CN103289962A - Mouse anti-human CD23 monoclonal antibody and hybridoma cell strain secreting monoclonal antibody - Google Patents
Mouse anti-human CD23 monoclonal antibody and hybridoma cell strain secreting monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a mouse anti-human CD23 monoclonal antibody and a hybridoma cell strain secreting the monoclonal antibody. The cloning number of the hybridoma cell strain secreting the CD23 monoclonal antibody is 1E9, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No: 6855. The invention also provides the monoclonal antibody produced by the hybridoma cell strain 1E9. The antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10. The invention also provides a DNA (Deoxyribonucleic Acid) molecule in SEQ ID NO: 7 for encoding the amino acid sequence of the heavy chain variable region in the SEQ ID NO: 9 and a DNA molecule in SEQ ID NO: 8 for encoding the amino acid sequence of the light chain variable region in the SEQ ID NO: 10. The antibody can be applied to the CD23 expression detection in a scientific research or a clinical immunohistochemistry assay.
Description
Technical field
The present invention relates to the hybridoma cell strain 1E9 of the secretion CD23 monoclonal antibody that obtains with the CD23 protein immunization mouse of prokaryotic expression, belong to technical field of bioengineering.
Background technology
CD23 also claims Fc ε RII, is people and vertebrate IgE low-affinity receptor, i.e. the II receptor of IgE; It is the transmembrane glycoprotein of surface of cell membrane such as a kind of B of being distributed widely in cell, eosinophilic granulocyte (EOS), the Monocytes that activates, thrombocyte, dendritic cells,follicular, Langerhans cell (Langhans Cells), epidermis horn cells, thymic epithelial cells, part T cell, natural killer cell, and molecular weight is 45kDa.
CD23 belongs to typical II type and wears membrane glycoprotein, is made up of 321 amino acid, comprise that transmembrane domains that N-end in the born of the same parents that are made up of 23 amino-acid residues, 21 amino-acid residues are formed and 277 amino-acid residues form the terminal film outskirt of C-; Its C-terminal is positioned at the extracellular, and N-terminal is positioned at cell, belongs to C type zoo-agglutinin superfamily.The human CD 23 gene is positioned on No. 19 karyomit(e), is single copy gene, has two types, because their promotor gene difference causes aminoterminal 6 the amino acid differences of coded CD23 molecule, has formed two kinds of CD23:CD23a and CD23b.CD23a mainly expresses in bone-marrow-derived lymphocyte and eosinophil, and CD23b has expression on the Monocytes, B cell, eosinophilic granulocyte, thrombocyte, dendritic cells,follicular, Langerhans cell, epidermis horn cells, thymic epithelial cells, part T cell, natural killer cell and some leukemia cell that activate.The scavenger cell high expression level CD23b in medullary space matrix source.Surface of cell membrane CD23 can be the multiple soluble fragments that differs in size by self or by proteolytic enzyme cleaves, is called solubility CD23 (sCD23), and these fragment major parts still can be combined with IgE, and shows the multiple function irrelevant with IgE.
CD23 is multifunctional receptor molecule/cytokine, its participate in to regulate IgE synthetic, promote B cell-stimulating, propagation and differentiation, and by with film CD21 in conjunction with mediation B-T cell interaction, strengthen the B cell and offer antigen to the T cell, promote the T cell maturation.Solubility CD23 (sCD23) also can work in coordination with IL-1 and promote thymocyte, bone marrow precursor, germinal center's B cell proliferation and differentiation, suppresses the monocyte migration, induces monocyte to discharge TNF-α, IL-1 and IL-6.CD23 is expressed in the B lymphoma cell line of some subgroup, bone-marrow-derived lymphocyte and the Epstein-Barr virus transfection of peripheral blood cells, in chronic lymphocytic leukemia and part centroblastic lymphoma expression is arranged also; May be by promoting B cell activation, differentiation, maturation, prevent the B of germinal center apoptosis, make the B cell hyperergy, synthetic and secrete autoantibody, cause autoimmunization occur.Solubility CD23 (sCD23) and bronchial asthma are in close relations, may play a significant role in the process such as transcribe of, asthma genes involved synthetic at activation, the IgE of the presenting of intercellular adhesion, antigen, T and B cell.
CD23 has multiple function in immunity system, all bringing into play to act at aspects such as cell-stimulating and propagation, antigen presentation and IgE are synthetic, and CD23 receives publicity more and more also at immunoregulation, signal transduction and with vital role that the aspects such as relation of disease play and is subjected to.
At present, obtained the monoclonal antibody of multiple CD23 both at home and abroad, but all needed expression amount more all the time, affinity is better, and extent of dilution is higher, has the monoclonal antibody of more good characteristics.The monoclonal antibody that obtains the high CD23 of activity is significant for clinical diagnosis and scientific research.
Summary of the invention
The purpose of this invention is to provide a kind of high-affinity, can be used for scientific research or clinical immunohistochemical methods and detect the mouse anti human CD23 monoclonal antibody that CD23 expresses and secrete the hybridoma cell strain of this monoclonal antibody.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
(1) according to the encoder block of the gene order (NM_001207019.2) of CD23, design a pair of special primer, TrizolReagent reagent extracts the total RNA of human fat tissue, total RNA reverse transcription become cDNA and be template pcr amplification CD23 gene with cDNA, make up recombinant expression vector PET28a-CD23, transformed into escherichia coli BL21 competence, the expression of IPTG inducible protein and affinity purification albumen are as antigen.
(2) adopt classical cell-fusion techniques to prepare the CD23 monoclonal antibody.The affinitive layer purification antibody protein, SDS-PAGE measures antibody purity, and directly the ELISA method is measured the titre of antibody purification.
(3) adopt immunohistochemical analysis CD23 monoclonal antibody dyeing people chronic tonsillitis paraffin section.
(4) according to the synthetic Auele Specific Primer of the constant region sequence of antibody gene, pcr amplification monoclonal antibody CD23 variable region of heavy chain and variable region of light chain, reclaim the purpose fragment, be cloned in the pGEM-T carrier, screening positive clone behind the transformed into escherichia coli TGl cell, heavy chain and the light chain variable region sequence of monoclonal antibody CD23 determined in order-checking behind the extracting plasmid.
CD23 albumen with prokaryotic expression provided by the invention is the hybridoma cell strain of the secretion CD23 monoclonal antibody of antigen, immune mouse acquisition, name is called 1E9, classification called after mouse anti human CD23 hybridoma cell line, this cell strain 1E9 has been preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on November 15th, 2012, Institute of Microorganism, Academia Sinica), deposit number is CGMCC No.6855.
The present invention also provides described hybridoma cell strain 1E9, the monoclonal antibody that CGMCC No.6855 produces.This antibody comprises variable region of heavy chain and variable region of light chain, and the weight chain variable region amino acid sequence is SEQ ID NO:9, and the light chain variable region amino acid sequence is SEQ ID NO:10.
The present invention also provides a kind of dna molecular SEQ ID NO:7 and dna molecular SEQ ID NO:8, dna molecular SEQ ID NO:7 encoding heavy chain variable region amino acid sequence SEQ ID NO:9; Dna molecular SEQ ID NO:8 encoded light chain variable region aminoacid sequence SEQ ID NO:10.
Advantage of the present invention and beneficial effect:
The CD23 albumen that the present invention uses recombinant human is immunizing antigen, immunity Balb/c mouse, adopt classical cell-fusion techniques, screen by ELISA, obtain the hybridoma cell strain of the anti-CD23 of strain energy stably excreting, called after 1E9, hypotype is accredited as IgG1, to collect supernatant after the hybridoma cell strain enlarged culturing, adopt Protein A affinity chromatography that the CD23 monoclonal antibody is carried out purifying.SDS-PAGE result shows that antibody purity is more than 95% behind the purifying; ELISA titer determination result shows that the titre of monoclonal antibody is all more than 1: 10000.People's chronic tonsillitis paraffin section can be observed the obvious yellow of after birth appearance to brown yellow granule in observing under the light microscopic through the dyeing of anti-CD23 antibody mediated immunity group under light microscopic, background is clear, does not have non-specific painted.On experimental result, the present invention has prepared height and has tired, and the CD23 monoclonal antibody of high specific confirms that with this antibody test it has high recognition capability to the CD23 albumen in the cell, can be used for scientific research or clinical immunohistochemical methods and detects the CD23 expression.
Description of drawings
Fig. 1 SDS-PAGE analyzes the CD23 monoclonal antibody behind the purifying.
Fig. 2 ELISA method is measured CD23 monoclonal antibody titre.
Fig. 3 is that immunohistochemistry detects analysis CD23 monoclonal antibody dyeing people chronic tonsillitis paraffin section.
Embodiment
Method therefor is normal applying method if no special instructions among the following embodiment.
Embodiment 1: the acquisition of the monoclonal antibody of hybridoma cell strain 1E9 and generation thereof
1, antigen prepd
(1) obtains goal gene
In this embodiment, the encoder block according to the gene order (NM_001207019.2) of CD23, design 1 pair of special primer:
Primer 1:5 '-GGATCCATGTTACCTCCAAGCCAGGAG-3 ' (SEQ ID NO:1)
Primer 2: 5 '-CTCGAGTCAAGAGTGGAGAGG-3 '; (SEQ ID NO:2)
Trizol Reagent reagent extracts the total RNA of human fat tissue, total RNA reverse transcription is become cDNA and is template pcr amplification CD23 gene with cDNA.
(2) make up recombinant expression vector
Reclaim behind the PCR product double digestion with step (1) acquisition, under the effect of T4DNA ligase enzyme, connect into expression vector PET28a, construction recombination plasmid PET28a-CD23.
(3) acquisition contains the expression bacterial classification of recombinant expression plasmid
With the connection product transformed into escherichia coli BL21 competence that step (2) obtains, with the solid medium screening that contains sulphuric acid kanamycin, picking list spot, alkaline lysis is the extracting plasmid in a small amount, the double digestion preliminary evaluation.The direction of insertion of sequence verification goal gene and reading frame are all correct, enter next-step operation.Transform the competent cell of expressive host bacterium with this recombinant plasmid dna.
(4) abduction delivering and protein purification
The plasmid that step (2) is extracted transforms the BL21 competence, with the solid medium screening that contains sulphuric acid kanamycin, select in mono-clonal contains sulphuric acid kanamycin to 10ml the LB liquid nutrient medium and cultivate, 37 ℃, 220rpm cultivates 10h, get in the big bottle that the 5ml liquid nutrient medium is transferred to 250ml and continue to cultivate, being cultured to the OD value is 0.8, adds 0.2mM IPTG abduction delivering, induces for 16 ℃ and spends the night, it is ultrasonic to collect bacterium liquid, gets supernatant Ni-NTA agarose affinity chromatography method purifying CD23 albumen.
2, the preparation of monoclonal antibody and purifying
(1), immune animal
Generally select female Balb/c mouse in 6-8 age in week for use, carry out three immunizations according to the immunization protocol that pre-establishes.
First immunisation.The recombinant protein c D23 of purifying (with an amount of physiological saline dilution)+complete Freund's adjuvant, 100 μ g/, the subcutaneous multi-point injection of nape portion;
Second immunisation (two weeks at interval).The recombinant protein c D23 of purifying (with an amount of physiological saline dilution)+incomplete Freund's adjuvant, 100 μ g/, the subcutaneous multi-point injection of nape portion;
Three immunity (two weeks at interval).The recombinant protein c D23 of purifying (with an amount of physiological saline dilution), incomplete Freund's adjuvant, 100 μ g/, the subcutaneous multi-point injection of nape portion;
The blood sampling in 7～10 days of three immunity backs is tired with the ELISA method detection of setting up, and the soprano that selects to tire is used for cytogamy.
Booster immunization (merging preceding 3 days) is with the recombinant protein 50 μ g abdominal injections of purifying.After 3 days, get spleen and merge.
(2), cytogamy
Adopt the eyeball excise depletion method to put to death mouse, spleen is taken out in aseptic technique, crush and grind in plate, preparation splenocyte suspension.Ready homology myeloma cell SP2/0 is mixed (1: 5～1: 10) by a certain percentage with mouse boosting cell, and add short fusogen polyoxyethylene glycol.Under the polyoxyethylene glycol effect, various lymphocytes can merge with the myeloma cell, form hybridoma.Adopt the HAT selective medium, carry out the selectivity of hybridoma and cultivate and screening.
Detect the Hybridoma Cell Culture supernatant with the ELISA method: with CD23 albumen (the 10 μ g/ml) coated elisa plate of purifying, every hole 100 μ l, 4 ℃ of wrapper sheets spend the night.Get rid of coating buffer, add the skim-milk of 200 μ l 5%, behind 37 ℃ of sealing 1h, wash 3 times, add Hybridoma Cell Culture and detect supernatant 100 μ l (negative control is PBS100 μ l), hatched 1 hour for 37 ℃.After washing 3 times, adding enzyme labelling two, anti-(sheep anti-mouse igg-HRP), hatched 1 hour for 37 ℃ removes ELIAS secondary antibody, washs 3 times, adds substrate developer 50 μ l, and room temperature left standstill 5 minutes, added stop buffer 50 μ l.Detect the OD value at 450nm wavelength place with microplate reader.The OD value apparently higher than negative control more than 2 times the person be decided to be the positive.Screening at last obtains the anti-CD23 hybridoma cell strain of strain secretion performance the best, called after 1E9, and hypotype is accredited as IgG1.
(limiting dilution assay) cultivated in the positive hybridoma cell strain 1E9 cloning of selecting, obtained to produce the hybridoma cell clone of the high monoclonal antibody of tiring.With the hybridoma cell strain enlarged culturing, and frozen guarantor's kind.Described positive hybridoma cell is anti-human CD 23 hybridoma cell line 1E9, and this clone has been stored in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.6855.
(3) a large amount of preparations and the purifying of monoclonal antibody
The cell strain 1E9 that step (2) is obtained is seeded to the Balb/c mouse peritoneal, and preparation ascites is extracted antibody then from ascites.
The purifying of monoclonal antibody CD23: adopt Protein A affinity chromatography.At first prepare protein A affinity column, behind PBS balance pillar, get the ascites of anti-CD23 and cross post, be washed till the OD value close to zero with PBS then, with glycine-HCl solution (PH) wash-out of 50mmol/LPH2.5, collect the elutriant of peak region, standby after concentrating through dialysing.SDS-PAGE result shows that antibody purity is at (referring to Fig. 1) more than 95% behind the purifying.
(4) direct ELISA method is measured the titre of antibody purification
With CD23 albumen (the 10 μ g/ml) coated elisa plate of purifying, every hole 100 μ l, 4 ℃ of wrapper sheets spend the night.Get rid of coating buffer, add the skim-milk of 200 μ l 5%, behind 37 ℃ of sealing 1h, wash 3 times, antibody purified was pressed 1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400, dilute at 1: 12800, add in the enzyme plate (negative control is PBS100 μ l) with every hole 100 μ l, hatched 1 hour for 37 ℃.After washing 3 times, adding enzyme labelling two, anti-(sheep anti-mouse igg-HRP), hatched 1 hour for 37 ℃ removes ELIAS secondary antibody, washs 3 times, adds substrate developer 50 μ l, and room temperature left standstill 5 minutes, added stop buffer 50 μ l.Detect the OD value at 450nm wavelength place with microplate reader.ELISA titer determination result shows that the titre of monoclonal antibody is all at (referring to Fig. 2) more than 1: 10000.
Embodiment 2: the evaluation of monoclonal antibody immunity group and application
Use the CD23 monoclonal antibody, method is done the pathology section routinely, and people's chronic tonsillitis paraffin section is carried out immunohistochemical staining.
Concrete grammar is:
(1) dewaxing
Section places dimethylbenzene I, II, III to dewax successively each 5 minutes, move to and respectively soaked among dehydrated alcohol I, the II 4 minutes, move in 95% alcohol and soaked 4 minutes, move in 85% alcohol and soaked 4 minutes, move to again in 70% alcohol and soaked 4 minutes, flowing water flushing 2 minutes.
(2) antigen retrieval
To rinse tissue slice well and put into pressure kettle, add the Citrate trianion antigen retrieval liquid about about 3000ml
(pH6.0), transfer to low fire after the high fiery heated and boiled and keep jet 3 minutes of boiling, turn off the electromagnetic oven switch, after two minutes pressure kettle moved in the cold water and cool off, after treating that pressure kettle endoantigen repair liquid cools off fully, open pressure kettle and tissue slice is rinsed well to move in the distilled water with flowing water soaked 2 minutes.
(3) blocking-up
3%H
2O
2The middle immersion 10 minutes, flowing water flushing distilled water flushing.Take out section, dry the water around the tissue, around tissue block, draw a circle with the immunohistochemical methods pen and note enclosing joint and will connect, prevent from that antibody from running out of to cause false negative outside enclosing.The flushing of PBS damping fluid.
(4) drip primary antibodie
Get rid of PBS unnecessary on the tissue slice to be measured, drip primary antibodie, wherein primary antibodie is the CD23 monoclonal antibody of embodiment 1 step (3) preparation and purifying, and extent of dilution is 1: 2500.Be placed on and hatch in the box 4 ℃ of refrigerator overnight and hatched about 12 hours.
(5) dripping two resists
To hatch box and take out from refrigerator, and return to and take out section after the room temperature, wash 3 times each 5 minutes with PBS.Drip universal two anti--HRP polymkeric substance.Section put into hatch wet box, cover lid connects and hatches box and put into 37 ℃ of thermostat containers together and hatched 30 minutes.
(6) colour developing, lining dye, mounting
Take out and cut into slices, wipe tissue unnecessary PBS on every side, colour developing is 3-5 minute in the adding DAB colour developing liquid, controls the intensity that dyes at microscopically.Treat that intensity puts color development stopping in the tap water with section after moderate, with flowing water flushing 5-10 minute, the Hematorylin dye liquor was redyed 1 minute again, and 0.5% hydrochloride alcohol broke up for 3 seconds, flowing water flushing 5-10 minute, dehydration, transparent, mounting, microscopy.
The result judges: according to the criterion of foreign scholar to CD23 in the tissue, the reaction of CD23 protein positive is positioned cytolemma for obvious brown yellow granule occurring.People's chronic tonsillitis tissue (referring to Fig. 3) is through the dyeing of anti-CD23 antibody mediated immunity group, can observe cytolemma under light microscopic occurs obviously yellow to brown yellow granule, background is clear, do not have non-specific paintedly, illustrate that this CD23 mouse monoclonal antibody can specificly be applied to immunohistochemical assay.
Embodiment 3: the variable region of mab order-checking
Constant region sequence according to antibody gene is synthesized following primer:
zh08 5′-GGGGATATCCACCATGRACTTCGGGYTGAGCTKGGTTTT-3′(SEQID NO：3)
zhr11 5′-GACHGATGGGGSTGTYGTGCTAGCTGNRGAGACDGTGA-3′(SEQID NO：4)
zl05 5′-GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG-3′(SEQ lDNO：5)
zlr05 5′-GGATACAGTTGGTGCAGTCGACTTACGTTTKATTTCCARCTT-3′(SEQID NO：6)
Trizol Reagent reagent extracts 5 * 10 respectively
6Total RNA of hybridoma 1E9 becomes cDNA with total RNA reverse transcription.Being that primer carries out pcr amplification monoclonal antibody CD23 variable region of heavy chain with zh08 and zhr11, is that primer carries out pcr amplification monoclonal antibody CD23 variable region of light chain with zl01 and zlr05, and warm start, reaction conditions are all adopted in the PCR reaction: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 60 ℃ 45 seconds, 72 ℃ 1 minute 10 seconds, 30 circulations; 72 ℃ 7 minutes.The PCR product reclaims purifying purpose fragment (light chain length 391bp, heavy chain length 420bp) after 1% agarose gel electrophoresis separates.Be cloned in pGEM-T (Promega) carrier, at the dull and stereotyped enterprising row filter of IPTGIX-gal, the extracting waste bacterial plaque is inoculated in the LB liquid nutrient medium that contains ammonia Wei penicillin and increases behind the transformed into escherichia coli TGl cell.Screening positive clone with the plasmid extraction test kit extracting plasmid of QIAGEN and check order, has been determined heavy chain and the light chain variable region sequence of monoclonal antibody CD23.
The dna sequence dna of monoclonal antibody CD23 variable region and aminoacid sequence:
Monoclonal antibody CD23 variable region of heavy chain dna sequence dna (5 ' → 3 ', 412bp) (SEQ ID NO:7);
The variable region of light chain dna sequence dna of monoclonal antibody CD23 (5 ' → 3 ', 392bp) (SEQ 1D NO:8);
The deduction aminoacid sequence of monoclonal antibody CD23 variable region of heavy chain (137 amino acid) (SEQ ID NO:9);
The deduction aminoacid sequence of monoclonal antibody CD23 variable region of light chain (130 amino acid) (SEQ 10ID NO:10).
Claims (5)
1. the hybridoma cell strain 1E9 of a secretion CD23 monoclonal antibody that obtains with the CD23 protein immunization mouse of prokaryotic expression, deposit number is CGMCC No.6855.
2. monoclonal antibody that is produced by the described hybridoma cell strain 1E9 of claim 1.
3. monoclonal antibody according to claim 2 comprises variable region of heavy chain and variable region of light chain, and the weight chain variable region amino acid sequence is SEQ ID NO:9, and the light chain variable region amino acid sequence is SEQ ID NO:10.
4. dna molecular SEQ ID NO:7, the weight chain variable region amino acid sequence SEQ ID NO:9 described in its coding claim 3.
5. dna molecular SEQ ID NO:8, the light chain variable region amino acid sequence SEQ ID NO:10 described in its coding claim 3.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104388391A (en) * | 2014-10-27 | 2015-03-04 | 武汉市工程科学技术研究院 | Paneth cell hybridoma cell strain of mice as well as preparation method and application paneth cell hybridoma cell strain |
| CN112442124A (en) * | 2020-12-09 | 2021-03-05 | 福州迈新生物技术开发有限公司 | anti-CD 23 protein monoclonal antibody, cell line, preparation method and application thereof |
| CN112646036A (en) * | 2021-01-21 | 2021-04-13 | 苏州百道医疗科技有限公司 | anti-CD 23 specific monoclonal antibody and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104388391A (en) * | 2014-10-27 | 2015-03-04 | 武汉市工程科学技术研究院 | Paneth cell hybridoma cell strain of mice as well as preparation method and application paneth cell hybridoma cell strain |
| CN104388391B (en) * | 2014-10-27 | 2017-03-29 | 武汉市工程科学技术研究院 | Mice paneth's cell hybridoma cell strain, preparation method and applications |
| CN112442124A (en) * | 2020-12-09 | 2021-03-05 | 福州迈新生物技术开发有限公司 | anti-CD 23 protein monoclonal antibody, cell line, preparation method and application thereof |
| CN112646036A (en) * | 2021-01-21 | 2021-04-13 | 苏州百道医疗科技有限公司 | anti-CD 23 specific monoclonal antibody and application thereof |
| CN112646036B (en) * | 2021-01-21 | 2022-05-31 | 苏州百道医疗科技有限公司 | anti-CD 23 specific monoclonal antibody and application thereof |
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