CN104164407A - Anti(pig erythrocyte CR1-like) monoclonal antibody and preparation thereof - Google Patents
Anti(pig erythrocyte CR1-like) monoclonal antibody and preparation thereof Download PDFInfo
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Abstract
The invention discloses an anti(pig erythrocyte CR1-like) monoclonal antibody and preparation thereof. The invention discloses a hybridoma cell strain CR-M01 with an accession number of CCTCC NO:C201475. The hybridoma cell strain can secrete the anti(pig erythrocyte CR1-like) monoclonal antibody capable of specific recognition of the pig erythrocyte CR1-like. The monoclonal antibody can specifically recognize CR1-like protein on a pig erythrocyte, and specifically interdict immune adherence of the pig erythrocyte to sensitizing latex particles, and has high application value in function research of the pig erythrocyte CR1-like.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of monoclonal antibody, be specifically related to a kind of anti-swine erythrocyte CR1-like monoclonal antibody, and the preparation of this monoclonal antibody, the invention still further relates to the hybridoma cell strain of secreting this anti-swine erythrocyte CR1-like monoclonal antibody.
Background technology
From American scholar Siegel etc. proposed " erythrocyte immune system (Red cell immune system; RCIS) " concept in 1981 since, people recognize that red corpuscle not only has the respiratory function that carries oxygen in blood and the pooling feature that regulates pH value of blood gradually, also there is immunologic function, and comprise complete self regulating and control system.At present, in physianthropy field, the research of relevant erythrocyte immune is very deep, red corpuscle wide participation specificity in vivo and non-specific immunity, immunity of organism regulate and control, and affect generation, the development of various diseases, and CR1 is the most important basic substance of HRBC immunologic function.
People CR1 is a kind of strand glycoprotein that is present in the relative molecular mass 205kDa of red corpuscle, lymphocyte, leukocyte surface, and its quantity on each red corpuscle is 950, to be dispersed in and to collect bunch distributions on cytolemma.In HRBC, have 50% CR1 to be collection bunch distribution, and on other cells, the collection of a CR1 bunch distributive law is less than 15%, this collection bunch distribution mode of red blood cell cr1 can make the binding site of the coated immunocomplex of it and C3b be polyvalency, and connection is more firm.Because CR1 most in blood circulation is present on erythrocyte membrane, than the large 500-1000 of white corpuscle doubly, therefore, with the erythrocyte immune on CR1 basis, stick is the most important immunologic function of HRBC to the probability that red corpuscle is removed blood circulation immunocomplex.When HRBC is restricted the combination of immunocomplex, immunocomplex will be deposited in sensitive organization, causes the generation of numerous disease.
Current studies confirm that, multiple Mammals, rodent, bird red corpuscle have immune function, but do not confirm that CR1 is the unique immune adherence receptor of animal erythrocyte, there are no the research report of this immune adherence of specific inhibition, from animal erythrocyte membrane, do not obtain adhesion receptor and proved its protein properties yet.Pig is as domestic animal and physianthropy and the most frequently used animal pattern of biomedical engineering field of China's number of animals raised maximum, research to swine erythrocyte CR1-like function, to likely improve and improve the present situation of pig cultivation and disease treatment, and offer reference for the research of other animal erythrocyte immune adherences, also by carrying out for the mankind, take disease treatment and the medicament research and development that CR1 is target spot Research foundation is provided.
Because research lacks effective test instrument, the particularly specific antibody of anti-swine erythrocyte CR1-like to swine erythrocyte CR1-like, make swine erythrocyte CR1-like progress comparatively slow.Therefore, provide the monoclonal antibody of a kind of anti-swine erythrocyte CR1-like, significant to further research swine erythrocyte CR1-like.
Summary of the invention
The object of this invention is to provide a kind of hybridoma cell strain of secreting the monoclonal antibody of specific recognition swine erythrocyte CR1-like, and the anti-swine erythrocyte CR1-like monoclonal antibody of above-mentioned hybridoma cell strain secretion.
The invention provides the hybridoma cell strain of the anti-swine erythrocyte CR1-like of strain monoclonal antibody, described hybridoma cell strain called after CR-M01, on June 6th, 2014, be preserved in Chinese Typical Representative culture collection center (CCTCC), address: Wuchang, Wuhan, China city Luo Jia Wuhan University, postcode 430072.Preserving number is CCTCC NO:C201475.
Hybridoma cell strain of the present invention is by hybridoma technology, and the polypeptide that synthesizes of usining is set up as immunogen.
The present invention also provides the anti-swine erythrocyte CR1-like monoclonal antibody by described hybridoma cell strain CR-M01 secretion, and the hypotype of described monoclonal antibody is IgG1.
After described monoclonal antibody purifying, through western blot and immunofluorescence dyeing, prove that it can specific recognition swine erythrocyte CR1-like albumen.
And then, the immune adherence effect that anti-swine erythrocyte CR1-like monoclonal antibody of the present invention can specific blocking-up swine erythrocyte.
The preparation method of anti-swine erythrocyte CR1-like monoclonal antibody provided by the invention comprises the steps:
1 antigen preparation
Swine erythrocyte CR1-like gene to GenBank login is analyzed, and chooses CDS district and translates corresponding protein sequence, and analysing protein sequence secondary structure, obtains following antigen site pep:NSFWSIPEGNCKRKT.By the synthetic polypeptide of antigen site, polypeptide is coupling KLH and BSA respectively, and wherein KLH-pep is for mouse immune, and BSA-pep screened for the ELISA later stage.
2 animal immunes
With KLH-pep immunity Balb/c mouse, ELISA detects the mouse that serum titer is greater than 10000, merges within first 3 days, with immunogen 50 μ g/, only to measure abdominal injection mouse to be merged and carry out booster immunization.
3 hybridoma preparations
Prepare immune spleen cell, impact fusion with the myeloma cell who cultivates, adopt HAT selective medium to filter out hybridoma, ELISA filters out the positive hybridoma cell that can produce required monoclonal antibody, clonal expansion is frozen, the monoclonal antibody in hybridoma supernatant is carried out to hypotype evaluation simultaneously.
4 monoclonal antibody preparations
Get Balb/c mouse, inoculation hybridoma in abdominal injection 0.5ml white oil 1-2 week pneumoretroperitoneum, mouse web portion expands and with syringe, extracts ascites afterwards, use Protein-A affinity purification, detection antibody purification purity, concentration and avidity.With Western Blot and immunofluorescence dyeing, prove the specificity of prepared monoclonal antibody.
The hybridoma cell strain CR-M01 that the present invention is prepared and the monoclonal antibody of secretion thereof the CR1-like albumen on can specific identification swine erythrocyte, the monoclonal antibody that obtains can the immune adherence of specific blocking-up swine erythrocyte to sensitization latex particle, in the functional study of swine erythrocyte CR1-like, there is very high using value.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE of anti-swine erythrocyte CR1-like monoclonal antibody.
Fig. 2 is the specific binding figure that Westem Blot analyzes anti-swine erythrocyte CR1-like monoclonal antibody and antigen.
Fig. 3 is the specific binding (1000 times of microscope magnifications) of immuning fluorescent dyeing analysis anti-swine erythrocyte CR1-like monoclonal antibody and antigen.The wherein negative contrast of A, B is for adding monoclonal antibody.
Fig. 4 is the immune adherence effect (1000 times of microscope magnifications) of anti-swine erythrocyte CR1-like monoclonal antibody blocking-up swine erythrocyte, and wherein A adds PBS, and B adds homotype antibody, and C adds monoclonal antibody.
Embodiment
The acquisition of embodiment 1, hybridoma cell strain CR-M01
The preparation of 1 antigen
Swine erythrocyte CR1-like gene (Gene ID:1640741) to GenBank login is analyzed, choose CDS district, utilize Translate(http: //web.expasy.org/translate/) translate corresponding protein sequence, the secondary structure of Geneious software analysis protein, hydrophilic and hydrophobic, antigenicity, cross-film district etc., selection secondary protein structure elasticity is large, wetting ability is strong, antigenicity is strong, the position in non-cross-film district; On NCBI, carry out and infraspecific homology analysis, avoid the region of homology, the following antigen site Pep:NSFWSIPEGNCKRKT of final acquisition simultaneously.
Adopt the polypeptide automatic DNA synthesizer DNA of American AB I company, with the antigen site sequence obtaining, adopt the synthetic polypeptide of solid-phase synthesis, synthesis program is according to ABI operational guidance and Fmoc chemosynthesis step.Process condensation → wash → go the operations such as protection → neutralization washing → next round condensation, until complete desired sequence, is used Kaiser method to becoming condensation reaction degree in peptide process to carry out qualitative monitoring.Utilize HPLC to carry out purifying to synthetic polypeptide, LCMS detects polypeptide purity ,-80 ℃ of preservations.
By synthetic polypeptide respectively with sulfo-GMBS method coupling for KLH and BSA.Weigh polypeptide, with 0.05M PBS, dissolve, final concentration is 6mg/ml, according to demand the good polypeptide of packing.KLH and BSA are dissolved to activation with sulfo-GMBS, " carrier proteins-sulfo-GMBS " solution having activated is added drop-wise in aforementioned polypeptides solution, with vertical vortex mixer, at room temperature rotate and mix 3h(or 4 ℃ of rotations are spent the night).With DTNB analytic liquid, detect the variation of coupling front and back free sulfhydryl group absorbance value, OD before coupling
413oD after-coupling
413> 2.0, and coupling success is described, otherwise failure.According to the dense amount of carrier proteins after activation, determine the concentration of " carrier proteins-polypeptide " after coupling.KLH-pep is for mouse immune, and BSA-pep screened for the ELISA later stage.
2 animal immunes
KLH-pep is pressed to the amount of a 60 μ g/ mouse, 4 SPF Balb/c female mices of the subcutaneous initial immunity of difference, immunity interval 14d, after just exempting from, strengthen 4 times, the each dosage 30 μ g/ of booster immunization, after the 4th is strengthened, eye socket blood sampling separation of serum, adopts indirect elisa method to measure immune serum and tires.
Get 20 μ g BSA-pep 1/2/3 polypeptide and be dissolved in 10ml 0.05M pH9.6 carbonate buffer solution, coated micro-96 orifice plates of polystyrene, 100 μ l/ holes, 4 ℃ are spent the night.With containing 0.05% Tween-20 PBS, wash plate 3 times, with 37 ℃ of sealing 2h of 1%BSA confining liquid, use 0.05% Tween-20 PBS to wash plate 3 times, by mouse immune serum since 200 times of 2 times of gradient dilutions, get 100 μ l and add 96 orifice plates, hatch 1h for 37 ℃, 0.05% Tween-20 PBS washes after plate 3 times, add 1000 times of dilution horseradish peroxidase-labeled goat anti-mouse iggs of 100 μ l 1 ﹕, hatch 1h for 37 ℃, 0.05% Tween-20 PBS washes after plate 3 times, add 100 μ l TMB colour developings, room temperature lucifuge 15min, add 50 μ l 1M HCl termination reactions, survey 450nm absorption value, the front mice serum of the immunity of usining is as negative control, so that ratio >=2.1 of measured value and control value are positive, judge that immune serum tires.
Get the mouse that serum titer is greater than 10000, merge first 3 days, with immunogen 50 μ g/ abdominal injection mouse to be merged, carry out booster immunization.
3 cytogamy
By soft the laying from culturing bottle wall blowing up of SP2/0 cell in good condition, be drawn in 50ml centrifuge tube; Mouse is plucked eyeball and gets blood, draws neck to put to death, and puts into 75% alcohol and soaks 5min; In plate, pour a small amount of serum-free IMDM into, cell sieve and plunger are put into plate, with scissors and tweezers, take off mouse spleen and be put into cell sieve above, with plunger, fully pulverize gently, the cell having ground is drawn in the centrifuge tube that SP2/0 is housed, the centrifugal 5min of 1500rad/min.With scissors and tweezers, take off mouse thymus, pulverize, the thymocyte having ground joins in 15ml centrifuge tube, then adds 1ml HAT, is placed in incubator standby.Centrifugal good cell is outwelled to supernatant, with serum-free IMDM, cell is carefully gently blown even, centrifugal (1500rad/min, 5min), abandon supernatant, abundant suspension cell at the bottom of beating centrifuge tube, centrifuge tube is put into 37 ℃ of warm water, in 1min, slowly add 1ml PEG, after adding, standing 1min in warm water.Then the serum-free IMDM that slowly adds 2ml in 2min then slowly adds the serum-free IMDM of 8ml in 2min.The centrifugal 5min of 1000rad/min, abandons supernatant, adds 10ml serum, carefully cell is blown evenly, pours ready thymocyte above into, then adds 25ml sterilizing semisolid medium (containing HAT), fully mixes.Evenly pour in 30 Tissue Culture Dishs, Tissue Culture Dish is put into wet box and in incubator, cultivate.
4 screening monoclonal cells
Indirect elisa method screening cells and supernatant, the positive colony hybridoma that selection is tired higher carries out subcloning, and with the continuous cloning of limiting dilution assay 2～3 times, until 100% cell positive rate, finally obtain hybridoma cell strain 1 strain of the anti-swine erythrocyte CR1-like of stably excreting monoclonal antibody, called after CR-M01.
Positive rate after cloning is reached to 100% the rear liquid nitrogen cryopreservation of cell amplification cultivation.Adopt ELISA to identify the hypotype of the secreted monoclonal antibody of positive hybridoma cell strain, the hypotype of monoclonal antibody is IgG1.
The preparation of embodiment 2, monoclonal antibody
The preparation of 1 antibody
Get Balb/c mouse, abdominal injection 0.5ml whiteruss or pristane pre-treatment 1～2 week, afterwards every mouse peritoneal inoculation 10
6individual hybridoma.Hybridoma is in mouse peritoneal internal breeding, and generation and secrete monoclonal antibody, and approximately 1～2 week, visible mouse web portion expanded.With syringe, extract ascites, can obtain a large amount of monoclonal antibodies.
The purifying of 2 antibody
With Protein A/G affinity column (Pierce Protein A/G Magnetic Beads, article No.: thermo 88802) antibody purification.Purification step proceeds as follows according to practical illustration book: with coupling buffer, with 1 ﹕ 3, dilute ascites, and trim, 4 ℃ of centrifugal 20min of 10000rpm, 0.22 μ m membrane filtration, except degrease, cell residue and finely ground particle substance.With the coupling buffer balance prepackage affinity column of 5～10 times of column volumes, keeping flow velocity is 4/s.With syringe, sample is injected to end interface on pillar, collect effluent liquid in 50ml centrifuge tube, keeping flow velocity is that 5s/ drips.With the coupling buffer of 5 times of column volumes, cross post, keeping flow velocity is 4/s.Near the required neutralizer of correspondence, get several values, add EP pipe; Drip elutriant to 1.0ml, mix rear detection pH value.The amount of adjusting neutralizer according to result, making neutralization results pH is 7.0.Get 5 EP pipes, according to neutralizer debug results, add respectively according to quantity neutralizer to maintain antibody biological activity, avoid antibody inactivation.With 5 times of column volume elution buffer wash-out antibody, be collected in above-mentioned EP pipe.With 5 times of column volume pH2.7 elution buffers, cross post, keeping flow velocity is 4/s.With 5～10 column volume G column coupling damping fluid balance pillars, to pH7.0, keeping flow velocity is 4/s.
The CHARACTERISTICS IDENTIFICATION of embodiment 3, monoclonal antibody
The purity testing of 1 antibody
The purity of measuring monoclonal antibody with SDS-PAGE, purity reaches more than 90%, as shown in Figure 1.
The concentration determination of 2 antibody
The concentration that adopts the BCA determination of protein concentration kit measurement monoclonal antibody of Beyotime company, the antibody concentration of hypotype IgG1 is 3.12mg/ml.
The titration of 3 antibody
Through ELISA, measure, the antibody titer of hypotype IgG1 is 10
8.
The Westem Blot of 4 antibody identifies
Extract the membranin of swine erythrocyte, after 12% SDS-PAGE electrophoresis, electrotransfer is to pvdf membrane, under 5% skim-milk room temperature, seal 2h, add 1 ﹕ 1000 dilution monoclonal antibodies, 4 ℃ of overnight incubation, the Tris-HCl damping fluid (pH7.4) of 0.1% Tween-20 is washed film 3 times, each 10min, the sheep anti-mouse igg that adds the horseradish peroxidase-labeled of 1 ﹕ 10000 dilutions, hatch 1h for 37 ℃, the Tris-HCl damping fluid (pH7.4) of 0.1% Tween-20 is washed film 3 times, each 10min, ECL luminescence reagent box colour developing post-exposure.There is single specific band in anti-swine erythrocyte CR1-like monoclonal antibody prepared by hybridoma, result as shown in Figure 2.
The identified by immunofluorescence of 5 antibody
Gather pig fresh blood, add isopyknic PBS, then blood is added to isopyknic lymphocyte separation medium upper strata, the centrifugal 15min of 1500rpm.With red corpuscle at the bottom of suction pipe suction pipe, add in cell washing lotion for 3～4ml (PBS, pH7.4, containing 2% foetal calf serum), the centrifugal 5min of 1500rpm, then wash 2 times by cell washing lotion.By tally counting, simultaneously observation of cell form after 100 times of dilutions of cell.Red corpuscle is diluted to 1 * 10
6individual/ml, adds the anti-swine erythrocyte CR1-like monoclonal antibody (1 ﹕ 100 dilution) of preparation, hatches 1h for 37 ℃, with PBS(pH7.4) negative contrast.PBS(pH7.4) after washed cell, add FITC mark sheep anti mouse two anti-(1 ﹕ 1000), hatch 1h for 37 ℃, PBS(pH7.4) after washed cell, fluorescence microscopy Microscopic observation, the cell observation dyeing through anti-swine erythrocyte CR1-like monoclonal antibody is to green fluorescence, as shown in Figure 3.Anti-swine erythrocyte CR1-like monoclonal antibody prepared by result proof hybridoma can be identified natural swine erythrocyte CR1-like albumen.
Embodiment 4, the immune adherence of monoclonal antibody blocking-up swine erythrocyte to latex particle
1 material and reagent
Latex particle (Invitrogen), BSA-Alexa Fluor 488(Invitrogen), coupling buffer(0.1M MES) and, activate buffer(10mg/ml EDAC), containing the coupling buffer of 2%BSA, 2.5mM EDTA solution, 30mM CaCl
2100mM MgCl
2solution.
2 steps
Get latex particle (Beads) stoste 10 μ l, resuspended with coupling buffer is 1ml.Resuspended Beads and 5 μ l BSA-Alexa Fluor 488 are mixed, and adjusting immediately pH is 6.5.The activation buffer 50 μ l that get pH6.5, mix with above-mentioned suspension, under room temperature condition, and darkroom reaction 15min, the centrifugal 5min of 10000rpm, abandons supernatant.With the coupling buffer containing 2% BSA, wash latex particle 2 times, the resuspended beads of PBS.In suspension, add appropriate human serum (containing Ca, Mg ion), making serum final concentration is 20%, and control serum adds enough EDTA, be placed in 37 ℃ of water-baths, sensitivity response 10min, adds enough EDTA to stop complementary reaction after reaction finishes simultaneously, the centrifugal 5min of 10000rpm, abandons supernatant.With the coupling buffer containing 2%BSA, wash latex particle 2 times, the resuspended beads of PBS.The preincubate 15min in 37 ℃ by swine erythrocyte and anti-swine erythrocyte CR1-like monoclonal antibody, the centrifugal 5min of 1200rpm, abandons supernatant, and PBS washed twice is also resuspended.Above-mentioned resuspended red corpuscle is reacted to 15min with sensitization beads darkroom, and to add PBS and to add homotype antibody respectively in contrast, the centrifugal 5min of 1200rpm, abandons supernatant, PBS washing three times, resuspended rear fluorescence microscope.
As shown in Figure 4, anti-swine erythrocyte CR1-like monoclonal antibody can the immune adherence effect of specific blocking-up swine erythrocyte to sensitization latex particle for result.
Claims (4)
1. anti-swine erythrocyte CR1-like monoclonal antibody hybridoma cell strain CR-M01, preserving number CCTCC NO:C201475.
2. by the monoclonal antibody of the hybridoma cell strain CR-M01 secretion of preserving number CCTCC NO:C201475.
Described in claim 2 monoclonal antibody as the application of identification swine erythrocyte CR1-like detection reagent.
Described in claim 2 monoclonal antibody as the application of blocking-up swine erythrocyte CR1-like immune adherence reagent.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107056944A (en) * | 2017-01-20 | 2017-08-18 | 新乡学院 | The anti-monoclonal antibodies of pig PD 1 of one plant of mouse and its application |
| CN108359007A (en) * | 2018-02-11 | 2018-08-03 | 山西农业大学 | Swine erythrocyte ECR1-like film combinations peptide fragment, polyclonal antibody, preparation method and application |
| CN113533134A (en) * | 2021-08-04 | 2021-10-22 | 山西农业大学 | Circulating detection system and method for detecting pig red blood cell immunoadhesion function |
| CN114703146A (en) * | 2022-01-19 | 2022-07-05 | 山西农业大学 | Hybridoma cell strain and application thereof |
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2014
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107056944A (en) * | 2017-01-20 | 2017-08-18 | 新乡学院 | The anti-monoclonal antibodies of pig PD 1 of one plant of mouse and its application |
| CN107056944B (en) * | 2017-01-20 | 2020-05-29 | 新乡学院 | Mouse anti-pig PD-1 monoclonal antibody and application thereof |
| CN108359007A (en) * | 2018-02-11 | 2018-08-03 | 山西农业大学 | Swine erythrocyte ECR1-like film combinations peptide fragment, polyclonal antibody, preparation method and application |
| CN108359007B (en) * | 2018-02-11 | 2021-11-23 | 山西农业大学 | Pig red blood cell ECR1-like membrane-bound peptide fragment, polyclonal antibody, preparation method and application |
| CN113533134A (en) * | 2021-08-04 | 2021-10-22 | 山西农业大学 | Circulating detection system and method for detecting pig red blood cell immunoadhesion function |
| CN114703146A (en) * | 2022-01-19 | 2022-07-05 | 山西农业大学 | Hybridoma cell strain and application thereof |
| CN114703146B (en) * | 2022-01-19 | 2023-03-10 | 山西农业大学 | Hybridoma cell strain and application thereof |
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