CN102199212A - Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof - Google Patents

Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof Download PDF

Info

Publication number
CN102199212A
CN102199212A CN2011101159774A CN201110115977A CN102199212A CN 102199212 A CN102199212 A CN 102199212A CN 2011101159774 A CN2011101159774 A CN 2011101159774A CN 201110115977 A CN201110115977 A CN 201110115977A CN 102199212 A CN102199212 A CN 102199212A
Authority
CN
China
Prior art keywords
mucus
immunoglobulin
lefteye flounder
monoclonal antibody
flounder
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2011101159774A
Other languages
Chinese (zh)
Other versions
CN102199212B (en
Inventor
战文斌
许国晶
绳秀珍
邢婧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ocean University of China
Original Assignee
Ocean University of China
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ocean University of China filed Critical Ocean University of China
Priority to CN 201110115977 priority Critical patent/CN102199212B/en
Publication of CN102199212A publication Critical patent/CN102199212A/en
Application granted granted Critical
Publication of CN102199212B publication Critical patent/CN102199212B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a monoclonal antibody against mucus immunoglobulin in flounder. The monoclonal antibody is secreted by a hybridoma cell which is named JF-mIg and collected in China Center for Type Culture Collection on April 25, 2011 with the collection number of CCTCCC201127. The monoclonal antibody can be specifically combined with the mucus immunoglobulin in flounder, and provides an important tool for studying the structure, source and function of the mucus immunoglobulin and application thereof in a relationship with lymphocyte; meanwhile, the monoclonal antibody can be used for detecting a specific antibody of a certain pathogen or aquatic vaccine so as to perform early diagnosis of the disease and the evaluation of use effect of the vaccine, and provides an important technical means for controlling diseases of the flounder and a reference for controlling diseases of other cultured fishes.

Description

Monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig) and preparation method thereof
Technical field
The present invention relates to a kind of monoclonal antibody and preparation method thereof, specifically be a kind of anti-lefteye flounder ( Paralichthys olivaceus) monoclonal antibody and preparation method thereof of mucus immunoglobulin (Ig), belong to fish molecular immunology technical field.
Background technology
Covering the mucous membrane sample Lymphoid tissue and the excretory mucus thereof on surfaces such as fish body skin, the gill and gi tract, constituted the mucosa-immune system of fish, is the first line of defence of body opposing cause of disease invasion.Be distributed with lymphocyte, scavenger cell and all kinds of granulocyte in the fish mucous membrane tissue, make it have the function of independently finishing local immune response.Except that containing nonspecific immunizing compositions such as N,O-Diacetylmuramidase and complement, also contain specific antibody in the mucus, in the immunoprotection process, play an important role.
Also there are many disputes at present in relation about fish mucus immunoglobulin (Ig) and serum immune globulin.Document shows, fish can detect specific antibody at the skin mucus behind the antigen immersion immunity, and in serum seldom or detect less than these antibody, infer that the mucus immunoglobulin (Ig) is not to be shifted by the immunoglobulin (Ig) in the serum, but secreted by the plasmocyte of mucous membrane self.The monoclonal antibody for preparing anti-lefteye flounder mucus immunoglobulin (Ig) can provide strong instrument for the structure of illustrating the mucus immunoglobulin (Ig), source, function and with serum immune globulin and lymphocytic relation etc.; In addition, after fish infected certain cause of disease or vaccination, fish was known from experience the specific antibody that produces at this cause of disease/vaccine.Therefore, utilize the monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig), by detecting the generation of this specific antibody in the lefteye flounder mucus, can carry out the early diagnosis of lefteye flounder disease and the evaluation of vaccine result of use, prevention and treatment to the lefteye flounder disease have most important theories and realistic meaning, and the control of other cultured fishes diseases is had reference value.
Summary of the invention
The purpose of this invention is to provide a kind of monoclonal antibody by the anti-lefteye flounder mucus of hybridoma excretory immunoglobulin (Ig); Another object of the present invention provides a kind of anti-lefteye flounder mucus immunoglobulin (Ig) MONOCLONAL ANTIBODIES SPECIFIC FOR method.
The objective of the invention is to realize: a kind of monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig) by following technical scheme, described monoclonal antibody is to be called by name: hybridoma JF-mIg, depositary institution is: Chinese typical culture collection center, preserving number is: CCTCC C201127, preservation date is: on 04 25th, 2011 hybridoma excretory.
Under inverted microscope, observe, this hybridoma division that growth conditions is good is vigorous, outward appearance is full, perfectly round, refractivity is strong, the big or small homogeneous of cell, adherent good; This hybridoma has unlimited division and proliferation ability; This hybridoma that grows fine is conventional to be cultivated 2-3 days, and its substratum changes yellow into by pink, wherein contains the monoclonal antibody of a large amount of anti-lefteye flounder mucus immunoglobulin (Ig)s of this hybridoma excretory.
A kind of described anti-lefteye flounder mucus immunoglobulin (Ig) MONOCLONAL ANTIBODIES SPECIFIC FOR method comprises the steps: the lefteye flounder mucus is obtained lefteye flounder mucus immunoglobulin (Ig) through the column purification by chromatography of saltouing; Lefteye flounder mucus immunoglobulin (Ig) with purifying is an antigen immune Balb/C mouse; Fusion is by the splenocyte of immune mouse and myeloma cell; Through the immunology detection screening method, filter out the hybridoma JF-mIg of the anti-lefteye flounder mucus immunoglobulin (Ig) monoclonal antibody of secretion, its excretory antibody is the monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig).
The described column chromatography of saltouing, be with the lefteye flounder mucus after saturated ammonium sulphate is saltoutd step by step, be further purified through Sephacryl S-300 gel permeation chromatography and DEAE Sepharose ion chromatography successively and obtain lefteye flounder mucus immunoglobulin (Ig).
Described immunology detection screening method is indirect enzyme-linked immunosorbent method and transfer printing immunoblotting; Wherein said indirect enzyme-linked immunosorbent method is that the lefteye flounder mucus immunoglobulin (Ig) with the Hybridoma Cell Culture supernatant liquor of gained after the cytogamy and purifying reacts in 96 hole enzyme plates; Then the sheep anti-mouse antibody that adds horseradish peroxidase (AP) mark; 405 nm operation wavelengths are measured each hole absorbance value, screen the monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig).Described transfer printing immunoblotting is with the monoclonal antibody of the anti-lefteye flounder mucus of hybridoma JF-mIg excretory immunoglobulin (Ig) and the lefteye flounder mucus immunoglobulin (Ig) reaction that is transferred to nitrocellulose filter, and the antigenic determinant of determining this monoclonal antibody is positioned on the heavy chain of lefteye flounder mucus immunoglobulin (Ig) that molecular weight is 72 kDa.
Described hybridoma JF-mIg excretory monoclonal antibody can be verified its characteristic through streaming immunofluorescence technique or immunoelectron microscopic method.
Described streaming immunofluorescence technique is that hybridoma JF-mIg excretory monoclonal antibody and lefteye flounder peripheral blood lymphocyte suspension react in cultivating plate hole; Then the sheep anti-mouse antibody that adds fluorescein isothiocyanate (FITC) mark; The monoclonal antibody that detects anti-lefteye flounder mucus immunoglobulin (Ig) with flow cytometer can combine with the peripheral blood lymphocyte specificity.
Described immunoelectron microscopic method is with hybridoma JF-mIg excretory monoclonal antibody and the reaction of lefteye flounder peripheral blood lymphocyte ultrathin section(ing); Then the sheep anti-mouse antibody that adds colloid gold label; The electricity consumption microscopy is surveyed the antigenic determinant that monoclonal antibody can specificity bind lymphocytes film surface.
Technology of preparing highway route design novelty of the present invention, it is saltoutd step by step by saturated ammonium sulphate and obtains highly purified lefteye flounder mucus immunoglobulin (Ig) as the antigen immune BALB/c mouse in conjunction with Sephacryl S-300 gel permeation chromatography and DEAE Sepharose ion chromatography purifying; Obtain the monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig) through cytogamy, indirect immunofluorescence screening, clone, screening; The monoclonal antibody of gained is verified its characteristic through streaming immunofluorescence technique and immuno-electron microscope again.Such technology of preparing route is tight reasonable and feasible, has given full play to the effect and the effect of existing immunology detection screening method.
Monoclonal anti physical efficiency by the anti-lefteye flounder mucus of hybridoma JF-mIg excretory immunoglobulin (Ig) combines with lefteye flounder mucus immunoglobulin (Ig) specificity.This hybridoma that grows fine has big or small homogeneous, and outward appearance is full, and is perfectly round bright, divides vigorously, can infinitely secrete characteristics single, pure antibody.This hybridoma is conventional the cultivation 2-3 days in substratum, promptly contained a large amount of highly sensitively in the substratum, tired the monoclonal antibody of the anti-lefteye flounder mucus of specificity immunoglobulin (Ig).The making structure for research mucus immunoglobulin (Ig), source, function and provide important tool of anti-lefteye flounder mucus immunoglobulin (Ig) monoclonal antibody with application in the lymphocyte relation; This monoclonal antibody can be used for certain cause of disease or aquatic products vaccine specific detection of antibodies simultaneously, thereby carry out the early diagnosis of disease and the evaluation of vaccine result of use, for the prevention and the treatment of lefteye flounder disease provides the important techniques means, for the control of other cultured fishes diseases provides reference.
Description of drawings
Fig. 1 is the transfer printing immunoblotting figure as a result of monoclonal antibody of the present invention.
Fig. 2 is the streaming immunofluorescence detected result figure of monoclonal antibody of the present invention.
Fig. 3 is monoclonal antibody Electronic Speculum detected result figure of the present invention.
Referring to Fig. 1-Fig. 3.
Shown in Figure 1: the 1st, the proteic coomassie brilliant blue staining result of standard molecular weight; The total lefteye flounder mucus albumen coomassie brilliant blue staining result of 2 expressions; The lefteye flounder mucus immunoglobulin (Ig) coomassie brilliant blue staining result that 3 expressions are purified; Lefteye flounder mucus immunoglobulin (Ig) reaction after 4 expression monoclonal antibodies of the present invention and the sex change, the albumen of identification is the heavy chain of lefteye flounder mucus immunoglobulin (Ig), molecular weight is 72 kDa.
Shown in Figure 2: A is the scatter diagram that lefteye flounder peripheral blood lymphocyte suspension streaming detects; A is the corresponding streaming histogram of A figure; B is the scatter diagram that lefteye flounder splenic lymphocyte suspension streaming detects; B is the corresponding streaming histogram of B figure; The wherein negative contrast of L1; The positive cell of L2.
Shown in Figure 3: A is a lefteye flounder peripheral blood lymphocyte immuno-electron microscope detected result; A-1 and A-2 are that A schemes local Radioactive colloidal gold enlarged view.
Embodiment
Further specify the present invention below in conjunction with accompanying drawing and by specific embodiment.
Embodiment 1: the MONOCLONAL ANTIBODIES SPECIFIC FOR of anti-lefteye flounder mucus immunoglobulin (Ig).
One, antigenic preparation
(1) the lefteye flounder mucous is collected
Get 10 tail lefteye flounders and scrape gently with clean slide and get fish surface skin, getting mucus is mixed, add equivalent 0.01 mol/L PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 8.09 mmol/L Na 2HPO 4, 1.47 mmol/L KH 2PO 4, pH 7.4), 15 000 g, 4 ℃ of centrifugal 30 min get supernatant, and-80 ℃ are frozen standby.
(2) purifying of lefteye flounder mucus immunoglobulin (Ig)
1. saturated ammonium sulphate is saltoutd step by step: get above-mentioned mucus sample, the saturated ammonium sulphate solution that slowly adds pH 7.0, make its final concentration reach 30%, 4 ℃ of standing over night, centrifugal 30 min of 15 000 g, getting supernatant, to continue to add saturated ammonium sulphate solution to final saturation ratio be 50%, the same processing, next day centrifugal collecting precipitation, resolution of precipitate is in 0.02 mol/L Tris-HCl damping fluid (pH 8.0), with identical damping fluid 24 h that dialyse, per 4 h change liquid once again, and the mucous membrane antibody crude extract of acquisition is in-80 ℃ of preservations.
2. Sephacryl-S300 column chromatography: the said extracted thing is further purified by Sephacryl S-300 gel chromatography column, and with 0.02 mol/L pH, 8.0 Tris-HCl wash-outs, flow velocity 0.5 mL/min collects first peak, and-80 ℃ of preservations are standby.
3. DEAE ion-exchange chromatography: first protein peak of gel chromatography merge concentrate after again through DEAE Sepharose column chromatography, be respectively 0.05 mol/L with concentration, 0.1 mol/L, 0.15 mol/L, 0.2 mol/L, 0.4 the NaCl solution stepwise elution of mol/L and 1 mol/L is collected the elution peak of 0.1 mol/L NaCl, dialysis, freeze-drying concentrates, be resuspended among the PBS (pH 7.4) of 0.01 mol/L, the adjustment protein content is 1mg/ml, and-80 ℃ of preservations are standby.
Two, immune mouse
With the lefteye flounder mucus immunoglobulin (Ig) of purifying as antigen.Each immunizing dose is 0.1 ml, and immunity is divided into 4 times to be carried out, and preceding 2 immunity are spaced apart 2 weeks, abdominal injection; 3 immunity in back are spaced apart 1 week, tail vein injection.
(1) fundamental immunity: the lefteye flounder mucus immunoglobulin (Ig) of purification and Fu Shi Freund's complete adjuvant equivalent (V/V) mixing are as antigen;
(2) booster immunization: the lefteye flounder mucus immunoglobulin (Ig) of purification and freund 's incomplete adjuvant equivalent (V/V) mixing are as antigen;
(3) secondary booster immunization: the lefteye flounder mucus immunoglobulin (Ig) of purification is as antigen;
(4) merge the amplification immunity of first three day: the lefteye flounder mucus immunoglobulin (Ig) of purification is as antigen.
Three, cytogamy
(1) takes off cervical vertebra and put to death immune mouse, behind aseptic taking-up spleen and the thymus gland, cross 100 order mesh screens respectively, form single cell suspension with the piping and druming of RPMI-1640 solution;
(2) respectively with splenocyte suspension and centrifugal 3 min of thymus cell suspension 1000 rpm, abandoning supernatant, the splenocyte precipitation is resuspended with RPMI-1640 solution, and the thymocyte precipitation is resuspended with RPMI-1640 (containing 10% foetal calf serum) the selecting cell nutrient solution that contains 1%HAT.
(3) get 3 * 10 7The individual P3-X63-Ag8U1 myeloma cell who is in logarithmic phase, centrifugal 3 min of 1000 rpm, remove supernatant liquor after, resuspended with RPMI-1640 solution;
(4) splenocyte suspension and tumor cell suspension are mixed after, centrifugal 3 min of 1000 rpm inhale fully and remove supernatant liquor, flick at the bottom of the centrifuge tube, make two kinds of cell precipitations fully be mixed into pasty state; With the polyglycol solution lml of the pre-temperature of suction pipe absorption to 37 ℃, be added drop-wise in the centrifuge tube, in 1 min, add; Leave standstill 5 min 37 ℃ of water-baths then;
(5) continue to have dripped temperature in advance to 37 ℃ RPMI-1640 solution 15 m1, PEG has been diluted and ineffective;
(6) add RPMI-1640 solution to 40 ml, through centrifugal 3 min of 1000 rpm, abandoning supernatant;
(7) sedimentary cell is resuspended with 3 ml37 ℃ RPMI-1640 (containing 10% foetal calf serum) cell culture fluid, frozen 2ml;
The 1 ml cell suspension that (8) will be left adds the thymus cell suspension that make (2), is added drop-wise in 96 well culture plates after mixing;
(9) culture plate is put into 37 ℃, CO 2Concentration is to cultivate in 4.5% the incubator, and inverted microscope observation of cell growing state after about two weeks, is got the Hybridoma Cell Culture supernatant liquor and detected.
Four, screening and clone
(1) screening: after the fusion, begin to detect when treating the long hole floorage 1/3 to 96 well culture plates of hybridoma group, adopt indirect enzyme-linked immunosorbent technology screening positive hybridoma cell.
1. envelope antigen: the lefteye flounder mucus immunoglobulin (Ig) of purifying with carbonate coating buffer (pH 9.6) 1:10 dilution, is added in (50 μ l/ hole) in the 96 hole enzyme plates, and 4 ℃ of bags are spent the night;
2. the sucking-off coating buffer washs with the phosphate buffered saline buffer (PBST) that contains 0.05% tween 20, each 5 min, and it is inferior to give a baby a bath on the third day after its birth;
3. every hole adds the bovine serum albumin (PBS joins) of 200 μ l 3%, 37 ℃ of sealing 1 h;
4. same 2. method washing three times;
5. the Hybridoma Cell Culture supernatant is added to enzyme plate as first antibody by every hole 50 μ l, 37 ℃ of incubation 1 h;
6. same 2. method washing three times;
7. the goat-anti mouse Ig(1:4000 of alkali phosphatase enzyme mark dilution) be added to enzyme plate as second antibody by every hole 50 μ l, 37 ℃ of incubation 1 h;
8. same 2. method washing three times; Every then hole adds 100 μ l 4-nitrophenol phosphoric acid salt (pNPP) and uses liquid, dark place reaction 5-20 min, and every hole adds the NaOH of 50 μ l 2M, and stablizing 3-5 min is that available 405 nm operation wavelengths are measured the OD value.
Each hole absorbance value when surveying wavelength and being 405 nm calculates the ratio (P/N) of each experimental port and negative control absorbance value, and this hole is positive when P/N 〉=2.1.
(2) clone: adopt limiting dilution assay that detected positive hybridoma cell is cloned, step is as follows:
1. take off cervical vertebra and put to death mouse, aseptic taking-up thymus gland grinds on 100 order mesh screens, forms single cell suspension with the piping and druming of RPMI-1640 solution;
2. centrifugal 3 min of thymus cell suspension 1000 rpm, abandoning supernatant, the thymocyte precipitation is resuspended with 10 ml RPMI-1640 (containing 10% foetal calf serum) cell culture fluid;
3. the cell in the positive cell hole that will clone is counted with blood cell counting plate, use nutrient solution then, take out 100 hybridomas, put into thymus cell suspension with 10 times of gradient dilutions;
4. cell suspension with dropper piping and druming evenly is added drop-wise in 96 well culture plates, every hole 100 μ l, and on average a hybridoma is contained in each hole;
5. put into CO 2Cultivate in the incubator;
6. pass through each hole Hybridoma Cell Culture supernatant of indirect enzyme-linked immunosorbent technology for detection after two weeks, the hybridoma in gained positive colony hole is cloned once as stated above again, to guarantee to form mono-clonal.
Five, frozen
It is vigorous to get growth, and the hybridoma that form is good is made cell suspension, and centrifugal 5 min of 200 g remove supernatant, add frozen storing liquid (9 parts of RPMI-1640 substratum+1 part methyl-sulphoxides), and making final cell density is 5 * 10 6Individual/ml, 1 ml cell suspension is loaded in the frozen pipe of 2 ml, tighten blind nut, frozen pipe is packed into fills in the capsule of cotton then, be put in spend the night in-80 ℃ of Ultralow Temperature Freezers (8-12 h) after, immerse prolonged preservation in the liquid nitrogen again.It is full that the present invention has obtained outward appearance, perfectly round bright, divides vigorous hybridoma, and its name is called: hybridoma JF-mIg, preserving number is: CCTCC C201127, preservation date is: on 04 25th, 2011.
Embodiment 2: the indirect enzyme-linked immunosorbent method of monoclonal antibody of the present invention is identified.
(1) envelope antigen: the lefteye flounder mucus immunoglobulin (Ig) of purifying is diluted with carbonate coating buffer (pH 9.6) 1:10, add in (50 μ l/ hole) in the 96 hole enzyme plates, 4 ℃ of bags are spent the night;
(2) sucking-off coating buffer, with the PBST washing, each 5 min wash 3 times;
(3) every hole adds 37 ℃ of sealings of bovine serum albumin (PBS joins), 1 h of 200 μ l 3%;
(4) same 2. method washing is 3 times;
(5) above-mentioned screening and the Hybridoma Cell Culture supernatant that clones are added to enzyme plate as first antibody by every hole 50 μ l, 37 ℃ of incubation 1 h;
(6) same 2. method washing is 3 times;
(7) the goat-anti mouse Ig(1:4000 of alkali phosphatase enzyme mark dilution) be added to enzyme plate as second antibody by every hole 50 μ l, 37 ℃ of incubation 1 h;
(8) same 2. method washing is 3 times; Every then hole adds 100 μ l pNPP and uses liquid, dark place reaction 5-20 min, and every hole adds the NaOH of 50 μ l 2M, and stablizing 3-5 min is that available 405 nm operation wavelengths are measured the OD value.
With bag by PBS as negative control, each hole absorbance value when surveying wavelength and being 405 nm calculates the ratio (P/N) of positive serum and PBS absorbance value, and is positive when P/N 〉=2.1.
Result: the positive: 0.462; Negative control: 0.092.This result confirm monoclonal antibody of the present invention can with lefteye flounder mucus immunoglobulin (Ig) generation specificity association reaction.
Embodiment 3: the transfer printing immunoblotting of monoclonal antibody of the present invention is identified.
(1) sodium laurylsulfonate-polyacrylamide gel electrophoresis:
1. the lefteye flounder mucus immunoglobulin (Ig) that extracts is added the sample buffer that equal proportion contains sodium laurylsulfonate, in boiling water, boil 3-5 min;
2. the sample that will 1. handle adds to be gone up in the sample hole, add sample 10 μ l in every hole, under constant current conditions, use low current (30-40 mA) when initial, treat that sample is after concentrated glue partial concentration becomes a line, strengthen electric current (50-70 mA), electrophoresis can stop electrophoresis when the tetrabromophenol sulfonphthalein indicator reaches bottom margin, takes out gel;
3. one of clip and the nitrocellulose filter (aperture 0.22 μ m) of the identical size of running gel are with electrotransfer damping fluid (electrotransfer damping fluid: 25 mmol/L Tris-Base, 192 mmol/L glycine, 20% methyl alcohol, pH 8.3) wetting, be placed on the gel behind the electrophoresis.On nitrocellulose filter, cut an angle, with the initiating terminal of sign sample order.With wetting filter paper support, second wetting filter paper is attached to the another side of gel film; By above-mentioned placement order, make blob of viscose and nitrocellulose filter and filter paper form sandwich " sandwich " of a cover;
4. " gel sandwich " inserted in the electrophoresis chamber that fills the electrotransfer damping fluid, with nitrocellulose filter towards anode; Electrophoresis constant current 200 mA switched on 5 hours;
5. shift and finish the taking-up nitrocellulose filter.
(2) immunoblotting:
1. nitrocellulose filter is washed 10 min with PBS, put 3% the middle sealing of bovine serum albumin solution (PBS joins) 1h then, 37 ℃;
2. wash 3 times each 5 min with PBST;
3. nitrocellulose filter is placed the Hybridoma Cell Culture supernatant, 37 ℃ are slowly shaken 1 h;
4. same 2. method washing 3 times;
5. nitrocellulose filter is added in the goat anti-mouse ig antibody (1:4000 dilution) of alkali phosphatase enzyme mark, 37 ℃ were slowly shaken 1 hour;
6. same 2. method washing 3 times;
7. nitrocellulose filter is put into alkaline phosphatase color development liquid (NBT-BCIP color development liquid) color development, till color is clear;
8. use deionized water wash, with termination reaction.Nitrocellulose filter is clipped between filter paper drying.Putting the dark place preserves.
The result: monoclonal antibody of the present invention and molecular weight are the heavy chain generation specific reaction of the lefteye flounder mucus immunoglobulin (Ig) of 72 kDa, show the puce band, and negative control does not have the band demonstration.
Embodiment 4: the streaming immunofluorescence technique of monoclonal antibody of the present invention detects.
(1) preparation of lefteye flounder peripheral blood lymphocyte suspension:
Place an amount of single-cell suspension medium eRPMI(65% RPMI-1640 distilled water dilution in the syringe in advance, contain 20 IU ml -1Heparin, 0.1% (w/v) NaN 3With the bovine serum albumin of 1% (w/v), pH 7.4,240 mOsm kg -1), gather healthy Paralichthys olivaceus blood, the blood of collection is placed 1 h for 4 ℃, and 4 ℃ of following centrifugal 10 min of 100 g go precipitation then, and supernatant is used to prepare the peripheral blood single cell suspension;
(2) flow cytometry:
1. the white corpuscle of slightly carrying about 1 * 10 7Individual usefulness 500 μ l hybridoma culture supernatant are resuspended, hatch 1 h for 4 ℃;
2. PBS 680 g, 4 ℃ of centrifuge washings 3 times, each 5 min;
3. the goat-anti mouse Ig(1:500 dilution that adds marked by fluorescein isothiocyanate), hatch 1 h for 4 ℃;
4. PBS 680 g, 4 ℃ of centrifuge washings 3 times, each 5 min;
5. PBS is resuspended, and cell suspension detects through flow cytometer after 200 mesh sieve tulles filter.
The result is as shown in Figure 3: the peripheral blood lymphocyte suspension is in negative control, and fluorescent value is lower; And behind the monoclonal antibody reactive of anti-lefteye flounder mucus immunoglobulin (Ig), fluorescent value significantly increases, and positive rate shows that up to 38.64% monoclonal antibody of the present invention can combine with the peripheral blood lymphocyte specificity.
Embodiment 5: the immuno-electron microscope of monoclonal antibody of the present invention detects.
(1) preparation of lefteye flounder peripheral blood lymphocyte suspension:
According to the method among the embodiment 4, preparation lefteye flounder peripheral blood lymphocyte suspension, 680 g, 4 ℃ are centrifugal, the collecting cell precipitation.
(2) immuno-electron microscope detects:
1. behind the ultrathin section(ing), golden net drags for sheet;
2. 1%H 2O 2After 200 ul handled 30 min, distilled water cleaned;
3. add 3%BSA 200 ul(and be dissolved in PBS), 37 ℃ of sealing 45 min;
4. with the PBST washing, each 5min washes 3 times;
5. add the Hybridoma Cell Culture supernatant as first antibody 200 ul, hatch 45 min for 37 ℃;
6. same 2. method washing 3 times;
7. colloid gold label goat anti-mouse igg 150 ul(1:100 that add 15-nm), hatch 45 min for 37 ℃ as second antibody;
8. distilled water embathes 3 times, dries, then 2% phospho-wolframic acid negative staining 1 min;
9. inhale and remove residual dye liquor, electron microscopic observation is taken pictures.
The result is as shown in Figure 4: visual field background clearance, there are not the gold grain or other pollutents that are dispersed in, and colloid gold particle is concentrated and is combined in around the lymphocyte film, and the colloid gold particle that does not have in the zone, extracellular scatters.But this result has directly proved the antigenic determinant on monoclonal antibody specificity bind lymphocytes film of the present invention surface.
The streaming immunofluorescence shows that with the immuno-electron microscope detected result mucus immunoglobulin (Ig) has similar antigenic characteristic to the peripheral blood lymphocyte surface membrane immunoglobulin, thereby confirms that monoclonal antibody of the present invention can be as the structure of research mucus immunoglobulin (Ig), the important tool that the source reaches the function in mucosal immune response.In addition, can be by the specific antibody of certain cause of disease or vaccine in the detection lefteye flounder mucus, carry out the diagnosis of lefteye flounder disease and the evaluation of vaccine result of use, make monoclonal antibody of the present invention can be used for preparing lefteye flounder disease early diagnosis kit or structure vaccine result of use appraisement system, for the prevention and the treatment of lefteye flounder disease provides instrument and technique means, for the control of other cultured fishes diseases provides reference.
Those of ordinary skill in the art can understand, and in protection scope of the present invention, makes amendment for the foregoing description, and it all is possible adding and replacing, and it does not all exceed protection scope of the present invention.

Claims (6)

1. the monoclonal antibody of an anti-lefteye flounder mucus immunoglobulin (Ig), it is characterized in that: described monoclonal antibody is to be called by name: hybridoma JF-mIg, depositary institution is: Chinese typical culture collection center, preserving number is: CCTCC C201127, preservation date is: on 04 25th, 2011 hybridoma excretory.
2. an anti-lefteye flounder mucus immunoglobulin (Ig) MONOCLONAL ANTIBODIES SPECIFIC FOR method as claimed in claim 1 is characterized in that described method comprises the steps: the lefteye flounder mucus is obtained lefteye flounder mucus immunoglobulin (Ig) through the column purification by chromatography of saltouing; Lefteye flounder mucus immunoglobulin (Ig) with purifying is an antigen immune Balb/C mouse; Fusion is by the splenocyte of immune mouse and myeloma cell; Through the immunology detection screening method, filter out the hybridoma JF-mIg of the monoclonal antibody of the anti-lefteye flounder mucus immunoglobulin (Ig) of secretion; Its excretory antibody is the monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig).
3. anti-lefteye flounder mucus immunoglobulin (Ig) MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 2, it is characterized in that the described column chromatography of saltouing be with the lefteye flounder mucus after saturated ammonium sulphate is saltoutd step by step, be further purified through Sephacryl S-300 gel permeation chromatography and DEAE Sepharose ion chromatography successively.
4. according to the described anti-lefteye flounder mucus immunoglobulin (Ig) MONOCLONAL ANTIBODIES SPECIFIC FOR method of claim 2, it is characterized in that described immunology detection screening method is indirect enzyme-linked immunosorbent method and transfer printing immunoblotting.
5. anti-lefteye flounder mucus immunoglobulin (Ig) MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 4 is characterized in that described indirect enzyme-linked immunosorbent method is that the Hybridoma Cell Culture supernatant liquor of gained after the cytogamy and the lefteye flounder mucus immunoglobulin (Ig) of purifying are reacted in 96 hole enzyme plates; Then the sheep anti-mouse antibody that adds horseradish peroxidase-labeled; 405 nm operation wavelengths are measured each hole absorbance value, screen the monoclonal antibody of anti-lefteye flounder mucus immunoglobulin (Ig), i.e. hybridoma JF-mIg excretory monoclonal antibody.
6. anti-lefteye flounder mucus immunoglobulin (Ig) MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 4, it is characterized in that described transfer printing immunoblotting is with hybridoma JF-mIg excretory monoclonal antibody and the lefteye flounder mucus immunoglobulin (Ig) reaction that is transferred to nitrocellulose filter, the antigenic determinant of determining this monoclonal antibody is positioned on the heavy chain of lefteye flounder mucus immunoglobulin (Ig) that molecular weight is 72 kDa.
CN 201110115977 2011-05-06 2011-05-06 Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof Active CN102199212B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201110115977 CN102199212B (en) 2011-05-06 2011-05-06 Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201110115977 CN102199212B (en) 2011-05-06 2011-05-06 Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof

Publications (2)

Publication Number Publication Date
CN102199212A true CN102199212A (en) 2011-09-28
CN102199212B CN102199212B (en) 2012-12-12

Family

ID=44660228

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201110115977 Active CN102199212B (en) 2011-05-06 2011-05-06 Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof

Country Status (1)

Country Link
CN (1) CN102199212B (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104311668A (en) * 2014-11-01 2015-01-28 中国海洋大学 Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof
CN108341875A (en) * 2018-04-02 2018-07-31 中国海洋大学 The monoclonal antibody and the preparation method and application thereof of anti-lefteye flounder T cell surface markers CD4-1
CN108484769A (en) * 2018-04-02 2018-09-04 中国海洋大学 The monoclonal antibody and the preparation method and application thereof of anti-lefteye flounder T cell surface markers CD4-2
CN114292812A (en) * 2021-12-28 2022-04-08 中国海洋大学 Flow cytometry sorting paralichthys olivaceus CD4+Method for T lymphocytes
CN115785243A (en) * 2022-10-30 2023-03-14 中国海洋大学 Specific antibody of paralichthys olivaceus M cell marker molecule GP2 and application thereof
CN116003562A (en) * 2022-12-11 2023-04-25 中国海洋大学 Preparation and application of paralichthys olivaceus mucin Muc2 specific antibody

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1683411A (en) * 2005-03-07 2005-10-19 中国海洋大学 Lefteye flonder lymphocystic virus neutralizing monoclonal antibody and its preparing method
CN101003572A (en) * 2006-07-19 2007-07-25 中国海洋大学 Monoclonal antibody of immunoglobulin of anti lymphocyst vitos of Pacific fluke, and preparation method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1683411A (en) * 2005-03-07 2005-10-19 中国海洋大学 Lefteye flonder lymphocystic virus neutralizing monoclonal antibody and its preparing method
CN101003572A (en) * 2006-07-19 2007-07-25 中国海洋大学 Monoclonal antibody of immunoglobulin of anti lymphocyst vitos of Pacific fluke, and preparation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
许国晶 等，: "牙鲆皮肤黏液免疫球蛋白的纯化及部分特征分析", 《水产学报》, vol. 35, no. 4, 30 April 2011 (2011-04-30) *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104311668A (en) * 2014-11-01 2015-01-28 中国海洋大学 Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof
CN104311668B (en) * 2014-11-01 2017-05-10 中国海洋大学 Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof
CN108341875A (en) * 2018-04-02 2018-07-31 中国海洋大学 The monoclonal antibody and the preparation method and application thereof of anti-lefteye flounder T cell surface markers CD4-1
CN108484769A (en) * 2018-04-02 2018-09-04 中国海洋大学 The monoclonal antibody and the preparation method and application thereof of anti-lefteye flounder T cell surface markers CD4-2
CN114292812A (en) * 2021-12-28 2022-04-08 中国海洋大学 Flow cytometry sorting paralichthys olivaceus CD4+Method for T lymphocytes
CN114292812B (en) * 2021-12-28 2024-04-05 中国海洋大学 Flow cytometry sorting flounder CD4 + T lymphocyte method
CN115785243A (en) * 2022-10-30 2023-03-14 中国海洋大学 Specific antibody of paralichthys olivaceus M cell marker molecule GP2 and application thereof
CN116003562A (en) * 2022-12-11 2023-04-25 中国海洋大学 Preparation and application of paralichthys olivaceus mucin Muc2 specific antibody

Also Published As

Publication number Publication date
CN102199212B (en) 2012-12-12

Similar Documents

Publication Publication Date Title
CN102199212B (en) Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof
CN100560602C (en) Monoclonal antibody of immunoglobulin of anti lymphocyst vitos of Pacific fluke and preparation method thereof
CN101915846A (en) ELISA kit for detecting porcine reproductive and respiratory syndrome virus and using method
CN108329378A (en) Senecan paddy virus VP 1 albumen, encoding gene, hybridoma cell strain and monoclonal antibody and its application
CN102099375B (en) Anti-idiotypic monoclonal antibody against envelope protein vp28 of shrimp white spot syndrome virus and the preparation method thereof
CN105085673A (en) Monoclonal antibody for detecting solid particles of coxsackievirus A16 and use of monoclonal antibody
CN107475203A (en) A kind of H7 avian influenza virus monoclonal antibody and application
CN103995126A (en) ELISA kit for detecting Salmonella pullorum antibody
CN108341875A (en) The monoclonal antibody and the preparation method and application thereof of anti-lefteye flounder T cell surface markers CD4-1
CN110079507A (en) The monoclonal antibody and its application of anti-lefteye flounder mucus immunoglobulin IgT
CN104311668B (en) Anti-paralichthys olivaceus immunoglobulin D monoclonal antibody as well as application and preparation method thereof
CN1312180C (en) Lefteye flonder lymphocystic virus neutralizing monoclonal antibody and its preparing method
CN103773736B (en) Secrete the foundation that anti-duck source NDV separates the hybridoma cell strain of strain monoclonal antibody
CN108484769A (en) The monoclonal antibody and the preparation method and application thereof of anti-lefteye flounder T cell surface markers CD4-2
CN102220285B (en) Monoclonal antibody of outer membrane protein of chlamydia abortus and application thereof
CN107831306A (en) A kind of H7 subtype avian influenza virus double-antibody sandwich elisa kit and its detection method
CN104130328B (en) Monoclonal antibody of anti-Eriocheir sinensis granular hemocyte cell membrane 30.9kDa albumen and preparation method thereof
CN103336121B (en) Swine influenza virus H3 subtype colloidal gold detection kit as well as preparation method thereof
CN108866008A (en) The monoclonal antibody and its cell strain of anti-Koi herpesvirus and application
CN1322009C (en) Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method
CN106754742A (en) The hybridoma cell strain of microcapsule algae toxin resistant antibody and its secreted monoclonal antibody
CN102206278B (en) Monoclonal antibody of anti-LCDV (lymphocystis disease virus) 27.8kDa receptor protein and preparation method thereof
CN103214572B (en) Anti-sterigmatocystin monoclonal antibody
CN108486066A (en) A kind of monoclonal antibody of the anti-current of hybridoma cell strain and its secretion production Chlamydia
CN101671392A (en) Monoclonal antibody resisting hemocyanin of China prawns as well as preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant