CN108866002A - For SOST albumen or the cell strain of antibody screening and its preparation method and application - Google Patents

For SOST albumen or the cell strain of antibody screening and its preparation method and application Download PDF

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CN108866002A
CN108866002A CN201710335781.3A CN201710335781A CN108866002A CN 108866002 A CN108866002 A CN 108866002A CN 201710335781 A CN201710335781 A CN 201710335781A CN 108866002 A CN108866002 A CN 108866002A
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sost
albumen
leu
ser
cell strain
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高贝
沈毅珺
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FUDAN ZHANGJIANG BIOLOGICAL MEDICINE Co Ltd SHANGHAI
Shanghai Fudan Zhangjiang Bio pharmaceutical Co Ltd
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FUDAN ZHANGJIANG BIOLOGICAL MEDICINE Co Ltd SHANGHAI
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Abstract

The invention discloses a kind of for SOST albumen or the cell strain of antibody screening and its preparation method and application.Cell strain is the HEK293 cell for stablizing expression wnt1 protein gene, LRP acceptor gene and luciferase gene, which can be used for the assessment or screening of SOST albumen or antibody activity.Cell strain disclosed by the invention can stablize passage, and fluorescence intensity is higher, and SOST albumen is to the inhibitory activity IC of wnt1 albumen50It is lower, and detection window is larger, can preferably embody the biological activity of different SOST antibody, improves stability, reduces the coefficient of variation.Have the advantages that in the assessment and/or screening of SOST albumen or antibody activity quick, sensitive and stable.

Description

For SOST albumen or the cell strain of antibody screening and its preparation method and application
Technical field
The present invention relates to field of biotechnology more particularly to it is a kind of for the cell strain of SOST albumen or antibody screening, should The preparation method of cell strain, the preparation comprising the cell strain and cell strain or preparation answering in SOST albumen or antibody screening With.
Background technique
Os osseum fibroin (Sclerostin, SOST) is encoded by SOST gene, is the secretion sugar egg of 213 amino acid It is white.Sclerostin is the member of knot containing cystine (cystine-knot) factor superfamily, with DAN/Cerberus protein man Race is related, by inhibiting bone morphogenetic protein (bone morphogenetic protein, BMP) and the combination of receptor direct BMP signal is interfered, and therefore interferes BMP signal cascade.Since sclerostin is the osteocyte specificity negative regulator of bon e formation, this So that it becomes the attractive medicine target for osteoporosis treatment.Amgen is used for osteoporosis treatment in exploitation The antibody for os osseum fibroin, to FDA submit application for quotation, Novartis and Eli Lily also exploitation sclerostin resistance Disconnected antibody is currently in clinical 2 phases.
Sclerostin by be bound to LDH receptor related protein 5 and 6 (LRP5/LRP6) and Frizzled by Body works as the negative regulatory factor of typical Wnt signal.Classical Wnt access describes Wnt albumen and cell surface LRP/ Frizzled receptor family combine after series reaction, activation including intracellular protein Dishevelled (Dsh) and final thin The variation of β-catenin level in karyon.Dishevelled is the key component of cell membrane correlation Wnt receptor complex, it with Wnt is activated after combining, and inhibits downstream protein compound, including axin, GSK-3 and APC albumen.axin/GSK-3/APC Complex can promote the degradation of endocellular signal molecule β-catenin.After " β-catenin degradation compound " is suppressed, born of the same parents β-catenin in slurry is stabilized presence, and part β-catenin enters nucleus and TCF-LEF transcription factor family acts on And promote the expression of specific gene.β-catenin is widely present in various types of thin as a kind of multi-functional protein Born of the same parents, in endothelial cell, fibroblast, osteoblast, participate in the proliferation of these cells, differentiation and in terms of play Important adjustment effect.The activation of Wnt/ β-catenin approach is that differentiation of mesenchymal cells is bone and fat cell, Yi Jicheng Necessary to the differentiation of osteocyte and cartilage cell.β-catenin has played important function in regulation bone amount, by increasing bone The expression of plain (OPG) is protected, so that the ratio of OPG and nuclear Factor-Kappa B ligand receptor activator (RANKL) are improved, it is net to imitate Fruit is the generation for reducing osteoclast.Hardened proteins SOST is the ligand of LRP5/LRP6 compound, prevents receptor and Wnt In conjunction with to block Wnt/ β-catenin signal path.GSK-3 β smoothly in conjunction with multiprotein complex, promotes β- The phosphorylation of catenin, causes it finally to degrade.
In the existing method with reporter gene detection SOST antibody screening, usually certain cis response element and the light of firefly Luciferase reporter gene (Luciferase) carries out expressing in series, by the variation of the expression of examining report gene, Reflect the variation of the cis element.According to the literature (Ettenberg, S.A., Charlat, O., Daley, M.P., Liu, S., Vincent,K.J.,&Stuart,D.D.,et al.(2010).Inhibition of tumorigenesis driven by different wnt proteins requires blockade of distinct ligand-binding regions by lrp6antibodies.Proceedings of the National Academy of Sciences of the United States of America,107(35),15473.Verena,B.,Maarten,V.D.,Stella,W., Katharina,V.P.,Eva-Maria,M.,&Peter,T.D.,et al.(2014).Mutational analysis of sclerostin shows importance of the flexible loop and the cystine-knot for Wnt-signaling inhibition.Plos One, 8 (11), e81710.), it is bis- by transiently transfecting wnt1 and TCF-LEF Luciferase reporter plasmid can detecte the activity that SOST inhibits wnt signal.Wherein luciferase reporter gene plasmid The luciferase reporter plasmid of TCF-LEF comprising 1 mediation signal path and 1 conduct internal reference are stabilized every time instantaneous The Renilla luciferase reporter gene plasmid of transfection efficiency.Its antibody that SOST is detected by the instantaneous method of testing of wnt reporter gene, But this method is to obtain test cell strain by way of transiently transfecting cell.In the cell of transient transfection, foreign gene is not It can be integrated into the genome of cell, do not participate in duplication and be lost in fission process, but the time of exogenous gene expression It is limited, it usually only takes several days, therefore use be required to be transiently transfected when cell every time, so the coefficient of variation is big, and It is extremely not quick and conveniently.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of stabilization, long-term expression wnt1 protein gene, LRP6 receptor bases Cause and luciferase gene can be used for the cell strain of SOST albumen or antibody activity screening, and provide a kind of quick, sensitive And the method for steadily evaluating SOST albumen or antibody biological activity.And provide the preparation method and the cell of the cell strain Application of the strain in SOST albumen or antibody activity assessment or screening.In addition, also providing a kind of preparation comprising the cell strain.
In order to solve the above technical problems, one of the technical solution that the present invention takes is：It provides a kind of for SOST albumen Or antibody assessment and/or the cell strain of screening, it is to stablize expression wnt1 protein gene, LRP acceptor gene and luciferase The HEK293 cell of gene.
According to the present invention, described " stablizing expression " refer in cell strain can the long period, generally at least pass on 20 generations with Interior energy expression alien gene, and expression quantity is without obvious decline.
Preferably, the cell strain cotransfection has the plasmid 1 of the wnt1 protein gene, comprising the LRP acceptor gene Plasmid 2 and plasmid 3 comprising luciferase gene.
Wherein, the wnt1 albumen can choose rmwnt1 albumen, the preferred hLRP6 of LRP6 receptor (people LRP6 by Body), the luciferase is preferably connected with the luc2P of TCF-LEF element.
Wherein, each plasmid can be constructed voluntarily, can also if yes buy existing commercialization plasmid.Institute of the present invention Existing business plasmid rmwnt1-pCMV6-Kan/Neo can be selected by stating plasmid 1；The plasmid 3 selects existing business plasmid pGL4.49[luc2P/TCF-LEF/Hygro]；LRP receptor in the plasmid 2 is hLRP6 (people LRP6, gene nucleotide Sequence is as shown in SEQ ID NO.1), the preferred pCDNA3.1/Hygro (-) of plasmid backbone.
Preferably, the relative intensity of fluorescence RLU of cell strain expression is at least 100,000, preferably 100,000~500, 000, more preferable 180,000~250,000；Inhibitory activity of the SOST albumen to cell strain of the present invention expression wnt1 albumen IC50Not higher than 110nM, generally in 25~110nM, it is more commonly in 26~45nM；It is described in a preferred embodiment of the present invention The relative intensity of fluorescence RLU of cell strain is 248,000, the inhibitory activity IC50For 43nM.
In order to solve the above technical problems, one of the technical solution that the present invention takes is：Provide the preparation side of the cell strain Method comprising following steps：
(1) plasmid containing LRP acceptor gene is prepared；
(2) by the plasmid in step (1) and the plasmid containing wnt1 protein gene and contain the matter of luciferase gene Grain liposome cotransfection HEK293 cell.
According to the present invention, " cotransfection " refers to HEK293 cell while transfecting different plasmids, makes it simultaneously It is integrated into the genome of cell, the protein stabilized expression of the plasmid of transfection is made by antibiotic pressurization screening.
Preferably, liposome is Lipofectamine2000；Preferably, by after cotransfection HEK293 cell kind plate, also plus Enter antibiotic pressurization screening；More preferably, antibiotic is hygromycin (Hygromycin) and Geneticin (Geneticin G418)； Most preferably, the final concentration of 400 μ g/ml of the final concentration of 300 μ g/ml of hygromycin and Geneticin.
Preferably, the amount ratio of three kinds of plasmids is 1:1:1, Lipofectamine2000 dosage is 2.8 μ g/ reaction；More Goodly, the time of the kind plate is 2 days and/or the time of pressurization screening is 2~3 weeks；Most preferably, pressurize time of screening is 3 weeks.
In order to solve the above technical problems, one of the technical solution that the present invention takes is：SOST albumen or antibody activity are provided Assessment or screening technique comprising following steps：
(1) gradient dilution SOST albumen to be measured；Or gradient dilution test antibodies, certain density SOST antibody is added and incubates It educates；
(2) it is co-cultured again with cell strain as described in any one of claims 1-3；
(3) then be added Luciferase Assay Reagent, be protected from light, react at room temperature after read；
(4) SOST albumen or antibody activity value are judged according to reading.
Preferably, the SOST protein concentration to be measured is 4.5 μM~80pM；The concentration of test antibodies is 850nM~1nM； The temperature of the incubation is 37 DEG C, and the time is 1 hour；Co-cultured cell strain number amount is every reaction 2 × 104It is a；The temperature of co-cultivation Degree is 37 DEG C, and the time is 18-20 hours；And/or be protected from light room temperature reaction time be 15 minutes.
In order to solve the above technical problems, one of the technical solution that the present invention takes is：Cell strain is in SOST albumen or antibody Application in Activity Assessment or screening.
In order to solve the above technical problems, one of the technical solution that the present invention takes is：Preparation comprising the cell strain.Preferably Ground, said preparation are SOST albumen or antibody test preparation.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that：Cell strain of the present invention is to stablize the thin of passage for SOST antibody screening Born of the same parents' strain stablizes expression wnt1 and LRP6, while stablizing expressing luciferase reporter plasmid, the fluorescence of cell strain expression Intensity height (>=100,000) and SOST albumen is to the inhibitory activity IC of wnt1 albumen50Low (≤110nM), and the detection of antibody Window it is larger (>2.15) biological activity of different SOST antibody, can be preferably embodied, stability is improved, reduces variation lines Number.The step of cell strain is used for the assessment and/or screening of SOST albumen or antibody biological activity, can simplify experiment, Accelerate the process of experiment and increase the stability of sensitivity and experiment, for the antibody and research SOST antibody for screening high activity Activity lay the foundation.
Detailed description of the invention
Fig. 1 is cotransfection plasmid map, wherein 1A.hLRP6-pcDNA3.1/Hygro：The eukaryotic expression of hLRP6 receptor carries Body map；1B.pGL4.49[luc2P/TCF-LEF/Hygro]：Luciferase reporter gene expression containing TCF-LEF element Vector map；1C.rmwnt1-pCMV6-Kan/Neo：The carrier for expression of eukaryon map of rmwnt1.
Fig. 2 is the expression of different cell strain composing type wnt signal reports genes.
Fig. 3 is the screening of biology Activity determination cell strain, and wherein 3A.SOST is living to the inhibition of constitutive expression signal Property dose-dependent effect curve；3B.Romosozumab standard antibody biological activity dosage in W18, W45 and W60 cell strain Rely on effect curve.
Fig. 4 is the detection of candidate antibodies biological activity.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.
The gene of nucleotide hLRP6 as shown in SEQ ID NO.1 entrusts Suzhou Jin Weizhi Biotechnology Co., Ltd Synthesis, gene both ends are separately added into the site Nhe I and Hind III, are connected in pUC57, obtain plasmid hLRP6-pUC57.It is logical It crosses Nhe I and Hind III (purchased from TaKaRa) and double digestion is carried out to hLRP6-pUC57, with the expression vector of identical digestion PcDNA3.1/Hygro (-) (being purchased from Invitrogen) is attached, and is used for cell transfection assays after sequencing identification is correct.
PGL4.49 [luc2P/TCF-LEF/Hygro] is the reporter plasmid containing TCF-LEF element, and purchase is certainly Promega company.
Rmwnt1-pCMV6-Kan/Neo is used for the expression of wnt1 albumen, buys from OriGene Technologies.
HEK293 cell strain is purchased from Chinese Academy of Sciences's cell bank, and SOST albumen is purchased from R&D systems.
Embodiment 1
Stablize the preparation of the HEK293 cell strain of expression
The determination of 1.1 HEK293 antibiotic-screening concentration
Dose-response analysis is carried out before transfection to HEK293 cell to determine that (Hygromycin is purchased from hygromycin ) and the most suitable screening concentration of Geneticin (G418, be purchased from A.G.Scientific) Generay.Prepare the complete of cell growth Culture medium：10% fetal calf serum (FBS) and 2mM glutamine (being purchased from Gibco) are added in without phenol red DMEM, it is thin in 24 holes 100 μ l cell suspensions (1 × 10 are added in every hole in born of the same parents' culture plate3A cell/ml), the complete culture of 900 μ l is added in every hole Base sets up the kind slat element of 100, every hole cell；The Hygromycin solution for sequentially adding 50mg/ml obtains final concentration point Not Wei 0,50,100,200,300,400,500,600,700,800 μ g/ml complete medium or 100mg/ml G418 it is molten Liquid obtains the complete medium that final concentration is respectively 0,50,100,200,300,400,500,600,700,800 μ g/ml, each dense Spend 3 multiple holes.Culture plate is put into 37 DEG C of -5%CO2Incubator stationary culture uses Hygromycin containing respective concentration in every 3-4 days Or the fresh complete medium of G418 replaces old culture medium, the Cmin of complete cell death turns sieve as steady after 10-15 days The optium concentration of choosing.As a result：For cell after 13 days in 24 orifice plates under 300 μ g/ml pressure of Hygromycin final concentration, cell is several All dead, i.e., HEK293 screening pressure is Hygromycin：300μg/ml；Under 400 μ g/ml pressure of G418 final concentration, Cell almost all is dead, i.e., HEK293 screening pressure is G418：400μg/ml.
The transfection of 1.2 HEK293 cells
A.HEK293 cell cotransfection rmwnt1, hLRP6 and TCF-LEF-luc2P
HEK293 cell culture is in without in phenol red DMEM, condition of culture is containing 10% fetal calf serum and 2mM glutamine 37 DEG C of -5%CO2Incubator.With 3.5 × 105A cell/ml density is inoculated in 24 well culture plates, and every hole is inoculated with 0.5ml.Kind plate 24 After hour, using liposome Lipofectamine2000 (Lifetech) cotransfection plasmid, according to hLRP6-pcDNA3.1/ Hygro：pGL4.49[luc2P/TCF-LEF RE/Hygro]：Rmwnt1-pCMV6-Kan/Neo=0.27 μ g：0.27μg： The parameter of the Lipofectamine2000 in the ratio of 0.27 μ g and 2.8 holes μ l/ is transfected.Transfection composite and cell are total Fresh culture is replaced after being incubated for 4-6 hours, next day plants 96 well culture plate of transfection cell inoculation plate 2 days with limiting dilution assay Hygromycin (final concentration is added afterwards：300 μ g/ml) and G418 (final concentration：400 μ g/ml) pressurization screening 2-3 weeks.Stand training It supports, is gradually expanded to 24 orifice plates, T25 culture bottle.
B. cell strain is compared：HEK293 cell cotransfection rmwnt1 and TCF-LEF-luc2P
According to the method for above-described embodiment part A, with HEK293 cell cotransfection rmwnt1 and TCF-LEF-luc2P, with Detect the fiting effect of HEK393 cell included LRP6 receptor and rmwnt1.
1.3 stablizing the screening of the HEK293 cell strain of expression
Pancreatin -0.25%EDTA handles transfection cell to be screened, after fresh culture is resuspended, with 2 × 10 after counting4It is a For the density kind plate of cells/well in 96 well culture plate of White-opalescent, each cell strain to be screened and ghost HEK293 are each 2 multiple holes of kind.After 37 DEG C of 18-20 hours of incubation, with Steady Glo Luciferase Assay System (Promega, E2520 it) detects, the detection reagent Steady Glo Luciferase Assay System of 100 μ l is added in every hole, is protected from light room temperature Reaction is placed on SpectraMax L microplate reader detection reading for 15 minutes.
The HEK293 cell strain of 21 plants of stable transfections is obtained altogether, this waits cell strains constructive expression wnt signal, shows as producing Raw stable fluorescence signal.Compare the fluorescence signal that different cell strains generate, i.e. Relative fluorescence units (Relative Luciferase Unit), see Fig. 2.Wherein W45 generates highest fluorescence signal (RLU>500,000), W41-43, W48, W60 The fluorescence signal of moderate strength is generated, W18, W23, W28, W40, W36, W44, W55, W58 generate lower but detectable fluorescence Signal (RLU≤100,000).Wherein, W18RLU=35,100, W41RLU=182,500, W42RLU=278,541, W43RLU= 239,202, W48RLU=264,279, and W60RLU=248,000.
The comparison cell strain that part B constructs in 1.2, after being handled with above-mentioned same method, after being protected from light room temperature reaction 15 minutes It is placed in SpectraMax L microplate reader detection reading, but fluorescence signal illustrates that rmwnt1 albumen can not be with lower than detection limit HEK293 cell combination, it is necessary to be introduced into hLRP6 and be integrated into HEK293 cell and could be combined with rmwnt1.
Embodiment 2
Stablize the functional evaluation of the HEK293 cell strain of expression rmwnt1, hLRP6, TCF-LEF-luc2
Prepared by embodiment 1 stablizes the cell strain for generating fluorescence signal, chooses high fluorescence signal, intermediate fluorescence letter respectively Number, cell strain W45, W60, W18 of low Poison signal carry out function assessment evaluation.
The measurement of 2.1 SOST (os osseum fibroin) half-inhibitory concentration
Pancreatin -0.25%EDTA handle it is to be screened surely turn cell, after fresh culture is resuspended, with 2 × 10 after counting4It is a For the density kind plate of cells/well in 96 well culture plate of White-opalescent, each cell strain (W18, W45, W60) to be screened is each respectively 16 holes of kind, are incubated for the diluted SOST solution of gradient concentration (4.5 μM~80pM), after 37 DEG C of 18-20 hours of incubation, The Steady Glo Luciferase Assay System detection reagent of 100 μ l is added in every hole, is protected from light room temperature reaction 15 minutes It is placed on SpectraMax L microplate reader detection reading.Compare the average fluorescent strength (MeanValue) that different cell strains generate, Calculate the relative activity (Relative Luciferase Activity, i.e. RLA) of luciferase.RLA=(RLU (gradient concentration SOST react mean value))/(RLU (composing type Wnt signal mean value)), using SoftMax software analysis data, with the dense of SOST Degree is used as x-axis, corresponding RLA value as y-axis, using quadruplex parameters models fitting SOST 4.5 μM~80pM concentration model Interior dose-dependent effect curve is enclosed, sees Fig. 3 A.Inhibitory activity IC of the SOST to W18 cell strain constitutive expression Wnt signal50= 26nM；Inhibitory activity IC of the SOST to W45 cell strain constitutive expression Wnt signal50=110nM；SOST forms W60 cell strain The inhibitory activity IC of type expression Wnt signal50=43nM.
The measurement of 2.2 SOST standard antibody dose-effect relationships
The preparation of Romosozumab standard antibody：Referring to patent US7,592,429B2, full genome synthesizes optimized core (heavy chain amino acid sequence is referring to the SEQ ID NO in US7,592,429B2 for the total length heavy chain and light chain of nucleotide sequence：145, gently Chain amino acid sequence is then the SEQ ID NO in US7,592,429B2：141) it after, is building up to very by HindIII and EcoRI Nuclear expression carrier pcDNA3.1/Zeo+ (is purchased from Lifetech).It is right using ExpiCHOS transient transfection system (being purchased from Gibco) The light chain and heavy chain of Romosozumab carries out cotransfection.The initial density for transfecting cell is 5 × 106A cell/ml, in 250ml Shaking flask (be purchased from NUNC) in 37 DEG C of -5%CO2Incubator, 140 revs/min are cultivated one day.1000 revs/min, 5 points are centrifuged to cell Zhong Hou is added 50ml fresh culture ExpiCHO Expression Medium and is resuspended to 1 × 107The density of a cell/ml, Vigor is more than or equal to 95%, and cell can be used for turning express express target protein in wink at this time.The light chain and heavy chain plasmid of transient transfection respectively take OptiproSFM (being purchased from Gibco) to final volume 4ml is added in 20 μ g.Take transfection reagent ExpiFectamine CHO 160 μ l of Tansfectamine is diluted to 4ml final volume with OptiproSFM (being purchased from Gibco), is added to the plasmid of 4ml and is suspended Liquid is incubated at room temperature after five minutes after mixing, is added in ready cell suspension, 37 DEG C of -5%CO2Incubator, 140 revs/min It is cultivated.
Standard antibody is captured using affine filler Mabselect (being purchased from GE), is washed in purification process using containing 2M Urea It washs, and is eluted under pH3.5 environment.High concentrate formulation buffer, adjustment preparation pH and aseptic filtration are added in elution samples, Obtain final preparation.Preparation system is：50mM PB, 100mM NaCl, pH7.0, protein concentration 3.35mg/ml.Purifying resists Body can be used for subsequent experimental.
With 2 × 10 after the counting of W18, W45 and W60 cell strain4The density kind plate of a cells/well is trained in 96 hole of White-opalescent Plate is supported, (W18 antibody is dense to carry out gradient concentration dilution without phenol red DMEM solution (containing 10%FBS) for Romosozumab standard antibody Spend range 500nM-0.6nM, W45 antibody concentration range 2000nM-2.5nM, W60 antibody concentration range 850nM~1nM) respectively With in the SOST of 57nM, 245nM and 100nM and be incubated for, after 37 DEG C of 1 hours of incubation, be added in the cell of 96 well culture plates, After 37 DEG C of 18-20 hours of incubation.The Steady Glo Luciferase of 100 μ l is added according to the every hole of explanation of kit Assay System detection reagent is protected from light room temperature reaction and is placed within 15 minutes SpectraMax L microplate reader detection reading.Compare The average fluorescent strength (MeanValue) that different cell strains generate, calculates the relative activity (RLA) of luciferase.Four parameter sides Journey models fitting Romosozumab dose-dependent effect curve, is shown in Fig. 3 B.W60 cell strain is when detecting antibody screening, four parameters The amount effect curve window of equation model curve is larger.The detection window that wherein detection window of W18 is 3.39, W45 is 2.15, and The detection window of W60 is 4.35.Detection window is larger, can preferably embody the biological activity of different SOST antibody, improves Stability reduces the coefficient of variation.
Function assessment evaluation through SOST albumen, SOST protein standard antibody, the cell strain of discovery expression intermediate fluorescence intensity, If W60 is most suitable for screening of the invention, detection SOST inhibits the activity curve fitting of wnt signal more excellent.When SOST low concentration, Wnt signaling protein14-3-3 is preferable.
The screening of the freshly prepd SOST protein antibodies candidate of embodiment 3
SOST protein antibodies are prepared with conventional hybridoma technology, it is (anti-to obtain totally 5 plants of H102, H106, H109, H207, H208 Body is commercially available in Yao Ming biotech firm；It can also voluntarily prepare, preparation method refers to Ozato Ket al., Hybridoma cell Lines secreting monoclonal antibodies to mouse h-2and ia antigens, Journal of Immunology, 1980,124 (2), 533-540；And Goding JW, Antibody production by hybridomas, Journal of Immunological Methods, 1980,39 (4), 285-308.), with the IgG antibody not in conjunction with SOST As negative control, activity rating is carried out to SOST protein antibodies with the function assessment evaluation method of embodiment 2.
To respectively with the SOST of 100nM, 37 DEG C are neutralized after the dilution of candidate antibodies progress gradient concentration (850nM~1nM) It is incubated for 1 hour, is added in the W60 cell of preparatory kind of plate, each 2 multiple holes of concentration processing neutralizes after being incubated for, every hole adds The detection reagent Steady Glo Luciferase Assay System for entering 100 μ l is read after being protected from light room temperature reaction 15 minutes. Compare the average fluorescent strength (MeanValue) that different cell strains generate, calculates the relative activity (RLA) of luciferase.Four ginsengs Number equation model fitted dose relies on effect curve, sees Fig. 4.Different candidate antibodies by gradient concentration dilute, with SOST into Stable cell strain of the present invention, the level of activity of the expression reflection antibody of luciferase are acted on after row neutralization reaction.It is dense with antibody Degree is X-axis, and the relative activity RLA of luciferase is Y-axis, and the IC50 of quadruplex parameters matched curve compares the activity of antibody, bent The slope of line reflects the difference of the candidate antibodies mode of action.
Stable cell strain of the present invention can be used for detecting the SOST albumen of different activities degree and the antibody of SOST.Similarly, no SOST and wnt1 protein competition with concentration is bound to stable cell strain surface receptor, to generate to classical wnt signal transduction It influences, passes through the expression reflection SOST albumen of detection luciferase or the activity of SOST antibody.
SEQUENCE LISTING
<110>Shanghai Fudan Zhangjiang biomedical Co., Ltd
<120>For SOST albumen or the cell strain of antibody screening and its preparation method and application
<130> P1710536C
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 4842
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(4842)
<223> hLRP6
<400> 1
atg ggg gcc gtc ctg agg agc ctc ctg gcc tgc agc ttc tgt gtg ctc 48
Met Gly Ala Val Leu Arg Ser Leu Leu Ala Cys Ser Phe Cys Val Leu
1 5 10 15
ctg aga gcg gcc cct ttg ttg ctt tat gca aac aga cgg gac ttg cga 96
Leu Arg Ala Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp Leu Arg
20 25 30
ttg gtt gat gct aca aat ggc aaa gag aat gct acg att gta gtt gga 144
Leu Val Asp Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val Val Gly
35 40 45
ggc ttg gag gat gca gct gcg gtg gac ttt gtg ttt agt cat ggc ttg 192
Gly Leu Glu Asp Ala Ala Ala Val Asp Phe Val Phe Ser His Gly Leu
50 55 60
ata tac tgg agt gat gtc agc gaa gaa gcc att aaa cga aca gaa ttt 240
Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr Glu Phe
65 70 75 80
aac aaa act gag agt gtg cag aat gtt gtt gtt tct gga tta ttg tcc 288
Asn Lys Thr Glu Ser Val Gln Asn Val Val Val Ser Gly Leu Leu Ser
85 90 95
ccc gat ggg ctg gca tgt gat tgg ctt gga gaa aaa ttg tac tgg aca 336
Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr Trp Thr
100 105 110
gat tct gaa act aat cgg att gaa gtt tct aat tta gat gga tct tta 384
Asp Ser Glu Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly Ser Leu
115 120 125
cga aaa gtt tta ttt tgg caa gag ttg gat caa ccc aga gct att gcc 432
Arg Lys Val Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala Ile Ala
130 135 140
tta gat cct tca agt ggg ttc atg tac tgg aca gac tgg gga gaa gtg 480
Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly Glu Val
145 150 155 160
cca aag ata gaa cgt gct gga atg gat ggt tca agt cgc ttc att ata 528
Pro Lys Ile Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe Ile Ile
165 170 175
ata aac agt gaa att tac tgg cca aat gga ctg act ttg gat tat gaa 576
Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp Tyr Glu
180 185 190
gaa caa aag ctt tat tgg gca gat gca aaa ctt aat ttc atc cac aaa 624
Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile His Lys
195 200 205
tca aat ctg gat gga aca aat cgg cag gca gtg gtt aaa ggt tcc ctt 672
Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly Ser Leu
210 215 220
cca cat cct ttt gcc ttg acg tta ttt gag gac ata ttg tac tgg act 720
Pro His Pro Phe Ala Leu Thr Leu Phe Glu Asp Ile Leu Tyr Trp Thr
225 230 235 240
gac tgg agc aca cac tcc att ttg gct tgc aac aag tat act ggt gag 768
Asp Trp Ser Thr His Ser Ile Leu Ala Cys Asn Lys Tyr Thr Gly Glu
245 250 255
ggt ctg cgt gaa atc cat tct gac atc ttc tct ccc atg gat ata cat 816
Gly Leu Arg Glu Ile His Ser Asp Ile Phe Ser Pro Met Asp Ile His
260 265 270
gcc ttc agc caa cag agg cag cca aat gcc aca aat cca tgt gga att 864
Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys Gly Ile
275 280 285
gac aat ggg ggt tgt tcc cat ttg tgt ttg atg tct cca gtc aag cct 912
Asp Asn Gly Gly Cys Ser His Leu Cys Leu Met Ser Pro Val Lys Pro
290 295 300
ttt tat cag tgt gct tgc ccc act ggg gtc aaa ctc ctg gag aat gga 960
Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu Asn Gly
305 310 315 320
aaa acc tgc aaa gat ggt gcc aca gaa tta ttg ctt tta gct cga agg 1008
Lys Thr Cys Lys Asp Gly Ala Thr Glu Leu Leu Leu Leu Ala Arg Arg
325 330 335
aca gac ttg aga cgc att tct ttg gat aca cca gat ttt aca gac att 1056
Thr Asp Leu Arg Arg Ile Ser Leu Asp Thr Pro Asp Phe Thr Asp Ile
340 345 350
gtt ctg cag tta gaa gac atc cgt cat gcc att gcc ata gat tac gat 1104
Val Leu Gln Leu Glu Asp Ile Arg His Ala Ile Ala Ile Asp Tyr Asp
355 360 365
cct gtg gaa ggc tac atc tac tgg act gat gat gaa gtg agg gcc ata 1152
Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg Ala Ile
370 375 380
cgc cgt tca ttt ata gat gga tct ggc agt cag ttt gtg gtc act gct 1200
Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val Thr Ala
385 390 395 400
caa att gcc cat cct gat ggt att gct gtg gac tgg gtt gca cga aat 1248
Gln Ile Ala His Pro Asp Gly Ile Ala Val Asp Trp Val Ala Arg Asn
405 410 415
ctt tat tgg aca gac act ggc act gat cga ata gaa gtg aca agg ctc 1296
Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr Arg Leu
420 425 430
aat ggg acc atg agg aag atc ttg att tca gag gac tta gag gaa ccc 1344
Asn Gly Thr Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu Glu Pro
435 440 445
cgg gct att gtg tta gat ccc atg gtt ggg tac atg tat tgg act gac 1392
Arg Ala Ile Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp Thr Asp
450 455 460
tgg gga gaa att ccg aaa att gag cga gca gct ctg gat ggt tct gac 1440
Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Ser Asp
465 470 475 480
cgt gta gta ttg gtt aac act tct ctt ggt tgg cca aat ggt tta gcc 1488
Arg Val Val Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly Leu Ala
485 490 495
ttg gat tat gat gaa ggc aaa ata tac tgg gga gat gcc aaa aca gac 1536
Leu Asp Tyr Asp Glu Gly Lys Ile Tyr Trp Gly Asp Ala Lys Thr Asp
500 505 510
aag att gag gtt atg aat act gat ggc act ggg aga cga gta cta gtg 1584
Lys Ile Glu Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val Leu Val
515 520 525
gaa gac aaa att cct cac ata ttt gga ttt act ttg ttg ggt gac tat 1632
Glu Asp Lys Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly Asp Tyr
530 535 540
gtt tac tgg act gac tgg cag agg cgt agc att gaa aga gtt cat aaa 1680
Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser Ile Glu Arg Val His Lys
545 550 555 560
cga agt gca gag agg gaa gtg atc ata gat cag ctg cct gac ctc atg 1728
Arg Ser Ala Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp Leu Met
565 570 575
ggc cta aag gct aca aat gtt cat cga gtg att ggt tcc aac ccc tgt 1776
Gly Leu Lys Ala Thr Asn Val His Arg Val Ile Gly Ser Asn Pro Cys
580 585 590
gct gag gaa aac ggg gga tgt agc cat ctc tgc ctc tat aga cct cag 1824
Ala Glu Glu Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg Pro Gln
595 600 605
ggc ctt cgc tgt gct tgc cct att ggc ttt gaa ctc atc agt gac atg 1872
Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser Asp Met
610 615 620
aag acc tgc att gtc cca gag gct ttc ctt ttg ttt tca cgg aga gca 1920
Lys Thr Cys Ile Val Pro Glu Ala Phe Leu Leu Phe Ser Arg Arg Ala
625 630 635 640
gat atc aga cga att tct ctg gaa aca aac aat aat aat gtg gct att 1968
Asp Ile Arg Arg Ile Ser Leu Glu Thr Asn Asn Asn Asn Val Ala Ile
645 650 655
cca ctc act ggt gtc aaa gaa gct tct gct ttg gat ttt gat gtg aca 2016
Pro Leu Thr Gly Val Lys Glu Ala Ser Ala Leu Asp Phe Asp Val Thr
660 665 670
gac aac cga att tat tgg act gat ata tca ctc aag acc atc agc aga 2064
Asp Asn Arg Ile Tyr Trp Thr Asp Ile Ser Leu Lys Thr Ile Ser Arg
675 680 685
gcc ttt atg aat ggc agt gca ctg gaa cat gtg gta gaa ttc ggc tta 2112
Ala Phe Met Asn Gly Ser Ala Leu Glu His Val Val Glu Phe Gly Leu
690 695 700
gat tat cca gaa ggc atg gca gta gac tgg ctt ggg aag aac ttg tac 2160
Asp Tyr Pro Glu Gly Met Ala Val Asp Trp Leu Gly Lys Asn Leu Tyr
705 710 715 720
tgg gca gac aca gga acg aat cga att gag gtg tca aag ttg gat ggg 2208
Trp Ala Asp Thr Gly Thr Asn Arg Ile Glu Val Ser Lys Leu Asp Gly
725 730 735
cag cac cga caa gtt ttg gtg tgg aaa gac cta gat agt ccc aga gct 2256
Gln His Arg Gln Val Leu Val Trp Lys Asp Leu Asp Ser Pro Arg Ala
740 745 750
ctc gcg ttg gac cct gcc gaa gga ttt atg tat tgg act gaa tgg ggt 2304
Leu Ala Leu Asp Pro Ala Glu Gly Phe Met Tyr Trp Thr Glu Trp Gly
755 760 765
gga aaa cct aag ata gac aga gct gca atg gat gga agt gaa cgt act 2352
Gly Lys Pro Lys Ile Asp Arg Ala Ala Met Asp Gly Ser Glu Arg Thr
770 775 780
acc tta gtt cca aat gtg ggg cgg gca aac ggc cta act att gat tat 2400
Thr Leu Val Pro Asn Val Gly Arg Ala Asn Gly Leu Thr Ile Asp Tyr
785 790 795 800
gct aaa agg agg ctt tat tgg aca gac ctg gac acc aac tta ata gaa 2448
Ala Lys Arg Arg Leu Tyr Trp Thr Asp Leu Asp Thr Asn Leu Ile Glu
805 810 815
tct tca aat atg ctt ggg ctc aac cgt gaa gtt ata gca gat gac ttg 2496
Ser Ser Asn Met Leu Gly Leu Asn Arg Glu Val Ile Ala Asp Asp Leu
820 825 830
cct cat cct ttt ggc tta act cag tac caa gat tat atc tac tgg acg 2544
Pro His Pro Phe Gly Leu Thr Gln Tyr Gln Asp Tyr Ile Tyr Trp Thr
835 840 845
gac tgg agc cga cgc agc att gag cgt gcc aac aaa acc agt ggc caa 2592
Asp Trp Ser Arg Arg Ser Ile Glu Arg Ala Asn Lys Thr Ser Gly Gln
850 855 860
aac cgc acc atc att cag ggc cat ttg gat tat gtg atg gac atc ctc 2640
Asn Arg Thr Ile Ile Gln Gly His Leu Asp Tyr Val Met Asp Ile Leu
865 870 875 880
gtc ttt cac tca tct cga cag tca ggg tgg aat gaa tgt gct tcc agc 2688
Val Phe His Ser Ser Arg Gln Ser Gly Trp Asn Glu Cys Ala Ser Ser
885 890 895
aat ggg cac tgc tcc cac ctc tgc ttg gct gtg cca gtt ggg ggt ttt 2736
Asn Gly His Cys Ser His Leu Cys Leu Ala Val Pro Val Gly Gly Phe
900 905 910
gtt tgt gga tgc cct gcc cac tac tct ctt aat gct gac aac agg act 2784
Val Cys Gly Cys Pro Ala His Tyr Ser Leu Asn Ala Asp Asn Arg Thr
915 920 925
tgt agt gct cct acg act ttc ctg ctc ttc agt caa aag agt gcc atc 2832
Cys Ser Ala Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser Ala Ile
930 935 940
aac cgc atg gtg att gat gaa caa cag agc ccc gac atc atc ctt ccc 2880
Asn Arg Met Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile Leu Pro
945 950 955 960
atc cac agc ctt cgg aat gtc cgg gcc att gac tat gac cca ctg gac 2928
Ile His Ser Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro Leu Asp
965 970 975
aag caa ctc tat tgg att gac tca cga caa aac atg atc cga aag gca 2976
Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln Asn Met Ile Arg Lys Ala
980 985 990
caa gaa gat ggc agc cag ggc ttt act gtg gtt gtg agc tca gtt ccg 3024
Gln Glu Asp Gly Ser Gln Gly Phe Thr Val Val Val Ser Ser Val Pro
995 1000 1005
agt cag aac ctg gaa ata caa ccc tat gac ctc agc att gat att 3069
Ser Gln Asn Leu Glu Ile Gln Pro Tyr Asp Leu Ser Ile Asp Ile
1010 1015 1020
tac agc cgc tac atc tac tgg act tgt gag gct acc aat gtc att 3114
Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys Glu Ala Thr Asn Val Ile
1025 1030 1035
aat gtg aca aga tta gat ggg aga tca gtt gga gtg gtg ctg aaa 3159
Asn Val Thr Arg Leu Asp Gly Arg Ser Val Gly Val Val Leu Lys
1040 1045 1050
ggc gag cag gac aga cct cga gcc gtt gtg gta aac cca gag aaa 3204
Gly Glu Gln Asp Arg Pro Arg Ala Val Val Val Asn Pro Glu Lys
1055 1060 1065
ggg tat atg tat ttt acc aat ctt cag gaa agg tct cct aaa att 3249
Gly Tyr Met Tyr Phe Thr Asn Leu Gln Glu Arg Ser Pro Lys Ile
1070 1075 1080
gaa cgg gct gct ttg gat ggg aca gaa cgg gag gtc ctc ttt ttc 3294
Glu Arg Ala Ala Leu Asp Gly Thr Glu Arg Glu Val Leu Phe Phe
1085 1090 1095
agt ggc tta agt aaa cca att gct tta gcc ctt gat agc agg ctg 3339
Ser Gly Leu Ser Lys Pro Ile Ala Leu Ala Leu Asp Ser Arg Leu
1100 1105 1110
ggc aag ctc ttt tgg gct gat tca gat ctc cgg cga att gaa agc 3384
Gly Lys Leu Phe Trp Ala Asp Ser Asp Leu Arg Arg Ile Glu Ser
1115 1120 1125
agt gat ctc tca ggt gct aac cgg ata gta tta gaa gac tcc aat 3429
Ser Asp Leu Ser Gly Ala Asn Arg Ile Val Leu Glu Asp Ser Asn
1130 1135 1140
atc ttg cag cct gtg gga ctt act gtg ttt gaa aac tgg ctc tat 3474
Ile Leu Gln Pro Val Gly Leu Thr Val Phe Glu Asn Trp Leu Tyr
1145 1150 1155
tgg att gat aaa cag cag caa atg att gaa aaa att gac atg aca 3519
Trp Ile Asp Lys Gln Gln Gln Met Ile Glu Lys Ile Asp Met Thr
1160 1165 1170
ggt cga gag ggt aga acc aaa gtc caa gct cga att gcc cag ctt 3564
Gly Arg Glu Gly Arg Thr Lys Val Gln Ala Arg Ile Ala Gln Leu
1175 1180 1185
agt gac att cat gca gta aag gag ctg aac ctt caa gaa tac aga 3609
Ser Asp Ile His Ala Val Lys Glu Leu Asn Leu Gln Glu Tyr Arg
1190 1195 1200
cag cac cct tgt gct cag gat aat ggt ggc tgt tca cat att tgt 3654
Gln His Pro Cys Ala Gln Asp Asn Gly Gly Cys Ser His Ile Cys
1205 1210 1215
ctt gta aag ggg gat ggt act aca agg tgt tct tgc ccc atg cac 3699
Leu Val Lys Gly Asp Gly Thr Thr Arg Cys Ser Cys Pro Met His
1220 1225 1230
ctg gtt cta ctt caa gat gag cta tca tgt gga gaa cct cca aca 3744
Leu Val Leu Leu Gln Asp Glu Leu Ser Cys Gly Glu Pro Pro Thr
1235 1240 1245
tgt tct cct cag cag ttt act tgt ttc acg ggg gaa att gac tgt 3789
Cys Ser Pro Gln Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp Cys
1250 1255 1260
atc cct gtg gct tgg cgg tgc gat ggg ttt act gaa tgt gaa gac 3834
Ile Pro Val Ala Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp
1265 1270 1275
cac agt gat gaa ctc aat tgt cct gta tgc tca gag tcc cag ttc 3879
His Ser Asp Glu Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe
1280 1285 1290
cag tgt gcc agt ggg cag tgt att gat ggt gcc ctc cga tgc aat 3924
Gln Cys Ala Ser Gly Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn
1295 1300 1305
gga gat gca aac tgc cag gac aaa tca gat gag aag aac tgt gaa 3969
Gly Asp Ala Asn Cys Gln Asp Lys Ser Asp Glu Lys Asn Cys Glu
1310 1315 1320
gtg ctt tgt tta att gat cag ttc cgc tgt gcc aat ggt cag tgc 4014
Val Leu Cys Leu Ile Asp Gln Phe Arg Cys Ala Asn Gly Gln Cys
1325 1330 1335
att gga aag cac aag aag tgt gat cat aat gtg gat tgc agt gac 4059
Ile Gly Lys His Lys Lys Cys Asp His Asn Val Asp Cys Ser Asp
1340 1345 1350
aag tca gat gaa ctg gat tgt tat ccg act gaa gaa cca gca cca 4104
Lys Ser Asp Glu Leu Asp Cys Tyr Pro Thr Glu Glu Pro Ala Pro
1355 1360 1365
cag gcc acc aat aca gtt ggt tct gtt att ggc gta att gtc acc 4149
Gln Ala Thr Asn Thr Val Gly Ser Val Ile Gly Val Ile Val Thr
1370 1375 1380
att ttt gtg tct gga act gta tac ttt atc tgc cag agg atg ttg 4194
Ile Phe Val Ser Gly Thr Val Tyr Phe Ile Cys Gln Arg Met Leu
1385 1390 1395
tgt cca cgt atg aag gga gat ggg gaa act atg act aat gac tat 4239
Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr Asn Asp Tyr
1400 1405 1410
gta gtt cat gga cca gct tct gtg cct ctt ggt tat gtg cca cac 4284
Val Val His Gly Pro Ala Ser Val Pro Leu Gly Tyr Val Pro His
1415 1420 1425
cca agt tct ttg tca gga tct ctt cca gga atg tct cga ggt aaa 4329
Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met Ser Arg Gly Lys
1430 1435 1440
tca atg atc agc tcc ctc agt atc atg ggg gga agc agt gga ccc 4374
Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser Ser Gly Pro
1445 1450 1455
ccc tat gac cga gcc cat gtt aca gga gca tca tca agt agt tct 4419
Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser Ser Ser Ser
1460 1465 1470
tca agc acc aaa ggc act tac ttc cct gca att ttg aac cct cca 4464
Ser Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn Pro Pro
1475 1480 1485
cca tcc cca gcc aca gag cga tca cat tac act atg gaa ttt gga 4509
Pro Ser Pro Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe Gly
1490 1495 1500
tat tct tca aac agt cct tcc act cat agg tca tac agc tac agg 4554
Tyr Ser Ser Asn Ser Pro Ser Thr His Arg Ser Tyr Ser Tyr Arg
1505 1510 1515
cca tat agc tac cgg cac ttt gca ccc ccc acc aca ccc tgc agc 4599
Pro Tyr Ser Tyr Arg His Phe Ala Pro Pro Thr Thr Pro Cys Ser
1520 1525 1530
aca gat gtt tgt gac agt gac tat gct cct agt cgg aga atg acc 4644
Thr Asp Val Cys Asp Ser Asp Tyr Ala Pro Ser Arg Arg Met Thr
1535 1540 1545
tca gtg gca aca gcc aag ggc tat acc agt gac ttg aac tat gat 4689
Ser Val Ala Thr Ala Lys Gly Tyr Thr Ser Asp Leu Asn Tyr Asp
1550 1555 1560
tca gaa cct gtg ccc cca cct ccc aca ccc cga agc caa tac ttg 4734
Ser Glu Pro Val Pro Pro Pro Pro Thr Pro Arg Ser Gln Tyr Leu
1565 1570 1575
tca gca gag gag aac tat gaa agc tgc cca cct tct cca tac aca 4779
Ser Ala Glu Glu Asn Tyr Glu Ser Cys Pro Pro Ser Pro Tyr Thr
1580 1585 1590
gag agg agc tat tct cat cac ctc tac cca ccg cca ccc tct ccc 4824
Glu Arg Ser Tyr Ser His His Leu Tyr Pro Pro Pro Pro Ser Pro
1595 1600 1605
tgt aca gac tcc tcc tga 4842
Cys Thr Asp Ser Ser
1610

Claims (10)

1. a kind of for SOST albumen or antibody assessment and/or the cell strain of screening, which is characterized in that it is to stablize expression wnt1 The HEK293 cell of protein gene, LRP6 acceptor gene and luciferase gene.
2. cell strain as described in claim 1, which is characterized in that its cotransfection has the plasmid 1 of the wnt1 protein gene, packet Plasmid 2 containing the LRP acceptor gene and the plasmid 3 comprising the luciferase gene；Preferably, the wnt1 albumen is Rmwnt1 albumen, the LRP6 receptor is hLRP6, and the luciferase is the luc2P for being connected with TCF-LEF element；More preferably, The plasmid 1 selects rmwnt1-pCMV6-Kan/Neo；Described its plasmid backbone of plasmid 2 is pCDNA3.1/Hygro (-)；It is described Plasmid 3 select pGL4.49 [luc2P/TCF-LEF/Hygro].
3. cell strain as claimed in claim 1 or 2, which is characterized in that the relative intensity of fluorescence of its expression of the cell strain (RLU) it is at least 100,000, preferably more than 500,000；The inhibition of the SOST protein on cells strain expression wnt1 albumen is living Property IC50Not higher than 110nM, more preferably at least 25nM；It is highly preferred that the relative intensity of fluorescence RLU is 248,000, it is described Inhibitory activity IC50For 43nM.
4. the preparation method of cell strain as described in any one of claims 1-3, it includes following steps：
(1) preparation contains the plasmid of the LRP acceptor gene；
(2) by the plasmid in step (1) and the plasmid containing the wnt1 protein gene and contain the luciferase gene Plasmid liposome cotransfection HEK293 cell.
5. the preparation method of cell strain as claimed in claim 4, which is characterized in that the liposome is Lipofectamine2000；Preferably, antibiotic pressurization screening will after cotransfection HEK293 cell kind plate, be additionally added；It is preferred that Ground, antibiotic are hygromycin and Geneticin；It is highly preferred that the end of the final concentration of 300 μ g/ml and Geneticin of hygromycin Concentration is 400 μ g/ml.
6. the preparation method of cell strain as claimed in claim 5, which is characterized in that plasmid amount ratio described in three kinds is 1:1: 1, the time of the kind plate is 2 days and/or the time of pressurization screening is 2~3 weeks；Preferably, pressurize screening time be 3 Week.
7. assessment or the screening technique of a kind of SOST albumen or antibody activity comprising following steps：
(1) gradient dilution SOST albumen to be measured；Or gradient dilution test antibodies, certain density SOST antibody incubation is added；
(2) it is co-cultured again with cell strain as described in any one of claims 1-3；
(3) then be added Luciferase Assay Reagent, be protected from light, react at room temperature after read；
(4) SOST antibody or protein activity value are judged according to reading.
8. assessment or the screening technique of SOST albumen as claimed in claim 7 or antibody activity, which is characterized in that described to be measured The concentration of antibody is 850nM~1nM；The SOST protein concentration to be measured is 4.5 μM~80pM；The temperature of the incubation is 37 DEG C, the time is 1 hour；The co-cultured cell strain number amount is every reaction 2 × 104It is a；The temperature of co-cultivation is 37 DEG C, the time It is 18~20 hours；And/or the room temperature reaction time that is protected from light is 15 minutes.
9. application of the cell strain as claimed in any one of claims 1 to 3 in SOST albumen or antibody activity assessment or screening.
10. a kind of preparation comprising the described in any item cell strains of claims 1 to 3, preferably, the preparation is SOST albumen Or antibody test preparation.
CN201710335781.3A 2017-05-12 2017-05-12 For SOST albumen or the cell strain of antibody screening and its preparation method and application Pending CN108866002A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090130113A1 (en) * 2007-10-12 2009-05-21 Michaela Kneissel Compositions and methods for use for antibodies against sclerostin

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090130113A1 (en) * 2007-10-12 2009-05-21 Michaela Kneissel Compositions and methods for use for antibodies against sclerostin

Non-Patent Citations (1)

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Title
VERENA BOSCHERT等: "Mutational Analysis of Sclerostin Shows Importance ofthe Flexible Loop and the Cystine-Knot for Wnt-SignalingInhibition", 《PLOS》 *

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