CN102190714A - ELISA (Enzyme-linked immunosorbent assay) kit capable of identifying natural infection and artificial immune of foot-and-mouth disease virus - Google Patents
ELISA (Enzyme-linked immunosorbent assay) kit capable of identifying natural infection and artificial immune of foot-and-mouth disease virus Download PDFInfo
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Abstract
The invention relates to a recombinant foot-and-mouth disease virus non-structural protein, which can be used as a coating antigen applied to an ELISA (Enzyme-linked immunosorbent assay) kit; and the ELISA kit can identify animals naturally infected with a foot-and-mouth disease virus and inoculated with a foot-and-mouth disease vaccine. The invention also relates to the application of the recombinant foot-and-mouth disease virus non-structural protein in identifying the animals naturally infected with the foot-and-mouth disease virus and inoculated with the foot-and-mouth disease vaccine.
Description
Technical field
The present invention relates to animal and veterinary field and biological technical field, relate to a kind of be used to differentiate foot and mouth disease virus natural infection and vaccination animal recombined foot-and-mouth disease virus Nonstructural Protein particularly, also relate to a kind of ELISA method of differentiating foot and mouth disease virus natural infection and vaccination animal.
Background technology
Foot and mouth disease (Foot-and-Mouth Disease, FMD) be a kind of by foot and mouth disease virus (Foot-and-Mouth DiseaseVirus, people who FMDV) causes and artiodactyl suffer from altogether acute, deadly infectious disease.This sick route of transmission is many, and infectivity is strong, and hazard rating is serious.(Office International Des Epizooties OIE) classifies it as the category-A transmissible disease in International Office of Epizootics.FMD is a kind of global transmissible disease, and successively more than 60 countries are popular in Europe, Latin America, Africa and Asia etc.
FMDV belongs to the Picornaviridae Hostis, virion diameter 20nm～30nm.FMDV has A, O, C, Asia I, SAT I, SAT II, seven kinds of serotypes of SATIII, and each serotype is divided into multiple hypotype.The neutralizing antibody that the virus structural protein of different serotypes induces does not have the cross protection reaction to another serotype virus.The genome of FMDV is strand underlying stock RNA.Under the guidance of FMDV gene, can produce 12 kinds of viral proteins.These viral proteins can be divided into structural protein (structuralproteins, SP) and Nonstructural Protein (nonstructural proteins, NSP).Structural protein have VP1, VP2, VP3 and VP4.Nonstructural Protein has L, 2A, 2B, 2C, 3A, 3B, 3C and 3D.In the process that generates Nonstructural Protein, 3A, 3B, 3C or 3A, 3B can be combined into 3ABC or 3AB albumen [Grubman MJ, et al.Biochemical map ofpolypeptides specified by foot-and-mouth disease virus.J.Virol 1984; 50 (2): 579-86].Nonstructural protein gene sequence high conservative, the non-neutral antibody that induces, there is not type specificity [Saiz M, NunezJI, et al.Foot-and-mouth disease virus:biology and prospects for disease control.Microbes Infect.2002,4 (11): 1183-92].
The main policies of developed country control FMD is that large-area catching and killing infected and doubtful infected animals, and is that big area is injected conventional vaccine in the main policies of the control FMD of developing country.Vaccination at present can only protect immune animal not fall ill, but can not stop inapparent infection.In the vaccinated fauna in epidemic-stricken area, have the animal of some amount to become the virus carrier because of inapparent infection, these animals might the short-term toxin expelling, forms persistent infection, and virus can be in ox, sheep body long-term existence.Therefore, the inapparent infection animal not only may cause new epidemic situation, also is to produce virus variation new strain environment is provided.The discriminating of natural infection foot and mouth disease virus and vaccination animal also is that OIE judges that a country has or not foundation [the OfficeInternational des Epizooties of epidemic situation, World Organization for Animal Health, 2000.Foot and mouth disease.In:OIE Standards Commission (Ed.), Manual of Standards for Diagnostic Tests and Vaccines, fourth ed.Office International des Epizooties, Paris, France, 1-27 (Chapter 2.1.1)].In addition, the inapparent infection animal brings huge hidden danger to food safety and foreign trade.The animal of vaccination and natural infection all can produce the foot and mouth disease virus specific antibody, and animal and vaccinated animal how to distinguish the natural infection foot and mouth disease virus are control and the important topic of eliminating FMD always.
The experimental results shows, can distinguish natural infection and vaccinated animal by foot and mouth disease virus Nonstructural Protein specific antibody in the detection serum.Virulent propagation in the animal body of natural infection, Nonstructural Protein participates in the virus multiplication process, therefore causes the generation of a large amount of Nonstructural Proteins.Nonstructural Protein has higher immunogenicity, can stimulate body to produce the specific antibody of Nonstructural Protein.And existing aftosa vaccine mainly is the foot and mouth disease virus inactivated vaccine.In process of production, the foot and mouth disease virus Nonstructural Protein in deactivation vaccine should be removed.The genetic vaccine of foot and mouth disease virus mainly adopts the structural protein development of foot and mouth disease virus, does not contain the composition of any foot and mouth disease virus Nonstructural Protein.Therefore, even repeatedly can not stimulate body to produce the specific antibody of Nonstructural Protein after the vaccination yet.
Summary of the invention
Adopt the reverse transcription-pcr technology to be separated to the encoding gene of one section foot and mouth disease virus Nonstructural Protein from foot and mouth disease virus.At expression in escherichia coli this encoding gene product, and then separation and purification arrived a kind of foot and mouth disease virus Nonstructural Protein, this recombinant protein is named as 3aB.3aB behind the purifying has been set up the animal that a kind of ELISA (enzyme linked immunosorbent assay) method is distinguished natural infection hostis pecoris and inoculation foot and mouth disease virus inactivated vaccine as detecting antigen.Detected serum with this method from 90 oxen, these serum from foot and mouth disease Asia 1 type and O C-type virus C infect, the two valency foot and mouth disease virus inactivated vaccines of inoculation, inoculation recombinant vaccine and do not infect the ox of foot and mouth disease virus.The result shows that the serum of all virus infection animals all shows antibody positive in this detection system, and inoculation foot and mouth disease virus vaccine and the animal serum that do not infect foot and mouth disease virus all show negative antibody.Illustrate that the ELISA that sets up with 3aB albumen has very high specificity and susceptibility, the animal that the ELISA method of setting up as envelope antigen with this albumen can be used to distinguish the natural infection foot and mouth disease virus and inoculate foot and mouth disease virus.This method has high specificity and susceptibility height, and few characteristics easy and simple to handle, consuming time are suitable for the examination of a large amount of serum.This method not only can be used for the negative animal of examination and carry out immunization, removes the animal of inapparent infection, eliminates potential contagium, helps control and eliminates FMD.Also can import and export the method that quarantine provides examination virus infection animal, provide safeguard for meat and milk preparation safety simultaneously for the livestock that lives.
Description of drawings
Fig. 1: reorganization Asia 1 type foot and mouth disease virus Nonstructural Protein 3aB dna fragmentation agarose gel electrophoresis
Swimming lane 1:2000DNA molecular weight scale
Swimming lane 2:3aB dna fragmentation has target DNA fragment at the 432bp place.3AB coding DNA size is 672bp, and the 3aB size behind the N end brachymemma 240bp is 432bp, and is consistent with theoretical size.
Fig. 2: pMD18T-3aB recombinant plasmid enzyme is cut the back agarose gel electrophoresis
The contrast of swimming lane 1:pMD18T-3aB recombinant plasmid
Swimming lane 2:2000DNA molecular weight scale
After cutting, swimming lane 3:pMD18T-3aB recombinant plasmid enzyme discharges the dna fragmentation of 432bp, as shown by arrows.
Fig. 3: pET28a (+)-3aB recombinant plasmid restriction enzyme digestion and electrophoresis
Swimming lane 1:2000DNA molecular weight scale
Swimming lane 2:pET28a (+)-3aB recombinant plasmid contrast
Swimming lane 3:pET28a (+)-3aB recombinant plasmid enzyme is cut the dna fragmentation that discharges 432bp after the back enzyme is cut, as shown by arrows.
Fig. 4: the SDS-PAGE of recombined foot-and-mouth disease virus Nonstructural Protein 3aB analyzes and Western blot identifies
A: the SDS-PAGE of recombined foot-and-mouth disease virus Nonstructural Protein 3aB analyzes
Swimming lane 1: molecular weight of albumen scale
Swimming lane 2: inductive bacterial lysate not
Swimming lane 3:IPTG induces the bacterial lysate behind the 3h, and recombined foot-and-mouth disease virus Nonstructural Protein 3aB as shown by arrows.
The molecular weight of recombined foot-and-mouth disease virus Nonstructural Protein 3aB is about 29KD.
B:Western blot identifies recombined foot-and-mouth disease virus Nonstructural Protein 3aB
Swimming lane 2: inductive bacterial lysate not, do not discerned by the foot and mouth disease virus specific antibody, there is not discernible band.
Swimming lane 3:IPTG inductive bacterial lysate, recombined foot-and-mouth disease virus Nonstructural Protein 3aB is discerned by the foot and mouth disease virus specific antibody, specific band occurs.As shown by arrows.
Fig. 5: the SDS-PAGE electrophoresis of identifying recombinant protein 3aB solubility expression
Swimming lane 1: inductive bacterial lysate not
Swimming lane 2:IPTG induces the bacterial lysate behind the 3h, and recombinant protein 3aB as shown by arrows.
Swimming lane 3: the resuspended liquid of the precipitation behind the ultrasonic degradation thalline contains small part recombinant protein 3aB.
Swimming lane 4: the supernatant liquor behind the ultrasonic degradation thalline contains most of recombinant protein 3aB.
Swimming lane 5: molecular weight of albumen scale
Fig. 6: the SDS-PAGE electrophoresis of recombinant protein 3aB purifying
Swimming lane 1: molecular weight of albumen scale
Swimming lane 2: the not purified bacterial lysate that contains recombinant protein 3aB
Swimming lane 3: through not following medium bonded foreign protein solution behind the nickel post
Swimming lane 4: the foreign protein solution that washs from the nickel post
Swimming lane 5: the solution that contains recombinant protein 3aB that elutes from the nickel post
Swimming lane 6: the solution process that contains recombinant protein 3aB is except that the solution behind the salt plug desalination
Arrow is depicted as the 3aB recombinant protein.
Fig. 7: the 3aB-ELISA method detects 3aB protein specific antibody in the bovine serum
Natural sera in the X-coordinate is not infected also the not bovine serum of artificial inoculation vaccine by foot and mouth disease virus, and FMDV infects the specific antibody feminine gender; It is the bovine serum of artificial inoculation FMDV Asia 1 type and O C-type virus C that FMDV infects serum, and FMDV infects the specific antibody positive; Vaccination serum is the bovine serum behind two valency deactivation vaccines of artificial inoculation and the recombinant vaccine, and FMDV infects the specific antibody feminine gender.Ordinate zou is represented the OD value of color reaction in the ELISA detection.Horizontal line infects the positive and negative threshold value of specific antibody for distinguishing FMDV.
The acquisition of embodiment 1 reorganization Asia 1 type foot and mouth disease virus Nonstructural Protein 3aB encoding gene
Material:
Foot and mouth disease Asia 1 C-type virus C (the biological Group Co.,Ltd of Inner Mongol gold space); Trizol reagent, DEPC (TakaRa company); Chloroform, Virahol, ethanol, CaCl2, glucose, Tris, EDTA, NaOH, SDS, Potassium ethanoate, Glacial acetic acid, phenol, chloroform, DMSO (Beijing Chemical Plant); AMV, Rnasin, dNTPs, Taq archaeal dna polymerase, Rnase, ECOR I, Sal I, solution I, T4 ligase enzyme, pMD18T, pET28a (+) (TakaRa company); Penbritin (worker's biotechnology company limited is given birth in Shanghai); LB substratum (OXOID LTD.ENGLAND); JM109 and BL21 (DE3) (Novagen company)
The PCR primer:
Primer 1:5 ' CCATGGAATTCAGATCT AAGAGACAGCAGATGGTGA 3 '
Primer 2: 5 ' AAGCTTCTAGTCGACGGATCCCTCAGTGACAATCAAGTTC 3 '
And Oligo dT (it is synthetic that worker's biotechnology company limited is given birth in Shanghai)
Method:
1. the extraction of foot and mouth disease virus RNA
Adopt the Trizol method from foot and mouth disease Asia 1 C-type virus C of deactivation, to extract RNA.Concrete steps are as follows:
1) gets 0.4ml inactivation of viruses stoste, add the 0.8ml dehydrated alcohol.Fully put upside down mixing, the centrifugal 15min of 11000rpm.
2) add 0.5ml Trizol reagent, dissolution precipitation acutely shakes mixing 30s, incubated at room 10min up and down.
3) add the 0.2ml chloroform, acutely put upside down mixing 15s, incubated at room 5min up and down; 11,000rpm, 4 ℃, centrifugal 15min.
4) upper water moves in another centrifuge tube mutually, adds the equal-volume Virahol.Room temperature is placed 10min, 11, the centrifugal 15min of 000rpm.
5) abandon supernatant, in slow adherent adding 1ml 75% ethanol in precipitation opposite (facing), 11000rpm, centrifugal 20s with preceding 10min preparation.Abandon supernatant, dry 3-15min.
6) stand-by with 9 μ l DEPC-water dissolution RNA.
2. reverse transcription
With Oligo dT is primer, and the RNA of above-mentioned gained is a template, and reverse transcription synthesizes cDNA.
In the 0.5ml centrifuge tube, add Oligo dT (100 μ M) 1 μ l, RNA 9 μ l.The rearmounted 70 ℃ of water-bath 10min of mixing add following reagent: 5 * AMV buffer, 4.0 μ l, RNasin (40U) 2.0 μ l, dNTPs (10mM) 2.0 μ l, AMV (5U/ μ l) 2.0 μ l at once after the ice bath 2min..Final volume is 20 μ l, the rearmounted 42 ℃ of water-bath 60min of mixing.70 ℃ of water-bath 15min are used for pcr amplification.
3.PCR amplification 3aB dna fragmentation
The cDNA that obtains with step 2 is a template, carries out pcr amplification with primer 1 and primer 2.50 μ l reaction systems add cDNA 2 μ l, 10 * PCR buffer, 5 μ l, dNTP (10 μ M) 1 μ l, Mgcl2 (50 μ M) 3 μ l, upstream primer (10 μ M) 0.5 μ l, downstream primer (10 μ M) 0.5 μ l, Taq archaeal dna polymerase (5U/ μ l) 0.25 μ l, ultrapure water 37.75 μ l.
In the rearmounted PCR instrument of centrifugal mixing, 94 ℃ of warm start 5min, carry out following circulation: 94 ℃, 30s; 57 ℃, 1min; 72 ℃, 2min; 25 circulations are extended 10min for back 72 ℃.The PCR product is identified through 1% agarose gel electrophoresis.
4.3aB the purifying of dna fragmentation reclaims
Earlier with the PCR product in 1% sepharose electrophoresis (120v 15min), after purpose fragment and assorted band separate, contains the segmental gel piece of purpose with the cleaning blade cutting-out.Plastic squeeze is reclaimed behind-20 ℃ of frozen 20min.
The result:
See Fig. 1.The size of 3aB dna fragmentation is 432bp.Arrow is depicted as the 3aB dna fragmentation among the figure, the about 432bp of size.
Material:
Ethanol, CaCl2, glucose, Tris, EDTA, NaOH, SDS, Potassium ethanoate, Glacial acetic acid, phenol, chloroform, DMSO (Beijing Chemical Plant); Rnase, ECOR I, Sal I, solution I, pMD18T (TakaRa company); Penbritin (worker's biotechnology company limited is given birth in Shanghai); JM109 (Novagen company); LB substratum (OXOID LTD.ENGLAND); Agarose (Beijing ancient cooking vessel state Bioisystech Co., Ltd)
Method:
1.3aB dna fragmentation is connected with linear pMD18T
3aB dna fragmentation after reclaiming is connected by T-A with linear pMD18T carrier, and linked system is 10 μ l, that is: dna fragmentation 4.7 μ l, and pMD18T carrier 0.3 μ l, solution I 5 μ l, 16 ℃ of connections are spent the night.
2. the preparation of competent cell
1) choose a single bacterium colony in 5mL LB substratum, 37 ℃, 225rpm shaking culture 12-16h.
2) get the above-mentioned culture of 1mL and be inoculated in the 100mL LB substratum, 37 ℃, it is about 0.5 that the 225rpm velocity fluctuation is cultured to the OD value.
3) with bacterium liquid ice bath 10min, 2500 * g then, 4 ℃ of centrifugal 20min collect thalline.
4) abandon supernatant, be inverted centrifuge tube 1min Ex-all remaining liq, add the ice-cold 100mmol/L CaCl of 10mL then
2Solution (high pressure degerming) suspension cell, ice bath 45min.
5) 2000 * g, 4 ℃ of centrifugal 10min collect thalline, abandon supernatant, add the ice-cold 100mmol/L CaCl of 4mL
2Solution, suspension cell.
6) hang cell with the CaCl2 solution that contains 0.05%DMSO and 15% glycerine, with sample injector mixing gently, packing 100 μ l/ manage, be stored in-70 spend standby.
3.3aB being connected product with linear pMD18T, dna fragmentation is transformed into the JM109 bacterium
1) in super clean bench, 10 μ l is connected product and add in the JM109 competent cell, flick mixing, place 30min on ice.
2) the Eppendorf pipe is transferred in 42 ℃ of water-baths 45s rapidly.Transfer to rapidly on ice, place 5min.
3) bacterium liquid is added in the 1ml LB substratum, and 37 ℃, 150rpm, 30min makes cell recovery.
4) get bacterium liquid and place the Eppendorf pipe, the centrifugal 5min of 5000rpm.Abandon 800 μ l supernatants, with the resuspended bacterium of remaining culture liq.
5) resuspended bacterium liquid moves into and has on the LB agar plate of amicillin resistance, smears evenly, is inverted into incubated overnight in the 37 degree thermostat containers.
4.pMD18T-3aB the extraction of recombinant plasmid
1) choose the mono-clonal bacterium colony and in 5ml LB substratum, (contain penbritin 50 μ g/ml), 37 ℃, 180r/m shaking culture 14-16 hour.
2) bacterium liquid is poured into branch centrifugal (11000rpm, 4 ℃, centrifugal 2min) in the 2ml pipe, abandoned nutrient solution.Recentrifuge exhausts nutrient solution.
3) bacterial precipitation is resuspended among the solution I (50mmol/L glucose, 25mmol/LTris.Cl (pH8.0), 10mmol/LEDTA (pH8.0)) of 300 μ l precoolings the vibration of vortex oscillation device, piping and druming mixing.
4) now join solution II (0.2mol/LNaOH, 1%SDS, 1: 1).The solution II that adds the new preparation of 300 μ l softly puts upside down the EP pipe up and down 6 times, with the cracking bacterium, uncaps and sees that the wire drawing phenomenon gets final product.
5) horse back adds solution III (5mol/L Potassium ethanoate 60ml, Glacial acetic acid 11.5ml, deionized water 28.5ml) the 300 μ l of precooling, and the mixing that turns upside down 5 times is put 5min on ice, and 4 ℃, the centrifugal 5min of 11000rpm.
6) supernatant is transferred in another EP pipe, recentrifuge is removed white precipitate.Add the saturated phenol of equal-volume TE: chloroform (1: 1), vibration mixing 1min, 4 ℃, the centrifugal 5min of 11000rpm.
7) upper water is moved in another Ep pipe mutually, add isopyknic chloroform, vibration mixing, 11000rpm, centrifugal 3min.
8) upper water is moved to mutually in another Ep pipe, add the dehydrated alcohol of 2-2.5 times of volume, put upside down mixing ,-20 ℃ of precipitation 20min, the centrifugal 10min of 11000rpm.
9) abandon clean supernatant, the centrifugal 2min of 11000rpm abandons dehydrated alcohol with the sample injector suction as far as possible.37 ℃ of thermostat container thorough drying plasmid DNA, about 10min.
10) contain the deionized water of RNase 25 μ g/ml with 20 μ l, the dissolving plasmid DNA ,-20 ℃ are frozen.
5.pMD18T-3aB the evaluation of recombinant plasmid
Enzyme is cut evaluation: with ECORI and Sal I recombinant plasmid is carried out double digestion, reaction system is 10 μ l: plasmid 2 μ l, ECOR I 0.5 μ l, Sal I 0.5 μ l, 10 * H buffer, 1 μ l, deionized water 6 μ l.37℃，45min。1% agarose gel electrophoresis is identified clip size.Enzyme is cut the positive clone bacterium in evaluation back serve the order-checking of sea living worker Bioisystech Co., Ltd.
The result:
See Fig. 2.The pMD18T-3aB recombinant plasmid should discharge the dna fragmentation of 432bp size in theory behind ECOR I and Sal I double digestion.Arrow is depicted as the dna fragmentation of release among the figure, and size is about 432bp, with consistent in theory.The result of sequencing is shown in sequence in the sequence table 2.
Material:
CaCl2, glucose, Tris, EDTA, NaOH, SDS, Potassium ethanoate, Glacial acetic acid, phenol, chloroform, DMSO (Beijing Chemical Plant); Rnase, ECOR I, Sal I, T4 ligase enzyme, pET28a (+) (TakaRa company); Kantlex (worker's biotechnology company limited is given birth in Shanghai); LB substratum (OXOID LTD.ENGLAND); BL21 (DE3) (Novagen company)
Method:
1.3aB dna fragmentation is connected with linear pET28a (+)
The enzyme of 3aB dna fragmentation and pET28a (+) carrier cuts back to close: correct pMD18T-3aB recombinant plasmid and pET28a (+) plasmid in back that will check order cut with ECOR I and Sal I enzyme respectively.The enzyme system of cutting is 20 μ l: plasmid 5 μ l, ECOR I 1 μ l, Sal I 1 μ l, 10 * H buffer, 2 μ l, deionized water 11 μ l.37℃，60min。1% agarose gel electrophoresis separates 3aB fragment and linearizing pET28a (+) carrier.Under ultraviolet lamp, downcut the purpose band, place-20 ℃ of refrigerator and cooled to freeze 20min, take out the back plastic squeeze and reclaim 3aB gene fragment and pET28a (+) linear carrier.Agarose gel electrophoresis is quantitative once more.
The 3aB gene fragment is connected with pET28a (+) carrier: linked system is 20 μ l, that is: 3aB gene fragment 14 μ l, and pET28a (+) carrier 3 μ l, T4 ligase enzyme 1 μ l, 16 ℃ of connections of 10 * buffer, 2 μ l are spent the night.
Transform: 10 μ l are connected product be transformed in BL21 (DE3) competent cell.Method for transformation as previously mentioned.
The extraction and the evaluation of (2.pET28a+)-3aB recombinant plasmid
Extract recombinant plasmid in a small amount with aforesaid alkaline lysis, enzyme is cut evaluation.With ECOR I and Sal I recombinant plasmid is carried out double digestion, reaction system is 10 μ l: plasmid 2 μ l, ECOR I 0.5 μ l, Sal I 0.5 μ l, 10 * H buffer, 1 μ l, deionized water 6 μ l.37℃，45min。1% agarose gel electrophoresis evaluation enzyme is cut the segmental size of back released dna.Enzyme is cut the positive clone bacterium in evaluation back serve the order-checking of sea living worker Bioisystech Co., Ltd.
The result:
See Fig. 3.PET28a (+)-3aB recombinant plasmid discharges the dna fragmentation of 432bp size after with ECOR I and Sal I double digestion in theory.Arrow is depicted as the dna fragmentation of release among the figure, and size is about 432bp, with consistent in theory.The result of sequencing is shown in sequence in the sequence table 2.
The abduction delivering of embodiment 4 recombinant protein 3aB
Material:
Glycerine (Beijing Chemical Plant); Kantlex (worker's biotechnology company limited is given birth in Shanghai); IPTG (Beijing ancient cooking vessel state Bioisystech Co., Ltd).
Method:
To be inoculated in 0.5～1/1000 ratio in the LB liquid nutrient medium of 20mL kalamycin resistance (50 μ g/mL) through the BL21 that contains the pET28-3aB recombinant plasmid (DE3) the glycerine bacterial classification after identifying, 37 ℃, 150r/m cultivates 12 hours as first order seed.All be inoculated in first order seed in the 1L LB substratum next day, 37 ℃ of shaking table 225r/m shaking culture, and monitoring A600, when reaching 0.5-0.8, adding final concentration is 1 μ mol/L IPTG, collects wet bacterium after inducing 3h ,-70 ℃ of the backs of weighing are frozen.
The result:
See Fig. 4 A.Swimming lane 2 is for inducing the bacterial lysate behind the 3h among the figure, and a visible higher protein band of expression amount is recombinant protein 3aB at the 29KD place, and the molecular weight size is consistent with software prediction.
The Western blot of embodiment 5 recombinant protein 3aB identifies
Material:
Xylene Brilliant Cyanine G, DAB (Beijing ancient cooking vessel state Bioisystech Co., Ltd); Skim-milk (Wanda Mountain dairy industry company limited); The anti-ox IgG of the rabbit of horseradish peroxidase-labeled (sigma); H
2O
2(Beijing Chemical Plant); NC film (Wuhan Boster Biological Technology Co., Ltd.);
Method:
With before inducing and BL21 (DE3) cellular lysate after inducing carry out SDS-PAGE and separate, the commentaries on classics film is pressed 0.65mA/cm according to the gel area
2Electrotransfer 2h.After electrotransfer finishes, gel is also decoloured by coomassie brilliant blue staining, to judge whether successfully to change film.To changeing the nitrocellulose filter (NC film) of film success, add confining liquid (5%[W/V] skim-milk) according to membrane area with the amount of 0.1mL/cm2, sealing is spent the night.Next day, the amount of pressing 0.1mL/cm2 adds first antibody (FMDV AsiaI virus infection serum, dilution in 1: 50), the NC film is lain on the shaking table that shakes gently, behind incubated at room 2h, with (10min/ time) after the PBS washing 3 times.The anti-ox IgG of rabbit (1/5000 dilution) that adds horseradish peroxidase-labeled is behind incubated at room 2h, with 3 times (10min/ time) of PBS washing.Diaminobenzidine (DAB) with dissolving 6mg in the PBS solution of 9mL adds 10mL 30%H
2O
2, immediately the NC film is put into colour developing behind the mixing.Observe the colour developing of NC film, after the color depth of discovery protein band reaches requirement, color development stopping.
The result:
See Fig. 4 B.Swimming lane 2 is for inducing the cellular lysate thing behind the 3h among the figure, has one to be infected the band that the back bovine serum is discerned by foot and mouth disease virus at 29KD place.This band is that recombinant protein 3aB is discerned by the foot and mouth disease virus specific antibody.
The evaluation of embodiment 6 recombinant protein 3aB solubility expressions
Method:
Ultrasonicly split bacterium: induce the BL21 thalline that contains the 3aB recombinant protein behind the 3h, mixing on ice by 1: 5 resuspended IPTG of (m/v) PBS.The ultrasonication instrument transfers to 800W, and work 5 ' at intermittence 5 ', ultrasonic 60 times, is total to the 10min supersound process.11000r/m, centrifugal 30min collects supernatant.To precipitate with after the PBS washing three times, use resuspended precipitation with the isopyknic PBS of supernatant.To go up and cleer and peacefully hang sedimentary liquid and carry out the SDS-PAGE electrophoresis and identify the proteic phraseology of 3aB.
The result:
See Fig. 5.Ultrasonic can not broken inclusion body, contained target protein is a recombinant protein 3aB solubility expression part in the supernatant liquor after therefore ultrasonic.Contained target protein is that inclusion body is expressed part in the precipitation.Visible about 70%3aB recombinant protein is at supernatant liquor among the figure, and therefore, 70% recombinant protein 3aB is a solubility expression.
The purifying of embodiment 7 solubility recombinant protein 3aB
Material:
Sepharose4B-Ni2+, Sephadex G25 (Pharmacia); Tris, NaCl (Beijing Chemical Plant); Imidazoles (Tianjin recovery fine chemistry industry institute); Lowry protein quantification reagent (sigma packing); BSA (LVSHENGYUANBIOTECHNOLOGY).
Method:
The proteic purifying of 3aB: will split the supernatant that obtains behind the bacterium and slowly be added on the nickel affinity chromatography post, treat that supernatant liquor all adds after, with lavation buffer solution (20mmol/LTris; 0.5mol/LNaCl; The 30mmol/L imidazoles; PH 7.8) slowly wash post bed 4h, all wash lower prop to foreign protein; With elutriant wash-out 3aB albumen (20mmol/L Tris; 0.5mol/L NaCl; 0.5mol/L imidazoles; PH 7.8); Collection contains the proteic elutriant of 3aB.Elutriant is added except that salt plug, remove salt buffer (20mmol/L Tris; 0.9%NaCl; PH 7.8) wash-out, collect 3aB albumen.SDS-PAGE analyzes, and identifies the purity of recombinant protein.
3aB is proteic quantitatively: employing Bradford method is carried out quantitative analysis to the recombinant protein of purifying, promptly does typical curve with BSA (BSA, human serum albumin) as standard substance, gets three lyophilized proteins at random, and 2 or 3 extent of dilution are also done in the ultrapure water dissolving.3 multiple holes use microplate reader to detect the A576 value.
The result:
See Fig. 7.Swimming lane 7 recombinant protein 3aB after the 29KD place has a tangible protein band to be purifying among the figure.Through software analysis, the purity of the 3aB recombinant protein behind the purifying reaches more than 90%.
The 3aB-ELISA method of setting up embodiment 8 detects anti-3aB antibody
Material:
Skimmed milk (Wanda Mountain dairy industry company limited), the anti-ox IgG of the rabbit of HRP mark (sigma); OPD (Beijing ancient cooking vessel state Bioisystech Co., Ltd) H
2O
2(Beijing Chemical Plant); Various serum (the biological Group Co.,Ltd of Inner Mongol gold space)
Method:
With 3aB albumen bag quilt, set up the indirect ELISA method (called after 3aB-ELISA) that detects anti-3aB antibody
1) bag quilt: with PBS solution (Nacl 8.0g, KCl 0.2g, Na
2HPO
412H
2O 3.58g, KH
2PO
40.24g PH 7.2) 3aB albumen is diluted to final concentration 2 μ g/mL.Add 0.1mL in the reacting hole of each polystyrene board, 4 ℃ are spent the night.Discard solution in the hole next day, with lavation buffer solution (PBST: the PBS that contains 0.05%Tween) wash 3 times, each 3 minutes.
2) sealing: confining liquid (PBST that contains 5% skimmed milk) 0.2mL in above-mentioned wrapped by reacting hole in, put 37 ℃ and hatched 1 hour.Do not wash.
3) anti-in conjunction with one: as in each reacting hole, to add test serum, positive serum and negative serum 0.1mL with sample diluting liquid (PBST that contains 5% skimmed milk) fresh dilution in 1: 100.Hatched 1 hour for 37 ℃, as above washing.
4) anti-in conjunction with two: as in each reacting hole, to add the anti-ox IgG0.1mL of rabbit with the HRP mark of sample diluting liquid fresh dilution in 1: 300.Hatched 1 hour for 37 ℃, as above washing.
5) colour developing: the OPD/H that in each reacting hole, adds interim preparation
2O
2Substrate solution 0.1mL, room temperature lucifuge colour developing 5～30 minutes.
6) stop: in each reacting hole, add 20% sulfuric acid 0.05mL.On the ELISA detector, survey each hole OD value in 492nm.
Negative decision content=NCX+3SD (NCX, the mean number of negative control A492 value; SD, standard deviation) positive decision content=PCX+3SD (PCX, the mean number of positive control A492 value; SD, standard deviation) sample A492 value 〉=positive decision content is positive ,≤negative decision content negative.Between positive decision content and negative decision content, be suspicious sample, need further to judge.
Detect the serum of 90 routine different immunological statuss with this method, comprised bovine serum (comprising that Asia 1 type and O type foot and mouth disease virus infect serum), 30 routine vaccinated bovine serums and the 20 routine healthy bovine serums of 40 routine FMDV natural infections.
The result:
The serum of all virus infectiones shows positive, and all vaccinated serum all negative (Fig. 7).This result shows that the specificity of this method and susceptibility are 100%.
Conclusion:
Above result shows, the method for the indirect ELISA of setting up as envelope antigen with reorganization 3aB albumen can be used to distinguish that foot and mouth disease virus infects and and the animal of inoculation aftosa vaccine.This method has specificity and susceptibility height, and few characteristics easy and simple to handle, consuming time are suitable for the examination of a large amount of serum.This method not only can be used for the negative animal of examination and carry out immunization, removes the animal of inapparent infection, eliminates potential contagium, helps control and eliminates FMD.Also can import and export the method that quarantine provides examination virus infection animal, provide safeguard for meat and milk preparation safety simultaneously for the livestock that lives.
Sequence table
＜110〉Beijing DiWeiHuaYu Biological Technology Co., Ltd
＜120〉the ELISA test kit of discriminating natural infection and artificial immunization foot and mouth disease virus
<160>2
<210>1
<211>144
<212>PRT
＜213〉artificial sequence
<400>1
Lys?Arg?Gln?Gln?Met?Val?Asn?Asp?Ala?Val?Asn?Glu?Tyr?Ile?Asp 15
Lys?Ala?Asn?Ile?Thr?Thr?Asp?Asp?Lys?Thr?Leu?Asp?Glu?Ala?Glu 30
Lys?Asn?Pro?Leu?Glu?Thr?Ser?Gly?Ala?Ser?Thr?Val?Gly?Phe?Arg 45
Glu?Arg?Thr?Leu?Pro?Gly?Arg?Lys?Thr?Ser?Asp?Asp?Val?Asn?Ser 60
Glu?Pro?Val?Lys?Pro?Val?Glu?Glu?Gln?Pro?Gln?Ala?Glu?Gly?Pro 75
Tyr?Ala?Gly?Pro?Leu?Glu?Arg?Gln?Lys?Pro?Leu?Lys?Val?Arg?Ala 90
Lys?Leu?Pro?Gln?Gln?Glu?Gly?Pro?Tyr?Ala?Gly?Pro?Met?Glu?Arg 105
Gln?Lys?Pro?Leu?Lys?Val?Lys?Ala?Lys?Ala?Pro?Val?Val?Lys?Glu 120
Gly?Pro?Tyr?Glu?Gly?Pro?Val?Lys?Lys?Pro?Val?Ala?Leu?Lys?Val 135
Lys?Ala?Lys?Asn?Leu?Ile?Val?Thr?Glu 144
<210>2
<211>432
<212>DNA
＜213〉artificial sequence
<400>2
AAGAGACAGC?AGATGGTGAA?TGATGCGGTG?AACGAGTACA?TCGACAAAGC?CAACATCACC 60
ACAGATGACA?AGACTCTTGA?CGAGGCGGAA?AAGAACCCTC?TGGAGACCAG?TGGTGCTAGC 120
ACCGTTGGTT?TCAGAGAGAG?AACCCTCCCG?GGGCGCAAGA?CGAGTGATGA?CGTGAACTCC 180
GAGCCCGTCA?AACCCGTGGA?GGAACAACCA?CAAGCTGAAG?GACCCTACGC?CGGGCCACTC 240
GAGCGTCAGA?AACCTCTGAA?AGTGAGAGCC?AAGCTCCCAC?AGCAAGAGGG?ACCCTACGCT 300
GGCCCGATGG?AGAGACAGAA?ACCACTGAAA?GTGAAAGCAA?AAGCCCCGGT?CGTTAAGGAA 360
GGGCCTTACG?AAGGACCGGT?GAAGAAACCT?GTCGCTTTGA?AAGTGAAAGC?GAAGAACTTG 420
ATTGTCACTG?AG 432
Claims (8)
1. recombined foot-and-mouth disease virus Nonstructural Protein, it has the aminoacid sequence shown in the SEQ ID NO1.
2. recombined foot-and-mouth disease virus Nonstructural Protein, its encoding gene has the nucleotide sequence shown in the SEQ ID NO 2.
3. an ELISA method is characterized in that, utilizes claim 1 or 2 arbitrary described recombined foot-and-mouth disease virus Nonstructural Proteins to differentiate the animal of foot and mouth disease virus natural infection and inoculation aftosa vaccine.It comprises following basic step:
1) utilize claim 1 or 2 arbitrary described recombined foot-and-mouth disease virus non-structural proteins envelope antigen bag in vain to be detected the culture plate of usefulness by ELISA;
2) seal the culture plate that ELISA detects usefulness with skimmed milk or other non-recombined foot-and-mouth disease virus Nonstructural Protein;
3) add animal serum to be detected;
4) add enzyme and mark second anti-antibody, this kind of enzyme is marked the IgG that second anti-antibody includes but not limited to horseradish peroxidase-labeled;
5) add the enzyme substrates colour developing, this kind of enzyme substrate includes but not limited to OPD;
6) stop the chromogenic enzyme substrate reaction;
7) use the microplate reader detected result.
4. identification reagent box and using method thereof is characterized in that application rights requires 1 or 2 arbitrary described albumen, and application rights requires 3 described steps.
5. the described test kit of claim 4 is used to differentiate the purposes of the foot and mouth disease virus natural infection and the animal of inoculation aftosa vaccine.
6. the described test kit of claim 5 is used to differentiate the purposes of foot and mouth disease correlated virus.
7. utilize claim 1 or 2 arbitrary described recombined foot-and-mouth disease virus Nonstructural Proteins to be used to differentiate the purposes of foot and mouth disease virus natural infection and inoculation aftosa vaccine animal.
8. described according to claim 3-7, animal wherein is that ox, sheep, pig and other can be by the foot and mouth disease virus infected animals.
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| CN102662063A (en) * | 2012-04-25 | 2012-09-12 | 中国农业科学院兰州兽医研究所 | Detection kit and method for non-structural protein antibody dot blots of foot and mouth disease viruses |
| CN103076451A (en) * | 2013-01-17 | 2013-05-01 | 中国农业科学院兰州兽医研究所 | O type foot-and-mouth disease 146S antigen quantitative ELISA detection kit and method for using same |
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| CN105348386A (en) * | 2015-11-06 | 2016-02-24 | 北京三联博悦生物技术有限公司 | Anti foot-and-mouth disease type O (O/GX/09-7) virus monoclonal antibody combination, ELISA kit and gold-labelled paper chromatography test paper for detection of the same |
| CN105348386B (en) * | 2015-11-06 | 2019-02-19 | 北京标驰泽惠生物科技有限公司 | The monoclonal antibody cocktail and detection kit of the O-shaped virus of resistant to foot and mouth disease |
| CN107513101A (en) * | 2017-09-18 | 2017-12-26 | 中牧实业股份有限公司 | Swine foot-and-mouth disease virus non-structural protein antibody ELISA immunity detection reagent |
| CN109765366A (en) * | 2019-01-31 | 2019-05-17 | 中国农业科学院兰州兽医研究所 | A kind of kit and its detection method detecting foot and mouth disease virus 3AB antibody |
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Application publication date: 20110921 |