CN103288955B - Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof - Google Patents
Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and the monoclonal antibody is secreted by hybridoma cell of which the name is hybridoma cell strain Crab, the preservation number is CCTCC NO: C2012196, the preservation unit is China Typical Culture Preservation Center, and the preservation data is Jan 7th, 2013. The monoclonal antibody has a characteristic of being specifically combined with blue crab particle hemocyte 26.7kDa protein. The preparation method comprises the steps of: obtaining particle hemocyte by continuous density gradient centrifugation of Percoll medium; purifying the particle hemocyte 26.7kDa protein as antigen to immunize mice by electrophoresis rubber cutting and recycling; preparing hybridoma cells by a cell fusion method; screening out the monoclonal antibody of the anti-blue crab particle hemocyte 26.7kDa protein by an immunological detection method. The monoclonal antibody disclosed by the invention provides an important tool for researching functions of particle hemocyte in nonspecific immune response of blue crab and in other physiological function aspects.
Description
Technical field
The present invention relates to monoclonal antibody of a kind of anti-Portunus trituberculatus Miers of being secreted by hybridoma (Neptunus trituberculatus) granular hemocyte 26.7kDa albumen and preparation method thereof, belong to crab class cellular immunology technical field.
Background technology
Crab class does not have immunoglobulin (Ig), lacks antibody-mediated immunity system, is non-specific immunity, and its immune defense reaction mainly depends on humoral factor in hemocyte and hemolymph and completes.Hemocyte by engulfing, packing, aggegation, tubercle formation, exocytosis, reparation etc. complete cellular immunization process; by synthetic release immune-regulating factor, as hemolysin, phenol oxidase etc., assistance completes body fluid immunologic process simultaneously, and hemocyte plays decisive role in crab class immunity of organism protection process.At present according to the form of cell, size and intracytoplasmic particle situation, crab class hemocyte is divided into two large classes: transparent hemocyte and granular hemocyte.In transparent hemocyte kytoplasm, do not contain particle or contain less particle, organoid rareness, nuclear-cytoplasmic ratio is large; Granular hemocyte kytoplasm includes more particle, organelles, and nuclear-cytoplasmic ratio is little.Studies have shown that in a large number transparent hemocyte and granular hemocyte all play a significant role in body immune system, but function difference, wherein transparent hemocyte mainly participates in the agglutination reaction of hemolymph, and granular hemocyte contains phenol oxidase and lytic enzyme, main participation engulfing and packing external foreign matter.But, there is no clear and definite conclusion for the generating process in exact function, the fetal development of this two classes hemocyte and the aspect such as mechanism that participates in immunity of organism regulation and control, major cause is to show its generation and participate in immunoreactive process without suitable molecular marked compound.Monoclonal antibody due to have high specificity, highly sensitive, without features such as intercrossing, in location, generation, the differentiation of higher animal immunocyte are studied, application is more extensive, the now research report of the existing monoclonal antibody for Hemocytes In Chinese Shrimp Penaeus Chinensis, chlamys ferreri blood cells etc., is mainly used in identification, the qualification of blood lymphocyte, the research of neurogenesis and development aspect.Therefore, the relation between further research granular hemocyte and other type hemocyte that is prepared as of Portunus trituberculatus Miers granular hemocyte protein monoclonal antibody, the distribution of granular hemocyte, generation, differentiation and immunologic function etc. in embryo development procedure provide new instrument.
Summary of the invention
One of the object of the invention is to provide a kind of monoclonal antibody of the anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa albumen of being secreted by hybridoma.
Another object of the present invention is to provide the preparation method of above-mentioned anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa protein monoclonal antibody.
The object of the invention is to be realized by following technical scheme:
A kind of monoclonal antibody of the anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa albumen of being secreted by hybridoma, it is characterized in that described monoclonal antibody is to be called by name: hybridoma cell strain Crab, preserving number is: CCTCC NO:C2012196, depositary institution is: Chinese Typical Representative culture collection center, preservation date is: the hybridoma secretion of on 01 07th, 2013.Under inverted microscope, observe this hybridoma size homogeneous that growth conditions is good, outward appearance is full, individuality is perfectly round, refractivity is strong, adherent well; This hybridoma has unlimited division growth ability; This hybridoma of cellar culture 2～3 days, its nutrient solution color changes yellow into by pink, contains the monoclonal antibody of the anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa albumen of this hybridoma secretion in this nutrient solution.
This monoclonal anti physical efficiency and Portunus trituberculatus Miers granular hemocyte generation specific binding, and with other histocyte no cross reaction, be positioned on the albumen that Portunus trituberculatus Miers granular hemocyte molecular weight is 26.7kDa with the antigenic determinant of described monoclonal antibody generation specific binding.
A preparation method for described anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa protein monoclonal antibody, its step is as follows: extract Haemolymph in Crab Portunus trituberculatus, the centrifugal complete blood cell that obtains; Complete blood cell obtains Portunus trituberculatus Miers granular hemocyte through Percoll medium continuous density gradient partition method; Carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis taking Portunus trituberculatus Miers granular hemocyte as sample, 26.7kDa protein band is carried out to albumen purification; Using purify Portunus trituberculatus Miers granular hemocyte 26.7kDa albumen as antigen, immune Balb/C mouse; Getting immune mouse spleen cell and SP2/0 myeloma cell merges; The strain of indirect immunofluorescence antibody method screening positive hybridoma cell, to positive hybridoma cell, strain is cloned, and finally screening obtains the hybridoma cell strain Crab of the anti-Portunus trituberculatus Miers granular hemocyte of strain energy stably excreting 26.7kDa protein monoclonal antibody; The antibody of its secretion is that the monoclonal antibody of anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa albumen (is called for short: monoclonal antibody Crab), verify its monoclonal antibody characteristic through streaming immunofluorescence technique and transfer printing immunoblotting.
Described Percoll medium continuous density gradient partition method is the top layer that Portunus trituberculatus Miers complete blood cell suspension is laid on to Percoll medium continuous density gradient, and top-down the 3rd confluent monolayer cells band of centrifugation gained is granular hemocyte band.
Described indirect immunofluorescence antibody method is that fusion Hybridoma Cell Culture supernatant liquor is reacted with Portunus trituberculatus Miers complete blood cell drop of blood sheet, transparent hemocyte drop of blood sheet and granular hemocyte drop of blood sheet respectively, then under lucifuge condition, add fluorescein isothiocyanate (FITC) mark goat anti-mouse igg antibody, fluorescence microscopy Microscopic observation, screening particle-resistant hemocyte monoclonal antibody, i.e. monoclonal antibody Crab.The indirect immunofluorescence antibody method reaction result that wherein drips sheet in conjunction with these three kinds of hemocytes of Portunus trituberculatus Miers complete blood cell, transparent hemocyte and granular hemocyte is comprehensively determined: this monoclonal antibody only reacts with Portunus trituberculatus Miers granular hemocyte generation specific binding.
Described Immunological Identification method is streaming immunofluorescence technique and transfer printing immunoblotting, streaming immunofluorescence technique is that the monoclonal antibody of preparation is reacted with the transparent blood cell suspension of Portunus trituberculatus Miers, granular hemocyte suspension respectively, then under lucifuge condition, add fluorescein isothiocyanate (FITC) mark goat anti-mouse igg antibody, monoclonal antibody prepared by flow cytometer detection validation is the monoclonal antibody of anti-Portunus trituberculatus Miers granular hemocyte; Transfer printing Diagnosis of Sghistosomiasis notation is that gel protein is carried out to electrotransfer to nitrocellulose filter, nitrocellulose filter is immersed in the monoclonal antibody of preparation, then immersed in the goat anti-mouse igg antibody of alkali phosphatase enzyme mark, band is observed in the colour developing of color development liquid, determines the molecular weight of antigenic determinant.Wherein the transparent blood cell suspension streaming of Portunus trituberculatus Miers immunofluorescence, granular hemocyte suspension streaming immunofluorescence result can determine that in conjunction with transfer printing immunoblotting result monoclonal antibody Crab only reacts with Portunus trituberculatus Miers granular hemocyte generation specific binding, and the antigenic determinant of this monoclonal antibody is only positioned on the Portunus trituberculatus Miers granular hemocyte albumen that molecular weight is 26.7kDa.
The invention has the advantages that: technology of preparing route of the present invention is to obtain Portunus trituberculatus Miers granular hemocyte by Percoll medium continuous density gradient partition method, cut glue through electrophoresis and reclaim purification granular hemocyte 26.7kDa albumen as antigen immune mouse, adopt cell fusion method to prepare hybridoma, filter out anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa protein monoclonal antibody through immunological detection method, then adopt Immunological Identification method to identify its characteristic, this technological line is reasonable in design, tight and feasible, give full play to the effect of panimmunity detection process for screening and identifying.The present invention has adopted indirect immunofluorescence antibody method screening hybridoma cell strain, indirect immunofluorescence antibody method utilizes fluorescence antibody as second antibody, reaction quick and precisely, result judgement is directly perceived, can effectively eliminate the false positive results that enzyme-linked immunosorbent assay and immunoenzyme etc. occur due to the existence of endogenous enzyme, and the present invention has utilized respectively complete blood cell in the time utilizing indirect immunofluorescence antibody method screening positive hybridoma cell, transparent hemocyte and granular hemocyte, so make to confirm that monoclonal antibody Crab only reacts necessary being with the specific binding that granular hemocyte occurs.Simultaneously, because the present invention has adopted streaming immunofluorescence technique, it can be fast, accurately, objectively special group feature is analyzed, and uncertainty and the experimental artifact that can avoid artificial in experimentation or subjective factor etc. to cause, in addition utilize fluorescence to do probe mark antibody, can quantitatively obtain the component proportions about antigen antibody reaction, the method shows that Portunus trituberculatus Miers granular hemocyte Fluorescence Ratio is 86.3%, and this result further confirms that monoclonal antibody Crab and granular hemocyte specific binding have occurred and reacted.The present invention the particularly important is and adopts transfer printing immunoblotting to confirm that the antigenic determinant of monoclonal antibody Crab combination is positioned on the albumen that Portunus trituberculatus Miers granular hemocyte molecular weight is 26.7kDa, and this antigenic determinant is linear antigenic determinant.Comprehensive indirect immunofluorescence antibody result of the present invention, streaming immunofluorescence result and transfer printing immunoblotting result are known to be only positioned on Portunus trituberculatus Miers granular hemocyte albumen with the linear antigenic determinant of monoclonal antibody Crab generation specific binding, and the molecular weight of this albumen is 26.7kDa, this achievement is providing important tool for studying granular hemocyte aspect Portunus trituberculatus Miers nonspecific immune response and other physiological function, for further studying the relation between granular hemocyte and other type hemocyte, the distribution of granular hemocyte, generation in embryo development procedure, differentiation and immunologic function etc. are laid a good foundation, in addition, monoclonal antibody Crab understands fully the location of this antigenic determinant in different Crustacean bodies or in cell, expression provides important technique means.
Brief description of the drawings
Fig. 1 is the result figure of the three confluent monolayer cells bands through Percoll medium continuous density gradient partition method gained of the present invention and the corresponding Giemsa staining of each confluent monolayer cells suspension.
Fig. 2 is the result figure that monoclonal antibody indirect immunofluorescence antibody method of the present invention detects.
Fig. 3 is the result figure of monoclonal antibody streaming immuno-fluorescence assay of the present invention.
Fig. 4 is the result figure that monoclonal antibody transfer printing immunoblotting of the present invention detects.
Shown in Fig. 1: A, B, is respectively top-down the first layer after the centrifugation of Percoll medium continuous density gradient shown in arrow in C, the second layer, the 3rd confluent monolayer cells band; A, b, c is respectively the first layer, the second layer, the Giemsa staining result of the drop of blood sheet that the 3rd confluent monolayer cells band suspension is made.Wherein H represents transparent hemocyte; G represents granular hemocyte.
Shown in Fig. 2: A is the result that after Portunus trituberculatus Miers complete blood cell drop of blood sheet reacts with monoclonal antibody Crab, fluorescent microscope detects; B is the result that after Portunus trituberculatus Miers granular hemocyte drop of blood sheet reacts with monoclonal antibody Crab, fluorescent microscope detects; C is the result that after the transparent hemocyte drop of blood of Portunus trituberculatus Miers sheet reacts with monoclonal antibody Crab, fluorescent microscope detects; D is the result (negative control) that after Portunus trituberculatus Miers complete blood cell drop of blood sheet reacts with myeloma cell's culture supernatant, fluorescent microscope detects.Wherein G represents granular hemocyte.
Shown in Fig. 3: a is the scatter diagram (negative control) through the transparent blood cell suspension flow cytometer detection of Portunus trituberculatus Miers of myeloma cell's culture supernatant reaction; B is the scatter diagram (negative control) through the Portunus trituberculatus Miers granular hemocyte suspension flow cytometer detection of myeloma cell's culture supernatant reaction; C is respectively through the transparent blood cell suspension of Portunus trituberculatus Miers of myeloma cell's culture supernatant reaction and the histogram (superposeing) (negative control) of granular hemocyte suspension flow cytometer detection; A is the scatter diagram through the transparent blood cell suspension flow cytometer detection of Portunus trituberculatus Miers of monoclonal antibody Crab reaction; B is the scatter diagram through the Portunus trituberculatus Miers granular hemocyte suspension flow cytometer detection of monoclonal antibody Crab reaction; C is respectively through the transparent blood cell suspension of Portunus trituberculatus Miers of monoclonal antibody Crab reaction and the histogram (superposeing) of granular hemocyte suspension flow cytometer detection; Wherein H represents transparent hemocyte, and G represents granular hemocyte; N is negative; P is positive; G=86.3% represents the Fluorescence Ratio of granular hemocyte.
Shown in Fig. 4: M is the ponceau dye liquor coloration result after standard molecular weight protein electrophoresis, transfer printing; A is the Xylene Brilliant Cyanine G dye liquor coloration result after Portunus trituberculatus Miers granular hemocyte electrophoresis; B is the Xylene Brilliant Cyanine G dye liquor coloration result after the Portunus trituberculatus Miers granular hemocyte protein electrophoresis of purifying; C is the granular hemocyte reaction result after monoclonal antibody Crab of the present invention and transfer printing; D is the transparent Blood cellreaction result after monoclonal antibody Crab of the present invention and transfer printing; E is the granular hemocyte reaction result (negative control) after myeloma cell's culture supernatant and transfer printing.
Embodiment
Below in conjunction with accompanying drawing and by specific embodiment, the invention will be further described.
Embodiment 1:Percoll medium continuous density gradient centrifugation Portunus trituberculatus Miers complete blood cell
(1) 0.01M phosphate buffered saline buffer (the 0.135M NaCl of commercially available Percoll stoste and 10 times of concentration; 2.7mM KCl; 1.5mM KH
2pO
4; 8mM K
2hPO
4; PH7.2) 9:1 is mixed with Percoll application liquid by volume, then Percoll application liquid is diluted to different gradients 60%, 50%, 40%, 30%, 20%(v/v with phosphate buffered saline buffer), every kind of gradient 2.5ml is placed in centrifuge tube, 4 DEG C of hold over night.
(2) get the Portunus trituberculatus Miers of 3～4 normal health, extract hemolymph from swimmeret base portion mantle with asepsis injector, with 4 DEG C of precooling antithrombotics (0.14M NaCl; 3mM KCl; 1.5mM KH
2pO
4; 8mM Na
2hPO
4; 20mM EDTA; PH7.3) 1:2 mixes by volume, and mixed solution is in 4 DEG C, 1500rpm, and centrifugal 6min, the complete blood cell obtaining precipitation is resuspended with phosphate buffered saline buffer, and adjusting complete blood cell suspension concentration is 10
9cells/ml.
(3) Portunus trituberculatus Miers complete blood cell suspension is laid on to the top layer of Percoll medium continuous density gradient, 4 DEG C, 2200rpm, centrifugal 40min.
(4) asepsis injector takes out respectively three confluent monolayer cells bands of centrifugal rear formation, respectively at 4 DEG C, and 1500rpm, centrifugal 5min, abandons supernatant, and cell precipitation uses respectively phosphate buffered saline buffer resuspended, and centrifuge washing 2 times is removed Percoll medium.
(5) to adjust each confluent monolayer cells concentration be 10 to phosphate buffered saline buffer
9cells/ml, gets each confluent monolayer cells suspension 30 μ l and is added drop-wise on slide glass, and sedimentation 4～5h in wet box, makes its self-assembling formation monolayer cell drop of blood sheet, and in the fixing 15min of acetone ,-20 DEG C save backup afterwards.
(6) the each layer of hemocyte drop of blood sheet of making carried out to Giemsa staining, the each confluent monolayer cells form of observation and comparison.
Three confluent monolayer cells bands after the centrifugation of Percoll medium continuous density gradient are as A in Fig. 1, and B, shown in C arrow: the first layer cell band is the cell mixing of granular hemocyte and transparent hemocyte; The second layer is transparent hemocyte; The 3rd layer is granular hemocyte.Giemsa staining result corresponding to each confluent monolayer cells band is as a in Fig. 1, and b, shown in c: the first layer cell band is granular hemocyte and transparent hemocyte Giemsa staining result; The second layer is transparent hemocyte Giemsa staining result; The 3rd layer is granular hemocyte Giemsa staining result.
Embodiment 2: the preparation of antigen
(1) by resuspended the granular hemocyte phosphate buffered saline buffer obtaining after centrifugal Percoll medium continuous density gradient (adjust concentration be 10
9cells/ml) after, with electrophoresis sample buffer (0.5M Tris-HCl pH6.8; 1% sodium laurylsulfonate; 1% dredges base ethanol; 10% glycerine; 0.01% tetrabromophenol sulfonphthalein) equal-volume mixes, and boils 4～5min, cooling.
(2) install electrophoresis apparatus (Mini-PROTEAN II, Bio-Rad), pour 12% separation gel preparing into gel maker, distilled water front cover, after glue polymerization to be separated, sucking-off distilled water, add 5% concentrated glue, insert immediately point sample comb, after glue polymerization to be concentrated, pour electrophoretic buffer (0.025M Tris-Base into; 0.25M glycine; 0.1% sodium laurylsulfonate; PH8.3), extract point sample comb.
(3) sample is added loading hole (every hole 15 μ l), with electrophoretic buffer in upper and lower electrophoresis chamber, current stabilization electrophoresis under 4 DEG C of conditions, initial current 30mA, until sample, after concentrated glue partial concentration is into a line, adjusting constant current is 60mA.
(4) when electrophoresis is to bromophenol blue indicator from bottom margin 1cm, stop electrophoresis, pry open sheet glass, take out gel.
(5) gel of taking-up is immersed in completely in the reversible staining fluid of 5% cupric chloride to vibration 5min.
(6) take out gel, under black background illumination condition, observe protein band, find and cut target protein band---26.7kDa protein band, put it in destainer (5% hydrochloric acid soln) vibration decolouring 20min and disappear to band color, repeatedly rinse band 5～6 times with distilled water.
(7) target protein band is packed in wash-out Glass tubing (electroelution instrument Model422, Bio-Rad), add elution buffer (50mM NH
4hCO
3; 0.1% sodium laurylsulfonate), constant current (10mA/ pipe) current value wash-out 5h is set.
(8) after wash-out, Glass tubing is taken out, elutriant is dialysed with distilled water, spends the night; Dialysis sample (being Portunus trituberculatus Miers granular hemocyte 26.7kDa albumen) freeze-drying in lyophilized preparation is concentrated, and-80 DEG C of Ultralow Temperature Freezers are preserved.
Embodiment 3: the preparation of anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa protein monoclonal antibody
1. immune mouse
(1) fundamental immunity: the granular hemocyte 26.7kDa albumen of purification mixes with Freund's complete adjuvant equal-volume as antigen, abdominal injection female Balb/C mouse in 4 week age.
(2) booster immunization: after 2 weeks, purifying protein mixes with Freund's incomplete adjuvant equal-volume, abdominal injection.
(3) secondary booster immunization: after 1 week, tail vein injection purifying protein.
(4) merge the amplification immunity of first three day: after 1 week, tail vein injection purifying protein.
2. cytogamy
(1) de-cervical vertebra is put to death immune mouse, the aseptic spleen of getting, and RPMI-1640 solution washing grinds, and lapping liquid is 1000rpm in centrifuge tube, and centrifugal 4min, abandons supernatant, and the resuspended splenocyte precipitation of RPMI-1640 solution is for subsequent use.
(2) de-cervical vertebra execution Kunming is nutrition mouse, the aseptic thymus gland of getting, and RPMI-1640 solution washing grinds, and lapping liquid is 1000rpm in centrifuge tube, and centrifugal 3min, abandons supernatant, and the resuspended thymocyte precipitation of RPMI-1640 solution is for subsequent use.
(3) get and cultivate the SP2/0 myeloma cell of growing fine, 1000rpm in centrifuge tube, centrifugal 3min, abandons supernatant.
(4) the outstanding myeloma cell of RPMI-1640 solution weight mixing with (1) splenocyte suspension, the centrifugal 4min of 1000rpm, abandons supernatant, falls dry remaining liq.
(5) touch centrifuge tube bottom, make two kinds of cell precipitations fully be mixed into pasty state, absorption is preheating to the polyoxyethylene glycol 1ml of 37 DEG C, in 1min, evenly splash into centrifuge tube bottom, then in 90s, splash into continuously the RPMI-1640 solution 15ml that is preheating to 37 DEG C, 37 DEG C of water-baths leave standstill 5min, through the centrifugal 5min of 800rpm, abandon supernatant afterwards.
(6) cell precipitation is resuspended containing the RPMI-1640 nutrient solution of 10% foetal calf serum with 3ml, frozen 2ml.
(7) take out (2) thymus cell suspension for subsequent use, join in (6) remaining suspension, add containing (2%HAT; 10% foetal calf serum) RPMI-1640 cell culture fluid, after mixing, be added drop-wise in 96 porocyte culture plates.
(8) culture plate is put into 37 DEG C, CO
2concentration is to cultivate 8～10 days in 5% incubator, and inverted microscope is observed the upgrowth situation of hybridoma.
3. indirect immunofluorescence antibody method screening positive hybridoma cell strain
(1) Hybridoma Cell Culture supernatant liquor (first antibody) 30 μ l are added drop-wise to respectively on the Portunus trituberculatus Miers complete blood cell drop of blood sheet for preparing, granular hemocyte drop of blood sheet, transparent hemocyte drop of blood sheet, myeloma cell's culture supernatant (negative control) 30 μ l are added drop-wise on complete blood cell drop of blood sheet simultaneously, in 37 DEG C of wet boxes, hatch 50min.
(2) phosphate buffered saline buffer washing drop of blood sheet 3 times, each 5min.
(3) will under fluorescein isothiocyanate (FITC) mark goat anti-mouse igg antibody (second antibody) 30 μ l lucifuge conditions, be added drop-wise on drop of blood sheet, in 37 DEG C of wet boxes, lucifuge is hatched 50min.
(4) phosphate buffered saline buffer lucifuge washing drop of blood sheet 3 times, each 5min, dry rear glycerine mounting, fluorescence microscopy Microscopic observation.
Result: Portunus trituberculatus Miers granular hemocyte and the combination of positive hybridoma cell supernatant, then in conjunction with fluorescein isothiocyanate (FITC) mark goat anti-mouse igg antibody, invention bright green fluorescence; The transparent hemocyte of Portunus trituberculatus Miers and positive hybridoma cell supernatant are hatched, then add fluorescein isothiocyanate (FITC) mark goat anti-mouse igg antibody, without fluorescence; Portunus trituberculatus Miers complete blood cell and myeloma cell's supernatant (negative control) are hatched, then add fluorescein isothiocyanate (FITC) mark goat anti-mouse igg antibody, without fluorescence (Fig. 2).
By positive hybridoma cell group record, and be further clone.
4. limiting dilution assay clone positive hybridoma cell strain
(1) de-cervical vertebra execution Kunming is nutrition mouse, the aseptic thymus gland of getting, and RPMI-1640 solution washing grinds, and lapping liquid is 1000rpm in centrifuge tube, and centrifugal 3min, abandons supernatant, with containing (2%HAT; 10% foetal calf serum) the resuspended thymocyte of RPMI-1640 cell culture fluid precipitation for subsequent use.
(2) break up hybridoma cell strain, blood counting chamber counting, is placed in 10ml containing (2%HAT by 100 positive hybridoma cells; 10% foetal calf serum) RPMI-1640 cell culture fluid in, after mixing, be added drop-wise in 96 porocyte culture plates, every hole 100 μ l, Tissue Culture Plate is in 37 DEG C, CO
2concentration is to cultivate in 5% incubator.
(3) cultivate after 8～10 days, adopt indirect immunofluorescence antibody method again to detect screening positive hybridoma cell.
Result: screening and cloning obtains the monoclonal antibody (monoclonal antibody Crab) of the anti-Portunus trituberculatus Miers granular hemocyte of a strain, this monoclonal antibody only with Portunus trituberculatus Miers granular hemocyte generation specific binding (Fig. 2).
5. frozen
Hybridoma cell strain Crab is transferred in 24 porocyte culture plates from 96 porocyte culture plates, mix with cell culture fluid during in logarithmic phase until it, cell suspension and dimethyl sulfoxide (DMSO) cells frozen storing liquid by volume 9:1 mix and are placed in cryopreservation tube, towel parcel cryopreservation tube ,-80 DEG C of Ultralow Temperature Freezers are frozen.
Embodiment 4: the characteristic of streaming immunofluorescence technique checking monoclonal antibody
(1) the Portunus trituberculatus Miers granular hemocyte after centrifugal Percoll medium continuous density gradient and transparent hemocyte being adjusted respectively to concentration is 10
9cells/ml, gets respectively 1ml and adds 50ml centrifuge tube bottom (containing negative control), then adds respectively monoclonal antibody Crab(first antibody) and myeloma cell's culture supernatant (negative control) 1ml, 37 DEG C of oscillation incubation 50min.
(2) centrifuge tube is through 1200rpm, and centrifugal 5min, abandons supernatant, and cell precipitation is resuspended with phosphate buffered saline buffer, centrifuge washing 3 times.
(3) adjusting each cell concn with phosphate buffered saline buffer is 10
9cells/ml is in 50ml centrifuge tube, and lucifuge adds fluorescein isothiocyanate (FITC) mark goat anti-mouse igg antibody (second antibody) 1ml respectively, 37 DEG C of lucifuge oscillation incubation 50min.
(4) under lucifuge condition, centrifuge tube is through 1200rpm, and centrifugal 5min, abandons supernatant, and cell precipitation is resuspended with phosphate buffered saline buffer, centrifuge washing 3 times.
(5) to adjust each cell concn be 10 to phosphate buffered saline buffer
9cells/ml, detects through flow cytometer.
Result: the transparent hemocyte of Portunus trituberculatus Miers and granular hemocyte respectively after myeloma cell's culture supernatant (negative control) reaction Fluorescence Ratio all lower; After monoclonal antibody Crab reaction, transparent hemocyte Fluorescence Ratio is still lower, and granular hemocyte Fluorescence Ratio significantly raises, and positive rate is up to 86.3%, show monoclonal antibody Crab of the present invention only with Portunus trituberculatus Miers granular hemocyte generation specific binding (Fig. 3).
Embodiment 5: transfer printing immunoblotting is analyzed the molecular weight of antigenic determinant
1. sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(1) adopt the method for (embodiment 2) to carry out the gel electrophoresis of Portunus trituberculatus Miers granular hemocyte, the two clotting glue that obtain wherein one dye with coomassie brilliant blue R250 dye liquor; Another piece is used for doing immunoblotting.
(2) take out for the gel that dyes, put it in front stationary liquid (2% methyl alcohol and 7.5% acetic acid mixed solution) and fix 30min, then put into the coomassie brilliant blue R250 dye liquor 2～3h that dyes.
(3) gel after dyeing is put into destainer (5% methyl alcohol and 7% acetic acid mixed solution) decolouring, until protein band is high-visible.
(4) destaining gel is scanned by full automatic gel imaging analysis system, take a picture (Fig. 4-A).
2. electrotransfer
(1) nitrocellulose filter of clip and gel formed objects, with electrotransfer damping fluid (0.025M Tris-Base; 0.20M glycine; 20% methyl alcohol; PH8.35) after wetting, be put on another clotting glue, in the middle of with two onesize filter paper, gel and nitrocellulose filter being sandwiched in, the bubble between attention eliminating each several part.
(2) foam pad of the transferring buffered liquid wetting of electricity consumption is supported above-mentioned system, and outermost layer supports with two poly (methyl methacrylate) plates, and whole system is placed in the electrophoresis chamber that electrotransfer damping fluid is housed.
(3) by nitrocellulose filter towards anode, constant current 200mA, electrotransfer 5h.
3. transfer printing immunoblotting
(1) take out the complete nitrocellulose filter of above-mentioned transfer, phosphate buffered saline buffer washing 15min, seals and be placed in 5% bovine serum albumin solution, and 4 DEG C are spent the night.
(2) take out nitrocellulose filter, with the phosphate buffered saline buffer washing containing 0.05% tween 20 3 times, each 5min.
(3) nitrocellulose filter is immersed in completely to monoclonal antibody Crab(first antibody) in, longitudinal clip wherein a part of nitrocellulose filter is immersed in myeloma cell's culture supernatant (negative control) completely, hatches 50min for 37 DEG C.
(4) take out nitrocellulose filter, with the phosphate buffered saline buffer washing containing 0.05% tween 20 3 times, each 5min.
(5) nitrocellulose filter is immersed in completely in the goat anti-mouse igg antibody (second antibody) of alkali phosphatase enzyme mark, hatches 50min for 37 DEG C.
(6) take out nitrocellulose filter, with the phosphate buffered saline buffer washing containing 0.05% tween 20 3 times, each 5min.
(7) acid cellulose film is placed in to alkaline phosphatase color development liquid (NBT-BCIP solution) color development, until band clear (Fig. 4-C), negative control (Fig. 4-E).
(8) use distilled water wash nitrocellulose filter, with termination reaction, dry, preserve dark place.
The transparent hemocyte of Portunus trituberculatus Miers is carried out to electrophoresis and transfer printing immunoblotting with above-mentioned same method, nitrocellulose filter, through monoclonal antibody Crab reaction, the results are shown in (Fig. 4-D).
Result: monoclonal antibody Crab of the present invention only identifies the polypeptide that molecular weight is 26.7kDa after Portunus trituberculatus Miers granular hemocyte electrophoresis, and transparent hemocyte nitrocellulose filter after the transfer printing of monoclonal antibody Crab reaction occurs without band; There is (Fig. 4) without band in the granular hemocyte nitrocellulose filter after the transfer printing of myeloma cell's reaction.
Claims (1)
1. the monoclonal antibody of an anti-Portunus trituberculatus Miers granular hemocyte 26.7kDa albumen, it is characterized in that described monoclonal antibody is to be called by name: hybridoma cell strain Crab, preserving number is: CCTCC NO:C2012196, depositary institution is: Chinese Typical Representative culture collection center, preservation date is: the hybridoma secretion of on 01 07th, 2013.
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| CN201310182093.XA CN103288955B (en) | 2013-05-16 | 2013-05-16 | Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof |
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| CN104198715A (en) * | 2014-09-12 | 2014-12-10 | 青岛农业大学 | Enzyme-linked immunoassay kit for portunustrituberculatus blood cell and preparing method thereof |
| CN104914155B (en) * | 2015-06-16 | 2017-07-21 | 河南省人口和计划生育科学技术研究院 | A kind of collection of Polyacrylamide Gel Electrophoresis collection of illustrative plates and store method |
| CN106841625A (en) * | 2017-02-07 | 2017-06-13 | 青岛农业大学 | Enzyme-linked immunologic detecting kit of Portunus trituberculatus Miers haemocyte and preparation method thereof |
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| JPS60185162A (en) * | 1984-03-03 | 1985-09-20 | Fujirebio Inc | Monoclone antibody to human erythrocyte |
| CN101863979A (en) * | 2010-06-03 | 2010-10-20 | 中国海洋大学 | Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof |
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| JPS60185162A (en) * | 1984-03-03 | 1985-09-20 | Fujirebio Inc | Monoclone antibody to human erythrocyte |
| CN101863979A (en) * | 2010-06-03 | 2010-10-20 | 中国海洋大学 | Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof |
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| 陈惠群等.三疣梭子蟹血淋巴细胞及血液生化指标的初步研究.《水产科学》.2004,(第06期), * |
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