoma cloning is cultivated and is undertaken by limiting dilution assay, after the hybridoma porocyte of selection antibody positive is done suitably to breed, and the accurate counting cell.The cell suspension inoculation that is diluted to 10/ml with complete 1640 substratum is in 96 hole nutrient solutions of existing feeder cell, every hole 0.1ml, observation of cell growing state after 7-10 days, and antibody in the detection supernatant liquor, the selection antibody titers is higher, the culture hole that is single clone cell growth is done cloning once more and is cultivated, and is 100% until mono-clonal hole antibody test positive rate.
(6) induce ascites: inoculating preceding 10 days of hybridoma, give every 0.5ml of BALB/C mice abdominal injection whiteruss, then every inoculation 1.0 * 10
6Positive hybridoma cell is collected ascites and is measured antibody titer after 10 days.
(7) Purification of Monoclonal Antibodies: add the acetate buffer solution of the 10mM of 1 times of volume in the ascites supernatant, it is sad to add 10 μ l in the stirring in every milliliter of supernatant, leaves standstill 4 ℃ of refrigerator overnight behind the mixing.Abandon precipitation after high-speed low temperature is centrifugal.Add in the supernatant and dropwise add saturated ammonium sulphate, the ammonium sulfate saturation ratio reaches 50%, and centrifugal (12000g, 10 minutes) discard supernatant, and taking precipitate is dissolved in a small amount of physiological saline.With above-mentioned physiological saline solution liquid 33% saturated ammonium sulphate, centrifugal (12000g, 10 minutes), collecting precipitation is dissolved in the PBS of PH=7.4.With the extract dialysis tubing of packing into, in physiological saline, dialyse, remove ammonium sulfate.
(8) classification of monoclonal antibody is identified: the cell culture supernatant after concentrating, immune double diffusion test-results is that ZMA1, ZMA6, ZMB5 strain secretory antibody are IgG.
This monoclonal antibody of embodiment 2. usefulness is carried out the evaluation of hMYADM molecule
The anti-hMYADM monoclonal antibody of the present invention preparation can be used for the hMYADM molecule (qualitative detection) of identification of cell surface expression, and authentication method can be by following two kinds of methods realization:
1. indirect immunofluorescence, key step: 1. suspend with 1640 behind the cell dissociation, the 100ul/ hole is in 96 orifice plates, 37 ℃ * 4h; 2. 0.01M PH7.2PBS washes plate three times; 3. positive control, negative control and blank, are established simultaneously in culture supernatant 100ul/ hole by 37 ℃ * 1.5h; 4. the same plate of washing; 5. add fluorescence antibody, 37 ℃ * 1h; 6. wash plate three times; 7. fluorescence microscopy.Also can adopt the indirect immunofluorescence test tube method, key step: 1. cultured cells digestion back joins in the eppendorf pipe after suspending with physiological saline, 2. every pipe 100 μ l, add monoclonal antibody 100 μ l (1: 100) incubated at room 2 hours, the centrifugal 5min of 4000rpm/min, abandon supernatant, it is inferior to give a baby a bath on the third day after its birth with 0.01M pH7.2PBS 3%FCS, 3. 100 μ l PBS suspend, and add fluorescently-labeled two anti-3 μ l, pure calf serum 100 μ l, 4. the room temperature lucifuge is 1 hour, the same give a baby a bath on the third day after its birth time, 400 μ l PBS suspend, 5. the fluorescence microscopy.Under fluorescent microscope, can see the fluorescence of cell surface, be accredited as hMYADM and express positive cells, the result is referring to accompanying drawing 1, wherein THP-1 cell of Pei Yanging and anti-hMYADM monoclonal antibody (ZMB5) incubated at room are 2 hours, washing back and two anti-room temperature lucifuges of FITC mark are hatched hour, compressing tablet fluorescence microscopy can be seen the fluorescence of cell surface, and it is positive to be that hMYADM expresses.
2.Western Blotting: the lysate of cell carries out Western Blotting with this monoclonal antibody and detects, and presents the purpose band in the corresponding position, shows the expression that detects the hMYADM molecule.Concrete steps:
(1) sds polyacrylamide gel electrophoresis: method is referring to " fine works molecular biology experiment guide " (Science Presses 1998) such as F. Ao Sibai.Adopt 10% separation gel and 5% to concentrate glue, deposition condition is voltage 100V, carries out Coomassie brilliant blue dyeing after electrophoresis finishes.
(2) electrotransfer: method is referring to " fine works molecular biology experiment guide " (Science Presses 1998) such as F. Ao Sibai.The electricity consumption tranfer system is transferred to it on pvdf membrane.
(3) immunoblotting: anti-as one after 4 ℃ of sealings of 5% skim-milk are spent the night with the monoclonal antibody of the present invention preparation, hatched under the room temperature 2 hours or 4 ℃ of reactions are spent the night, with TST (TBS adds 0.5%tween) washing 3 times, each 10min, again with TBS washing 3 times, each 10min.Sheep anti-mouse igg with horseradish peroxidase-labeled is anti-as two, and room temperature reaction 2h with behind the ECL effect 1min, uses the X film development with aforesaid method washing back in the darkroom.
Embodiment 3, with this monoclonal antibody carry out cell surface hMYADM molecule quantitatively
Detect the intensity of cell surface expression hMYADM molecule with flow cytometer (FACS) method: collect PBS (contain 2%FCS) washing 3 time (1000g/5min/4 ℃) of cultured cells with precooling, abandon supernatant, cell is broken the every pipe in even back and is added 2-5 * 10
5Individual cell, the monoclonal antibody that adds our preparation is anti-as one, and test tube is put on ice, dark place reaction 30min, with PBS washed cell 3 times, the centrifugal supernatant of abandoning, beat even back and hatch 30min with the sheep anti-mouse igg of FITC mark, cell precipitation is resuspended among the PBS of 250 μ l, detect on flow cytometer, can obtain the fluorescence intensity and the positive cell rate of cell surface expression hMYADM molecule, the result is referring to accompanying drawing 2, the negative contrast of A, the fluorescence intensity of B figure is represented the expression amount of hMYADM.
Should understand, the present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (5)
1. the monoclonal antibody of the 266-283 amino acids of anti-people's medullary system differentiation sign is the hybridoma generation of CCTCC-C200615, CCTCC-C200616 or CCTCC-C200617 by preserving number.
2. the monoclonal antibody of anti-people's medullary system differentiation sign according to claim 1 is used in the qualitative detection that human myeloblast differentiation marker gene is expressed.
3. the monoclonal antibody of anti-people's medullary system differentiation sign according to claim 2 is used in the evaluation that human myeloblast differentiation marker gene is expressed, and it is characterized in that: the expression product of the method identifier's myeloblast differentiation marker gene by western blotting method or immunofluorescence.
4. the monoclonal antibody of anti-people's medullary system differentiation sign according to claim 1 is used in the detection by quantitative that human myeloblast differentiation marker gene is expressed.
5. the monoclonal antibody of anti-people's medullary system differentiation sign according to claim 4 is used in the detection by quantitative that human myeloblast differentiation marker gene is expressed, and it is characterized in that: undertaken quantitatively by intensity and positive cell rate that the flow cytometer method is expressed human myeloblast differentiation marker gene.
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| CN104277095B (en) * | 2013-07-10 | 2018-12-11 | 中国人民解放军第二军医大学 | The monoclonal antibody FMA1 of anti-human medullary system correlation differentiation marker protein and its application |
| CN104277096B (en) * | 2013-07-10 | 2019-10-25 | 中国人民解放军第二军医大学 | The monoclonal antibody DS6 of anti-human medullary system correlation differentiation marker protein and its application |
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| Title |
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| HL-60细胞表面分化抗原及细胞内髓过氧化物酶的电镜双重定位. 杨怡等.军事医学科学院院刊，第19卷第1期. 1995 |
| HL-60细胞表面分化抗原及细胞内髓过氧化物酶的电镜双重定位. 杨怡等.军事医学科学院院刊，第19卷第1期. 1995 * |
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