CN102174475B - Preparation and application of hIL-17RD-ECD monoclonal antibody - Google Patents
Preparation and application of hIL-17RD-ECD monoclonal antibody Download PDFInfo
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Abstract
The invention discloses preparation and application of a hIL-17RD-ECD monoclonal antibody. The invention provides a mus musculus hybridoma 1F8, the collection number of which is CGMCC No.4576. The invention also provides a monoclonal antibody generated by the mus musculus hybridoma 1F8. Proved by experiments, a mus musculus hybridoma 1F8 is obtained by screening; and biological property identification is performed on the mus musculus hybridoma 1F8 (the collection number of which is CGMCC No.4576) to provide proof and lay a foundation for further researching the possible interrelationship between the hIL-17RD-ECD gene and the autoimmune disease such as rheumatoid arthritis and deeply researching the functions of the mus musculus hybridoma 1F8 and prepare potential medicament proteins for inhibiting the autoimmune diseases such as the rheumatoid arthritis and the like.
Description
Technical field
The present invention relates to a kind of preparation and application of monoclonal antibody, relate in particular to a kind of preparation and application of hIL-17RD-ECD monoclonal antibody.
Background technology
The IL-17 of Th17 emiocytosis comprises IL-17A and these two inflammation molecules of IL-17F, they are by IL-17 receptor family transmission of signal, thereby affects downstream gene expression.Interleukin-17 acceptor family (IL-17receptor) is a kind of immunity acceptor that extensively is present on the immunologically competent cell, can mediate the IL-17 signal path, has a lot of important physiological functions.Had been found that at present mouse and human il-17 RD can transmit the conduction of IL-17 signal, and and IL-17RA formation heterozygosis dimer.
At present, in gene clone, a large amount of research has all been carried out in the aspects such as protein expression to IL-17RA, and has obtained remarkable progress, and relatively less to the research of IL-17RD.IL-17RD once is being found in the large-scale in situ hybridization, and this time in situ hybridization is intended to filter out space restriction gene in zebrafish development.These genes in similarly room and time expression form mutually regulation and control interactional family in co-channel.The IL-17RD expression pattern is similar to the fibroblast growth factor gene family, and its expression also is subject to the FGFs regulation and control.Since IL-17RD is found from zebra fish first, chicken, mouse, its lineal system homology is all out identified among the mankind.The IL-17RD gene only is present in the vertebrates, is positioned single locus.Yet, formed the IL-17RD isomer by variable connection mechanism and had different biological chemistries and biological characteristics.
Therefore, further the activity of IL-17RD and the research of mechanism of action are called focus.
Summary of the invention
Purpose of the present invention provides the mouse hybridoma cell that a kind of preserving number is CGMCC No.4576 (musmusculus hybridoma) 1F8.
The invention provides the mouse hybridoma cell that a kind of preserving number is CGMCC No.4576 (mus musculushybridoma) 1F8.
It is the monoclonal antibody of mouse hybridoma cell (musmusculus hybridoma) the 1F8 generation of CGMCC No.4576 that another object of the present invention provides by preserving number.
The invention provides by preserving number is the monoclonal antibody of mouse hybridoma cell (mus musculushybridoma) the 1F8 generation of CGMCC No.4576.
The application of described monoclonal antibody in preparation interleukin-17 acceptor D detection kit also is the scope of protection of the invention.
The application of described monoclonal antibody in the colloidal gold fast detecting test paper strip of preparation detection interleukin-17 acceptor D also is the scope of protection of the invention.
The test kit that contains the detection interleukin-17 acceptor D of described monoclonal antibody also is the scope of protection of the invention.
The colloidal gold fast detecting test paper strip that contains the detection interleukin-17 acceptor D of described monoclonal antibody also is the scope of protection of the invention.
Obtain the hybridoma cell strain 1F8 of the monoclonal antibody of energy stably excreting antihuman interleukin 17 acceptor D (hIL-17RD) through screening, the Classification And Nomenclature of this hybridoma cell strain 1F8 is mouse hybridoma cell mus musculushybridoma, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2011 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4576.
Of the present invention experimental results show that, the present invention's screening obtains the hybridoma of the monoclonal antibody of 1 strain energy stably excreting antihuman interleukin, 17 acceptor D (hIL-17RD), and it is carried out Identification of Biological Characteristics, foundation is provided and lays the foundation the potential drug albumen of the autoimmune diseases such as preparation inhibition rheumatoid arthritis in the hope of further inquiring into the mutual relationship that may exist between hIL-17RD gene and autoimmune disease such as the rheumatoid arthritis and the further investigation of function thereof.
Description of drawings
Fig. 1 is that the SDS-PAGE of hIL-17RD-ECD albumen analyzes
The positive hybridoma specificity screening of Fig. 2
Fig. 3 is the type identification of the monoclonal antibody of anti-hIL-17RD
Fig. 4 is the mensuration that ascites antibody is tired
Fig. 5 is that monoclonal antibody purity and molecular mass are identified
Fig. 6 is the result that Western blot detects endogenous hIL-17RD
Fig. 7 is hIL-17RD and the hIL-17RD-ECD that Western blot detects external source
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The preparation of embodiment 1, monoclonal antibody
1, animal immune
Extracellular region protein (the hIL-17RD-ECD of synthetic hIL-17RD, its nucleotides sequence is classified as shown in the sequence 1 in the sequence table, its aminoacid sequence is shown in the sequence 2 in the sequence table, consistent with the 1-739 position Nucleotide of GenbankNM_017563.3) nucleotide sequence as template, take IL-17RD-F-BamHI:atgcggatcccgccgacacctgtggc and IL-17RD-R-PstI:aactgcagtcagtgatggtgatg as primer, carry out pcr amplification, the PCR product that obtains is cut through BamHI and PstI enzyme, be connected with PFASTBAC-DUAL EXP VECTOR carrier (available from the Invitrogen products catalogue 10712024) large fragment of cutting through same enzyme, obtain connecting product, transform escherichia coli DH5a, obtain transformant, the transfection step is as follows:
A, choose the plasmid that hickie extracts transformant;
B, transfection: in the dish of diameter 8cm, spread uniformly (2.5-3) * 10
6Individual sf9 cell (available from Wuhan virus institute, sf9 cell), the cytogamy degree is 70%-80%.Prepare the EP pipe of two 2ml, each adds the sf900 serum free medium (Invitrogen of 100ul, products catalogue #10902096), add in the pipe wherein 15ulCellfectin (transfection reagent, available from Invitrogen, article No.: G4B2996936FB53), add the 2mg plasmid in another pipe, mixing gently, room temperature (25 ℃) leaves standstill 5min and then two pipes is mixed, and room temperature (25 ℃) leaves standstill 30min.Add the soft mixing of 1.8ml sf900 substratum, drip at the cell surface of completing, leave standstill 5h in the incubator.Cell is substantially adherent behind 5h, and the sucking-off substratum adds the new serum-free sf900 of 8ml.27 ℃ leave standstill cultivation 10 days, and the centrifugal supernatant that stays is P0 virus.
C, virus amplification: get 50ul P0 and join 50ml density for (1.5-2.5) * 10
6The sf9 cell in, 27 ℃ of 100rpm cultivated 6 days, centrifugal receipts supernatant is P1 virus, this moment, virus titer can cells infected and great expression albumen.
D, infection and expressing protein: cultivate 800ml sf9 cell with large Tissue Culture Flask, when cell density is (1.5-2.5) * 10
6And when in good condition, with 1: 100 adding P1 virus, 27 ℃ of 100rpm cultivated 48-72h, receive cell.
E, protein purification: with the centrifugal 10min of cell culture 4000rpm, concentrated behind the supernatant suction filtration, in this process, use lysis buffer (10mM HEPES instead, 150mM NaCl, pH 7.5) be concentrated to 200ml, centrifugal 15000rpm35min collects supernatant and adds 1.5ml Ni-NTA, 4 ℃ are shaken and hatch 3h, make the abundant combination of target protein and Ni-NTA.After hatching, clear liquid is flow through, add the lysis buffer that contains the 20mM imidazoles and wash 800ml, the lysis buffer wash-out target protein that contains the 500mM imidazoles with 15ml, be concentrated into 800ul, use AKTA and Superdex200 molecular sieve chromatography (the products catalogue G4A90FD2ACFF64 of GE company) to carry out further polishing purification, the SDS-PAGE detected result as shown in Figure 1 behind the purifying, about can finding out that this albumen size is for 35KDa, namely obtain target protein hIL-17RA-ECD (hIL-17RD extracellular region protein; External source hIL-17RD-ECD).
Select 6 8-12 Balb/C mouse in age in week to carry out the intraperitoneal immunity, initial immunity adds Freund's complete adjuvant with 80ug hIL-17RD extracellular region protein, after two weeks, add the Freund's incomplete adjuvant reinforced immunological 2 times with 40ug hIL-17RD-ECD, every 2 weeks of minor tick, adopt intravenous injection reinforced immunological (not containing adjuvant) with hIL-17RD-ECD for the last time, afterbody blood sampling after 3 days, ELISA detects serum antibody titer.
2, indirect elisa method
With coated 96 orifice plates in antigen (hIL-17RD-ECD albumen) the 100ul/ hole of 2ug/ml, 4 ℃ spend the night after, with washing lotion washing 3 times, add 150ul/ hole confining liquid at 37 ℃ of sealing 2h, wash 3 times, pat dry.Add testing sample (serum), the dilution in 1: 1000 of the first hole, down 1: 3 gradient doubling dilution is hatched 30min for 37 ℃, washes plate 4 times, pats dry.Get the sheep anti-mouse igg (bse-0296G Shanghai biotechnology company of large alliance) of horseradish enzyme labelling according to 1: 5000 times of dilution, the 100ul/ hole adds plank, hatches 30min for 37 ℃, washs 4 times, pats dry.Add at last 1 * TMB 100ul/ hole, 37 ℃ of colour developing 15-30min.Then add stop buffer (2M H
2SO
4) the 50ul/ hole.Measure each hole OD value with the single wavelength of 450nm, and with negative control hole PBS (GIBCO 21600-044, it is for subsequent use that weighing dry powder 4.875g dissolves in 500ml ultrapure water sterilization) ratio (P/N) of OD value is limited greater than 2.1, as the stagnation point of judging the positive or determining to tire.
The result is that serum titer is 1: 720000 positive mouse, can be used for cytogamy.
3, hybridoma merges screening
1), merge front 4 days with positive mouse reinforced immunological, mouse boosting cell and myeloma cell SP2/0 ratio are 10: 1, PEG (molecular weight 4000) merges, the selection substratum is the RPMI640 substratum that contains HAT.After 10 days, ELISA screening hybridoma supernatant is cloned with limiting dilution assay antagonist secretion positive hybridoma cell, finally determines the positive cell of 13 strain cells through twice screening.
2), specific assay:
Whether indirect elisa method detects all positive 13 strain cells can specific recognition hIL-17RD, (its nucleotides sequence is classified the 1-897 position Nucleotide of Gene ID:NM_017563 as, and the hIL-17RD-ECD among the preparation method and 1 is basic identical with hIL-17RD-ECD (nucleotides sequence is classified the sequence 1 in the sequence table as) and hIL-17RA-ECD.) be antigen, the result can find out as described in Figure 2, only have a strain called after 1F8 positive cell can with the hIL-17RD-ECD specific combination, illustrate that 1F8 can specific recognition hIL-17RD
Obtain the hybridoma cell strain 1F8 of the monoclonal antibody of energy stably excreting antihuman interleukin 17 acceptor D (hIL-17RD) through screening, the Classification And Nomenclature of this hybridoma cell strain 1F8 is mouse hybridoma cell mus musculushybridoma, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2011 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4576.
4, the production of monoclonal antibody and purifying
1) preparation of Syngenic mice monoclonal antibody ascites:
Adopt the method for producing monoclonal antibody in the animal body.Every Balb/C mouse peritoneal injection sterilising liq paraffin 0.5ml, after 7 days, every mouse peritoneal injection 1 * 10
6Individual mouse hybridoma cell (mus musculus hybridoma) 1F8CGMCC No.4576.Observe the mouse ascites condition of production after 7 days, obviously expand such as belly, can extract ascites.The centrifugal 10min of 13000RPM collects supernatant liquor except degrease and precipitation after the ascites collection, is the ascites monoclonal antibody.
2) purifying
With ascites monoclonal anti body and function Binding Buffer (LS6510-10 supplier: Biovision) balance protein G (LS6510-10 supplier: Biovision) steady to baseline, collect stream and wear liquid, then stream is worn again upper prop of liquid, balance is steady to baseline.Adding Eluting Buffer (the LS6510-10 supplier: Biovision) wash-out, collect elution peak, be the ascites monoclonal antibody of purifying.
Two, monoclonal antibody CHARACTERISTICS IDENTIFICATION
1, the evaluation of antibody class and subclass
Identify the subclass of monoclonal antibody with adopting Southern Biotech test kit (5300-01 southernbiotech), the result as shown in Figure 3, the heavy chain of the monoclonal antibody of hybridoma cell strain 1F8CGMCC No.4576 secretion is IgG1, and light chain is the Kappa type.
2, the ELISA mensuration of tiring
Adopt indirect ELISA to measure the ascites monoclonal antibody of purifying, coated 96 orifice plates in the antigen of 2ug/ml (hIL-17RD-ECD albumen) 100ul/ hole are ELISA, and the result can find out that as shown in Figure 4 tiring behind the purifying is 1: 81000.
3. the molecular mass of antibody is identified:
Adopt SDS-PAGE to measure monoclonal antibody, the result as shown in Figure 5, the heavy chain of monoclonal antibody is about 46KD, light chain is about 25KD.
4. monoclonal antibody concentration determination:
Ultraviolet spectrometry is measured monoclonal antibody at absorbance A280 and the A260 at 280nm and 260nm place.
Protein content calculates according to following formula: protein content (g/L)=1.45 * A280-0.74 * A260.
The result is that protein content is 1.62mg/ml.
The application of embodiment 2, monoclonal antibody
Get respectively brain, kidney, the lung of Kunming white mouse (Beijing Vital River Experimental Animals Technology Co., Ltd. please offer for sale company and catalog number), extract respectively the albumen of brain, kidney, lung;
Synthetic hIL-17RD (the 1-739 position Nucleotide of GenbankNM_017563.3), take IL-17RD-F-BamHI:atgcggatcccgccgacacctgtggc and IL-17RD-R-PstI:aactgcagtcagtgatggtgatg as primer, the PCR product that obtains is cut through BamHI and PstI enzyme, with being connected of the PFASTBAC-DUAL EXP VECTOR carrier large fragment of cutting through same enzyme, obtain connecting product, transform escherichia coli DH5a, obtain transformant, the transfection step is as follows:
A, choose the plasmid that hickie extracts transformant;
B, transfection: in the dish of diameter 8cm, spread uniformly (2.5-3) * 10
6Individual sf9 cell, cytogamy degree are 70%-80%.Prepare the EP pipe of two 2ml, each adds the sf900 serum free medium of 100ul, to wherein adding 15ul Cellfectin in the pipe, add the 2mg plasmid in another pipe, mixing gently, room temperature (25 ℃) leaves standstill 5min and then two pipes is mixed, and room temperature (25 ℃) leaves standstill 30min.Add the soft mixing of 1.8ml sf900 substratum, drip at the cell surface of completing, leave standstill 5h in the incubator.Cell is substantially adherent behind 5h, and the sucking-off substratum adds the new serum-free sf900 of 8ml.27 ℃ leave standstill cultivation 10 days, and the centrifugal supernatant that stays is P0 virus.
C, virus amplification: get 50ul P0 and join 50ml density for (1.5-2.5) * 10
6The sf9 cell in, 27 ℃ of 100rpm cultivated 6 days, centrifugal receipts supernatant is P1 virus, this moment, virus titer can cells infected and great expression albumen.
D, infection and expressing protein: cultivate 800ml sf9 cell with large Tissue Culture Flask, when cell density is (1.5-2.5) * 10
6And when in good condition, with 1: 100 adding P1 virus, 27 ℃ of 100rpm cultivated 48-72h, receive cell.
E, protein purification: with the centrifugal 10min of cell culture 4000rpm, concentrated behind the supernatant suction filtration, in this process, use lysis buffer (10mM HEPES instead, 150mM NaCl, pH 7.5) be concentrated to 200ml, centrifugal 15000rpm35min collects supernatant and adds 1.5ml Ni-NTA, 4 ℃ are shaken and hatch 3h, make the abundant combination of target protein and Ni-NTA.After hatching, clear liquid is flow through, add the lysis buffer that contains the 20mM imidazoles and wash 800ml, the lysis buffer wash-out target protein that contains the 500mM imidazoles with 15ml, be concentrated into 800ul, use AKTA and Superdex200 molecular sieve chromatography to carry out further polishing purification, the SDS-PAGE detected result namely obtains purer target protein purifying and obtains external source hIL-17RD.
The albumen of brain, kidney, lung, external source hIL-17RD, external source hIL-17RD-ECD (the hIL-17RD extracellular region protein that embodiment 1 obtains) carry out Western blot and analyze, with albumen through the SDS-PAGE electrophoresis, be transferred on the NC film, embodiment 1 is obtained ascites monoclonal antibody (1: the 10000) dilution of purifying as primary antibodie, the sheep anti-mouse igg of HRP mark (bse-0296G, Shanghai biotechnology company of large alliance) is anti-as two.
The result is shown in Fig. 6 and 7, and wherein 6 is the result of the albumen of brain, kidney, lung, as can be seen from the figure 1 brain, and there is hIL-17RD in 2 kidneys in 3 lungs; Size is 130KD, the in the same size of this and hIL-17RD (the 1-897 position Nucleotide of Gene ID:NM_017563).
7 is the detected result of the hIL-17RD-ECD of external source IL-17RD and external source, wherein 1 is the hIL-17RD of external source, 2 is the hIL-17RD-ECD of external source, as seen from the figure, can detect external source hIL-17RD (100KD) and hIL-17RD-ECD (130KD).
The size that detection obtains and the in the same size of known albumen prove that monoclonal antibody of the present invention is correct.
Claims (6)
1. preserving number is mouse hybridoma cell (the mus musculus hybridoma) 1F8 of CGMCC No.4576.
2. be the monoclonal antibody of mouse hybridoma cell (mus musculus hybridoma) the 1F8 generation of CGMCC No.4576 by preserving number.
3. the application of monoclonal antibody claimed in claim 2 in preparation interleukin-17 acceptor D detection kit.
4. the application of monoclonal antibody claimed in claim 2 in the colloidal gold fast detecting test paper strip of preparation detection interleukin-17 acceptor D.
5. the test kit that contains the detection interleukin-17 acceptor D of monoclonal antibody claimed in claim 2.
6. the colloidal gold fast detecting test paper strip that contains the detection interleukin-17 acceptor D of monoclonal antibody claimed in claim 2.
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Citations (3)
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| US20030180255A1 (en) * | 2000-08-24 | 2003-09-25 | Genentech, Inc. | IL-17 homologous polypeptides and therapeutic uses thereof |
| CN1467222A (en) * | 2002-07-10 | 2004-01-14 | 清华大学 | Human interleukin-17 receptor sampling molecule fusion protein and coding gene and expression cell line |
| WO2008156709A1 (en) * | 2007-06-13 | 2008-12-24 | Amgen Inc. | Il-17 heteromeric receptor complex |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030180255A1 (en) * | 2000-08-24 | 2003-09-25 | Genentech, Inc. | IL-17 homologous polypeptides and therapeutic uses thereof |
| CN1467222A (en) * | 2002-07-10 | 2004-01-14 | 清华大学 | Human interleukin-17 receptor sampling molecule fusion protein and coding gene and expression cell line |
| WO2008156709A1 (en) * | 2007-06-13 | 2008-12-24 | Amgen Inc. | Il-17 heteromeric receptor complex |
Non-Patent Citations (2)
| Title |
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| Flachsbart F..interleukin-17 receptor D precursor [Homo sapiens].《GeneBank》.2010,ORIGIN. * |
| M.S. Rahman.IL-17R activation of human airway smooth muscle cells induces CXCL-8 production via a transcriptional-dependent mechanism.《Clinical Immunology》.2005,第115卷268-276. * |
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Application publication date: 20110907 Assignee: Saiertaihe Biomedicine Tech Co., Ltd., Beijing Assignor: Tsinghua University Contract record no.: 2013990000428 Denomination of invention: Preparation and application of hIL-17RD-ECD monoclonal antibody Granted publication date: 20130320 License type: Exclusive License Record date: 20130724 |
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