CN102690787A - Hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody and preparation method thereof - Google Patents
Hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody and preparation method thereof Download PDFInfo
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Abstract
The invention discloses hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody. By prokaryotically expressing human [beta]-actin fusion protein in Escherichia coli and taking the purified human [beta]-actin protein as antigen, BALB/c murines are immunized. Through fusion and selection of cells, hybridoma cells capable of stably secreting anti human [beta]-actin protein McAb are obtained and named as 2B4. Determination of the specificity, stability and applicable range of the McAb is done. The fusion protein has a relative molecular mass of 43 kDa and is soluble in 8M urea. The antibody titer of a supernatant of the hybridoma cells is 1 x 105 while the antibody titer of ascetic fluid is 1 x 107. Results of Westernblot show that the monocional antibody recognizes and generates specific reactions with the [beta]-actin protein of human, murines, rabbits and fish. The monocional antibody secreted by 2B4 can be widely applied to cell biology and immunology tests and has good application value.
Description
Technical field
The invention belongs to biological technical field, it relates to a kind of hybridoma, is specifically related to a kind of hybridoma of secreting anti-people β-actin protein monoclonal antibody.
Background technology
Actin i.e. " Actin muscle " is a kind of medium sized protein, and outward appearance is dumbbell shaped, is made up of 375 amino acid of genes encoding of a high conservative.The relative molecular mass of monomer Actin muscle is 43kDa, and three binding sites are arranged, and one of them is an ATP-binding site.Actin muscle is not only the chief component of cytoskeleton, but also has constituted the tissue that has contractile function among the myocyte.Actin muscle plays very big effect for cellular activity; Such as the transfer and the division of cell, protoplastis flow the motion of animal cyst and organ; The transmission of iuntercellular information; And (Sellers JR.Fifty years of contractility research post sliding filament hypothesis [J] .Journal of muscle research and cell motility, 2004,25 (6): 475-482 such as foundation of the form of cell; Winograd-Katz SE; Brunner MC; Mirlas N; Geiger B.Analysis of the signaling pathways regulating Src-dependent remodeling of the actin cytoskeleton [J] .European journal of cell biology, 2011,90 (2-3): 143-156.).Actin does not exist only in the tenuigenin; And more and more evidences shows that Actin also has important effect (Primal de Lanerolle in nucleus; Leonid Serebryannyy.Nuclear actin and myosins:Life without filaments [J] .Nature cell biology; 2011,13 (11): 1282-1288.).Except the threadworms spermatid, in the middle of all eukaryotic cells, all finding has Actin muscle to exist.In the biomolecules evolutionary process; One of protein molecule that Actin muscle is highly remained; From alga cells to the human body cell Actin muscle only less than 20% variation (Berepiki A, Lichius A, Read ND.Actin organization and dynamics in filamentous fungi [J] .Nature review microbiology; 2011,9 (12): 876-87.).Actin can be divided into 6 kinds, and β-actin and γ-non-muscle actin is distributed widely in the various tissues, and all the other 4 kinds have different muscle tissue specificitys.β-actin accounts for 10% of total protein in the myocyte, even in non-myocyte, Actin muscle also accounts for 1%～5% of total protein of cell.The expression of β-actin in each tissue and cell is constant relatively, when detecting the variation of protein expression level, uses it always and makees object of reference.
Summary of the invention
To the deficiency of prior art, the inventor utilizes the protokaryon recombination and expression techniques through TE, and the people β-actin albumen that gives expression to mainly exists with the form of inclusion body, dissolves in 8M urea.People β-actin albumen behind the electroelution purifying has very high purity, and Western blot experiment shows that this albumen has very strong immunoreactivity.People β behind the purifying-actin protein immunization BALB/c mouse through hybridoma technology, screens the hybridoma that anti-people β-actin protein monoclonal antibody is secreted in 10 strains, the hybridoma that wherein 1 strain character is good is carried out biological characteristics identify.The good characteristic of hybridoma of the present invention is that hybridoma of the prior art is incomparable; Species to reaction have broad spectrum; And β-actin albumen is had specificity, promptly β-actin the albumen that comprises people, mouse, rabbit and fish is all had good atopic.
Technical problem to be solved by this invention provides a kind of hybridoma of secreting anti-people β-actin protein monoclonal antibody, and its excretory monoclonal antibody can be widely used in cytobiology and immunological testing, has excellent application value.
Technical scheme provided by the invention is: a kind of hybridoma of secretion anti-people β-actin protein monoclonal antibody (McAb); It is that preserving number is the hybridoma of CGMCC 5908, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center.
(the preservation address was this hybridoma: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) for the preservation of common micro-organisms center C GMCC institute of China Committee for Culture Collection of Microorganisms on March 13rd, 2012; Its preserving number is CGMCC 5908, through detecting survival.This cell is circular half attached cell (can cell blown down from the bottle wall with bend pipe, collecting cell need trysinization, is normal if there is cell suspension to exist yet), and the time of cell fission multiplication is 16-18 hour.
Simultaneously, the present invention also provides the preparation method of said hybridoma, and its step is following:
(1) people β-actin albumen and Freund's complete adjuvant is fully emulsified, adopt subcutaneous immunization method, with the dosage immunity BALB/c mouse in age in 6 6～8 weeks of every 50 μ g;
Carry out the 2nd immunity after (2) two weeks, people β-actin albumen and Freund's incomplete adjuvant is fully emulsified, same dosage and method booster immunization BALB/c mouse in employing and the step (1);
Carry out respectively the 3rd time after (3) 2 weeks and 4 weeks and the 4th immunity, immunizing dose and method are with the 2nd immunity;
(4) merge preceding 3d with the people β-actin antigen that does not add adjuvant, adopt subcutaneous booster immunization, every dosage is 100 μ g;
(5) merge preceding 1d with the people β-actin antigen booster immunization that does not add adjuvant, every dosage 50 μ g;
(6) blood sampling of tail point is carried out in the mouse docking, and 4 ℃, the centrifugal 5min of 4000g gets supernatant, and indirect elisa method detects serum titer, chooses serum titer greater than 1,000, and 000 mouse prepares to carry out the cytogamy test;
(7) select the good SP2/0 myeloma cell of growth conditions to mix with 1: 5～1: 10 with the spleen cell of immune mouse, the centrifugal 5min of 1500g abandons supernatant;
(8) the jog centrifuge tube makes the cell homodisperse, slowly adds the PEG 1500 of 37 ℃ of preheatings of 1mL and mixing 1.5min gently, adds 20mL 1640 substratum then;
(9) abandon supernatant behind the centrifugal 5min of 800g, add HAT-1640 substratum and the mixing that contains 20% serum in the cell;
(10) the mixing cell evenly is laid in 96 orifice plates, at 37 ℃, CO
2Concentration is to observe syncretizing effect and change liquid after cultivating 5～7d in 5% the cell culture incubator;
(11) change liquid after 4d detect hybridoma supernatant with indirect ELISA method, select positive value height and the few hole of cell colony number, carry out subclone through limiting dilution assay, every porocyte number theoretical value is 1～2;
(12) through behind 3～4 subclones, promptly obtain can stably excreting antibody hybridoma cell strain.
Described hybridoma can be used for preparing anti-people β-actin protein monoclonal antibody.
The present invention has following beneficial effect:
The inventor is through prokaryotic expression people β-actin fusion rotein in intestinal bacteria, with the people β-actin albumen of purifying as the antigen immune BALB/c mouse.Through the fusion of cell and the hybridoma of the anti-people β of screening acquisition ability stably excreting-actin albumen McAb, called after 2B4.Adopt indirect ELISA and Western blot method that specificity, stability and the scope of application of McAb are identified.The result shows: the relative molecular mass of fusion rotein is 43kDa, dissolves in 8M urea.The antibody titer of hybridoma supernatant is 1 * 10
5, the antibody titer of ascites is 1 * 10
7Indirect ELISA is the result show, hybridoma is after external 20 generations of biography or frozen 3 months, and the excretory antibody titer is constant.Behind 37 ℃ of preservation 24h, tiring of antibody begins to descend.Western blot result shows that β-actin albumen of monoclonal antibody identification people, mouse, rabbit and fish is with its generation specific reaction.2B4 excretory monoclonal antibody can be widely used in cytobiology and immunological testing, has excellent application value.
Description of drawings
Fig. 1. the people β-pcr amplification of actin gene and the enzyme of expression vector are cut evaluation; Wherein, Figure 1A: from cDNA pcr amplification people β-actin gene, lane M:DNA relative molecular mass standard; Lane 1: blank, and lane 2: people β-actin PCR product; Figure 1B: the enzyme of expression vector pTO-T7-β-actin is cut evaluation, lane M:DNA relative molecular mass standard, the Nde I/EcoR I double digestion electrophoresis of lane 1:pTO-T7-β-actin.
Fig. 2. the prokaryotic expression and the evaluation of people β-actin fusion rotein, wherein, Fig. 2 A: the SDS-PAGE collection of illustrative plates of expressing protein; Lane M: protein relative molecular mass standard; Lane 1: the negative control strain protein, and lane 2,3: the recombinant bacterial strain albumen of abduction delivering; Fig. 2 B: the Western blot collection of illustrative plates of expressing protein.Lane M: protein relative molecular mass standard, lane 1: the negative control strain protein, lane 2,3: the recombinant bacterial strain albumen of abduction delivering.
Fig. 3. the purifying of people β-actin fusion rotein, wherein, Fig. 3 A:SDS-PAGE analyzes proteic expression body form of recombinant human β-actin and the solvability in urea thereof; Lane M: protein relative molecular mass standard, lane 1: suspension after the cytoclasis, lane 2: the centrifugal back of cytoclasis supernatant; Lane 3: the cytoclasis centrifuged deposit; Lane 4-6:2mol/L, 4mol/L, 8mol/L urea dissolving people β-actin inclusion body; Fig. 3 B:SDS-PAGE analyzes proteic purity behind the electroelution purifying, Lane M: protein relative molecular mass standard, lane 1: electroelution albumen for the first time, lane 2: electroelution albumen for the second time.
Fig. 4. the SDS-PAGE on the hybridoma behind the cleer and peaceful mouse ascites antibody purification; Wherein, Fig. 4 A: cell conditioned medium is through the SDS-PAGE behind the ammonium sulfate precipitation, lane M: protein relative molecular mass standard; Lane 1: before the cell conditioned medium purifying, lane 2: behind the cell conditioned medium purifying; Fig. 4 B: the SDS-PAGE after mouse ascites antibody process ammonium sulfate precipitation and the IX, lane M: protein relative molecular mass standard, lane 1: behind the ascites antibody thiamines deposition and purification, lane 2: behind the ascites antibody ion exchange chromatography purifying.
The Detection of Stability that Fig. 5 ascites monoclonal antibody is preserved under differing temps.
Fig. 6 2B4 antibodies specific detect and and to the different animals proteic immunoreactivity of originating, lane ck: blank, lane 1,4,6; 8 and 10: antigen is respectively the HeLa total protein of cell, the rabbit cerebral tissue, and the mouse nephridial tissue, fish heart tissue and e. coli total protein, one anti-is the 15B2 monoclonal antibody; Lane 2,5, and 7,9 and 11: antigen is respectively the HeLa total protein of cell; The rabbit cerebral tissue, the mouse nephridial tissue, fish heart tissue and e. coli total protein, one anti-is 2B4; Lane 3:Hela total protein of cell, one anti-is the Bioworld Company products.
Embodiment
Come further to illustrate the present invention through the detailed description of embodiment below, but be not limitation of the present invention, only make example description.
The present embodiment material therefor is following:
Bacterial classification and cell strain: intestinal bacteria E.coli Top 10, ER2566 bacterial strain are preserved by this laboratory, and the S/P20 murine myeloma cell is so kind as to give by professor Xia Ningshao of HSPH of Xiamen University.
Main agents: pTO-T7,15B2 monoclonal antibody, HRP-sheep anti mouse are so kind as to give by professor Xia Ningshao of Xiamen University; Trizol and reverse transcription test kit are available from Ferments company; PMD-18T carrier, Taq enzyme, T4 ligase enzyme, EcoR I and Nde I restriction enzyme are available from TAKARA company; DNA glue reclaims test kit and plasmid extraction kit is given birth to the worker available from Shanghai; Standard β-actin monoclonal antibody is available from Bioworld company; Protein relative molecular mass standard is available from Genestar company; HRP-DAB substrate colouring reagents box is available from TIANGEN company; ECL chemoluminescence colouring reagents box is available from Thermo company; PEG1500 is available from SIGMA company; 1640 substratum are available from Gibco company.
Prepare the hybridoma of secretion anti-people β-actin protein monoclonal antibody through following method
1. people β-actin encoding sequence is cloned
Adopt Trizol reagent from the HepG2 cell, to extract total RNA, get the up-to-standard RNA of 1.0 μ g and carry out the synthetic of people β-actin cDNA by Ferments company reverse transcription test kit specification sheets.Utilize oligo 6.0 softwares, according to people β-actin gene coded sequence design pcr amplification primer (introduce Nde I site at upstream primer, FP:5 '-
CATATGGCTACATATGGATGATG ATATCGCCG-3 '; Downstream primer is introduced EcoR I site, RP:5 '-
GAATTCACAGAATTCCTAGAAG CATTTGCGGTG-3 '); Be template amplification people β-actin gene with cDNA then, reaction system is: 10 * PCR Buffer, 2.5 μ L, dNTPs (10mmol/L) 2 μ L, each 1 μ L of upstream and downstream primer (10 μ mol/L), Taq archaeal dna polymerase (5U/ μ L) 0.2 μ L, cDNA 2 μ L, deionized water are supplied 25 μ L.Reaction conditions: 94 ℃ of 5min; 94 ℃ of 40sec, 58 ℃ of 40sec, 72 ℃ of 1min 20sec, 30 circulations; 72 ℃ of 10min.The PCR product identifies that through 1.5% agarose gel electrophoresis glue reclaims the PCR product.Reclaim product and is connected with pMD18-T and spends the night and transform the Top10F` competent cell, the extraction recombinant plasmid is also served Hai Shenggong and is checked order.Identify sequence correct after again subclone to expression vector pTO-T7, through kantlex screening and EcoR I/Nde I double digestion evaluation recombinant clone.
CDNA is through behind the pcr amplification, and product carries out electrophoresis detection with 1.5% agarose, the result between 1000bp～2000bp, find unique one clearly, size is about the DNA band (Figure 1A) of 1200bp, conform to people β-actin gene theory value size.Serve the sea after being connected on the T carrier and give birth to the public testing preface, the result shows that people β-actin gene order is in full accord among this sequence and the Genebank.Then with people β-actin gene subclone in the pTO-T7 expression plasmid; Recombinant plasmid pTO-T7-β-actin is after Nde I and EcoR I double digestion and electrophoresis detection; The result observes the DNA band in the 1200bp position, explains that people β-actin gene successfully has been cloned into (Figure 1B) on the expression vector.
2. the Western-blot of target protein abduction delivering and product identifies
The mono-clonal bacterial strain of picking screening is inoculated in the 5mL LB liquid nutrient medium and (contains 100 μ g/mL sulphuric acid kanamycin (Kan
+) 37 ℃ be cultured to OD
600Value reaches 0.6, and the adding final concentration is that the IPTG of 0.4mmol/L carries out abduction delivering, does not induce as negative control with the bacterial strain that transforms recombinant expression plasmid simultaneously, and other culture condition is consistent with experimental group.37 ℃ of inducing culture 4h, the centrifugal collection thalline of the centrifugal 10sec of room temperature 12000g.The preparation protein sample utilizes 12% SDS-PAGE to analyze the Recombinant Protein Expression situation.People β-actin protein sample is through behind the electrophoresis, and electrotransfer is to pvdf membrane.With the TBS that contains 5% skimmed milk (100mmol/L TrisHCl, 150mmol/L NaCl, pH7.5) damping fluid sealing 2h.Ratio in 1: 1000 is diluted monoclonal antibody with skimmed milk, adds one and resists room temperature reaction 2h.(0.05%Tween-20 pH7.5) washes film three times for 100mmol/L TrisHCl, 150mmol/L NaCl, and each 10min adds HRP enzyme mark sheep anti mouse, room temperature reaction 2h with the TBST damping fluid.Wash film three times with TBST, each 10min develops the color with HRP-DAB colouring reagents box.
The recombinant bacterial strain of inducing and control group bacterial strain are prepared protein sample to carry out SDS-PAGE and detects; The result can observe a relative thicker protein band in the test group of people β-actin recombinant bacterial strain abduction delivering; Its size and theoretical basically identical; And this band does not appear in control group, explain that recombinant human β-actin albumen possibly obtain high expression level in recombinant bacterial strain.Western blot result shows; The recombinant protein of expressing and the β-actin mouse resource monoclonal antibody generation specific reaction of Bioworld company; Person of good sense β-actin gene has obtained to efficiently express in intestinal bacteria furtherly, and has good antigenicity (Fig. 2).
3. target protein great expression and inclusion body protein purifying
From picking list colony inoculation on the flat board to 50mL LB (Kan
+) cultivate 12h in the substratum, inoculate 1% reorganization bacterium then to 2L LB substratum (Kan
+) the middle cultivation.Treat bacterium liquid OD
600Value reaches that to be added to final concentration after 0.6 be that the IPTG of 0.4mmol/L carries out abduction delivering.Behind the 4h, room temperature, the centrifugal 8min of 12000g collects thalline.Adding 100mL lysate (50mmol/L TrisHCl, 1mmol/L EDTA, 100mmol/L NaCl, pH 8.0), 10mm probe 200W power ultrasonic smudge cells.With 50mL buffer I (20mmol/L TrisHCl; 5mmol/L EDTA; 100mmol/L NaCl) and 0.5%TritonX 100-buffer I wash inclusion body respectively 2 times, the urea with the 2mol/L of 50mL buffer I preparation, 4mol/L, 8mol/L dissolves inclusion body successively then.
Get the maximum component of solubilization of inclusion bodies degree and carry out big plate SDS-PAGE (130mm * 180mm * 1.5mm), first 90V electrophoresis 40min, 120V electrophoresis 12h then.From blob of viscose, downcut the target protein band, dialysis tubing is placed with the electrophoresis direction is vertical after placing dialysis tubing, reverse electroelution 30sec behind the 100V electroelution 2h repeats 2 times.Merge in the albumen elutriant and the dialysis tubing of packing into, each dialysis 2h repeats to dialyse 3 times in the PBS of 10 times of volumes.Collect dialysis albumen and identify purity of protein with SDS-PAGE.
The SDS-PAGE detected result shows that target protein mainly is distributed in the deposition, explains that proteic expression is that form with inclusion body exists.Bandscan software analysis people β-actin albumen accounts for 65.8% of total protein in deposition.Inclusion body protein after the washing can only minimal amounts of dissolved in 2mol/L and 4mol/L urea, and meltage is very big in 8mol/L urea, accounts for 87.7% (Fig. 3 A) of 8mol/L urea dissolving total protein.Behind people β-actin albumen process electroelution purifying, on the SDS-PAGE electrophorogram, only observe a protein band, explain that purity is greatly improved, surpassed 99% with the proteic purity of Bandscan software analysis.SDS-PAGE result's demonstration, so that improve the proteic recovery, need be through at least twice electrophoresis elution, just can obtain higher protein recovery (Fig. 3 B)
4. animal immune and serum titer detect
People β-actin albumen and Freund's complete adjuvant is fully emulsified, adopt subcutaneous immunization method, with the dosage immunity BALB/c mouse in age in 6 6～8 weeks of every 50 μ g.Carry out the 2nd immunity after two weeks, people β-actin albumen and Freund's incomplete adjuvant is fully emulsified, adopt same dosage and method booster immunization BALB/c mouse.Carry out respectively the 3rd time after 2 weeks and 4 weeks and the 4th immunity, dosage and method are with the 2nd immunity.After carrying out the 4th immunity, 1 week detecting mice serum tires.3d adopts subcutaneous and the four limbs booster immunization with the people β-actin antigen that does not add adjuvant before merging, and every dosage is 100 μ g.1d is with the people β-actin antigen intravenous injection booster immunization that does not add adjuvant, every dosage 50 μ g before merging.
The blood sampling of tail point is carried out in the mouse docking, and 4 ℃, the centrifugal 5min of 4000g gets supernatant.Indirect elisa method detects serum titer, chooses serum titer greater than 1,000, and 000 mouse carries out the cytogamy test.
5. cytogamy, positive hybridoma cell screen and subclone
Select the good SP2/0 myeloma cell of growth conditions to mix with 1: 5～1: 10 with the spleen cell of immune mouse, the centrifugal 5min of 1500g abandons supernatant.The jog centrifuge tube makes the cell homodisperse, slowly adds the PEG 1500 of 37 ℃ of preheatings of 1mL and mixing gently, adds 20mL 1640 substratum then.Abandon supernatant behind the centrifugal 5min of 800g, add HAT-1640 substratum and the mixing that contains 20% serum in the cell precipitation.
The mixing cell evenly is laid in 96 orifice plates, at 37 ℃, and CO
2Concentration is to observe syncretizing effect and change liquid after cultivating 5～7d in 5% the cell culture incubator.4d detects hybridoma supernatant with indirect ELISA method after changing liquid, selects positive value height and the few hole of cell colony number, carries out subclone through limiting dilution assay, and every porocyte number theoretical value is 1～2.Through behind 3～4 subclones, promptly obtain can stably excreting antibody hybridoma cell strain, this cell strain partly is used for the production of monoclonal antibody, part is frozen in liquid nitrogen container.
The fusion rate that merges behind the 7d through observing and calculate this cell is 82.1%, merges that to record the positive colony rate through indirect elisa method behind the 10d be 32.2%.Adopt indirect ELISA and limiting dilution assay screening positive hybridoma cell; Through behind 3～4 subclones, obtained the cell strain that 10 strains can the anti-people β of stably excreting-actin protein monoclonal antibody, wherein the output of a strain antibody is high; Specificity and reactivity are had outstanding performance, with its called after 2B4.
The 2B4 cell strain is cultivated, cleer and peaceful hybridoma on the collecting cell respectively behind the 6d, hybridoma immune mouse abdominal cavity, about 8～10d extracts ascites after treating that mouse ascites reaches certain antibody titers.The Hybridoma Cell Culture supernatant just can reach very high purity (Fig. 4 A) behind ammonium sulfate precipitation; After the mouse ascites of preparation passes through ammonium sulfate precipitation preliminary purification and anion-exchange chromatography purifying respectively; The purity of antibody protein obviously increases, and can very clearly see the heavy chain of 50kDa and the light chain of 25kDa.Through the protein concentration before and after the Bradford method measurement ascites monoclonal antibody purifying, the recovery that the ascites purifying is described is 23.3% (Fig. 4 B)
6. the generation of monoclonal antibody and purifying
Adopt the method manufacture order clonal antibody that induces ascites in the mouse body.Get 10 8～10 the week age BALB/c mouse, the aseptic Yellow Protopet 2A of first abdominal injection, every 0.5mL.Inject hybridoma to intraperitoneal behind the 3d, every approximately injection 10 of mouse
6Individual cell promptly begins to gather ascites behind 8～10d.Carry out preliminary purification with gathering 1: 1 earlier adding saturated ammonium sulphate of good ascites, 4 ℃, abandon supernatant behind the centrifugal 5min of 8000g.With PBS damping fluid (20mmol/L NaCl, 2.68mmol/L KCl, 10mmol/L Na
2HPO
4, 1.76mmol/L KH
2PO
4, pH7.4) resuspended deposition is put into the PBS damping fluid of 10 times of volumes and is dialysed 2 times, each 2h.4 ℃, the centrifugal 10min of 8000g collects supernatant.Dialysis antibody directly carries out the anion-exchange chromatography purifying with the DE-52 resin, last kind after, continue to penetrate chromatography column with the PBS of 5 times of column volumes, the PBS that contains 50mmol/L NaCl with 5 times of column volumes again carries out wash-out.Collect PBS elution peak antibody, the effect of SDS-PAGE purification Identification.
7. the evaluation of the tiring of monoclonal antibody, specificity and the scope of application
People β-actin albumen with 1 μ g/mL encapsulates 96 hole enzyme plates, detects antibody purification with indirect ELISA method and tires.Confirm as antibody titer with the positive value and the ratio of negative value greater than 2.0 maximum antibody dilution multiple.The result shows that the antibody titer of Hybridoma Cell Culture supernatant is 1 * 10
5, the antibody titer of ascites is 1 * 10
7, ascites is tired and is tired apparently higher than supernatant.
With HeLa cell, rabbit cerebral tissue, mouse nephridial tissue, fish heart tissue and colibacillary total protein as antigen; The linear monoclonal antibody 15B2 (LIS of hepatitis E virus; TANG X; SEETHARAMAN J; Et al.Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus-host interaction [J] .PLoS Pathogens, 2009,5 (8): e1000537.) as negative control; By " molecular cloning experiment guide " (J Sa nurse Brooker; E.F. Ritchie not, the graceful girl's Asti of T. molecular cloning experiment guide [M]. Beijing: Science Press (J Sam brooke.E.F.Fritz ' s.T.Rasmani Christina o.Molecular cloning:A Laboratory Manual [M], Beijing:Science Press); 2002.889-897) method carry out SDS-PAGE and Western blotting and detect, the ultimate density of an anti-15B2,2B4 and Bioworld company antibody incubation is 100ng/mL.Mouse source β-actin monoclonal antibody that the result shows 2B4 and Bioworld company all only with the HeLa cell in β-actin albumen generation specific reaction, and the reaction band is obvious especially, explain that 2B4 has specificity highly and good susceptibility.
8. the evaluation of hybridoma cell line and monoclonal antibody stability
2B4 is carried out external 20 generations of biography and liquid nitrogen cryopreservation recovery processing again after 3 months respectively, and anti-people β-actin McAb's tires in the indirect ELISA detection culture supernatant.The ascites monoclonal antibody places the environment of 37 ℃ and 4 ℃ respectively.Every 8h gets once appearance, and mensuration is tired, and as positive control, mice serum is as negative control with the sample of-20 ℃ of preservations.
The present invention screens the external biography of hybridoma of acquisition after 20 generations, adopts indirect ELISA to detect tiring of monoclonal antibody in the cell conditioned medium, and it is tired and remains unchanged basically, still can reach 1 * 10
5Frozen hybridoma recovery in liquid nitrogen is cultivated the production monoclonal antibody, and the result finds that also its also basic change that there is not of tiring can reach 1 * 10 after frozen 3 months
5, the hybridoma cell line good stability that obtains is described.The ascites monoclonal antibody places 37 ℃ and 4 ℃ of incubation 24h respectively, and it is tired and-20 ℃ of control group indifferences, surpasses that the antibody titer of 37 ℃ of groups just begins to have decline (Fig. 5) behind the 24h.The good stability of monoclonal antibody is described, is met the shelf stable property requirement of commercially available antibody fully, have a good application prospect.
β-actin albumen is a very conservative albumen, between different plant species, has high homology.In order to study the scope of application of 2B4; Carry out immunoblot experiment with HeLa cell, rabbit cerebral tissue, mouse nephridial tissue and fish heart tissue total protein; With the blank of e. coli protein, hepatitis E virus monoclonal antibody 15B2 is as negative antibody control simultaneously.Western blot result shows, the 2B4 monoclonal antibody can with the β-actin albumen generation specific reaction in four kinds of different animals sources, specificity colour developing band promptly occurs, and blank and negative control group have no band appearance (Fig. 6) at 43kDa place.When carrying out immunoblot experiment, it is sheep anti-mouse igg that two of use resists, and therefore can also judge that the hypotype of mouse-anti 2B4 is the IgG type.
Monoclonal antibody technique is an important breakthrough in the medical science eighties in 20th century, and it has fundamentally solved the medium-term and long-term problems such as specificity, susceptibility and poor repeatability that exist of immunology.From nineteen eighty-two Fukushi H
[10]Since the AIV hypotype H4 monoclonal antibody that has prepared first, obtained using widely with its distinctive high specific and susceptibility.The factor that influences cytogamy has a lot, and immune effect is particularly important, utilizes the immunity system of the people β-subcutaneous multidigit point immunostimulation of the actin albumen mouse behind the purifying.After four subcutaneous immunity, the millionfold obviously test positive of tiring still of mice serum dilution is carried out abdominal injection immunity and tail vein injection immunity subsequently, so just makes the intravital bone-marrow-derived lymphocyte of mouse keep the active state of a kind of height.The present invention has guaranteed positive colony rate preferably through the assurance of this committed step of animal immune.Grow into 1/2 of hole floorage～1/3 after the cytogamy and o'clock clone, avoided the generation of positive cell omission and chromosome elimination situation.Adopt limiting dilution assay easy and simple to handle, stability and the antibody-secreting of the having guaranteed hybridoma cell line high characteristics of tiring.Through 4 time cloningizations, the hybridoma positive rate of acquisition is 100%.When carrying out the cell subclone, preferably use the HT-1640 substratum that is added with feeder cell or adds ESC to cultivate, in case hybridoma is dead because of out of order in culturing process.
At present, the Purification of Monoclonal Antibodies method has a lot, like sad-ammonium sulfate method
[11], ion exchange chromatography
[12], affinity chromatography
[13], gel filtration method and high performance liquid chromatography (HPLC) etc.This research is adopted and is used the ion exchange chromatography antibody purification again with the ammonium sulfate precipitation preliminary purification earlier, and the odd contradictive hydroperitoneum antibody titer behind the purifying reaches 1 * 10
7The Hybridoma Cell Culture supernatant just can reach very high purity behind ammonium sulfate precipitation, can satisfy laboratory request for utilization and commercialization requirement fully, because emiocytosis mainly is monoclonal antibody.Because the price comparison of cell cultures serum is high; The antibody titer that produces is lower again; Therefore in the process of producing antibody, can cultivate a certain amount of hybridoma earlier, utilize mouse to induce ascites to produce antibody then; The antibody of producing the like this height of not only tiring, and its production cost can reduce greatly.Utilize the β-actin sequence of Blast contrast people and Chinese hamster, ctenopharyn odon idellus fish and european rabbits, homology is respectively 93%, 88% and 88%.β-actin the albumen of these 4 kinds of biologies of 2B4 excretory monoclonal anti physical efficiency specific recognition.2B4 excretory McAb has wide spectrum suitability preferably, can be widely used as the confidential reference items antibody of gene expression regulation researchs such as people, mouse, rabbit and fish.Hybridoma cell line and monoclonal antibody Detection of Stability presentation of results, the hybridoma cell line of monoclonal antibody and the stability of monoclonal antibody all are very good, prolonged preservation can not make antibody titer reduce.Multinomial data show that 2B4 excretory McAb can be widely used in multiple biology and cells of tissues biology and immunological testing, has applications well prospect and commercial value.
Claims (3)
- One kind the secretion anti-people β-actin protein monoclonal antibody hybridoma, it is that preserving number is the hybridoma of CGMCC 5908, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center.
- 2. the preparation method of the described hybridoma of claim 1, it is characterized in that: this method comprises the steps:(1) people β-actin albumen and Freund's complete adjuvant is fully emulsified, adopt subcutaneous immunization method, with the dosage immunity BALB/c mouse in age in 66 ~ 8 weeks of every 50 μ g;Carry out the 2nd immunity after (2) two weeks, people β-actin albumen and Freund's incomplete adjuvant is fully emulsified, same dosage and method booster immunization BALB/c mouse in employing and the step (1);Carry out respectively the 3rd time after (3) 2 weeks and 4 weeks and the 4th immunity, immunizing dose and method are with the 2nd immunity;(4) merge preceding 3 d with the people β-actin antigen that does not add adjuvant, adopt subcutaneous booster immunization, every dosage is 100 μ g;(5) merge preceding 1 d with the people β-actin antigen booster immunization that does not add adjuvant, every dosage 50 μ g;(6) blood sampling of tail point is carried out in the mouse docking, and 4 ℃, centrifugal 5 min of 4000 g get supernatant, and indirect elisa method detects serum titer, chooses serum titer greater than 1,000, and 000 mouse prepares to carry out the cytogamy test;(7) select the good SP2/0 myeloma cell of growth conditions to mix with 1:5 ~ 1:10 with the spleen cell of immune mouse, centrifugal 5 min of 1500 g, the collecting cell deposition is abandoned supernatant;(8) the jog centrifuge tube makes the cell homodisperse, slowly adds the PEG 1500 of 37 ℃ of preheatings of 1 mL and mixing 1.5 min gently, adds 20 mL, 1640 substratum then;Centrifugal 5 min of (9) 800 g, the collecting cell deposition is abandoned supernatant, adds HAT-1640 substratum and the mixing that contains 20% serum in the cell;(10) the mixing cell evenly is laid in 96 orifice plates, at 37 ℃, CO 2Concentration is to observe syncretizing effect and change liquid after cultivating 5 ~ 7 d in 5% the cell culture incubator;(11) change liquid after the 4th d detect hybridoma supernatant with indirect ELISA method, select positive value height and the few hole of cell colony number, carry out subclone through limiting dilution assay, every porocyte number theoretical value is 1 ~ 2;(12) through behind 3 ~ 4 subclones, promptly obtain can stably excreting antibody hybridoma cell strain.
- 3. the described hybridoma of claim 1 is in the application of preparation anti-people β-actin protein monoclonal antibody.
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| CN106405085A (en) * | 2016-08-31 | 2017-02-15 | 天津市泌尿外科研究所 | ELISA kit used for detecting castration-resistant prostate cancer and using method thereof |
| CN108517011A (en) * | 2018-04-26 | 2018-09-11 | 成都百奥克林生物科技有限公司 | A kind of nano antibody and its coded sequence for cytoskeletal protein β-actin |
| CN109265544A (en) * | 2018-09-19 | 2019-01-25 | 吉林省养蜂科学研究所（吉林省蜂产品质量管理监督站、吉林省蜜蜂遗传资源基因保护中心） | The antibody and preparation method thereof of ground bumblebee actin albumen |
| CN114324897A (en) * | 2022-01-10 | 2022-04-12 | 亚科因(武汉)生物技术有限公司 | Method for producing human reference protein for western-blot experiment and application thereof |
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| CN109265544A (en) * | 2018-09-19 | 2019-01-25 | 吉林省养蜂科学研究所（吉林省蜂产品质量管理监督站、吉林省蜜蜂遗传资源基因保护中心） | The antibody and preparation method thereof of ground bumblebee actin albumen |
| CN114324897A (en) * | 2022-01-10 | 2022-04-12 | 亚科因(武汉)生物技术有限公司 | Method for producing human reference protein for western-blot experiment and application thereof |
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