CN101831407B - Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof - Google Patents
Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof Download PDFInfo
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- CN101831407B CN101831407B CN2010101540475A CN201010154047A CN101831407B CN 101831407 B CN101831407 B CN 101831407B CN 2010101540475 A CN2010101540475 A CN 2010101540475A CN 201010154047 A CN201010154047 A CN 201010154047A CN 101831407 B CN101831407 B CN 101831407B
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Abstract
The invention discloses a hybridoma cell line of a monoclonal antibody against African swine fever virus and the secreted monoclonal antibody thereof. The preparation method of the invention comprises the following steps: preparing a recombined P30 soluble antigen by prokaryotic expression; immunizing a BALB/c mouse; and finally fusing, screening and cloning by a hybridoma technology to obtain the hybridoma cell line which can stably secrete the monoclonal antibody against African swine fever virus P30 protein. The invention further discloses a method for preparing the monoclonal antibody with the cell line, an antibody purification method and a labeling method for horseradish peroxidase of the antibody. The monoclonal antibody can be used in detecting the African swine fever viral antibody in pig serum.
Description
Technical field
The invention belongs to biotechnology and cell engineering field; Relate to secretion anti-African swine fever virus monoclonal antibody hybridoma cell line and excretory monoclonal antibody thereof; And said Purification of Monoclonal Antibodies and horseradish peroxidase-labeled, can be applicable to detect African swine fever virus antibody in the porcine blood serum.
Background technology
(African swine fever ASF) is a kind of acute, hot, the strong communicable disease of contact highly of the pig that caused by African swine fever virus (ASFV) to African swine fever.It is characterized by course of disease weak point, case fatality rate high rate, can be up to 100%, clinical symptom and pathological change all are similar to acute swine fever, very easily mistaken diagnosis when diagnosis, the high heat of performance, dermohemia cyanosis, miscarriage, oedema and internal organs are hemorrhage.(William; Hess Adv.African swine fever:a reassessment [J] .Vet Sci Comp Med, 1981,25:39～69) world animal tissues (OIE) classifies the category-A eqpidemic disease as; China is defined as one type of disease of animal; Receive countries in the world great attention (Sun Huaichang. Chinese Preventive Veterinary Medicine newspaper, 1999,21 (2): 117～119).
This disease from nineteen twenty-one since Kenya finds; Be present in the African country on the south the Sahara, nineteen fifty-seven successively spreads to West Europe and Latin American countries always, and majority is in time put out; Singly in Portugal; The Spain west and south and gondola Sardinia still have popular, and be popular in dozens of countries such as Africa, Europe and America so far, and continuous spreading trend is arranged.2007, Armenia recurred six African swine fevers, and China does not still have should disease.
African swine fever belongs to Iridoviridae in the 4th report of ICTV, in the 5th report of this council, it is listed under the Poxviridae, places outside the Chordopoxvirinae and Entomopoxvirinae of this section.But dna sequence analysis shows; ASF virus has the characteristic between poxvirus and irido virus; This characteristic of ASFV shows any section that it does not belong to ICTV and is appraised and decided, and is new, the 6th report of nineteen ninety-five the 9th international virus taxis committee member; African swine fever virus is listed in " type African swine fever virus belongs to ", African swine fever is unique known representative species.
African swine fever virus is a kind of big, double-stranded DNA virus that cyst membrane is arranged, is unique entomophila dna virus.Its genome is terminal covalence closed unit molecule wire double-stranded DNA, and the viral genome total length is 170kb～190kb, and there is the conserved regions about 125kb in central authorities; Two ends are the variable region; Contain terminal counter-rotating Tumor-necrosis factor glycoproteins, the increase of these Tumor-necrosis factor glycoproteinss or disappearance are major cause (Rafacl, the Yancz that causes different isolates genome difference in length; Javier M; Et al.Analysis of the completeNucleotide Sequence of Afican Swine Fever Virus [J] .Virology, 1995,208:249～279).The ASF viral genome has 5 encoding soxs, comprises putative membrane protein, secreted protein, participation nucleic acid and nucleic acid metabolism (DNA reparation) and protein modified enzyme, and whole genome contains 151 ORF; 150～200 kinds of protein of can encoding, isolation identification goes out 86 kinds of viral protein polypeptide (Qu Liandong, Yu Kangzhen from the cell that ASFV infects; The African swine fever progress; China animal doctor science and technology, 1998,28 (11): 42～43).
The virulence of the most of strains of African swine fever virus is all very strong, but immunogenicity is very low, has only a few albumen to have immunogenicity.Experiment showed, P30 albumen for having one of better antigenic albumen, molecular weight of albumen is about 36KD.
Summary of the invention
The purpose of this invention is to provide a kind of hybridoma cell line of secreting the monoclonal antibody of specific recognition African swine fever virus.Another object of the present invention provides above-mentioned clone excretory anti-African swine fever virus monoclonal antibody, and said monoclonal antibody has specificity preferably to African swine fever structural protein P30 antigen.Another object of the present invention provides the enzyme labelling thing of above-mentioned purifying antibody and HRPO mark.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of hybridoma cell line of anti-African swine fever virus monoclonal antibody, the preserving number of said clone are CGMCCNO.3771.
Described hybridoma cell line is set up as immunogen with prokaryotic expression recombinant soluble proteins P30 through hybridoma technology.
Said hybridoma cell line excretory anti-African swine fever virus monoclonal antibody.
Described monoclonal antibody purifying is after the sodium periodate legal system is equipped with the enzyme labelling thing of horseradish peroxidase.
The invention has the beneficial effects as follows: hybridoma cell strain ASFV:P30Mab and excretory monoclonal antibody thereof that this invention is prepared can be discerned the ASFV natural antigen; The monoclonal antibody that is obtained is not only in the ASFV fundamental research, and in the immunoreactive detection reagent of ASFV antigen-antibody, all has very high practical value.
Description of drawings
Fig. 1 is SDS-PAGE figure behind the reorganization P30 protein purification.
Fig. 2 is western blotting figure behind the reorganization P30 protein purification.
Embodiment
The hybridoma cell line of anti-African swine fever virus monoclonal antibody is preserved in the common micro-organisms center C GMCC NO.3771 of China Committee for Culture Collection of Microorganisms, preservation day: 2010-04-22, classification name: hybridoma cell strain.
Below in conjunction with embodiment the present invention is done further explain:
Technical scheme of the present invention is following:
One, sets up clone
1. antigen prepd
A) the proteic expression of African swine fever P30
According to African swine fever among the GenBank (ASFV) P30 gene order; 1 pair of primer has been synthesized in design; Adopt PCR method from ASFV DNA, to amplify the P30 gene fragment, it is cloned among the expression vector pET28b, made up recombinant plasmid pET-P30; Be transformed into expressive host bacterium BL21 (DE3) after the sequence verification, through the IPTG abduction delivering.
Designed primer is:
P30-1-5’-GC
GGATCCTAATTTTAAAATTGAATGGAT-3’
P30-2-5’-GT
CTCGAGCCCAATCAAAATTAGATAACT-3’
Underscore is the restriction enzyme site for introducing partly, and P30-1 is an ASFV P30 upstream region of gene amplimer, and the restriction enzyme site of introducing is BamHI; P30-2 is ASFV P30 gene downstream amplimers, and the restriction enzyme site of introducing is XhoI.
B) the proteic purifying of African swine fever P30
After inducing end, the back thalline is induced in centrifugal collection, washs resuspended thalline with PBS, ultrasonic degradation (ultrasonic 1s, 1s, 10min altogether at interval), and 12000rpm is centrifugal, collects respectively and goes up cleer and peaceful deposition, carries out SDS-PAGE and analyzes.Use the nickel ion affinity chromatograph post during protein purification, behind the purifying, identify, get access to single band, and possess antigenicity (seeing accompanying drawing 1,2) preferably through SDS-PAGE, western blotting.Protein content behind the use BCA method mensuration purifying, standard substance concentration and corresponding OD value thereof are seen table 1.The proteic OD value of P30 is 2.25 behind the purifying, with standard substance relatively after, its concentration is 1700 μ g/ml.
Table 1BCA method bioassay standard article protein content
| The sample title | A (μg/ml) | B (μg/ml) | C (μg/ml) | D (μg/ml) | E (μg/ml) | F (μg/ml) | G (μg/ml) | H (μg/ml) | 0 (μg/ml) |
| Concentration | 2000 | 1500 | 1000 | 750 | 500 | 250 | 125 | 25 | 0 |
| The OD value | 2.6894 | 2.2378 | 1.6493 | 1.3278 | 1.0414 | 0.6454 | 0.4062 | 0.1893 | 0.1251 |
2. antigen immune
African swine fever virus P30 albumen with purifying is antigen, ordinary method immunity BALB/c mouse in 6 age in week, and the antigen of fundamental immunity subcutaneous injection first 200 μ g use complete Freund's adjuvant; Whenever immune at a distance from carrying out in 3 weeks once, totally three times, 50 μ g//inferior, 3 all backs intrasplenic injection booster immunizations after the last fundamental immunity, 25 μ g/, the immune completion.
3. the preparation of hybridoma
A) preparation of feeder cell
With the BALB/c mouse peritoneal macrophage as feeder cell.Merged preceding 1 day, and drew neck to put to death mouse, the body surface sterilization and fixing after, cut tweezer with sterilization and start skin of abdomen, the exposure peritonaeum from venter posterior.Sterilize with cotton ball soaked in alcohol wiping peritonaeum.To the abdominal cavity, washing fluid is reclaimed in flushing repeatedly with injector to inject 10ml RPMI RPMI-1640, and centrifugal 10 minutes of 1000r/min abandons supernatant.With the resuspended deposition of RPMI RPMI-1640 that contains 20% foetal calf serum, the adjustment cell concn is 2 * 10
5/ ml.Above-mentioned cell suspension is added 96 orifice plates, and every hole 0.1ml puts 37 ℃, 6%CO
2Incubator in overnight cultures.
B) preparation of immune spleen cell
Get the BALB/c mouse after immunity is accomplished, through the neck dislocation mouse that causes death, be soaked in 75% alcohol 5 minutes, aseptic condition takes out spleen down, and in plate, the RPMI RPMI-1640 cleans 1 time.Spleen is moved in another plate that fills 10ml RPMI RPMI-1640, make splenocyte get into nutrient solution.With suction pipe piping and druming for several times.Filter, the results splenocyte suspension, centrifugal 10 minutes of 1000r/min, then that cell is resuspended with RPMI RPMI-1640 centrifuge washing 2 times, count, be used for cytogamy.
C) myeloma cell
Before merging 48-36 hour, with myeloma cell's enlarged culturing, cell is blown down from the bottle wall gently on fusion same day with the elbow dropper, be collected in the 50ml centrifuge tube.Centrifugal 10 minutes of 1000r/min, supernatant discarded.Add the incomplete substratum of 30ml, once with the method centrifuge washing.Then cell is resuspended to the incomplete substratum of 10ml, mixing.Get myeloma cell's suspension, counting is used for cytogamy.
D) cytogamy and HAT select hybridoma
Myeloma cell and immune spleen cell are pressed 1: 10 mixed, in the 50ml centrifuge tube, wash 1 time with the RPMI1640 nutrient solution, 1200r/min, centrifugal 10min abandons supernatant, with the careful sucking-off residual liquid of dropper.At the bottom of touching fusion pipe on the palm, make sedimentation cell loose evenly, put preheating in 40 ℃ of water-baths.Adding is preheated to 40 ℃ 50%PEG (PH 8.0) 1ml in the time of 45 seconds, acts on 90 seconds.Add 20ml and be preheated to 37 ℃ RPMI RPMI-1640, room temperature left standstill 10 minutes.1000r/min, centrifugal 5min, supernatant discarded.Add 5ml HAT substratum, the pressure-vaccum sedimentation cell suspends and mixing it gently, add then contain feeder cell the HAT substratum to 80ml.Packing 96 porocyte culture plates, every hole 0.1ml puts 37 ℃ with culture plate, 6%CO then
2Cultivate in the incubator.
With HAT substratum 1/2 substratum that swaps out, change liquid after 48 hours fully after 24 hours.Two week backs are with the HT substratum HAT substratum that swaps out, kept for two weeks again after, use the RPMI RPMI-1640 instead.
4. hybridoma screening
After hybridoma merged for two weeks, carry out hybridoma screening according to the routine immunization zymotechnic.Filter out the hybridoma hole that to secrete anti-African swine fever virus antibody.
5. the cloning of hybridoma and frozen
A) clone of hybridoma
The cloning scheme is a limiting dilution assay, carries out according to conventional hybridization oncocyte cloning process, and the clone carries out three times with method.
B) hybridoma is frozen
Cells frozen storing liquid: 50% calf serum+40%RPMI RPMI-1640+10% DMSO 99.8MIN.
Hybridoma is centrifugal, be suspended in again in the frozen storing liquid of precooling, concentration is 10
6-10
7/ ml is transferred in the frozen pipe every bottle of 1ml.Place-70 ℃ of refrigerators, change in the liquid nitrogen next day.
The hybridoma cell line of preparation anti-African swine fever virus monoclonal antibody is preserved in the common micro-organisms center C GMCC NO.3771 of China Committee for Culture Collection of Microorganisms, preservation day: 2010-04-22, classification name: hybridoma cell strain.
Two, Monoclonal Antibody
Among the present invention, the scheme of mass production monoclonal antibody is the mouse ascites method, and step is following:
This programme is inoculated in the hybridoma of above-mentioned acquisition in the mouse peritoneal, the hybridoma of in mouse peritoneal, growing, and produce ascites, obtain a large amount of ascites monoclonal antibodies.
Concrete grammar is: BALB/c mouse abdominal cavity inoculation liquid paraffin body, every mouse 0.5ml.After two weeks, the abdominal cavity inoculation is with the hybridoma of serum free medium dilution, every mouse 5 * 10
5/ 0.2ml.At interval after 5 days, observe the mouse ascites production every day, when treating that the many as far as possible and mouse of ascites is on the verge of death, put to death mouse, under the aseptic condition with the ascites sucking-off.The static 30min of room temperature, 1000r/min, centrifugal 10min collects supernatant, packing ,-70 ℃ are frozen subsequent use.
Three, monoclonal antibody purifying and HRPO mark
1. Purification of Monoclonal Antibodies
The purification schemes of the ascites monoclonal antibody of above-mentioned acquisition is sad-ammonium sulfate precipitation method.
Get 1 part of pretreated ascites and add 2 parts of 0.06mol/L pH5.0 acetate buffer solutions, transfer pH to 4.8 with 1mol/LHCl; Add the sad ratio of 11ul in every milliliter of dilution ascites, it is sad dropwise to add under the stirring at room, in 30 minutes, adds, and 4 ℃ left standstill 2 hours, took out the centrifugal 30min of 12000r/min, abandoned deposition; Supernatant filters (125um) through nylon mesh, adds the 0.01mol/L PBS of 1/10 volume, transfers pH to 7.2 with 1mol/L NaOH; Add saturated ammonium sulphate to 45% saturation ratio down at 4 ℃, mixing 30min left standstill 1 hour gently; Centrifugal 30 minutes of 12000r/min abandons supernatant; Deposition is dissolved among an amount of PBS, and to the PBS dialysis of 50-100 times of volume, 4 ℃ are spent the night; Take out the centrifugal 30min of 12000r/min, remove infusible precipitate, packing, frozen subsequent use.
2. the HRPO mark of monoclonal antibody
The preparation scheme of the enzyme labelling thing of the monoclonal antibody behind the purifying is the sodium periodate method, and concrete steps are following:
Taking by weighing 5mg HRP is dissolved in the 1ml zero(ppm) water; To wherein adding the 0.1M NaIO that 0.2ml newly joins
4Solution, the room temperature lucifuge stirred 20 minutes; Dialyse in the sodium-acetate buffer to 1mM pH4.4,4 ℃ are spent the night; Carbonate buffer solution to wherein adding 20 μ l 0.2M pH9.5 again makes the pH of above hydroformylation thing be elevated to 9.0, adds the pure article 5mg of monoclonal antibody (10mg/ml) that is dissolved in the 0.01M carbonate buffer solution then immediately, and the room temperature lucifuge is soft to be stirred 2 hours; Add the 4mg/ml NaBH that 0.1ml newly joins
4, mixing was placed 2 hours for 4 ℃; Products therefrom is to 4 ℃ of dialysed overnight among the 0.15M pH7.4PBS.
After dialysis is accomplished, in stirring, dropwise add the equal-volume saturated ammonium sulphate, put 4 ℃ 1 hour.The centrifugal 30min of 3000r/min abandons supernatant.Throw out is washed secondary with semi-saturation ammonium sulfate, and last throw out is dissolved among the PBS of a small amount of 0.15M pH 7.4.Above-mentioned solution is packed in the dialysis tubing, to the PBS damping fluid dialysis of 0.15M pH7.4, take out ammonium ion, the centrifugal 30min of 10000r/min removes deposition, and supernatant is enzyme conjugates, and is after the packing, frozen.
Four, the preparation of standard A SFV positive serum and standard A SFV negative serum
In the ELISA testing process, can there be certain error between different operation personnel and different detection batch, operate miss can cause the error of test sample OD value.The present invention has developed standard A SFV antibody positive control serum and standard A SFV negative antibody control serum, is used for the optimization of testing conditions and the judgement of detected result.
A) preparation of standard positive serum
Get the healthy adult rabbit, body weight 2-3kg cuts off the part rabbit hair of two hind paws, uses iodine disinfection skin.Immunity is drawn not formula Freund's complete adjuvant (FCA) emulsive antigen (P30 recombinates behind the purifying) (to call FCA-P30 in the following text) liquid 1ml with syringe for the first time, the subcutaneous 0.5ml that injects of every batter palm.At interval after 10-14 days, carry out immunity second time, and inject not formula Freund (FIA-P30) in the lymphoglandula of Liang Ce popliteal nest and groin enlargement, each lymphoglandula 0.1ml, all the other inject near the subcutaneous 1ml of being total to lymphoglandula.After 7-10 days, from ear vein blood sampling 0.5-1.0ml, separation of serum adopts the ELISA method to detect serum titer at interval, and antibody titer can reach more than 1: 100.
Adopt the blood sampling of heart blood-collecting method, the blood that extracts is injected aseptic Erlenmeyer flask immediately.The blood of Erlenmeyer flask is put 37 ℃ of incubators 1 hour, put 4 ℃ of refrigerators again 3 hours.After treating the blood coagulation blood clot retraction, draw serum with dropper, the centrifugal 15min of 3000r/min gets supernatant, and it is anticorrosion to add final concentration and be 0.01% Thiomersalate, after the packing, and-20 ℃ of preservations.
B) preparation of standard female serum
The SPF rabbit of learning from else's experience and being up to the standards, heart blood sampling, separation of serum, add ten thousand/ Thiomersalate anticorrosion.Be sub-packed in the sterile tube-20 ℃ of preservations with 0.5ml.
Five, the foundation of test kit
A) the detection principle of test kit of the present invention
Adopt competition law, the African swine fever virus structural protein P30 that recombinates is encapsulated in microwell plate, with 1%BSA enzyme plate is sealed then, add sample to be tested and standard positive control, negative control.The P30 antigen-reactive that encapsulates in African swine fever virus antibody capable in sample or the standard substance and the enzyme plate adds to the competition of participating in combining epi-position behind the monoclonal antibody linked with peroxidase of P30.Subsequently; Add horseradish peroxidase substrate TMB colour developing, the stop buffer termination reaction, through ELIASA under the 450nm wavelength; Measure each hole absorbance, the content of African swine fever virus antibody is inversely proportional in size of OD value (depth of color development stopping reaction back color) and the sample to be tested.
B) composition of test kit of the present invention
A) the best preparation method of enzyme plate
Carbonate buffer solution conduct with pH9.60.05M encapsulates damping fluid, after reorganization P30 albumen dilution behind the purifying of above-mentioned preparation, presses the 100ul/ hole and adds in the microwell plate, guarantees that the P30 content in every hole is 0.2ug.4 ℃ encapsulate and spend the night, and discard coating buffer next day, press the confining liquid that the 200ul/ hole adds 1%BSA, and 37 ℃ left standstill 2 hours, and washing dries.The packing bag of packing into after the drying at room temperature adds siccative, and vacuum is preserved.
B) configuration of work reagent
Washings (pH 7.4,0.15M PBS): KH
2PO
40.2g, Na
2HPO
4-12H
2O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 (0.05%) 0.5ml, adding distil water is to 1000ml.Be condensed into 25 times as storage liquid.
Serum dilution: bovine serum albumin 0.1g adds lavation buffer solution to 100ml
Substrate buffer solution (pH 5.0 phosphoric acid Hydrocerol As): 0.2M Na
2HPO
425.7ml, 0.1M Hydrocerol A 24.3ml, adding distil water 50ml
TMB (TMB) uses liquid: TMB (10mg/5ml absolute ethyl alcohol) 0.5ml, substrate buffer solution 10ml, 0.75%H
2O
232ul
Stop buffer (2M H
2SO
4): zero(ppm) water 178.3ml dropwise adds the vitriol oil (98%) 21.7ml
C) establishment of the competitive ELISA kit of detection African swine fever
Set up the enzyme linked immunological kit that detects African swine fever, comprise following component:
96 hole enzyme plates
The monoclonal antibody of horseradish peroxidase-labeled
Standard positive control
Standard negative control
Concentrated cleaning solution
Serum dilution
Tmb substrate
Stop buffer
Product description
Six, African swine fever virus detection of antibodies in the sample
A) test kit with above-mentioned preparation detects
1) test serum, positive control and negative control are diluted with antibody diluent in proportion, every hole 100 μ l add enzyme plate, hatch 1 hour for 37 ℃.Washings cleans 3-5 time.
2) every hole adds dilution back monoclonal antibody linked with peroxidase 100 μ l, hatches 30min for 37 ℃, and washings cleans 3-5 time.
3) every hole adds tmb substrate liquid 100 μ l, room temperature lucifuge reaction 10-15 minute.
4) every hole adds 100 μ l 2M H
2SO
4Termination reaction, ELIASA detects the 450nm absorbance.
B) detected result analysis
When the OD value of negative control sera (NC) is 4 times of positive control serum (PC) at least in i. surveying,
Detection is considered to effectively.
NC/PC≥4
Positive Cut Off=NC-[(NC-PC) * 0.5]
Negative Cut Off=NC-[(NC-PC) * 0.4]
NC=negative control sera OD value wherein
PC=positive control serum OD value
Use multiple hole when ii. detecting, finally using the OD value is two MV.
When the OD of serum sample value was lower than positive Cut Off value, this sample was the ASFV antibody positive.
When the OD of serum sample value is higher than negative Cut Off value is that this sample is the ASFV negative antibody.
When the OD of serum sample value is between two Cut Off values, be considered to suspiciously, should test again for this sample, perhaps adopt other detection methods to confirm.
In sum, content of the present invention is not confined in the above embodiments, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment is included within the scope of the present invention.
Claims (3)
1. the hybridoma cell line of an anti-African swine fever virus monoclonal antibody is characterized in that, the preserving number of said clone is CGMCC NO.3771.
2. the said hybridoma cell line excretory of claim 1 anti-African swine fever virus monoclonal antibody.
3. the described monoclonal antibody purifying of claim 2 is after the enzyme labelling thing of the horseradish peroxidase of sodium periodate method preparation.
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