CN102690787B - 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-human 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 comprised 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 ATP-binding site.Actin muscle is not only the chief component of cytoskeleton, but also has consisted of the tissue that has contractile function among the myocyte.Actin muscle plays very large effect for cellular activity, transfer and division such as cell, flowing of protoplastis, the motion of animal cyst and organ, the transmission of iuntercellular information, and the foundation of the form of cell etc. (Sellers JR.Fifty years of contractility research post sliding filament hypothesis[J] .Journal of muscle research and cell motility, 2004,25 (6): 475-482; 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 increasing evidence 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, all finding in the middle of all eukaryotic cells 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 dynamic s 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.β-actin is relative with the expression in the cell constant at each tissue, commonly uses it and make object of reference when detecting the variation of protein expression level.
Summary of the invention
For the deficiencies in the prior art, the inventor utilizes the RT-PCR expression technology through repetition test, and the people β that gives expression to-actin albumen mainly exists with the form of inclusion body, dissolves in 8M urea.People β behind the electroelution purifying-actin albumen 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 by hybridoma technology, screens the hybridoma that anti-human β-actin protein monoclonal antibody is secreted in 10 strains, and the hybridoma that wherein 1 strain character is good is carried out Identification of Biological Characteristics.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, namely the β that comprises people, mouse, rabbit and fish-actin albumen is all had good atopic.
Technical problem to be solved by this invention provides a kind of hybridoma of secreting anti-human β-actin protein monoclonal antibody, and the monoclonal antibody of its secretion can be widely used in cytobiology and immunological testing, has good using value.
Technical scheme provided by the invention is: a kind of hybridoma of secretion anti-human 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, after testing survival.This cell is circular half attached cell (cell can be got off from bottle wall blowing up with bend pipe, collecting cell does not 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 described hybridoma, and its step is as follows:
(1) people β-actin albumen and Freund's complete adjuvant is fully emulsified, adopt the subcutaneous inoculation 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, adopt with step (1) in same dosage and method booster immunization BALB/c mouse;
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 front 3d with the people β that does not add adjuvant-actin antigen, adopt subcutaneous booster immunization, every dosage is 100 μ g;
(5) merge before 1d with the people β that does not add adjuvant-actin antigen booster immunization, 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 Uniform Dispersion, slowly adds the PEG 1500 of 37 ℃ of preheatings of 1mL and mixing 1.5min gently, then adds 20mL 1640 substratum;
(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 by limiting dilution assay, every porocyte number theoretical value is 1～2;
(12) through behind 3～4 subclones, namely obtain can stably excreting antibody hybridoma cell strain.
Described hybridoma can be used for preparing anti-human β-actin protein monoclonal antibody.
The present invention has following beneficial effect:
The inventor is by prokaryotic expression people β-actin fusion rotein in intestinal bacteria, with the people β of purifying-actin albumen as the antigen immune BALB/c mouse.Through the fusion of cell and the hybridoma of the anti-human β of screening acquisition energy 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
7Result of indirect ELISA shows that hybridoma is after external 20 generations of biography or frozen 3 months, and the antibody titer of secretion is constant.Behind 37 ℃ of preservation 24h, tiring of antibody begins to descend.Western blot result shows that the β of monoclonal antibody identification people, mouse, rabbit and fish-actin albumen is with its generation specific reaction.The monoclonal antibody of 2B4 secretion can be widely used in cytobiology and immunological testing, has good using 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. 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 recombinant human β-actin protein expression bodily form formula and solvability in urea thereof, lane M: protein relative molecular mass standard, lane 1: suspension after the cytoclasis, lane 2: the centrifugal rear supernatant of cytoclasis, 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 the purity of albumen 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 ion-exchange, 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 detects and reaches the immunoreactivity of different animals being come source protein, lane ck: blank, and lane 1,4,6,8 and 10: antigen is respectively the HeLa total protein of cell, the rabbit cerebral tissue, mouse nephridial tissue, fish heart tissue and e. coli total protein, primary antibodie is the 15B2 monoclonal antibody, and lane 2,5,7,9 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, primary antibodie is 2B4; Lane 3:Hela total protein of cell, primary antibodie are Bioworld company product.
Embodiment
Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does the example explanation.
The present embodiment material therefor is as follows:
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 by the following method the hybridoma of secretion anti-human β-actin protein monoclonal antibody
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, (introduce Nde I site, FP:5 ' at upstream primer according to people β-actin gene coded sequence design pcr amplification primer
CATATGGCTACA TATGGATGATG ATATCGCCG-3 '; Downstream primer is introduced EcoR I site, RP:5 '-
GAATTCACAGA ATTCCTAGAAG CATTTGCGGTG-3 '), then take cDNA as template amplification people β-actin gene, 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 that sequence is subcloned on the expression vector pTO-T7 after correct again, identifies recombinant clone by kantlex screening and EcoR I/Nde I double digestion.
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 order, the result shows that people β-actin gene order is in full accord among this sequence and the Genebank.Then people β-actin gene is subcloned 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, illustrates 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 expression of recombinant protein.People β-actin protein sample is through behind the electrophoresis, and electrotransfer is to pvdf membrane.With the TBS(100mmol/L TrisHCl that contains 5% skimmed milk, 150mmol/L NaCl, pH7.5) damping fluid sealing 2h.Ratio in 1:1000 is diluted monoclonal antibody with skimmed milk, adds primary antibodie, room temperature reaction 2h.(100mmol/L TrisHCl, 150mmol/L NaCl, 0.05%Tween-20, pH7.5) washes film three times with the TBST damping fluid, and each 10min adds HRP enzyme mark sheep anti mouse, room temperature reaction 2h.Wash film three times with TBST, each 10min is with the colour developing of 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 relatively thick protein band in the test group of people β-actin recombinant bacterial strain abduction delivering, its size is with theoretical basically identical, and this band does not appear in control group, illustrate that recombinant human β-actin albumen may obtain high expression level in recombinant bacterial strain.Western blot result shows, the recombinant protein of expressing and the β of Bioworld company-actin mouse resource monoclonal antibody generation specific reaction, 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, then inoculate 1% recombinant bacterium 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 respectively inclusion body 2 times, then dissolve successively inclusion body with 2mol/L, the 4mol/L of 50mL buffer I preparation, the urea of 8mol/L.
The component of getting solubilization of inclusion bodies degree maximum is carried out large plate SDS-PAGE(130mm * 180mm * 1.5mm), first 90V electrophoresis 40min, then 120V electrophoresis 12h.Downcut the target protein band from blob of viscose, dialysis tubing and electrophoresis perpendicular direction are placed after placing dialysis tubing, reverse electroelution 30sec repeats 2 times behind the 100V electroelution 2h.Merge in 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 precipitation, illustrates that protein expression is that form with inclusion body exists.Bandscan software analysis people β-actin albumen accounts for 65.8% of total protein in precipitation.Inclusion body protein after the washing can only dissolve in 2mol/L and 4mol/L urea on a small quantity, and meltage is very large in 8mol/L urea, accounts for 87.7%(Fig. 3 A of 8mol/L urea dissolving total protein).People β-actin albumen through the electroelution purifying after, on the SDS-PAGE electrophorogram, only observe a protein band, illustrate that purity is greatly improved, surpassed 99% with the purity of Bandscan software analysis albumen.SDS-PAGE result's demonstration, so that improve the rate of recovery of albumen, need to 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 the subcutaneous inoculation 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.1 week detecting mice serum after carrying out the 4th immunity tires.3d adopts subcutaneous and the four limbs booster immunization with the people β that does not add adjuvant-actin antigen before merging, and every dosage is 100 μ g.1d is with the people β that does not add adjuvant-actin antigen intravenous injection booster immunization, 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 Uniform Dispersion, slowly adds the PEG 1500 of 37 ℃ of preheatings of 1mL and mixing gently, then adds 20mL 1640 substratum.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 by limiting dilution assay, and every porocyte number theoretical value is 1～2.Through behind 3～4 subclones, namely 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 by observing and calculate this cell is 82.1%, merges that to record the positive colony rate by 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-human 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 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 respectively ammonium sulfate precipitation preliminary purification and anion-exchange chromatography purifying, the purity of antibody protein obviously increases, and can very clearly see the heavy chain of 50kDa and the light chain of 25kDa.By the protein concentration before and after the Bradford method measurement ascites monoclonal antibody purifying, the rate of 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 Mice Body.Get 10 8～10 the week age BALB/c mouse, the aseptic paraffin oil of first abdominal injection, every 0.5mL.The backward intraperitoneal of 3d injects hybridoma, every approximately injection 10 of mouse
6Individual cell namely begins to gather ascites behind 8～10d.The 1:1 of ascites elder generation that is collected is added saturated ammonium sulphate carry out preliminary purification, 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 precipitation 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, after the loading, continues to penetrate chromatography column with the PBS of 5 times of column volumes, and 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
With the coated 96 hole enzyme plates of the people β of 1 μ g/mL-actin albumen, detect antibody purification with indirect ELISA method and tire.Be defined as antibody titer with the ratio of positive value and negative value greater than 2.0 maximum antibody dilution multiple.The result shows that the antibody titer of Hybridoma Cell Culture supernatant liquor is 1 * 10
5, the antibody titer of ascites is 1 * 10
7, titer of ascites is tired apparently higher than supernatant.
Use the HeLa cell, the rabbit cerebral tissue, the mouse nephridial tissue, fish heart tissue and colibacillary total protein are as antigen, the linear monoclonal antibody 15B2(LI S 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 Pehanorm 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 primary antibodie 15B2, the ultimate density of 2B4 and Bioworld company antibody incubation is 100ng/mL.The result show the mouse source β of 2B4 and Bioworld company-actin monoclonal antibody all only with the HeLa cell in β-actin albumen generation specific reaction, and the reaction band is obvious especially, illustrate that 2B4 has specificity highly and good susceptibility.
8. the evaluation of hybridoma cell line and monoclonal antibody stability
2B4 is carried out respectively external 20 generations of biography and liquid nitrogen cryopreservation again recovery processing after 3 months, and anti-human β-actin McAb's tires in the indirect ELISA detection culture supernatant.The ascites monoclonal antibody places respectively the environment of 37 ℃ and 4 ℃.Every 8h gets sample one time, 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 substantially remains unchanged, and 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 no change 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 respectively 37 ℃ and 4 ℃ of incubation 24h, 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, meets the shelf stable requirement of commercially available antibody fully, have a good application prospect.
β-actin albumen is a very conservative albumen, has high homology between different plant species.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, E type hepatitis virus monoclonal antibody 15B2 is as negative antibody control simultaneously.Western blot result shows, the 2B4 monoclonal antibody can with the β in four kinds of different animals sources-actin albumen generation specific reaction, specificity colour developing band namely occurs at 43kDa place, and blank and negative control group are without any band appearance (Fig. 6).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, be widely used with its distinctive high specific and susceptibility.The factor that affects cytogamy has a lot, and immune effect is particularly important, utilizes the immunity system of multidigit point immunostimulation mouse under people β behind the purifying-actin protein skin.Behind four subcutaneous inoculations, 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 so that the bone-marrow-derived lymphocyte in the Mice Body keeps the active state of a kind of height.The present invention has guaranteed preferably positive colony rate by 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 the undetected and chromosome elimination situation of positive cell.Adopt limiting dilution assay easy and simple to handle, guaranteed the stability of hybridoma cell line and the antibody-secreting high characteristics of tiring.Through 4 time clonings, 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 cell growth factor to cultivate, in case hybridoma in culturing process because of out of order death.
At present, the purification process of monoclonal antibody has a lot, such as caprylic acid-ammonium
[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 with the ammonium sulfate precipitation preliminary purification first again, 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 service requirements 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 first a certain amount of hybridoma, then utilize mouse to induce ascites to produce antibody, the antibody of producing the like this height of not only tiring, and its production cost can reduce greatly.Utilize the β of Blast contrast people and Chinese hamster, ctenopharyn odon idellus fish and European rabbits-actin sequence, homology is respectively 93%, 88% and 88%.The β of these 4 kinds of biologies of monoclonal anti physical efficiency specific recognition of 2B4 secretion-actin albumen.The McAb of 2B4 secretion has preferably wide spectrum suitability, can be widely used as the confidential reference items antibody of the 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, and prolonged preservation can not make antibody titer reduce.Multinomial data show that the McAb of 2B4 secretion can be widely used in cytobiology and the immunological testing of multiple biology and tissue, has applications well prospect and commercial value.
＜110〉Hunan Normal University
＜120〉a kind of hybridoma of secreting anti-human β-actin protein monoclonal antibody and preparation method thereof
<160> 2
<210> 1
<211> 32
<212> DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the sequence of synthetic
<400> 1
CATATG GCTA CATATGGATG ATGATATCGC CG 32
<210> 2
<211> 33
<212> DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the sequence of synthetic
<400> 2
GAATTC ACAG AATTCCTAGA AGCATTTGCG GTG 33
Claims (2)
- One kind the secretion anti-human 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 application of hybridoma claimed in claim 1 in preparation anti-human β-actin protein monoclonal antibody.
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| CN 201210108863 CN102690787B (en) | 2012-04-13 | 2012-04-13 | Hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody and preparation method thereof |
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| CN 201210108863 CN102690787B (en) | 2012-04-13 | 2012-04-13 | Hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody and preparation method thereof |
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| CN102690787A CN102690787A (en) | 2012-09-26 |
| CN102690787B true CN102690787B (en) | 2013-03-13 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106405085B (en) * | 2016-08-31 | 2018-03-27 | 天津市泌尿外科研究所 | For detecting the ELISA kit and application method of castration-resistant prostate cancer |
| CN108517011B (en) * | 2018-04-26 | 2020-08-25 | 成都百奥克林生物科技有限公司 | Nano antibody aiming at cytoskeleton protein beta-actin and coding sequence thereof |
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1294194A (en) * | 1999-10-26 | 2001-05-09 | 上海博道基因技术有限公司 | Actin-binding protein and polynucleotide sequence for coding it |
| CN1302879A (en) * | 1999-10-28 | 2001-07-11 | 上海博容基因开发有限公司 | Polypeptide-human F-actin binding factor 75 and polynucleotide for coding it |
| CN1318552A (en) * | 2000-04-19 | 2001-10-24 | 上海生元基因开发有限公司 | New actin and polynucleotide sequence for encoding it |
| CN102206253A (en) * | 2010-10-25 | 2011-10-05 | 深圳华大基因科技有限公司 | Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof |
-
2012
- 2012-04-13 CN CN 201210108863 patent/CN102690787B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1294194A (en) * | 1999-10-26 | 2001-05-09 | 上海博道基因技术有限公司 | Actin-binding protein and polynucleotide sequence for coding it |
| CN1302879A (en) * | 1999-10-28 | 2001-07-11 | 上海博容基因开发有限公司 | Polypeptide-human F-actin binding factor 75 and polynucleotide for coding it |
| CN1318552A (en) * | 2000-04-19 | 2001-10-24 | 上海生元基因开发有限公司 | New actin and polynucleotide sequence for encoding it |
| CN102206253A (en) * | 2010-10-25 | 2011-10-05 | 深圳华大基因科技有限公司 | Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 人β-actin蛋白原核表达及其单克隆抗体制备;吴俊文;《激光生物学报》;20120630(第3期);238-244 * |
| 吴俊文.人β-actin蛋白原核表达及其单克隆抗体制备.《激光生物学报》.2012,(第3期),238-244. |
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| CN102690787A (en) | 2012-09-26 |
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