CN1322009C - Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method - Google Patents
Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method Download PDFInfo
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- CN1322009C CN1322009C CNB2004100755296A CN200410075529A CN1322009C CN 1322009 C CN1322009 C CN 1322009C CN B2004100755296 A CNB2004100755296 A CN B2004100755296A CN 200410075529 A CN200410075529 A CN 200410075529A CN 1322009 C CN1322009 C CN 1322009C
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Abstract
The present invention relates to monoclonal antibodies II for resisting melanodermia virus receptors, which is secreted by hybridoma cells. The preservation number of the hybridoma cells is CCTCC-C200418, the secretory monoclonal antibodies II have the function that the monoclonal antibodies II are combined with virus receptors on the cell membranes of the hemocytes of prawns in a specific mode to enclose the receptors and to block viruses to enter the cells, and thus, the viruses can not infect the hemocytes of prawns; the color of the hybridoma cells is changed from peachblow to yellow when the hybridoma cells are conventionally cultured for 2 to 3 days in culture solution, and the culture solution contains a large number of the monoclonal antibodies II secreted by the hybridoma cells at this moment; the monoclonal antibodies II are combined with the receptors on the cell membranes of the hemocytes in a specific mode by the detection of an indirect immunofluorescence method, and the hemocyte membrane presents yellowish green fluorescent under the observation by a fluorescence microscope; by the detection of an enzyme-linked immunosorbent assay method, OD value is detected by an enzyme-labeling instrument, and the ratio value of the OD value to negative control OD value is not less than 2. The monoclonal antibodies II can be suitable for mass industrial production, and perform a positive effect on preventing or treating melanodermia virus diseases of prawns.
Description
Technical field
The present invention relates to the improvement of the utilisation technology of antibody, specifically be a kind of anti-from spot syndrome virus (White Spot Syndrome Virus by the hybridoma excretory, WSSV) monoclonal antibody of acceptor and preparation method thereof, it belongs to molecular immunology and virusology interleaving techniques field.
Background technology
At present, the Leucodermia virus disease is to endanger one of serious disease in the prawn culturing, causes very high prawn mortality ratio, has caused tremendous loss to shrimp culture industry.Cause that this sick Leucodermia virus can extensively infect the subcutis of prawn, hemopoietic tissue, reticular tissue, lymphatic organ official rank, hemocyte is the main target cell of this virus infection, so has the acceptor of Leucodermia virus on the prawn hemocyte.The more employing of the screening of virus receptor virus overlay protein blot assay (virusoverlay protein blot assay) technology.Use the precondition of this technology screening virus receptor: receptor active must be by single expression of polypeptides, and this polypeptide can not have other cell membrane component to combine with viral under helping simultaneously, and does not lose its activity in washing agent.But some Virus receptors is after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation, transfer printing, the space structure of receptor protein is destroyed and lost the help of other cell membrane component, lost actively, can't screen this receptoroid with virus overlay protein blot assay's technology with combining of virus.The screening that is applied as virus receptor of monoclonal antibody technique, discriminating provide new technique means.At first prepare the monoclonal antibody of anti-cell surface antigen and set up the clone responsive virus; again by the sealing effect of monoclonal antibody to acceptor on the viral sensitive cells; filter out and to protect cell to avoid the antibody of virus infection, then with this antibody discriminating, isolated viral acceptor.At present, not only successfully do not set up clone both at home and abroad as yet, make and adopt aforesaid method screening Leucodermia virus acceptor to be restricted, and do not appear in the newspapers yet with the research of monoclonal antibody technology screening, discriminating Leucodermia virus acceptor to the Leucodermia virus sensitivity.
Summary of the invention
The objective of the invention is to provide a kind of monoclonal antibody by hybridoma excretory virus receptor of anti leukoplakia disease and preparation method thereof.The present invention intends the small white mouse with Chinese prawn blood cell immunity balb/c, learn a skill by biological immune, screening and clone obtain secreting the hybridoma of the monoclonal antibody of virus receptor of anti leukoplakia disease, and then develop the using value of this monoclonal antibody, for coming out early, the medicine of prevention and treatment prawn white spot syndrome virus or vaccine perform technology pad shop.Task of the present invention is finished by following technical scheme, has developed a kind of monoclonal antibody by hybridoma excretory virus receptor of anti leukoplakia disease.The preserving number of this hybridoma: CCTCC-C200418, the classification name: mouse hybridoma cell strain WSSV-R, preservation date: 2004/12/14, the CCTCC of depositary institution, preservation address: in the Chinese Wuhan University; The monoclonal antibody of its excretory specificity virus receptor of anti leukoplakia disease (hereinafter to be referred as: monoclonal antibody II) the Leucodermia virus receptor-specific that has on the cytolemma with the prawn hemocyte combines, thereby sealing this receptor, blocking virus enters cell, makes virus can not infect the function of prawn hemocyte; This hybridoma that grows fine is conventional in its nutrient solution cultivates 2---and 3 days, foster liquid promptly changed yellow into by pink, contained the oozy monoclonal antibody of a large amount of hybridomas---monoclonal antibody II in this nutrient solution; Detect through indirect immunofluorescence method, the receptor-specific on the cytolemma of this monoclonal antibody II and hemocyte combines, and observes under fluorescent microscope, and the hemocyte film presents bright yellow-green fluorescence, and endoglobar nuclear area does not have fluorescence; Detect through the enzyme linked immunological adsorption method, microplate reader is surveyed the OD value: the receptor-specific on the cytolemma of monoclonal antibody II and hemocyte combines, and the ratio of the OD value of its OD value and negative control is not less than 2.
Described MONOCLONAL ANTIBODIES SPECIFIC FOR method by hybridoma excretory virus receptor of anti leukoplakia disease is with Chinese prawn blood cell immunity balb/c small white mouse, merge through oncocyte and by the splenocyte of immunized mice, screening and clone obtain the monoclonal antibody of anti-Chinese prawn hemocyte---and be monoclonal antibody I; From this monoclonal antibody I, select the method for screening monoclonal antibody II again, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
Described this monoclonal antibody II by the transfer printing western blotting method, can determine the molecular weight of Leucodermia virus acceptor on the cytolemma of prawn hemocyte.
The molecular weight of the receptor antigen of described Leucodermia virus is 175-200kDa.
One of method of described selection screening monoclonal antibody II is: prawn hemocyte film is combined external with this monoclonal antibody I, adopt spot immune trace method, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
Two of the method for described selection screening monoclonal antibody II is: prawn hemocyte film is combined external with this monoclonal antibody I, adopt the enzyme linked immunological adsorption method, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
Three of the method for described selection screening monoclonal antibody II is: the monoclonal antibody method that adopts former generation prawn hemocyte cultivation screening virus receptor of anti leukoplakia disease, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
The invention has the advantages that: developed a kind of monoclonal antibody by hybridoma excretory virus receptor of anti leukoplakia disease.The preserving number of this hybridoma: CCTCC-C200418, the Leucodermia virus receptor-specific that the monoclonal antibody of its excretory specificity virus receptor of anti leukoplakia disease has on the cytolemma with the prawn hemocyte combines, thereby sealing this receptor, blocking virus enters cell, makes virus can not infect the function of prawn hemocyte.Shown in Figure 1 as the functional schematic of monoclonal antibody II of the present invention: the Leucodermia virus acceptor 2 on the Chinese prawn hemocyte film 1 at first combines with monoclonal antibody II3 specificity, Leucodermia virus 4 just can not be adsorbed in the Leucodermia virus acceptor and combine with it, and Leucodermia virus just can not infect the prawn hemocyte by this receptor like this.Therefore monoclonal antibody II of the present invention has and prevents that Leucodermia virus from infecting the function of prawn hemocyte.This hybridoma can be observed under inverted phase contrast microscope in external unlimited breeding: the growth conditions good cell, and full, perfectly round, bright, the big or small homogeneous of its outward appearance, adherent property are good; The hybridoma that grows fine is conventional the cultivation 2-3 days in its nutrient solution, and this nutrient solution promptly changes yellow into by pink, contains the oozy monoclonal antibody of a large amount of hybridomas---monoclonal antibody II in this nutrient solution.The genetic engineering antibody that this monoclonal antibody II or process are transformed becomes the medicine or the vaccine of prawn white spot syndrome virus probably, enters the effect of playing prevention or treatment prawn white spot syndrome virus disease in the prawn body by modes such as oral or injections.Detect through indirect immunofluorescence method, the receptor-specific on the cytolemma of this monoclonal antibody II and hemocyte combines, and observes under fluorescent microscope, and the hemocyte film presents bright yellow-green fluorescence, and endoglobar nuclear area does not have fluorescence; Detect through the enzyme linked immunological adsorption method, microplate reader is surveyed the OD value: the receptor-specific on the cytolemma of monoclonal antibody II and hemocyte combines, and the ratio of the OD value of its OD value and negative control is not less than 2.Monoclonal antibody II of the present invention can mass industrialized production, for prevention or treatment prawn white spot syndrome virus disease have a positive effect.Because monoclonal antibody II of the present invention has determined the receptor protein of a prawn white spot syndrome virus, can obtain the aminoacid sequence of this receptor protein and then obtain its gene order through order-checking, remove the intravital acceptor gene of prawn by genetically engineered again, the shrimp seedling of anti-Leucodermia virus might be cultivated, thereby the puzzlement of Leucodermia virus can be thoroughly solved world's shrimp farming.In a word, monoclonal antibody II has very big potentiality to be exploited in research and production from now on.
Description of drawings and embodiment thereof
Fig. 1 is the functional schematic of monoclonal antibody II of the present invention.
Fig. 2 is the technological process block-diagram of monoclonal antibody II preparation of the present invention.
Fig. 3 is external in conjunction with Experiment Preparation monoclonal antibody II employing spot immune trace method The selection result figure.
Fig. 4 is external in conjunction with Experiment Preparation monoclonal antibody II employing enzyme linked immunological adsorption method The selection result figure.
Fig. 5 is applied to identify the figure as a result of the Leucodermia virus acceptor molecule amount on the Chinese prawn hemocyte film for monoclonal antibody II of the present invention.
Fig. 6 is applied to identify the figure as a result of the Leucodermia virus acceptor molecule amount on other shrimps hemocyte film for monoclonal antibody II of the present invention.
Fig. 7 is applied to identify the figure as a result of the Leucodermia virus acceptor molecule amount on the crab class hemocyte film for monoclonal antibody II of the present invention.
Shown in Fig. 1: the Leucodermia virus acceptor 2 on the Chinese prawn hemocyte film 1 at first combines with monoclonal antibody II3 specificity, Leucodermia virus 4 just can not be adsorbed in the Leucodermia virus acceptor and combine with it, and Leucodermia virus just can not infect the prawn hemocyte by this receptor like this.
Screening method 1 among Fig. 2 is: spot immune trace method screening monoclonal antibody II.Screening method 2 is: enzyme linked immunological adsorption method screening monoclonal antibody II.Screening method 3 is: former generation prawn hemocyte cultivation screening monoclonal antibody II.
Among Fig. 3: A. is the monoclonal antibody I reaction of prawn hemocyte film and non-blocking, hatches the result who is obtained by the spot immune trace again with the Leucodermia virus of the high zinc mark in ground.B. for prawn hemocyte film with the monoclonal antibody I reaction of sealing process is arranged, hatch the result who obtains by the spot immune trace again with the Leucodermia virus of the high zinc mark in ground.C. the result who obtains by the spot immune trace for contrast (prawn hemocyte film and the monoclonal antibody I reaction of sealing process is arranged is hatched with the Leucodermia virus of high zinc mark deactivatedly again).Among the figure 1 and 2 be two groups parallel.
Among Fig. 4: 1. be the monoclonal antibody I reaction of prawn hemocyte film and non-blocking, the Leucodermia virus with the high zinc mark in ground reacts again, detects the OD value result who obtains by enzyme linked immunological absorption.2. be prawn hemocyte film and the monoclonal antibody I reaction that sealing process is arranged, the Leucodermia virus with the high zinc mark in ground reacts again, detects the OD value result who obtains by enzyme linked immunological absorption.3. detect the OD value result who obtains for contrast (prawn hemocyte film and the monoclonal antibody I reaction that sealing process is arranged are again with the Leucodermia virus reaction of high zinc mark deactivatedly) by enzyme linked immunological absorption.
Fig. 5: adopt the transfer printing western blotting method to determine the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II discerns on Chinese prawn hemocyte film.1 is the proteic Solargentum coloration result of standard molecular weight; 2 is that the Chinese prawn blood cell protein is separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, through the painted result of Solargentum; 3 molecular weight of discerning on Chinese prawn hemocyte film for monoclonal antibody II of the present invention from spot syndrome virus acceptor are 175kDa.
Fig. 6: adopt the transfer printing western blotting method to determine the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II discerns on Penaeus vannamei, japonicus and Procambius clarkii hemocyte film.1 is the proteic Solargentum coloration result of standard molecular weight; 2 is that the japonicus blood cell protein is separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, through the painted result of Solargentum; The molecular weight of the 3 Leucodermia virus acceptors of discerning on japonicus hemocyte film for monoclonal antibody II of the present invention is 175kDa; 4 is that the Penaeus vannamei blood cell protein is separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, through the painted result of Solargentum; 5 molecular weight of discerning on Penaeus vannamei hemocyte film for monoclonal antibody II of the present invention from spot syndrome virus acceptor are 200kDa; 6 is that the former whole shrimp blood cell protein of Ke Shi is separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, through the painted result of Solargentum; The molecular weight of the 7 Leucodermia virus acceptors of discerning on Procambius clarkii hemocyte film for monoclonal antibody II of the present invention is 200kDa.
Fig. 7: adopt the transfer printing western blotting method to determine the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II discerns on mitten crab and swimming crab hemocyte film.1 is the proteic Solargentum coloration result of standard molecular weight; 2 is that the mitten crab blood cell protein is separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, through the painted result of Solargentum; The molecular weight of the 3 Leucodermia virus acceptors of discerning on mitten crab hemocyte film for monoclonal antibody II of the present invention is 200kDa; 4 is that the swimming crab blood cell protein is separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, through the painted result of Solargentum; The molecular weight of the 5 Leucodermia virus acceptors of discerning on swimming crab hemocyte film for monoclonal antibody II of the present invention is 200kDa.
A kind of monoclonal antibody referring to Fig. 1-7 the present invention development by hybridoma excretory virus receptor of anti leukoplakia disease.The preserving number CCTCC-C200418 of this hybridoma, the Leucodermia virus receptor-specific that the monoclonal antibody of its excretory specificity virus receptor of anti leukoplakia disease has on the cytolemma with the prawn hemocyte combines, thereby sealing this receptor, blocking virus enters cell, makes virus can not infect the function of prawn hemocyte; This hybridoma can be observed under inverted phase contrast microscope in external unlimited breeding: full, perfectly round, bright, the big or small homogeneous of growth conditions good cell outward appearance, adherent property are good; The hybridoma that grows fine is conventional the cultivation 2-3 days in its nutrient solution, and nutrient solution promptly changes yellow into by pink, contains the oozy monoclonal antibody of a large amount of hybridomas---monoclonal antibody II in this nutrient solution; Detect through indirect immunofluorescence method, the receptor-specific on the cytolemma of this monoclonal antibody II and hemocyte combines, and in the fluorescence microscopy combination, observes under fluorescent microscope, and the hemocyte film presents bright yellow-green fluorescence, and endoglobar nuclear area does not have fluorescence; Detect through the enzyme linked immunological adsorption method, microplate reader is surveyed the OD value: the receptor-specific on the cytolemma of monoclonal antibody II and hemocyte combines, and the ratio of the OD value of its OD value and negative control is not less than 2.
Described this preparation method is with Chinese prawn blood cell immunity balb/c small white mouse, merges through oncocyte and by the splenocyte of immunized mice, and screening and clone obtain the monoclonal antibody of anti-Chinese prawn hemocyte---be monoclonal antibody I; From this monoclonal antibody I, select the method for screening monoclonal antibody II again, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
Described this monoclonal antibody II by the transfer printing western blotting method, can determine the molecular weight of identification Leucodermia virus acceptor on the cytolemma of prawn hemocyte.
The molecular weight of described Leucodermia virus acceptor is 175-200kDa.As Fig. 5-shown in Figure 7.
One of described method of screening monoclonal antibody II from monoclonal antibody I is: prawn hemocyte film is combined external with this monoclonal antibody I, adopt spot immune trace method, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
Described from monoclonal antibody I screening monoclonal antibody II method two be: prawn hemocyte film is combined external with this monoclonal antibody I, adopt the enzyme linked immunological adsorption method, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
Described from monoclonal antibody I screening monoclonal antibody II method three be: adopt former generation prawn hemocyte monoclonal antibody method of cultivating the screening virus receptor of anti leukoplakia disease, screening can be sealed the monoclonal antibody of virus receptor---the monoclonal antibody of virus receptor of anti leukoplakia disease, i.e. monoclonal antibody II.
Embodiment 1. development monoclonal antibody I:
1) antigenic preparation:
Get the healthy sea of the long 18-20cm of body and catch Chinese prawn, with cotton ball soaked in alcohol sterilization prawn head cuirass trailing edge, with drawing precooling antithrombotics (sodium-chlor 8.2g/L, citric acid 5.5g/L, glucose 19.8g/L, Trisodium Citrate 8.8g/L, pH5.6) 5ml Dispoable medical syringe inserts prawn heart equal proportion and extracts blood, centrifugal 400g, 10 minutes, 4 ℃.
Remove supernatant liquor, with the resuspended hemocyte precipitation of antithrombotics, centrifugal again.So repeat 3 times, obtain pure prawn hemocyte.With the 0.01M phosphate buffered saline buffer (137mM sodium-chlor, 2.7mM Repone K, the 8.09mM Sodium phosphate dibasic, the 1.47mM potassium primary phosphate, pH7.4) concentration of adjusting hemocyte reaches 2 * 10
8Individual/milliliter.
2) immunity:
With the Chinese prawn hemocyte of purifying, the female small white mouse of immunity 4 week balb/c in age, each immunizing dose is 0.1ml, amounts to 2 * 10
7Individual hemocyte.Immunity divides to be carried out for 4 times:
For the first time, fundamental immunity: with the right belly of cotton ball soaked in alcohol sterilization small white mouse, the 1ml syringe of drawing the 0.1ml blood cell suspension is inserted intraperitoneal mouse, injection finishes, and sterilizes the injection site in case infect with cotton ball soaked in alcohol.
For the second time, booster immunization: carry out the immunity second time after 2 of immunity weeks for the first time,, the 1ml syringe of drawing the 0.1ml blood cell suspension is inserted intraperitoneal mouse with the right belly of cotton ball soaked in alcohol sterilization small white mouse, injection finishes, and sterilizes the injection site in case infect with cotton ball soaked in alcohol.
For the third time, secondary booster immunization: carry out immunity for the third time after 1 of immunity week for the second time,, the 1ml syringe of drawing the 0.1ml blood cell suspension is inserted the small white mouse tail vein with cotton ball soaked in alcohol sterilization small white mouse afterbody, injection finishes, and sterilizes the injection site in case infect with cotton ball soaked in alcohol.
The 4th time, amplification immunity before merging: carry out the 4th immunity after Mian Yi 1 week for the third time,, the 1ml syringe of drawing the 0.1ml blood cell suspension is inserted the small white mouse tail vein with cotton ball soaked in alcohol sterilization small white mouse afterbody, injection finishes, and sterilizes the injection site in case infect with cotton ball soaked in alcohol.
3) merge:
Through the 3rd day after 4 immunity, put to death small white mouse, take out splenocyte, carry out cytogamy with P3U1 myeloma cell.The cytogamy operation steps is as follows:
(1) the etherization immunity behind aseptic taking-up spleen and the thymocyte (feeder cell), is crossed 100 order mesh screens for the spleen mouse respectively, blows down the formation single cell suspension with RPMI1640 (-) solution.
(2) splenocyte suspension and 1000 rev/mins of thymus cell suspensions is centrifugal 3 minutes, abandoning supernatant, the splenocyte precipitation is resuspended with RPMI1640 (-) solution, and the thymocyte precipitation is resuspended with RPMI1640 (-) selective medium (containing 10% calf serum and 1%HAT).
(3) get 3 * 10
7The individual P3U1 myeloma cell who is in logarithmic phase, 1000 rev/mins centrifugal 3 minutes, remove supernatant liquor after, with the resuspended P3U1 myeloma cell's precipitation of RPMI1640 (-) solution.
(4) splenocyte suspension and P3U1 myeloma cell's suspension are mixed after, 1000 rev/mins centrifugal 3 minutes, 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.
(5) centrifuge tube is placed the pre-warm beaker that is filled with water of 37 ℃ of water-baths,, and slowly be added drop-wise in the cell precipitation, in water-bath, left standstill then 5 minutes with the polyglycol solution 1ml of the pre-temperature of suction pipe absorption to 37 ℃.
(6) continue to have dripped temperature in advance to 37 ℃ RPMI1640 (-) solution 15ml, polyoxyethylene glycol has been diluted and ineffective.The method that adds is: preceding half a minute, add 1ml; Back half a minute, add 3ml; In the 2nd minute, add 15ml then.
(7) add RPMI1640 (-) solution to 40ml, through 1000 rev/mins centrifugal 5 minutes, abandoning supernatant.
(8) sedimentary cell gently is suspended from 3ml RPMI1640 (-) selective medium, behind the frozen 2ml, adds RPMI1640 (-) selective medium and feeder cell, mix every hole, back 100 microlitres and be added drop-wise in 96 orifice plates, be placed on CO
2Concentration is the CO of 4.5% and 37 ℃ of fixed temperature and humidity condition
2Culturing cell in the incubator.
4) screening and clone:
Merge about two weeks of back, hybridoma group head is when accounting for the hole floorage 1/3 of 96 well culture plates, and the employing indirect immunofluorescence method screens positive hybridoma cell.
The step of indirect immunofluorescence method screening positive hybridoma cell:
(1) antigenic fixing:
(1) with cotton ball soaked in alcohol sterilization prawn head cuirass trailing edge, inserts Chinese prawn heart equal proportion with the 5ml disposable syringe of drawing the precooling antithrombotics and extract blood.
(2) the centrifugal 400g of blood cell suspension that extracts, 10 minutes, 4 ℃.
(3) remove supernatant liquor, with the resuspended hemocyte precipitation of antithrombotics, centrifugal again.So repeat 3 times, obtain pure hemocyte, with the resuspended hemocyte of 0.01M phosphate buffered saline buffer.
(4) blood cell suspension is dropped on the clean slide glass, sedimentation is 1 hour in the at room temperature wet box.
(5) air-dry under the taking-up slide glass room temperature from wet box.
(6) put into acetone and fix 20 minutes, take out air-dry.
(7) be kept in-20 ℃ the refrigerator standby.
(2) indirect immunofluorescence reaction:
(1) is first antibody with hybridoma supernatant (hybridoma nutrient solution), is added on the hemocyte sample of slide glass.
Hatched 45 minutes in (2) 37 ℃ of wet boxes.
(3) take out slide glass, it is inferior to give a baby a bath on the third day after its birth with the 0.01M phosphate buffered saline buffer, each 5 minutes.
(4) goat anti-mouse antibody with marked by fluorescein isothiocyanate is a second antibody, is added on the hemocyte sample.
Hatched 45 minutes in (5) 37 ℃ of wet boxes.
(6) take out slide glass, it is inferior to give a baby a bath on the third day after its birth with the 0.01M phosphate buffered saline buffer, each 5 minutes.
(7) glycerine mounting.
(8) fluorescent microscope is observed down.
(3) adopt limiting dilution assay that the positive hybridoma cell that filters out is cloned, clone's step is as follows:
(1) etherization mouse behind the aseptic taking-up thymocyte (feeder cell), is ground on 100 order mesh screens, blows down the formation single cell suspension with RPMI1640 (-) solution.
(2) centrifugal 3 minutes of 1000 rev/mins of thymus cell suspensions, abandoning supernatant, the thymocyte precipitation is resuspended with RPMI1640 (-) substratum (adding 10% foetal calf serum).
(3) cell in the positive cell hole that will clone is counted with blood cell counting plate, use RPMI1640 (-) substratum (adding 10% foetal calf serum) with 10 whole time doubly dilution then, take out 100 hybridomas, put into RPMI1640 (-) substratum (the adding 10% foetal calf serum) suspension of thymocyte.
(4) cell suspension with dropper piping and druming evenly splashes into one 96 porocyte culture plate, and on average a hybridoma is contained in each hole.
(5) cell is placed on CO
2Concentration is the CO of 4.5% and 37 ℃ of fixed temperature and humidity condition
2Cultivate in the incubator.
After (6) two weeks, detect, the hybridoma in gained positive colony hole is cloned one time again, to guarantee to form monoclonal hybridoma with indirect immunofluorescence method.
5) frozen:
Treat that hybridoma is in logarithmic phase, blow and beat into the hybridoma suspension with dropper, add methyl-sulphoxide (9 parts of complete culture solution+1 part methyl-sulphoxides, methyl-sulphoxide is a cryoprotectant), making final cell density is 3-5 * 10
6Individual/milliliter.Be sub-packed in the frozen pipe of 2ml with the 1ml cell suspension, tighten blind nut, frozen pipe is packed into fills in the capsule of cotton then, place spend the night in-70 ℃ of Ultralow Temperature Freezers (8-12 hour) after, immerse prolonged preservation in the liquid nitrogen again.
Embodiment 2. screens monoclonal antibody II from monoclonal antibody I:
1) purification of Leucodermia virus:
(1) get the shrimp head of the Chinese prawn that Leucodermia virus infects, remove the liver pancreas, take by weighing 10 gram pathological material of diseases, add 2ml25% sucrose, quartz sand grinds 3-5 minute (4 ℃), makes cytoclasis, and virus is free in solution, adds 100ml25% sucrose then, mixing.
(2) centrifugal 3000 rev/mins of pathological material of diseases that grind, 15 minutes.
(3) get supernatant, 5000 rev/mins are centrifugal, and 15 minutes, 4 ℃.
(4) get supernatant, 8000 rev/mins are centrifugal, and 15 minutes, 4 ℃.
(5) get supernatant, 25000 rev/mins (P70AT rotors) are centrifugal, and 2 hours, 4 ℃.
(6) get precipitation, resuspended with 8ml 25% sucrose solution, magnetic stirring apparatus slowly stirred 1 hour, 4 ℃.
(7) the viral crude extract that stirs is laid on above the discontinuous sucrose density gradient, saccharose gradient is 33%, 40%, 46%, 52%, 57%, 62% (W/W), ultracentrifugation 25000 rev/mins (P40ST rotor) 2 hours, 4 ℃.
(8) get virus band between 46% and 52%, be the virus of purifying.
2) Leucodermia virus of the high zinc mark purification of land used:
(1) get the suspension (protein concentration is 1 mg/ml) of the Leucodermia virus that 1ml purifies, add the high zinc solution in 7 microlitre methyl-sulphoxide dissolved ground, room temperature was slowly stirred 2 hours.
(2) bovine serum albumin solution of the 2-3% that is made into 10ml 0.01M phosphate buffered saline buffer sealing Sephadex-G25 gel chromatography column.
(3) with 30ml 0.01M phosphate buffered saline buffer flushing pillar.
(4) the viral mixed solution of the high zinc mark in ground is crossed the Sephadex-G25 gel chromatography column, carry out sieve chromatography.
(5) liquid that chromatography is gone out is collected with the 0.5ml centrifuge tube, 5/pipe, collects 50 pipes altogether.
(6) from every pipe, take out 0.5 microlitre point respectively on nitrocellulose filter, dry naturally.So prepare the nitrocellulose filter of 2 Zhang Pings row, be labeled as No. 1 nitrocellulose filter and No. 2 nitrocellulose filters.
(7) 2 nitrocellulose filters are placed in the bovine serum albumin of the 2-3% that the 0.01M phosphate buffered saline buffer is made into hatched 37 ℃ 45 minutes.
(8) take out 2 nitrocellulose filters, wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(9) No. 1 nitrocellulose filter is put into the monoclonal antibody of anti-Leucodermia virus, No. 2 nitrocellulose filters are put into the high anti-ly zinc antibody of alkali phosphatase enzyme mark, hatched 37 ℃ 45 minutes.
(10) take out 2 nitrocellulose filters respectively, wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(11) No. 1 nitrocellulose filter is put into the goat anti-mouse igg antibody (be diluted at 1: 30000 0.01M phosphate buffered saline buffer) of alkali phosphatase enzyme mark, hatched 37 ℃ 45 minutes.No. 2 nitrocellulose filters are put into alkaline phosphatase color development liquid (NBT-BCIP color development liquid) color development, till color (blueness) is clear.
(12) take out No. 1 nitrocellulose filter, wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(13) No. 1 nitrocellulose filter is put into alkaline phosphatase color development liquid (NBT-BCIP color development liquid) color development, till color (blueness) is clear.
From the 50 pipe solution that step (5) is collected, filter out and to detect the viral solution that Leucodermia virus can detect the high zinc in ground again, be the good Leucodermia virus of the high zinc mark in ground.
3) purification of Chinese prawn hemocyte film:
(1) with putting forward film homogenate buffer (10mM hydroxyethyl piperazine ethanesulfonic acid, the 2mM disodium ethylene diamine tetraacetate, the 2mM phenylmethylsulfonyl fluoride, pH7.4) Chinese prawn hemocyte (it is the same to extract the hemocyte method) is caught in the sea of resuspended purification, put into manually homogenate of glass homogenizer, smudge cells.
(2) centrifugal hemocyte homogenate 200g, 20 minutes, 4 ℃.
(3) get centrifuged supernatant, 8000g is centrifugal, and 20 minutes, 4 ℃.
(4) get centrifuged supernatant, 100,000g ultracentrifugation, 20 minutes, 4 ℃.
(5) get hemocyte film precipitation, resuspended with the 0.01M phosphate buffered saline buffer, put-80 ℃ frozen standby.
4) spot immune trace method screening monoclonal antibody II:
(1) get the Chinese prawn hemocyte film suspension that 0.5 microlitre is purified, point dries on nitrocellulose filter naturally, and the bovine serum albumin solution of putting into the 2-3% that the 0.01M phosphate buffered saline buffer is made into sealed 37 ℃ 45 minutes.Take out nitrocellulose filter, wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(2) nitrocellulose filter is put into monoclonal antibody I and hatched 37 ℃ 45 minutes.Take out nitrocellulose filter, wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(3) the Leucodermia virus liquid that nitrocellulose filter is put into the high zinc mark in ground is hatched 4 hours (Leucodermia virus that replaces high activatedly zinc mark with the Leucodermia virus of the high zinc mark in ground of deactivation in contrast), 4 ℃.Take out nitrocellulose filter, wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(4) the high anti-ly zinc antibody that nitrocellulose filter is put into alkali phosphatase enzyme mark was hatched 37 ℃ 45 minutes.Take out nitrocellulose filter and wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(5) nitrocellulose filter is put into alkaline phosphatase color development liquid (NBT-BCIP color development liquid) color development, till color (blueness) is clear.
Result: the monoclonal antibody I reaction of prawn hemocyte film and non-blocking, hatch with the Leucodermia virus of the high zinc mark in ground again, because the Leucodermia virus of the high zinc mark in ground and all receptors bind on the prawn hemocyte film are very dark by the color as a result that the spot immune trace obtains; Prawn hemocyte film and the monoclonal antibody I reaction that sealing process is arranged are hatched with the Leucodermia virus of the high zinc mark in ground again, because the part acceptor is closed the Leucodermia virus of the high zinc mark in ground can't be combined with it, and be more shallow by the color as a result that the spot immune trace obtains; Contrast (prawn hemocyte film and the monoclonal antibody I reaction that sealing process is arranged, hatch with the Leucodermia virus of high zinc mark deactivatedly again) and since the Leucodermia virus of the high zinc mark in ground of inactivation can not with the receptors bind on the prawn hemocyte film, by the result that the spot immune trace obtains almost colourless (as Fig. 3).
5) enzyme linked immunological adsorption method screening monoclonal antibody II:
(1) gets the Chinese prawn hemocyte film suspension that 100 microlitres are purified, on enzyme plate, wrap and spent the night, the bovine serum albumin solution sealing of the 2-3% that adding 0.01M phosphate buffered saline buffer is made into 45 minutes, 37 ℃.Remove confining liquid, wash enzyme plate 3 times with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20), each 5 minutes.
(2) hatching 37 ℃ 45 minutes in the monoclonal antibody I adding enzyme plate.Wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(3) the Leucodermia virus liquid of the high zinc mark in ground is added hatch 4 hours (Leucodermia virus that replaces high activatedly zinc mark with the Leucodermia virus of the high zinc mark in ground of deactivation in contrast), 4 ℃ in the enzyme plate.Wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(4) hatching 37 ℃ 45 minutes in the high anti-ly zinc antibody adding enzyme plate of alkali phosphatase enzyme mark.Wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
(5) alkaline phosphatase color development liquid (NBT-BCIP color development liquid) was added the enzyme plate color development 20 minutes, 2M NaOH stops color development, and microplate reader is surveyed the OD value.As shown in Figure 4.
Result: the monoclonal antibody I reaction of prawn hemocyte film and non-blocking, hatch with the Leucodermia virus of the high zinc mark in ground again, because the Leucodermia virus of the high zinc mark in ground and all receptors bind on the prawn hemocyte film, it is bigger to detect the OD value that obtains by enzyme linked immunological absorption; Prawn hemocyte film and the monoclonal antibody I reaction that sealing process is arranged are hatched with the Leucodermia virus of the high zinc mark in ground again, because the part acceptor is closed the Leucodermia virus of the high zinc mark in ground can't be combined with it, and be less by the OD value that enzyme linked immunological absorption detection obtains; Contrast (prawn hemocyte film and the monoclonal antibody I reaction that sealing process is arranged, hatch with the Leucodermia virus of high zinc mark deactivatedly again) and since the Leucodermia virus of the high zinc mark in ground of inactivation can not with the receptors bind on the prawn hemocyte film, detect the OD value very little (as Fig. 4) that obtains by enzyme linked immunological absorption.
6) with the former breeding method screening monoclonal antibody II that is commissioned to train of Chinese prawn hemocyte:
(1) experiment is Leibovitz ' s 2 * L-15 with minimum medium, adds 2.4% sodium-chlor, 2% glucose, 100UI/mL penicillin and 100 μ g/ml Streptomycin sulphates, pH7.2-7.4, and 0.22 μ m membrane filtration, bacteria culture medium is cultivated, the aseptic length of being born.Add 20% calf serum.
(2) 75% alcohol disinfecting Chinese prawn body surfaces, the sterilization of heart place cotton ball soaked in alcohol.
(3) insert prawn heart equal proportion with the 5ml Dispoable medical syringe of drawing the precooling antithrombotics and extract blood.Centrifugal 400g 10 minutes, abandons supernatant.
(4) the hemocyte precipitation is resuspended with substratum, splashes into 96 porocyte culture plates, 50 microlitres/hole, and 29 ℃ of constant temperature culture, after 2 hours, the phase microscope observation of cell is adherent, adds cell culture fluid, 100 microlitres/hole.
(5) after the hemocyte of Pei Yanging is stablized 3 days, absorb nutrient solution, add monoclonal antibody I to be screened and hatched 1 hour, change to the viral crude extract of 0.45 μ m membrane filtration degerming, hatched 1 hour, and changed to cell culture fluid for 29 ℃, compare with the culturing cell that does not add virus, each gradient is established 4 repetitions.
(6) use the pathological change of inverted phase contrast microscope observation of cell every day.
The result: infective virus former be commissioned to train nourish blood the pathological change of cell show as cell in heaps, hang, cell debris occurs in the nutrient solution.The former foster hemocyte of being commissioned to train of hatching with the monoclonal antibody I of non-blocking just showed pathological change on the 3rd day; It is later that pathological change appears in the former foster hemocyte of being commissioned to train of hatching with the monoclonal antibody I that sealing process is arranged (being monoclonal antibody II), and the 5th talent has shown pathological change (seeing Table 1).
The protection effect of table 1. monoclonal antibody I
| First day | Second day | The 3rd day | The 4th day | The 5th day | The 6th day | The 7th day | |
| Non-blocking monoclonal antibody I has sealing process monoclonal antibody I | - - | - - | + - | ++ - | +++ + | +++ ++ | ++++ +++ |
| Contrast | - | - | - | - | - | - | - |
Note: "-" representative does not show pathological change; The degree of "+" representative performance pathological change, "+" the existing pathological change of multilist is serious more more.
Embodiment 3. monoclonal antibody II of the present invention one of use, and adopt the transfer printing western blotting method to determine the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II discerns on Chinese prawn hemocyte film:
Employed separation gel of table 2 sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concentrated glue composition:
| Separation gel 10% concentration 16ml | Concentrate glue 4.75% concentration 7.5ml |
| Distilled water 6.45ml | Distilled water 4.33ml |
| 30% acrylamide 5.3ml| | 30% acrylamide 1.19ml |
| 15M Tris-HCl 4ml | 0.5M Tris-HCl 1.88ml |
| 10% sodium laurylsulfonate 160ul | 10% sodium laurylsulfonate 75ul |
| 100% ammonium persulphate 60ul | 100% ammonium persulphate 60ul |
| Tetramethyl Ethylene Diamine (TEMED) 15ul | Tetramethyl Ethylene Diamine (TEMED) 15ul |
| Fall behind the glue to solidify in 10 minutes | Fall behind the glue to solidify in 2 minutes |
1) the Chinese prawn hemocyte of purifying is separated with sodium laurylsulfonate-polyacrylamide gel electrophoresis.
Sodium laurylsulfonate-polyacrylamide gel electrophoresis operation steps:
(1) glue:
The electrophoresis chamber that use is bought from BIO-RAD company.Gel is formed (seeing Table 2) by separation gel (Running gel) and concentrated glue (Stackinggel) two portions.Earlier separation gel is poured in the glass sandwich, top distilled water front cover, keep the glue face smooth, after the glue polymerization to be separated, with the distilled water on syringe (syringe is mixed dentistry minute hand head No. 5) sucking-off separation gel surface, add concentrated glue then, insert the point sample comb, after waiting to concentrate the glue polymerization, take out the point sample comb.
(2) the Chinese prawn hemocyte is handled:
The Chinese prawn hemocyte is resuspended with the 0.01M phosphate buffered saline buffer, add the equivalent sample buffer, boiled cooling in the boiling water 1-2 minute.
(3) electrophoresis and aftertreatment:
The doubling glass plate is fixed in the electrophoresis chamber, and syringe adds the distilled water in the minute hand head absorption point sample hole No. 5, and arrangement point sample duct is drawn sample to be analyzed respectively with micro sample adding appliance, adds sample 10 microlitres in every hole.Add the electrophoresis damping fluid and go into up and down in the storage tank, connect power supply, under the current stabilization condition, use low current (30-40mA) when initial, treat sample after concentrated glue partial concentration becomes a line, strengthen electric current (50-70mA), when the tetrabromophenol sulfonphthalein indicator reaches bottom margin, can stop electrophoresis.Electrophoresis finishes, and gently pries open layer glass, takes out gel.Downcut the gel of a runway, Solargentum dyeing.In order to identify whether sodium laurylsulfonate-polyacrylamide gel electrophoresis is successful.
2) one of clip and the nitrocellulose filter (0.22 micron in aperture) of the identical size of running gel are with electrotransfer damping fluid (electrotransfer damping fluid: 25mmol/L Tris-Base, the 192mmol/L glycine, 20% methyl alcohol, pH8.3) 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, note in the entire operation at any time with glass stick and remove bubble between nitrocellulose filter or filter paper and the gel.By above-mentioned placement order, make blob of viscose and nitrocellulose filter and filter paper form sandwich " sandwich " of a cover.
3) be ready to electrotransfer groove and accessory thereof (comprising foam pad and supporting plate etc.).Above-mentioned " sandwich " is placed between two abundant wetted foam pads, and outermost layer is supported with two synthetic glass supporting plates again, and aperture is arranged on the supporting plate, and the electrotransfer damping fluid is freely entered in " gel sandwich ", makes liquid stream by this system.
4) " gel sandwich " inserted in the electrophoresis chamber that fills the electrotransfer damping fluid.With nitrocellulose filter towards anode.
5) electrophoresis current stabilization 200mA switched on 5 hours.
6) electrophoresis finishes, and takes out nitrocellulose filter, and half nitrocellulose filter is dyeed with the bright orchid of Kao Masi-R250, and 7% acetate is sloughed background color, to show protein band.
7) nitrocellulose filter that second half is had repeat samples was washed 10 minutes with the 0.01M phosphate buffered saline buffer, put in the bovine serum albumin solution of the 2-3% that the 0.01M phosphate buffered saline buffer is made into sealing then 45 minutes, 37 ℃.
8) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
9) nitrocellulose filter is put among the monoclonal antibody II, 37 ℃ are slowly shaken and spent the night in 1 hour or 4 ℃.
10) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
11) nitrocellulose filter is added the goat anti-mouse igg antibody (being diluted in 0.01M phosphate buffered saline buffer at 1: 30000) of alkali phosphatase enzyme mark, 37 ℃ were slowly shaken 1 hour.
12) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
13) nitrocellulose filter is put into alkaline phosphatase color development liquid (NBT-BCIP color development liquid) color development, till color (blueness) is clear.
14) use deionized water wash, with termination reaction.Nitrocellulose filter is clipped between filter paper drying.Putting the dark place preserves.
The result: the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II of the present invention discerns on Chinese prawn hemocyte film is the 175kDa (see figure 5).
Two of embodiment 4. monoclonal antibody II of the present invention application, adopt transfer immunity trace method to determine the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II discerns on japonicus, Penaeus vannamei and Procambius clarkii hemocyte film:
1) the former whole shrimp hemocyte of japonicus, Penaeus vannamei and Ke Shi of purifying is separated with sodium laurylsulfonate-polyacrylamide gel electrophoresis.
Sodium laurylsulfonate-polyacrylamide gel electrophoresis operation steps:
(1) glue:
The electrophoresis chamber that use is bought from BIO-RAD company.Gel is formed (seeing Table 2) by separation gel (Running gel) and concentrated glue (Stackinggel) two portions.Earlier separation gel is poured in the glass sandwich, top distilled water front cover, keep the glue face smooth, after the glue polymerization to be separated, with the distilled water on syringe (syringe is mixed dentistry minute hand head No. 5) sucking-off separation gel surface, add concentrated glue then, insert the point sample comb, after waiting to concentrate the glue polymerization, take out the point sample comb.
(2) the prawn hemocyte is handled:
The prawn hemocyte is resuspended with the 0.01M phosphate buffered saline buffer, add the equivalent sample buffer, boiled cooling in the boiling water 1-2 minute.
(3) electrophoresis and aftertreatment:
The doubling glass plate is fixed in the electrophoresis chamber, and syringe adds the distilled water in the minute hand head absorption point sample hole No. 5, and arrangement point sample duct is drawn sample to be analyzed respectively with micro sample adding appliance, adds sample 10 microlitres in every hole.Add the electrophoresis damping fluid and go into up and down in the storage tank, connect power supply, under the current stabilization condition, use low current (30-40mA) when initial, treat sample after concentrated glue partial concentration becomes a line, strengthen electric current (50-70mA), when the tetrabromophenol sulfonphthalein indicator reaches bottom margin, can stop electrophoresis.Electrophoresis finishes, and gently pries open layer glass, takes out gel.Downcut the gel of a runway, Solargentum dyeing.In order to identify whether sodium laurylsulfonate-polyacrylamide gel electrophoresis is successful.
2) one of clip and the nitrocellulose filter (0.22 micron in aperture) of the identical size of running gel are with electrotransfer damping fluid (electrotransfer damping fluid: 25mmol/L Tris-Base, the 192mmol/L glycine, 20% methyl alcohol, pH8.3) 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, note in the entire operation at any time with glass stick and remove bubble between nitrocellulose filter or filter paper and the gel.By above-mentioned placement order, make blob of viscose and nitrocellulose filter and filter paper form sandwich " sandwich " of a cover.
3) be ready to electrotransfer groove and accessory thereof (comprising foam pad and supporting plate etc.).Above-mentioned " sandwich " is placed between two abundant wetted foam pads, and outermost layer is supported with two synthetic glass supporting plates again, and aperture is arranged on the supporting plate, and the electrotransfer damping fluid is freely entered in " gel sandwich ", makes liquid stream by this system.
4) " gel sandwich " inserted in the electrophoresis chamber that fills the electrotransfer damping fluid.With nitrocellulose filter towards anode.
5) electrophoresis current stabilization 200mA switched on 5 hours.
6) electrophoresis finishes, and takes out nitrocellulose filter, and half nitrocellulose filter is dyeed with the bright orchid of Kao Masi-R250, and 7% acetate is sloughed background color, to show protein band.
7) nitrocellulose filter that second half is had repeat samples was washed 10 minutes with the 0.01M phosphate buffered saline buffer, put in the bovine serum albumin solution of the 2-3% that the 0.01M phosphate buffered saline buffer is made into sealing then 45 minutes, 37 ℃.
8) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
9) nitrocellulose filter is put among the monoclonal antibody II, 37 ℃ are slowly shaken and spent the night in 1 hour or 4 ℃.
10) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
11) nitrocellulose filter is added the goat anti-mouse igg antibody (being diluted in 0.01M phosphate buffered saline buffer at 1: 30000) of alkali phosphatase enzyme mark, 37 ℃ were slowly shaken 1 hour.
12) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
13) nitrocellulose filter is put into alkaline phosphatase color development liquid (NBT-BCIP color development liquid) color development, till color (blueness) is clear.
14) use deionized water wash, with termination reaction.Nitrocellulose filter is clipped between filter paper drying.Putting the dark place preserves.
The result: the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II of the present invention discerns on japonicus hemocyte film is 175kDa; The molecular weight of the Leucodermia virus acceptor of discerning on Penaeus vannamei hemocyte and the former whole shrimp hemocyte film of Ke Shi is 200kDa.(see figure 6).
Three of embodiment 5. monoclonal antibody II of the present invention application, adopt transfer immunity trace method to determine the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II discerns on whole crab of Chinese suede and swimming crab hemocyte film:
1) mitten crab of purifying is separated with sodium laurylsulfonate-polyacrylamide gel electrophoresis with the swimming crab hemocyte, when noting adding sample, on same clotting glue, do a application of sample that repeats, adopt two kinds of method colour generations during in order to last colour developing, be convenient to analytical results by the application of sample order.
Protein sodium laurylsulfonate-polyacrylamide gel electrophoresis:
(1) glue:
The electrophoresis chamber that use is bought from BIO-RAD company.Gel is formed (seeing Table 2) by separation gel (Running gel) and concentrated glue (Stackinggel) two portions.Earlier separation gel is poured in the glass sandwich, top distilled water front cover, keep the glue face smooth, after the glue polymerization to be separated, with the distilled water on syringe (syringe is mixed dentistry minute hand head No. 5) sucking-off separation gel surface, add concentrated glue then, insert the point sample comb, after waiting to concentrate the glue polymerization, take out the point sample comb.
(2) mitten crab and swimming crab hemocyte are handled:
Use the 0.01M phosphate buffered saline buffer resuspended respectively mitten crab and swimming crab hemocyte, add the equivalent sample buffer, boiled in the boiling water 1-2 minute, cooling.
(3) electrophoresis and aftertreatment:
The doubling glass plate is fixed in the electrophoresis chamber, and syringe adds the distilled water in the minute hand head absorption point sample hole No. 5, and arrangement point sample duct is drawn sample to be analyzed respectively with micro sample adding appliance, adds sample 10 microlitres in every hole.Add the electrophoresis damping fluid and go into up and down in the storage tank, connect power supply, under the current stabilization condition, use low current (30-40mA) when initial, treat sample after concentrated glue partial concentration becomes a line, strengthen electric current (50-70mA), when the tetrabromophenol sulfonphthalein indicator reaches bottom margin, can stop electrophoresis.Electrophoresis finishes, and gently pries open layer glass, takes out gel.Downcut the gel of a runway, Solargentum dyeing.In order to identify whether sodium laurylsulfonate-polyacrylamide gel electrophoresis is successful.
2) one of clip and the nitrocellulose filter (0.22 micron in aperture) of the identical size of running gel are with electrotransfer damping fluid (electrotransfer damping fluid: 25mmol/L Tris-Base, the 192mol/L glycine, 20% methyl alcohol, pH8.3) 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, note in the entire operation at any time with glass stick and remove bubble between nitrocellulose filter or filter paper and the gel.By above-mentioned placement order, make blob of viscose and nitrocellulose filter and filter paper form sandwich " sandwich " of a cover.
3) be ready to electrotransfer groove and accessory thereof (comprising foam pad and supporting plate etc.).Above-mentioned " sandwich " is placed between two abundant wetted foam pads, and outermost layer is supported with two synthetic glass supporting plates again, and aperture is arranged on the supporting plate, and the electrotransfer damping fluid is freely entered in " gel sandwich ", makes liquid stream by this system.
4) " gel sandwich " inserted in the electrophoresis chamber that fills the electrotransfer damping fluid.With nitrocellulose filter towards anode.
5) electrophoresis current stabilization 200mA switched on 5 hours.
6) electrophoresis finishes, and takes out nitrocellulose filter, and half nitrocellulose filter is dyeed with the bright orchid of Kao Masi-R250, and 7% acetate is sloughed background color, to show protein band.
7) nitrocellulose filter that second half is had repeat samples was washed 10 minutes with the 0.01M phosphate buffered saline buffer, put in the bovine serum albumin solution of the 2-3% that the 0.01M phosphate buffered saline buffer is made into sealing then 45 minutes, 37 ℃.
8) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
9) nitrocellulose filter is put among the monoclonal antibody II, 37 ℃ are slowly shaken and spent the night in 1 hour or 4 ℃.
10) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
11) nitrocellulose filter is added the goat anti-mouse igg antibody (being diluted in 0.01M phosphate buffered saline buffer at 1: 30000) of alkali phosphatase enzyme mark, 37 ℃ were slowly shaken 1 hour.
12) wash 3 times each 5 minutes with 0.01M phosphate buffered saline buffer (containing 0.05% tween 20).
13) nitrocellulose filter is put into alkaline phosphatase color development liquid (NBT-BCIP color development liquid) color development, till color (blueness) is clear.
14) use deionized water wash, with termination reaction.Nitrocellulose filter is clipped between filter paper drying.Putting the dark place preserves.
The result: the molecular weight of the Leucodermia virus acceptor that monoclonal antibody II discerns on mitten crab hemocyte film and swimming crab hemocyte film is the 200kDa (see figure 7).
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.
Agents useful for same of the present invention and instrument are as follows:
Ground high zincon box (purchasing company) in Roch; The high anti-ly zinc antibody of alkali phosphatase enzyme mark (purchasing company) in Roch; The goat-anti mouse Ig of phosphatase enzyme mark (; Antibody (purchasing company) in Sigma; Nitrocellulose filter (purchasing company) in PALL; Alkaline phosphatase color development liquid (NBT-BCIP color development liquid) (purchasing company) in Sigma; Bovine serum albumin (purchasing company) in Sigma; The goat anti-mouse antibody of marked by fluorescein isothiocyanate (purchasing company) in Sigma; Microplate reader (purchasing company) in MOLEULAR; Leibovitz ' s L-15 (purchasing company) in GIBCO; Foetal calf serum (purchasing company) in Hyclone; RPMI (-) 1640 (purchasing company) in GIBCO; Electrophoresis chamber (purchasing company) in BIO-RAD; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (purchasing company) in Sigma; Acrylamide (purchasing company) in Sigma; Methylene fork bisacrylamide (purchasing company) in Sigma; Tris-Base (purchasing company) in Sigma; Sodium laurylsulfonate (purchasing company) in Sigma; Ammonium persulphate (purchasing company) in Sigma; Tetramethyl Ethylene Diamine (TEMED) (purchasing company) in Sigma; Dredge basic ethanol (purchasing company) in Sigma; Glycerine (purchasing company) in Sigma; Bromjophenol blue (purchasing company) in Sigma; Tris-Base (purchasing company) in Sigma; Glycine (purchasing company) in Sigma; Sodium laurylsulfonate (purchasing company) in Sigma; Xylene Brilliant Cyanine G-R250 (purchasing company) in Sigma; 4-nitrophenol phosphoric acid salt (pNPP) (purchasing company) in Sigma.
Claims (2)
1, a kind of monoclonal antibody II of virus receptor of anti leukoplakia disease is characterized in that: described monoclonal antibody II is by preserving number: the hybridoma excretory of CCTCC-C200418.
2, according to the monoclonal antibody II of the described virus receptor of anti leukoplakia disease of claim 1, it is characterized in that: the sick acceptor of described hickie disease, be the receptor antigen on the cytolemma of prawn hemocyte, the antigenic molecular weight of this receptor is 175-200kDa.
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