CN1683411A - Lefteye flonder lymphocystic virus neutralizing monoclonal antibody and its preparing method - Google Patents
Lefteye flonder lymphocystic virus neutralizing monoclonal antibody and its preparing method Download PDFInfo
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Abstract
The present invention is neutralizing monoclonal antibodies A and B of lymphocystis viruses secreted separately by two strains of hybridoma cells. These two hybridoma culturing cells, named separately as mouse hybridoma cell strain LCDV-E1 and mouse hybridoma cell strain LCDV-E2, have the preservation numbers of CCTCC-C200419 and CCTCC-C200420. The neutralizing monoclonal antibodies A and B have the function of combining with adherent cell on the lymphocystis virus to close the adherent cell, to block the combination between the adherent cell and the virus acceptor of lefteye flounder gill cell as the virus sensitive cell and to resist the virus from invading the cell. The neutralizing monoclonal antibodies A and B are prepared through establishing lymphocystis virus resisting monoclonal antibody library immunologically with purified lymphocystis virus as antigen and neutralizing test method to screen, and has important lymphocystis virus diagnosing and treating application.
Description
Technical field
The present invention relates to the improvement of antibody utilisation technology, specifically is a kind of lefteye flonder lymphocystic virus neutralizing monoclonal antibody and preparation method thereof.This neutralizing monoclonal antibody is that it belongs to immunology and virusology interleaving techniques field by hybridoma excretory lefteye flounder (Paralichthys olivaceus) lymphocystis disease virus (Lymphocystis virus) neutralizing monoclonal antibody.
Background technology
In the biotic factor of harm fish existence growth, virus be harm maximum, also be the cause of disease of the most difficult strick precaution.Parasitize the interior acellular type ultra micro microbial population of host cell because virus is a class, can not kill and wound the active drug that virus is not damaged host cell again so also work out so far.In a single day in addition, because the inapparent infection and the special aquatic environment of fish of virus make the infection of virus be difficult to perceive, usually find symptom, promptly to the stage that can't save.The lefteye flonder lymphocystic disease is China's seawater fish virus disease of large-scale outbreak first.The epidermis of ill fish, fin and afterbody etc. locate to occur many tumours.Since the nineties in 20th century, the occurrence frequency of this disease increases gradually, and annual cultured fishes are therefore sick mass mortality all, and the direct and indirect economic loss that causes reaches 10,000,000,000 yuan.Treatment has the potential practical value to the specific aim of virus disease to have the active monoclonal antibody of neutralization.Neutralizing monoclonal antibody at epitope can induce protective effect, this epitope synthetic peptide vaccine can effectively prevent virus infection.This is in the fowl poultry kind animal, and existing precedent as the medicine " the capsule spirit of going out " of commercialization treatment infections chicken cloacal bursa virus, promptly is to be made by neutralizing monoclonal antibody.From the clone antibody stock of lefteye flonder lymphocystic virus, filter out this viral neutralizing monoclonal antibody,, and then prevent and treat this virus disease that very most important theories and realistic meaning are arranged development lymphocystis disease virus vaccine.
Summary of the invention
The objective of the invention is to fill up the blank of relevant fish lymphocystis disease virus neutralizing monoclonal antibody research, provide a kind of by two strain of hybridoma difference excretory lefteye flonder lymphocystic virus neutralizing monoclonal antibody.Thereby reach the purpose of prevention and control flounder lymphocyst.
Task of the present invention is realized by following technical scheme, developed a kind of neutralizing monoclonal antibody by two strain of hybridoma difference excretory lymphocystis disease virus, the title of this two hybridomas culturing cell of described secretion lymphocystis disease virus neutralizing monoclonal antibody is respectively: mouse hybridoma cell strain LCDV-E1 and mouse hybridoma cell strain LCDV-E2, the preserving number of this two hybridoma is respectively: CCTCC-C200419 and CCTCC-C200420, its preservation date: on December 14th, 2004; Its excretory specificity lymphocystis disease virus neutralizing monoclonal antibody (be called for short: neutralization monoclonal antibody A and neutralization monoclonal antibody B) has with attachment proteins on the lymphocystis disease virus and combines, thereby seal this attachment proteins, block this viral attachment proteins and this virus sensitive cells---this virus receptor on the cytolemma of paralichthys olivaceus gilled cell combines, and stops the function of this poisoning intrusion cell; 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, contains a large amount of lymphocystis disease virus neutralizing monoclonal antibody of this hybridoma excretory in this substratum; In cell and in the experiment, this neutralization monoclonal antibody A and neutralization monoclonal antibody B neutralization are obvious, have the characteristic that the protection paralichthys olivaceus gilled cell is not infected by lymphocystis disease virus.
The preparation method of lymphocystis disease virus neutralizing monoclonal antibody of the present invention, its technology of preparing route is: from being antigen with the lefteye flonder lymphocystic virus, adopt immunological method to set up the clone antibody stock of antiangiogenic tumour virus; With the measuring method, from this clone antibody stock, detect neutralization monoclonal antibody A and the neutralization monoclonal antibody B that filters out lymphocystis disease virus in the selection.
In described with the measuring method be: the paralichthys olivaceus gilled cell to the lefteye flonder lymphocystic virus sensitivity is the neutralization experiment of material, adopt the virus dilution method, promptly at first, different dilution lymphocystis disease virus respectively with this virus monoclonal antibody storehouse in the monoclonal antibody equal-volume hatch; And then with virus---mixtures of antibodies is inoculated in paralichthys olivaceus gilled cell, treat that effect fully appears infecting in vaccinated paralichthys olivaceus gilled cell, promptly with reference to the paralichthys olivaceus gilled cell of contrast culture in metainfective pathology and death condition, can stop cytopathic generation according to monoclonal antibody, determine to have the active monoclonal antibody of neutralization.
Concrete steps with the measuring method in described are:
(1) cell is prepared: eugonic paralichthys olivaceus gilled cell is inoculated in the 96 porocyte culture plates every hole 3～5 * 10
4Individual, 15～25 ℃, 2%CO
2Cultivate;
(2) virus is prepared: is the membrane filtration of 0.45 μ m with viral suspension with the aperture, and seven continuous gradients are set up in 2 times or 10 times serial dilutions of MEM nutrient solution;
(3) contrast is prepared: 3 groups of contrasts are set: virus control, the contrast of monoclonal antibody toxicity; The paralichthys olivaceus gilled cell blank;
(4) virus and antibody incubation: the monoclonal antibody of anti lymphocyst virus is mixed with different gradient lymphocystis disease virus liquid equal-volumes respectively, hatch 1h～2h for 25～37 ℃;
(5) inoculation: mixed solution, each 8～10 hole of viral gradient inoculation of the virus that finishes and monoclonal antibody are hatched in inoculation on paralichthys olivaceus gilled cell in good condition, every hole inoculum size 25～200 μ l, and 25～37 ℃ adsorb 1h～2h, then 15～25 ℃, 2%CO
2Cultivate;
(6) result judges: the prerequisite of judgement occurs being the paralichthys olivaceus gilled cell bunching, being become round by the typical cells pathology phenomenon that lymphocystis disease virus causes for the virus control group, vacuolation is serious, cell granulations increases, the later stage sick cell takes off wall and forms plaque, cytopathic effect appears, and monoclonal antibody toxicity control group, the full fusiformis that is of paralichthys olivaceus gilled cell blank group cell, bottom, confluent culture hole, the iuntercellular tight, cell state is good; Can stop cytopathic generation from anti lymphocyst virus monoclonal antibody storehouse, to filter out neutralizing monoclonal antibody A and neutralizing monoclonal antibody B according to monoclonal antibody.
The invention has the advantages that: utilization of the present invention is that the neutralization of material experimental results show that monoclonal antibody has tangible neutralizing effect to virus infection to the paralichthys olivaceus gilled cell of lefteye flonder lymphocystic virus sensitivity.In the cell and experiment be a kind of antigen antibody reaction experimental technique commonly used in immunology and the virusology, it is in order to measure in the antibody and the biological effect of viral infection.In the cell and experiment have the susceptibility and the immunologic opsonin of height, there is not the individual difference problem in and stable and consistent as a result, is a kind of experimental technique of very reliable, favorable repeatability.The present invention obtains has the active lymphocystis disease virus monoclonal antibody A of neutralization; monoclonal antibody B; can further be applied in the further investigation of lymphocyst; for identifying the protective antigen of lymphocystis disease virus; distinguish different regions; different hosts' lymphocystis disease virus; analysis to the variation of lymphocystis disease virus specific antigens determinant; the preparation antiidiotypic antibody; utilize the synthetic peptide of neutralizing antibody epitope to carry out diagnosis research; and the research of vaccine effective constituent; extraction purifying etc. provides the most effective instrument; simultaneously, to the lymphocystis disease virus recombinant vaccine; synthetic peptide vaccine; the development of subunit vaccine; the theory and practice basis has been established in virus diagnose reagent production etc.
Description of drawings
Embodiments of the invention are as follows by description of drawings:
Fig. 1 is a technological process block-diagram of the present invention;
Fig. 2 is for amplifying 4 times of inverted microscopes viable cell form of the paralichthys olivaceus gilled cell of observation down;
Cytopathic paralichthys olivaceus gilled cell form appears in Fig. 3 behind the virus inoculation;
Fig. 4 is virus inoculation and monoclonal antibody mixed solution, cytopathic paralichthys olivaceus gilled cell form do not occur.
Specific embodiments of the invention are not only limited to protection scope of the present invention.
The neutralizing monoclonal antibody of a kind of lymphocystis disease virus of being secreted respectively by two strain of hybridoma of the present invention development. Described branch The title of secreting this two hybridomas cultured cell of lymphocystis disease virus neutralizing monoclonal antibody is respectively: mouse hybridoma cell strain LCDV-E1 and mouse hybridoma cell strain LCDV-E2, the preserving number of this two hybridoma is respectively: CCTCC-C200419 and CCTCC-C200420, its preservation date: on December 14th, 2004; The specificity lymphocystis disease virus neutralizing monoclonal antibody (letter of its secretion Title: neutralization monoclonal antibody A and neutralization monoclonal antibody B) attachment proteins that has on lymphocystis disease virus is combined, thereby seals this attachment proteins, Block this viral attachment proteins and this virus sensitive cells---this virus receptor on the cell membrane of paralichthys olivaceus gilled cell is combined, and stoping should The function of poisoning intrusion cell; Under inverted microscope, observe, this hybridoma division that growth conditions is good is vigorous, outward appearance is full, Perfectly round, refractivity is strong, cell size homogeneous, adherent good; This hybridoma has unlimited division and proliferation ability; What grow fine should Hybridoma cellar culture 2-3 days, its culture medium changes yellow into by pink, contains this hybridoma secretion in this culture medium A large amount of lymphocystis disease virus neutralizing monoclonal antibodies; In the neutralization experiment, this neutralization monoclonal antibody A and neutralization monoclonal antibody B neutralization are bright Aobvious, have the characteristic that the protection paralichthys olivaceus gilled cell is not infected by lymphocystis disease virus.
The preparation method of lymphocystis disease virus neutralizing monoclonal antibody of the present invention such as technology path shown in Figure 1 of the present invention is: from Begin take lefteye flonder lymphocystic virus as antigen, adopt immunological method to set up the clone antibody stock of antiangiogenic tumour virus; In the selection And experimental determining method, from this clone antibody stock, detect neutralization monoclonal antibody A and the neutralization monoclonal antibody B that filters out lymphocystis disease virus.
Embodiment is as follows:
One), the monoclonal antibody of development anti lymphocyst virus:
1) virus is purified:
(1) gets ill lefteye flounder, with 70% cotton ball soaked in alcohol sterilization affected part, downcut tumour with the sterilization knife blade more earlier, add quartz sand and TNE (50mM Tris, 100mM NaCl, 1mM EDTA, pH7.4) damping fluid, homogenate;
(2) homogenate is centrifugal 4 ℃, 500g, and 20min gets supernatant;
(3) supernatant liquor is centrifugal 4 ℃, 1800g, and, 20min gets supernatant;
(4) supernatant liquor and sucrose are made into the sucrose solution of 30% (W/W), and 4 ℃, the centrifugal 120min of 78500g abandons supernatant;
(5) add TNE to 1ml in precipitation, mixing gently places saccharose gradient liquid (37%, 40%, 47%, 52%, 57%, 62%) top, 4 ℃, the centrifugal 120min of 78500g with it;
(6) the virus band in the sucking-off saccharose gradient, the adjustment protein content is 1mg/ml ,-80 ℃ of preservations are standby.
2) immunity:
Immunity is divided into 4 times to be carried out, and each immunizing dose is 0.1ml.
(1) fundamental immunity, abdominal injection, the virus of purification and complete freund adjuvant equivalent (V/V) mixing are as antigen;
After (2) two liang of weeks, booster immunization, abdominal injection, the virus of purification and incomplete freund adjuvant equivalent (V/V) mixing are as antigen;
After (3) three weeks, the secondary booster immunization, tail vein injection, the virus of purification is as antigen;
(4) merge the amplification immunity of first three day, tail vein injection, the virus of purification is as antigen.
3) cytogamy:
(1) take off cervical vertebra and put to death immune mouse, blood drawing, 4 ℃ of placements are spent the night, and get serum; Behind aseptic taking-up spleen and the thymocyte, cross 100 order mesh screens respectively, form single cell suspension with RPMI-1640 piping and druming;
(2) respectively with splenocyte suspension and the centrifugal 3min of thymus cell suspension 200g, abandoning supernatant, the splenocyte precipitation is resuspended with RPMI-1640, and the thymocyte precipitation is resuspended with 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, the centrifugal 3min of 200g, remove supernatant liquor after, with RPMI-1640 resuspended;
(4) splenocyte suspension and tumor cell suspension are mixed after, the centrifugal 3min of 200g inhales and removes supernatant liquor, flicks at the bottom of the centrifuge tube, makes the abundant mixing of precipitation.Draw pre-temperature to 37 ℃ PEG liquid 1ml, be added drop-wise in the centrifuge tube;
(5) add RPMI-1640 liquid to 40ml, through the centrifugal 5min of 200g, abandoning supernatant;
(7) cell precipitation gently is suspended from the 3ml cell culture fluid, frozen 2ml;
(8) add the thymus cell suspension that make (2) in the Sheng Xia cell suspension, be added drop-wise to after mixing in 96 well culture plates, 37 ℃, 4.5%CO
2Incubator is cultivated, and after about two weeks, gets supernatant liquor and detects
4) detect:
(1) get fresh tumour, be cut into the square fritter of about 3mm, physiological saline cleans, and behind the suck dry moisture, with freezing embedding medium OCT embedding, places 30min, frozen section, slice thickness 5 μ m for-20 ℃.Acetone fixed 20min, drying at room temperature ,-20 ℃ are frozen standby;
(2) be first antibody with immune serum and hybridoma supernatant respectively, be added in the section;
Hatch 45min in (3) 37 ℃ of wet boxes;
(4) take out slide glass, it is inferior to give a baby a bath on the third day after its birth with 0.01M PBS, each 5min;
(5) the goat anti-mouse igg second antibody of marked by fluorescein isothiocyanate is added in the section;
Lucifuge is hatched 45min in (6) 37 ℃ of wet boxes;
(7) take out slide glass, it is inferior to give a baby a bath on the third day after its birth with 0.01M PBS, each 5min;
(8) glycerine mounting, fluorescent microscope is observed down, takes pictures;
5) clone:
Adopt limiting dilution assay that detected positive hybridoma cell is cloned;
(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 RPMI-1640 piping and druming;
(2) the centrifugal 3min of thymus cell suspension 200g, abandon supernatant liquor, precipitation is resuspended with 10ml cell complete culture solution;
(3) positive cell that will clone of blood cell counting plate numeration with 10 times of gradient dilutions, takes out 100 positive hybridoma cells with nutrient solution, puts into the thymus cell suspension after resuspended;
(4) cell suspension piping and druming evenly is added drop-wise in 96 well culture plates every hole 100 μ l;
(5) immuno-fluorescent antibody technique detects monoclonal cell hole supernatant liquor, positive porocyte enlarged culturing.
6) frozen:
It is vigorous to get growth, and form is good treats freeze-stored cell, makes cell suspension, and the centrifugal 5min of 200g removes supernatant, adds frozen storing liquid (cell culture fluid that contains 10%DMSO), and making final cell density is 5 * 10
6Individual/ml, the 1ml cell suspension is loaded in the frozen pipe of 2ml, 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 after, immerse prolonged preservation in the liquid nitrogen again.
7) result:
The positive hybridoma cell size homogeneous that the present invention is resulting grows fine, perfectly round, bright, divide vigorously, unlimited division and proliferation ability is arranged; The size homogeneous, perfectly round, bright, divide vigorous; The block strong positive signal of distribution appears in indirect immunofluorescence detected result showed cell matter edge, and positive signal is wedge-like, bulk, or several chaining rounds arrangements, and concentrates on the edge section of cell mostly.And do not find fluorescence in the healthy tissues.
Two), the cultivation of sensitive cell line-paralichthys olivaceus gilled cell system:
1) liquid dosage:
Preparation cell culture fluid: MEM/EBSS one bag (9.5g) is dissolved in the 1L sterilization tri-distilled water, adds NaHCO
32.2g, penbritin 1 * 10
5IU, Streptomycin sulphate 1 * 10
5μ g, adjustment pH is 7.0-7.4.By 0.22 μ m millipore filtration bacteriological filtration, packing, 4 ℃ of preservations.Before facing usefulness, add 10% calf serum (V/V).
The preparation cell dissociation buffer: liquid 0.25% trypsin Trypsin 1: 250), (0.01M, pH7.4) damping fluid preparation is by 0.22 μ m millipore filtration bacteriological filtration, packing ,-20 ℃ of preservations for aseptic PBS.
Preparation cell scavenging solution: (0.01M pH7.4) adds 0.02%EDTA, autoclaving, room temperature preservation in the damping fluid at PBS.
2) paralichthys olivaceus gilled cell is cultivated:
(1) recovery: from liquid nitrogen, take out frozen pipe, drop into immediately in the 40-42 ℃ of water and rock fast, dissolve fully until frozen storing liquid; The centrifugal 5min of 200g abandons supernatant; Add nutrient solution 1ml to precipitation, pressure-vaccum is even gently; Cell suspension is moved into 25cm
2In the culturing bottle, replenish nutrient solution 4ml, 20 ℃ of cultivations.
(2) go down to posterity: after cell covers with bottom, outwell old nutrient solution, add 3ml cell scavenging solution, remove the dead cell on upper strata, outwell scavenging solution, add the 0.3ml cell dissociation buffer then, treat that cellular contraction becomes circle, gently shake culturing bottle with have gentle hands, cell is split away off from the culturing bottle bottom, add fresh medium 2ml, behind the mixing, be divided into two bottles of every bottle each additional fresh medium 4ml, 20 ℃ of cultivations.
(3) morphological specificity of gill cell and growth rhythm
Gill cell is the epithelioid cell, adherent distribution.Cell is observed under inverted microscope and is fusiformis, and form is full, and size is even, and major axis is 30-40 μ m.Behind the gill passage, preceding 3 days is the quick rise period, and cell quantity increases about one times; Cell density reaches 7-8 * 10
5Behind the cells/ml, the cell growth tendency slows down, and quantity does not increase substantially.
Three), in the cell and the experiment:
1) virus is purified:
(1) gets ill lefteye flounder, with 70% cotton ball soaked in alcohol sterilization affected part, downcut tumour with the sterilization knife blade more earlier, add quartz sand and TNE (50mM Tris, 100mM NaCl, 1mM EDTA, pH7.4) damping fluid, homogenate;
(2) homogenate is centrifugal 4 ℃, 500g, and 30min gets supernatant;
(3) supernatant liquor is centrifugal 4 ℃, 1800g,, 30min gets supernatant, and-80 ℃ of preservations are standby;
2) virus infection paralichthys olivaceus gilled cell experiment:
(1) eugonic paralichthys olivaceus gilled cell is inoculated in 96 orifice plates 100 μ l (3-5 * 10, every hole
5Cells/ml), 20 ℃, 2%CO
2Cultivate;
(2) treat that cell grows up to individual layer, with PBS buffer solution for cleaning 1-2 time, every hole adds the viral suspension 40 μ l of the membrane filtration of 0.45 μ m, and 20 ℃ of absorption 1h (or light shaking) add and keep liquid (Eagle MEM, 2% calf serum) 100 μ l, 20 ℃, 2%CO
2Cultivate, every day is observation of cell pathology situation under inverted microscope, takes pictures.Cellular control unit adds aseptic PBS damping fluid 40 μ;
(3) result: referring to Fig. 2---shown in Figure 4;
Behind the virus inoculation 1-2 days, cytopathy appearred in individual layer gill cell.Sick cell bunching, poly-heap, take off wall, along with lesion degree is deepened, cell vacuolation and disintegration gradually form tangible plaque;
3) cell neutralization test: adopt the virus dilution method;
(1) cell is prepared: eugonic paralichthys olivaceus gilled cell is inoculated in 96 orifice plates every hole 3-5 * 10
4Cells, 20 ℃, 2%CO
2Cultivate;
(2) virus is prepared: will slightly carry the membrane filtration of virus with 0.45 μ m, the dilution of MEM nutrient solution is set up seven continuous gradients, 2
-1, 2
-22
-3, 2
-4, 2
-5, 2
-6, 2
-7
(3) contrast is prepared: 3 groups of contrasts are set: virus control, and each concentration virus liquid is all established contrast; The contrast of monoclonal antibody toxicity; The paralichthys olivaceus gilled cell blank;
(4) virus and antibody incubation: the monoclonal antibody of anti lymphocyst virus is mixed with different gradient lymphocystis disease virus liquid equal-volumes respectively, hatch 1h (or light shaking) for 25 ℃;
(5) inoculation: when 96 porocytes grow up to individual layer, use PBS buffer solution for cleaning 1～2 time, inoculate virus and the mixed solution of monoclonal antibody and viral liquid, monoclonal antibody, the MEM of control group of having hatched respectively, 8 holes of every kind of each gradient inoculation, every hole 40 μ l.20 ℃ of absorption 1h (or light shaking) add and keep liquid (Eagle MEM, 2% calf serum) 100 μ l, 20 ℃, 2%CO
2Cultivate.
(6) observe: under inverted microscope, observe and write down inoculation 24h every day, 48h, 72h, 96h, the cellular change behind the 120h is taken pictures.
(7) result: observed continuously 5 days, the virus infection positive control hole of virus inoculation liquid, obvious cytopathic effect appears behind 48h; sick cell bunching, change circle; vacuolation is serious; cell granulations increases, last cell take off wall and form plaque, the typical cells pathology effect that lymphocystis disease virus causes occurs.The monoclonal antibody toxicity control wells and the gill cell control well of virus inoculation not, the full fusiformis that is of cell, confluent culture hole, iuntercellular tight.Cell state in inoculation monoclonal antibody A or monoclonal antibody B and the viral mixed solution hole is good, according to Reed---and the Muench method is calculated median infective dose and is respectively: the virus infection positive control is 2
-2.9, promptly should equal 1 TCID by 7.5 times of diluents of virus, 40 μ l
50,The median infective dose of among the monoclonal antibody A and back virus is 2
-2, promptly this virus-4 times diluent 40 μ l equal 1 TCID
50The median infective dose of among the monoclonal antibody B and back virus is 2
-2.5Promptly .7 times of diluent of this virus 5 40 μ l equal 1 TCID
50Repeatedly neutralization experiment is all measured this 2 strain monoclonal antibody and is had better neutralization activity, can suppress the appearance of cell CPE behind the virus inoculation.According to above result as can be known, monoclonal antibody A and monoclonal antibody B are and have the active monoclonal antibody of neutralization.
Used instrument of the present invention and reagent: substratum MEM/EBSS (available from Hyclone company); Calf serum (available from Hyclone company); Trypsinase Trypsin 1: 250 (available from Amersco company); Methyl-sulphoxide (DMSO) (available from Sigma company); Freezing microtome (available from Leica company); Fluorescent microscope (available from Olympus company); Inverted phase contrast microscope (available from Olympus company); 1640 (available from Gibco companies); Foetal calf serum (available from Hyclone company); HAT (available from Gibco company); The goat anti-mouse antibody of marked by fluorescein isothiocyanate (available from Sigma company).
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 (4)
1, a kind of neutralizing monoclonal antibody by two strain of hybridoma difference excretory lymphocystis disease virus, it is characterized in that: the title of this two hybridomas culturing cell of secretion lymphocystis disease virus neutralizing monoclonal antibody is respectively: mouse hybridoma cell strain LCDV-E1 and mouse hybridoma cell strain LCDV-E2, the preserving number of this two hybridoma is respectively: CCTCC-C200419 and CCTCC-C200420, its preservation date: on December 14th, 2004; Its excretory specificity lymphocystis disease virus neutralizing monoclonal antibody (be called for short: neutralization monoclonal antibody A and neutralization monoclonal antibody B) has with attachment proteins on the lymphocystis disease virus and combines, thereby seal this attachment proteins, block this viral attachment proteins and this virus sensitive cells---this virus receptor on the cytolemma of paralichthys olivaceus gilled cell combines, and stops the function of this poisoning intrusion cell; 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, contains a large amount of lymphocystis disease virus neutralizing monoclonal antibody of this hybridoma excretory in this substratum; In the neutralization experiment, this neutralization monoclonal antibody A and neutralization monoclonal antibody B neutralization are obvious, have the protection paralichthys olivaceus gilled cell and are not subjected to lymphocystis disease virus to infect characteristic.
2, a kind of preparation method by the described lymphocystis disease virus neutralizing monoclonal antibody of claim 1, its technology of preparing route is: from being antigen with the lefteye flonder lymphocystic virus, adopt immunological method to set up the clone antibody stock of antiangiogenic tumour virus; It is characterized in that: with the measuring method, from this clone antibody stock, detect neutralization monoclonal antibody A and the neutralization monoclonal antibody B that filters out lymphocystis disease virus in the selection.
3, according to the preparation method of the described lymphocystis disease virus neutralizing monoclonal antibody of claim 2, it is characterized in that: in described with the measuring method be: the paralichthys olivaceus gilled cell to the lefteye flonder lymphocystic virus sensitivity is the neutralization experiment of material, adopt the virus dilution method, promptly at first, different dilution lymphocystis disease virus respectively with this virus monoclonal antibody storehouse in the monoclonal antibody equal-volume hatch; And then with virus---mixtures of antibodies is inoculated in paralichthys olivaceus gilled cell, treat that effect fully appears infecting in vaccinated paralichthys olivaceus gilled cell, promptly with reference to the paralichthys olivaceus gilled cell of contrast culture in metainfective pathology and death condition, can stop cytopathic generation according to monoclonal antibody, determine to have the active monoclonal antibody of neutralization.
4, according to the preparation method of claim 2 or 3 described lymphocystis disease virus neutralizing monoclonal antibodies, it is characterized in that: the concrete steps with the measuring method in described are:
(1) cell is prepared: eugonic paralichthys olivaceus gilled cell is inoculated in the 96 porocyte culture plates every hole 3～5 * 10
4Individual, 15～25 ℃, 2%CO
2Cultivate;
(2) virus is prepared: is the membrane filtration of 0.45 μ m with viral suspension with the aperture, and seven continuous gradients are set up in 2 times or 10 times serial dilutions of MEM nutrient solution;
(3) contrast is prepared: 3 groups of contrasts are set: virus control, the contrast of monoclonal antibody toxicity; The paralichthys olivaceus gilled cell blank;
(4) virus and antibody incubation: the monoclonal antibody of anti lymphocyst virus is mixed with different gradient lymphocystis disease virus liquid equal-volumes respectively, hatch 1h～2h for 25～37 ℃;
(5) inoculation: mixed solution, each 8～10 hole of viral gradient inoculation of the virus that finishes and monoclonal antibody are hatched in inoculation on paralichthys olivaceus gilled cell in good condition, every hole inoculum size 25～200 μ l, and 25～37 ℃ adsorb 1h～2h, then 15～25 ℃, 2%CO
2Cultivate;
(6) result judges: the prerequisite of judgement occurs being the paralichthys olivaceus gilled cell bunching, being become round by the typical cells pathology phenomenon that lymphocystis disease virus causes for the virus control group, vacuolation is serious, cell granulations increases, the later stage sick cell takes off wall and forms plaque, cytopathic effect appears, and monoclonal antibody toxicity control group, the full fusiformis that is of paralichthys olivaceus gilled cell blank group cell, bottom, confluent culture hole, the iuntercellular tight, cell state is good; Can stop cytopathic generation from anti lymphocyst virus monoclonal antibody storehouse, to filter out neutralizing monoclonal antibody A and neutralizing monoclonal antibody B according to monoclonal antibody.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100404554C (en) * | 2006-05-26 | 2008-07-23 | 中国海洋大学 | Antifish lymphocystis virus capsid protein monoclonal antibody, and its preparing method |
| CN101893632A (en) * | 2010-07-29 | 2010-11-24 | 中国海洋大学 | Test paper for detecting fish lymphocystis disease virus and preparation method thereof |
| CN102199212A (en) * | 2011-05-06 | 2011-09-28 | 中国海洋大学 | Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof |
| CN102206278A (en) * | 2011-05-06 | 2011-10-05 | 中国海洋大学 | Monoclonal antibody of anti-LCDV (lymphocystis disease virus) 27.8kDa receptor protein and preparation method thereof |
| CN101629955B (en) * | 2009-08-07 | 2012-09-26 | 中国海洋大学 | Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof |
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| CN105859879B (en) * | 2016-06-03 | 2019-02-19 | 青岛农业大学 | A kind of single-chain antibody of Antifish lymphocystis virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1322008C (en) * | 2004-12-17 | 2007-06-20 | 中国海洋大学 | Monoclonal antibody of anti lymphocyst virus of and preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100404554C (en) * | 2006-05-26 | 2008-07-23 | 中国海洋大学 | Antifish lymphocystis virus capsid protein monoclonal antibody, and its preparing method |
| CN101629955B (en) * | 2009-08-07 | 2012-09-26 | 中国海洋大学 | Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof |
| CN101893632A (en) * | 2010-07-29 | 2010-11-24 | 中国海洋大学 | Test paper for detecting fish lymphocystis disease virus and preparation method thereof |
| CN102199212A (en) * | 2011-05-06 | 2011-09-28 | 中国海洋大学 | Monoclonal antibody against mucus immunoglobulin in flounder and preparation method thereof |
| CN102206278A (en) * | 2011-05-06 | 2011-10-05 | 中国海洋大学 | Monoclonal antibody of anti-LCDV (lymphocystis disease virus) 27.8kDa receptor protein and preparation method thereof |
| CN102206278B (en) * | 2011-05-06 | 2013-04-17 | 中国海洋大学 | Monoclonal antibody of anti-LCDV (lymphocystis disease virus) 27.8kDa receptor protein and preparation method thereof |
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