CN104356232A - Preparation method of specific IgY for resisting bovine viral diarrhea virus protein E2 - Google Patents
Preparation method of specific IgY for resisting bovine viral diarrhea virus protein E2 Download PDFInfo
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Abstract
The invention discloses a preparation method of specific IgY for resisting bovine viral diarrhea virus protein E2 (BVDV-E2). The preparation method comprises the steps: (1) designing a pair of primers according to the BVDV-E2 gene sequence, performing polymerase chain reaction (PCR) amplification on the BVDV-E2 genes, connecting the genes to a carrier pMD18T, converting competent cells DH5a, performing blue-white selection on the competent cells, extracting plasmids, performing enzyme digestion analysis on the extracted plasmids, sequencing positive plasmids, and analyzing the sequenced results; (2) expressing and purifying E2 proteins; (3) preparing a E2-resisting IgY antibody by purifying immune E2 protein laying hens, extracting specific IgY antibodies by using a PEG6000 precipitation method, and performing SDS-PAGE analysis, ELISA potency monitoring, Western blotting specificity analysis on the antibodies. By using the preparation method disclosed by the invention, the E2-resisting IgY antibody can be greatly combined with the E2 protein, but not generate a cross reaction with degradation segments.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of Synthesis and applications of specific IgY, be specifically related to a kind of preparation method of anti-bovine viral diarrhea virus albumen E2 specific IgY.
Background technology
Bovine viral diarrhea (Bovine viral diarrhea, BVD), also known as bovine viral diarrhoea. mucosal disease (Bovine viral diarrhea-mucosal disease, BVD-MD) be cause the various clinical symptom of ox as high heat, oligoleukocythemia, depressed, diarrhea by bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV), salivate in a large number, subtract milk down to dry up, to ruminate stopping, conjunctivitis, oral mucosa hemorrhage and occur a kind of disease of ulcer symptoms etc.Calver may be miscarried, be produced stillborn foetus and monster etc.Also can cause immunological tolerance and the persistent infection of ox, immunosuppression.
This disease is worldwide widely current, and causes serious financial loss to countries in the world livestock industry, and die from this ox infected every year and be no less than 5,000,000, the indirect loss simultaneously caused due to its persistent infection etc. also seriously hinders the development of livestock industry.
Detection method main both at home and abroad has at present:
Round pcr has that high specificity, susceptibility are high, the feature such as rapidly and efficiently, has been widely used in multiple field.In recent years, new development was had again.RT-PCR sensitivity can detect BVDV from tissue, movement, serum, cell culture etc. and can distinguish swine fever and sheep border disease virus rapidly, utilizes the method can also carry out the research of BVDV nucleotide sequence.
Virus isolation techniques be one the most substantially, detection method the most accurately.The cell being commonly used to be separated BVDV is nose of an ox tracheal cell (BT) and bovine kidney cells passage cell strain (MDBK).
Electron microscopy becomes the indispensable means of virological investigation, quick diagnosis.This method is quick and easy, directly perceived.But electron microscopic observation needs the virus particle of high-content, need to concentrate virus.
The detection of antibody can provide foundation for the formulation of immunization program and clinical diagnosis.Compare other antibody detection method, colloidal gold strip and ELISA kit easy and simple to handle, cheap, be widely used clinically, colloidal gold strip is compared with traditional HI, and its Sensitivity and Specificity reaches 97.1%, and 76.6%, and ELISA kit positive rate reaches 85%, and HI coincidence rate reaches 80%.
IgY technology, namely by bird (chicken) immunization specific antigen, obtains specific polyclonal antibody from yolk.Relative to mammalian antibody, IgY technology avoids conventional animal blood taking, meets animal welfare requirement, and antibody production is large, and preparation is relatively simple, and economic advantages are obvious, what is more important, and IgY also has a series of biology and antibody characteristic and advantage.In recent years, IgY antibody all presents good growth momentum at medicine, biological technical field.IgY has been widely used in the pathogen detection such as bacterium, virus, and specificity, susceptibility are better, can reduce BVDV treatment cost and lay the foundation based on the development of IgY detection kit simultaneously.
Summary of the invention
The object of the invention is to for deficiency of the prior art, provide a kind of preparation method of anti-bovine viral diarrhea virus albumen E2 specific IgY, for use in detection and the treatment of BVDV virus.
In order to solve the problems of the technologies described above, the present invention adopts following technical scheme to realize:
A preparation method for anti-bovine viral diarrhea virus albumen E2 specific IgY, is characterized in that, said method comprising the steps of:
(1) cloned and sequenced of bovine viral diarrhea virus raq gene: according to BVDV-E2 gene order, utilize primer prime 5.0 software, design pair of primers, pcr amplification BVDV-E2 gene (1040bp), is connected to pMD18-T carrier, transforms DH5 α competent cell, after blue hickie screening, extract plasmid, restriction analysis, positive plasmid checks order.
(2) expression of E2 albumen in intestinal bacteria, purifying.PMD18-T-E2 and pET-32a carrier uses BamH I and Xho I double digestion, and object fragment connects, and builds pET-32a-E2 expression vector, transform Bal21 (DE3) pLysS competence bacterium, enzyme is optimized IPTG induced concentration and time, is carried out a large amount of abduction delivering after cutting and identifying with PCR.Expression product is analyzed through SDS-PAGE and Western blot, uses Ni+ affinity column to carry out purifying to recombinant protein.Result shows, successfully constructs pET-32a-E2 expression vector, and restructuring E2 albumen induces 5h at the IPTG of 0.75mmol/L, and mainly exist with inclusion bodies, occur fragment near 43ku, expression product is after Ni+ column purification.Western blot shows that recombinant protein has good reactionogenicity.
(3) preparation of anti-E2-IgY antibody.The E2 protein immunization laying hen of purifying, uses PEG6000 to extract specific IgY antibody, and carries out SDS-PAGE analysis.Indirect ELISA measures anti-E2-IgY antibody titer after immunity, Western blot identify obtain the specificity of anti-E2-IgY.Result shows, IgY antibody contains two chains through SDS-PAGE analysis package, heavy chain (65ku) and light chain (19ku); Four exempt from after, anti-E2-IgY tires and reaches 1: 128000, Western blot and show, prepared IgY antibody can specific binding E2 albumen.
Described preparation immune egg method is as follows: carry out initial immunity injection to bird in 20 week age, immunizing dose be 300 μ g/ only, after initial immunity 21 days, carry out 4 booster immunizations, interval time is 21 days, immunizing dose be 250 μ g/ only, affiliated bird is chicken.
The method of described extraction IgY is as follows;
(1) egg of described collection is carried out yolk to be separated with egg white, collect yolk, used by yolk PBS (pH 7.4) to dilute according to volume ratio 1: 2;
(2) add to diluent the PEG-6000 that mass volume ratio is 3.5%, fully stir, room temperature effect 30 minutes on shaking table;
(3) mixed solution is centrifugal, collect supernatant, filter, filtrate adds the PEG-6000 that mass volume ratio is 8.5%, fully stirs, room temperature effect 30 minutes on shaking table;
(4) by centrifugal for the mixed solution after stirring, abandoning supernatant, the PBS of precipitation use 10 milliliters suspends, and adds the PEG-6000 that mass volume ratio is 12%, fully stirs, room temperature effect 30 minutes on shaking table, then carry out centrifugal, obtains throw out;
(5), after being used by described throw out 1.2 milliliters of PBS to dissolve, use dialysis band to dialyse, use PBS dialysed overnight, obtain IgY, and be kept at-20 DEG C for subsequent use.
Beneficial effect of the present invention:
(1) recombinant protein that method of the present invention is extracted has good reactionogenicity;
(2) the anti-E2-IgY antibody that the present invention extracts can preferably in conjunction with E2 albumen, and with degradation fragment no cross reaction;
(3) extracting method is relatively simple, and antibody production is large, and preparation cost is low.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is elaborated.
A preparation method of anti-bovine viral diarrhea virus E2 protein I gY, said method comprising the steps of:
(1) cloned and sequenced of bovine viral diarrhea virus raq gene.According to the relevant BVDV-E2 gene order of GenBank report, utilize primer prime 5.0 software, design pair of primers, pcr amplification BVDV-E2 gene (1040bp), is connected to pMD18-T carrier, transforms DH5 α competent cell, after blue hickie screening, extract plasmid, restriction analysis, positive plasmid checks order.
(2) expression of E2 albumen in intestinal bacteria, purifying.PMD18-T-E2 and pET-32a carrier uses BamHI and XhoI double digestion, and object fragment connects, and builds pET-32a-E2 expression vector, transform Bal21 (DE3) pLysS competence bacterium, enzyme is optimized IPTG induced concentration and time, is carried out a large amount of abduction delivering after cutting and identifying with PCR.Expression product is analyzed through SDS-PAGE and Western blot, uses Ni+ affinity column to carry out purifying to recombinant protein.Result shows, successfully constructs pET-32a-E2 expression vector, and restructuring E2 albumen induces 5h at the IPTG of 0.75mmol/L, mainly exists with inclusion bodies, near 43ku, occurs fragment.Expression product is after Ni+ column purification, and Western blot shows that recombinant protein has good reactionogenicity.
(3) preparation of anti-E2-IgY antibody.The E2 protein immunization laying hen of purifying, uses PEG6000 to extract specific IgY antibody, and carries out SDS-PAGE analysis.Indirect ELISA measures anti-E2-IgY antibody titer after immunity, Western blot identify obtain the specificity of anti-E2-IgY.Result shows, IgY antibody contains two chains through SDS-PAGE analysis package, heavy chain (65ku) and light chain (19ku); Four exempt from after, anti-E2-IgY tires and reaches 1: 128000, Western blot and show, prepared IgY antibody can specific binding E2 albumen.
Embodiment
The present embodiment is intended cloning BVDV-E2 structural protein, builds prokaryotic expression carrier pET-32a-E2, expression and purification E2 albumen, and it can be used as immunogen, immunization laying hen, produce anti-E2 recombinant protein specific IgY antibody.
1, design of primers and synthesis
According to bovine viral diarrhea virus Reference Strains (FJ011098) sequence of GenBank registration, software Primer Prime 5.0 is used to design pair of primers, for the raq gene full length DNA sequence (11040bp) that increases.Upstream primer P:5 ,-AT
gGATCCaATATAGACTCGGCG-3, (underscore place is BamH I restriction enzyme site), downstream primer R:5 ,-CCG
cTCGAGaCCATGGTATGTCTA-3, (underscore place is Xho I restriction enzyme site).Primer is synthesized by Shanghai Sheng Gong company, and distilled water dilution is 10pmol/ μ L ,-20 DEG C of preservations.
2, the pcr amplification of raq gene
Template preparation: get viral supernatants 50 μ L, after boiling water boiling 10min, the centrifugal 10min of 12000rpm, gets supernatant.
PCR reaction system is as follows:
Reaction parameter: 95 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 55 DEG C of renaturation 1min, 72 DEG C extend 1min, 35 circulations, and 72 DEG C extend 10min.
3, the retrieve and purification of PCR primer
PCR primer carries out 1% agarose gel electrophoresis, 100V, 50min.Gel imaging system is observed, and takes pictures.
Under ultraviolet lamp, use blade cuts the gel containing object fragment, after thieving paper blots, is placed in centrifuge tube, weighs.Operation is carried out as follows according to the sepharose DNA recovery test kit process specifications of Beijing hundred Tyke Bioisystech Co., Ltd:
(1) the long-pending colloidal sol of triploid/in conjunction with liquid is added;
10min (dissolving completely to glue) is placed in (2) 56 DEG C of water-baths, and vortex concussion in 2-3 minute for several times, helps accelerate dissolution;
(3) dissolved gum solution is joined the adsorption column in collection tube, the centrifugal 30-60s of 12000rpm;
(4) 700 μ L rinsing liquids are joined in adsorption column, the centrifugal 1min of 12000rpm;
(5) adsorption column is placed in sky collection tube, the centrifugal 2min of 12000rpm;
(6) adsorption column is placed in 1.5ml sterile centrifugation tube, joins in the middle of adsorption film by 50 μ L elution buffers, room temperature places the centrifugal 1min of 2min, 12000rpm, and gleanings is stored in-20 DEG C.
4, reclaim product be connected with pMD18-T-E2 carrier and transform
Connect
By the precious biological pMD18-T carrier operation test kit specification sheets in Dalian, PCR is reclaimed product to be connected with pMD18-T carrier.Linked system is as follows:
Mix rear 4 DEG C of connections to spend the night.
The preparation of competent cell
(1) inoculating needle picking intestinal bacteria (-80 DEG C of preservations), in the line of LB culture medium flat plate, 37 DEG C of cultivations; Picking list bacterium colony, be inoculated in LB substratum (5ml), shaking culture is spent the night;
(2) bacterium liquid is inoculated in 100ml LB (1: 100) liquid nutrient medium, and 3h is cultivated in concussion, to OD600 ≈ 0.3-0.8;
(3) 100ml inoculum is transferred in an ice-cold 50ml centrifuge tube under aseptic condition, place 20min on ice;
(4) 4 DEG C centrifugal, 5000rpm, 10min;
(5) abandon supernatant, centrifuge tube is inverted 1min, flow to end to make the trace nutrient solution of final residual;
(6) CaCl of 10ml precooling is added
2(0.1mol/L) solution, places 30min on ice;
(7) 4 DEG C centrifugal, and 5000rpm, 10min, abandon supernatant;
(8) CaCl of 10ml precooling is added
2(0.1mol/L) solution re-suspended cell, adds 20% glycerine ,-80 DEG C of storages.
Transform
(1) take out the DH5 α competence bacterium of preparation from-80 DEG C, put on ice, after thawing completely, add 10 μ L and connect product, mix gently, place 30min on ice;
(2) by after 42 DEG C, mixture heating 45s, 1min is on ice put;
(3) 890 μ L LB substratum are added, 37 DEG C of shaking culture 60min;
(4) bacterium liquid is applied to the LB nutrient agar containing X-gal, IPTG, Amp, after forward places 30min, is inverted overnight incubation, carries out blue hickie screening for 37 DEG C;
(5) select 3 white colonies, the LB liquid nutrient medium be inoculated in containing penbritin (100 μ g/ml) is cultivated.
5, the extraction of recombinant plasmid
DH5 α bacterium containing recombinant plasmid, after the LB liquid nutrient medium containing Amp is cultivated, proposes plasmid enzyme restriction qualification:
With reference to prestige lattice Lars plasmid Mini Kit, extract pMD18-T-E2 plasmid, operate as follows
(1) receive bacterium: the DH5 α bacterium 2ml getting incubated overnight, be placed in 2ml centrifuge tube, centrifugal, 12000rpm, 2min, abandon supernatant, gets precipitation;
(2) resuspended: to add 200 μ L P1 damping fluids, abundant suspendible concussion bacterial sediment 10-15s, makes it scatter completely, to existing without floss;
(3) cracking: add 200 μ L P2 damping fluids, arrives centrifuge tube 3-5 time gently, and room temperature places 2-3min, and make the complete cracking of bacterium, solution is transparent, and pyrolysis time is no more than 5min;
(4) neutralize: add 300 μ L P3 damping fluids, put upside down centrifuge tube 4-6 time gently, fully mix, room temperature places 1-5min, the cotton-shaped generation of visible white, centrifugal 12000rpm, 8min;
(5) column equilibration: add 300 μ L PE damping fluids, 12000rpm, 30s in the centrifugal column of insertion sleeve pipe;
(6) DNA combines: the careful sucking-off of plasmid crude extract supernatant centrifugal after neutralization, and be transferred in the centrifugal column of balance liquid process, centrifugal, 12000rpm, 30s, discard cover liquid in pipe, centrifugal column is turned back to sleeve pipe;
(7) clean: in centrifugal column, add 500 μ L PWT damping fluids, centrifugal, 12000rpm, 30s, discard waste liquid in sleeve pipe;
(8) clean again: add 700 μ L PW PWT damping fluids, centrifugal, 12000rpm, 30s, discard waste liquid in sleeve pipe, recentrifuge 2min;
(9) centrifugal column is received: carefully take out centrifugal column, be not stained with the waste liquid in sleeve pipe, discard sleeve pipe;
(10) eluted dna; Centrifugal column is inserted a new 1.5ml centrifuge tube, in centrifuge tube, pellosil central position adds 100 μ L elutriants, and 12000rpm, 1min, namely obtain the plasmid DNA solution of purifying in centrifuge tube ,-20 DEG C of preservations.
6, the enzyme of recombinant plasmid cuts qualification
Recombinant plasmid uses BamH I, XhoI to carry out enzyme and cuts qualification, and it is as follows that enzyme cuts system.Digestion products carries out sepharose (0.8%) DNA electrophoresis, 100V, 50min.After electrophoresis, gel is put gel imaging system and is observed.Object fragment uses DNA gel to reclaim test kit and reclaims (operation as above) ,-20 DEG C of preservations.
37 DEG C of endonuclease reaction 2h
7, expression plasmid pET-32a increases
Get that laboratory preserves containing Bal 21 (DE3) bacterial strain of pET-32a carrier, be inoculated in LB substratum containing penbritin (100 μ g/ml), overnight incubation by 1: 100.Plasmid uses test kit to extract, and-20 DEG C of storages are for subsequent use.
8, the structure of recombinant expression plasmid pET-32a-E2 and qualification
Cloning vector pMD 18-T-E2 and expression vector pET-32a uses BamH I and Xho I double digestion respectively, and it is described above that enzyme cuts system.Digestion products is carried out electrophoresis, and object fragment uses sepharose to reclaim test kit and reclaims, and operation as mentioned above.According to DNA ligation kit operation instructions, enzyme is cut back to close fragment pET-32a carrier and be connected with E2 fragment, linked system is as follows.
Connect product (10 μ L) and transform DH5 α competent cell (conversion process is described above), bacterium liquid coats the LB solid medium containing penbritin (100 μ g/L), 37 DEG C of overnight incubation.Picking list bacterium colony, the LB substratum be inoculated in containing penbritin (100 μ g/L) is cultivated.Extract plasmid, through enzyme cut with PCR qualification after, positive plasmid called after pET-32a-E2, and positive plasmid is transformed Bal 21 (DE3) competent cell.
Linked system is as follows
pET-32a 3μL
E2 2μL
Solution I 5μL
4 DEG C of connections are spent the night.
9, the determination of raq gene abduction delivering time
Bal21 bacterium containing pET-32a-E2 is inoculated in the LB liquid nutrient medium containing penbritin (100 μ g/mL), when 37 DEG C of shaking culture reach 0.6-1.0 to OD600, add IPTG (1mmol/L) abduction delivering, collect rear 0h, 2h, 3h, 4h, 5h and 6h bacterium liquid 1ml of induction respectively, centrifugal, 8000rpm, 5min, collecting precipitation, carries out SDS-PAGE electrophoretic analysis.4h before the induction of pET-32a empty carrier, after induction, respectively gets 1ml, in contrast.
SDS-PAGE electrophoresis:
Glue: clean sheet glass, be fixed on gel maker, by separation gel formula, add each reagent successively, finally add ammonium persulphate, after mixing of turning upside down, injects between two sheet glass fast, glue covers one deck distilled water, leave standstill 30min.Incline upper strata distilled water, and thieving paper blots residual distilled water, and add the 5% concentrated glue configured, level inserts comb, leaves standstill 2h.Vertically remove comb, remove gel maker, move into electrophoresis chamber, add electrophoretic buffer at inside and outside groove, electrophoresis liquid there was not glue hole, used syringe to draw electrophoresis liquid, rinsed removing residual glue in glue hole.
Loading: sample adds electrophoresis sample-loading buffer according to 2: 1 ratios, vortex mixes, and the centrifugal 1min of boiling water bath 10min, 8000rpm, gets supernatant.Use liquid-transfering gun loading, 20 μ L/ holes.Electrophoresis, concentrated glue voltage is 90V, when tetrabromophenol sulfonphthalein enters separation gel, improves voltage to 120V, 1cm bottom tetrabromophenol sulfonphthalein to glue, stops electrophoresis.
Dyeing: after electrophoresis terminates, take off gel, use blade to cut away concentrated glue, after distilled water flushing, be placed in gel-colored liquid, room temperature shaker dyes 5h.
Decolouring: after dyeing terminates, get gel and be placed in destainer, shaking table decolours, changes staining fluid 3-4 time therebetween, until gel background is thin out.
10, the determination of raq gene IPTG induced concentration
Cultivate the Bal21 bacterium containing pET-32a-E2, when OD600 reaches 0.6-1.0, the IPTG adding 0.5mmol/L, 0.75mmol/L, 1mmol/L, 1.25mmol/L, 1.5mmol/L and 2mmol/L respectively induces.Collect each group of induction 4h bacterium liquid, carry out SDS-PAGE analysis.
11, recombinant protein soluble analysis
Under the condition of the best induced concentration of IPTG and induction time, inducing culture contains the Bal21 bacterium (200ml) of pET-32a-E2,4 DEG C of collected by centrifugation bacterium liquid, 8000rpm, 5min.Thalline ultrasonication in ice bath (120W, 10s/ time, interval 5s, 20min), centrifugal, 12000rpm, 5min, get cleer and peaceful precipitation respectively and carry out SDS-PAGE analysis.The forward and backward cleer and peaceful insoluble part of pET-32a empty carrier induction, respectively gets 1ml in contrast.
12, the Western blot of fusion rotein analyzes
SDS-PAGE electrophoretic separation target protein, operation as above, after electrophoresis, is carried out Western blot qualification, is operated as follows:
(1) prepare before transferring film: gel is placed in transfering buffering liquid after using rinsed with deionized water.Put on one's gloves, use blade 6 filter paper that cutting is identical with gel length and width respectively, 1 pvdf membrane.Pvdf membrane is placed in methyl alcohol (100%) to soak, is dipped in transfering buffering liquid and balances together with 6 filter paper.
(2) transferring film: according to the order of 3 filter paper, pvdf membrane, gel, 3 filter paper, be stacked on the graphite cake of half-dried transfer device, pvdf membrane is placed in positive pole.Use glass stick except striping and film, the bubble between film and glue, filter paper adds 1ml transfering buffering liquid, shifts, transfer parameters: about 2V, 100mA, 50min.Gel after transferring film is transferred in the pallet filling Xylene Brilliant Cyanine G dye liquor, dyes, and to check that whether Protein transfer is complete, cuts the lower left corner of filter membrane as mark simultaneously.
(3) close: bring gloves, pvdf membrane is placed in the plastics bag that can add heat sealing, add 10ml Block buffer (0.1ml/cm
2), get rid of bubble, incubation at room temperature 1 hour on shaking table.
(4) combination of primary antibodie and target protein: discard confining liquid, adds the anti-histidine-tagged primary antibodie (1: 5000) of Block buffer dilution, seals sack, lie against shaking table, incubation at room temperature 1-2 hour after getting rid of bubble.
(5) wash film: cut off plastics bag, discard confining liquid and antibody, TBST rinsing pvdf membrane 3 times, each 10 minutes.
(6) two resist the combination with primary antibodie: after TBST washs for the last time, pvdf membrane is transferred to plastics bag, the goat against murine two anti-(1: 5000) of the horseradish peroxidase-labeled of confining liquid dilution is added, 37 DEG C of shake, incubations 1 hour gently by filter membrane area.
(7) wash: pvdf membrane is transferred to TBST solution, room temperature washing 3 times, 10min/ time.
(8) develop the color: in darkroom, Rumi azoles and hydrogen peroxide are mixed according to 1: 1 ratio, makes luminescent solution.Pvdf membrane is placed in luminescent solution develop the color, until band clear after, use tweezers take out, be placed in distilled water rinsing, be put in stop bath, fixing 5min.
13, the purifying of expressing protein
Under best IPTG induced concentration and induction time, inducing culture contains the Bal21 bacterium (1000ml) of pET-32a-E2, the centrifugal 5min of 8000rpm, collects bacterial sediment.The ultrasonication buffer solution of precipitation use precooling 3 times, the centrifugal 10min of 12000rpm, collecting precipitation, the ultrasonic disruption damping fluid of precooling is added in the 3ml/g bacterium ratio that wets, after resuspended, ultrasonication (120W in ice bath, 10s/ time, interval 5s, 20min).4 DEG C centrifugal, and 12000rpm, 10min, get precipitation.Precipitation is resuspended in the inclusion body lavation buffer solution I of 9 times of volumes, suspend rear 4 DEG C centrifugal, 12000rpm, 10min, get precipitation.After precipitation uses inclusion body lavation buffer solution II, III washing respectively, 4 DEG C centrifugal, and 12000rpm, 10min, get precipitation.Inclusion body after washing is resuspended in (10 times of volumes) in solubilization of inclusion bodies liquid, and room temperature is placed, abundant cracking.Inclusion body after cracking 4 DEG C is centrifugal, and 12000rpm, 20min, get supernatant, after 0.22 μm of membrane filtration, uses Ni+ affinity chromatography column purification, collects elutriant, carry out SDS-PAGE analysis.Purge process is as follows:
(1) Ni+ affinity column, syringe, nucleic acid-protein detector are connected;
(2) with 5 times of column volume distillation washing affinity columns, make absorption photometric value A reach minimum, and remain unchanged;
(3) at least 5 times of column volume binding buffer liquid balance affinity columns, flow rate control is 1ml/min, stablizes to absorption photometric value A;
(4) the sample loading handled well, 5mi/ time;
(5) loading is complete, uses binding buffer liquid to wash affinity column, stablizes to absorption photometric value A;
(6) use elution buffer to carry out wash-out, collect each component of elutriant and carry out SDS-PAGE electrophoretic analysis;
(7), after wash-out, use distillation washing affinity column, be placed in 15% ethanol, 4 DEG C of preservations.
14, inclusion body protein renaturation
Dialysis tubing process: dialysis tubing boils 10min in large volume 2% (W/V) NaHCO3, after distilled water wash, is placed in 1mmol/L EDTA (pH 8.0) and boils 10min, after cooling, deposit in 4 DEG C.
Eluted protein is placed in dialysis tubing (interception is 8000-10000) and carries out dialysis renaturation.Dialysis tubing is placed in the PBS solution that urea concentration reduces gradually, and urea concentration is followed successively by 6mol/L, 5mol/L, 4mol/L, 3mol/L, 2mol/L, 0mol/L, changes liquid 2h interval time.
The amplification of raq gene
Pcr amplification BVDV-E2 full length gene sequence, amplified production, after 0.8% agarose gel electrophoresis is analyzed, obtains an electrophoretic band conformed to expectation size (1040bp).
15, pMD18-T-E2 enzyme cuts qualification
Recombinant clone plasmid pMD18-T-E2 is after BamH I and Xho I double digestion analyze, electrophoresis obtains two bar segment, conform to pMD18-T carrier (2672bp) with E2 goal gene (1040bp), illustrate that cloned sequence connects into pMD18-T carrier.Recombinant expression plasmid pET-32a-E2, after BamH I and Xho I double digestion Analysis and Identification, uses PCR to identify.There are two band in digestion products, conform to carrier segments (5900bp) size with E2 goal gene (1040bp) after electrophoresis, and show that E2 goal gene is connected to pET-32a carrier, expression vector pET-32a-E2 successfully constructs.
16, the determination of the best induction time of E2 fusion rotein
Under the IPTG induction of 1mmol/L, near 43ku, there is object band in pET-32a-E2.Under the different time of induction, SDS-PAGE electrophoretic analysis is carried out in sampling, shows to induce 5h, can obtain and express relatively preferably.
17, the determination of E2 fusion rotein IPTG induced concentration
Under the induction time (5 ~ 8h) optimized, induce pET-32a-E2 to express with the IPTG of 1mmol/L, SDS-PAGE electrophoretic analysis can obtain and express relatively preferably, found that the induced concentration increasing IPTG, and expressing output does not significantly increase.
18, the soluble analysis of E2 fusion rotein and qualification
Precipitation and supernatant carry out SDS-PAGE electrophoresis respectively.Result is near 43ku, and pET-32a-E2, after IPTG induction, occurs band in precipitation, and without corresponding object band in supernatant.Illustration purpose albumen is mainly present in precipitation with inclusion bodies.
19, Western blot analyzes
Use the primary antibodie of anti-His label to carry out Western blot qualification, near 43ku, occur band, reactionless band in supernatant, surperficial expressing fusion protein is correct, with histidine-tagged, and has good reactionogenicity.
20, the purifying of E2 fusion rotein
E2 fusion rotein uses Ni+ chromatographic column to carry out purifying, collects elutriant.Band is there is in result near 43ku.
21, Braford method measures recombinant protein content
(1) protein standard substance (1mg/ml) is dissolved in (0.5mg/ml) in PBS solution completely;
(2) standard substance are added in each hole respectively by 0,1,2,4,8,12,16,20 μ L, add PBS diluent and supply 20 μ L;
(3) sample is diluted, join in each hole, add standard dilutions to 20 μ L;
(4) each hole adds 200 μ L Bradford staining fluids, and even with sample loading gun piping and druming gently, room temperature places 3-5min;
(5) the absorption photometric value of OD630 is measured by microplate reader, drawing standard curve.
Add sample (restructuring E2 albumen) according to above step, measure the absorption photometric value of A630nm, use Origin 8.0 Software on Drawing typical curve, calculate recombinant protein content according to typical curve.
22, immunogen preparation and immunity
With aseptic physiological saline, antigen is diluted, get antigenic solution, add isopyknic Freund's complete adjuvant and carry out emulsification, treat that it forms good water-in-oil-type, carry out initial immunity injection to bird in 20 week age, immunizing dose is 200 ~ 800 μ g/, after initial immunity 21 days, carry out 4 booster immunizations, interval time is 21 days, and immunizing dose is 200 ~ 800 μ g/.Collect egg before and after immunity, 75% alcohol disinfecting, label, 4 DEG C of preservations (15 ~ 60 days).
23, IgY antibody extraction and identification
Polyoxyethylene glycol (PEG-6000) precipitator method extract IgY antibody, and operation steps is as follows:
(1) separator is separated yolk and egg white;
(2) yolk and PBS damping fluid (pH 7.0 ~ 7.8) 1: 2 mix, and vortex, fully mixes;
(3) PEG-6000 of 3.5% (W/V, lower same) is added, vortex mixed, room temperature effect 10 ~ 30min on shaking table;
(4) centrifugal (10000rpm, 4 DEG C, 20min, lower with) after, occurring layering, is from top to bottom yellow fatty layer, limpid layer (IgY) and semi-solid layer;
(5) liquid is through filter paper filtering, discards throw out, and record gained filtrate volume, adds 8.5%PEG-6000, room temperature effect 10 ~ 30min on shaking table;
(6) centrifugal, as above, abandoning supernatant, throw out adds 10ml PBS and dissolves, and adds 12% (1.2g) PEG 6000, room temperature effect 10 ~ 30min on shaking table;
(7) centrifugal, as above, abandoning supernatant, throw out adds 1.2ml PBS and dissolves;
(8) lysate is transferred in dialysis tubing, (1000ml) dialysed overnight in Large Copacity PBS solution;
(9) taken out by IgY antibody liquid in dialyzer every other day, mark ,-20 DEG C store for future use.
Purification IgY antibody through SDS-PAGE electrophoretic analysis, to identify IgY antibody DNA purity.24, the monitoring of IgY antibody titer
Indirect elisa method measures anti-BVDV-E2IgY antibody titer, and operation steps is as follows:
(1) recombinant protein uses bag to be buffered liquid (CBS, pH 9.0) and to carry out diluting (10 μ g/ml), and bag quilt is carried out in 100 μ L/ holes, 4 DEG C of overnight incubation;
(2) PBST-20 washs 3 times, 5min/ time;
(3) Block buffer (2%PBS-BSA) 200 μ L/ hole is added, 37 DEG C of closed 2h;
(4) PBST-20 washing, the same;
(5) IgY antibody uses PBSM, and doubling dilution 1: 1000, until 1: 128000; Non-specific IgY antibody, PBS (pH7.2), respectively as negative and blank, hatches 1h for 37 DEG C;
(6) PBST-20 washing, the same;
(7) add the antibody (1: 5000) of the anti-chicken of goat of HRP mark, hatch 1h for 37 DEG C;
(8) PBST washing, the same;
(9) every hole adds 100 μ L nitrite ions (10mL substrate buffer solution, 150 μ LTMB, the H of 30 μ L
2o
2), 37 DEG C of effect 15min;
(10) every hole adds the 2M H of 50 μ L
28O
4stop, read value OD450.
25, IgY antibody Western blot identifies
SDS-PAGE electrophoretic separation E2 recombinant protein, carry out Western blot qualification after transferring film, method as above.IgY antibody is as primary antibodie, and 1: 2000 dilution, the goat-anti chicken two of HRP mark is anti-dilutes, and extent of dilution is 1: 6000.
26, result
(1) extraction and appraisement of IgY antibody
Purification IgY, after SDS-PAGE electrophoresis, mainly comprises two bands, 65ku and 19ku, and occur low molecular weight fraction near 40ku, shows that its purity is very high with the purifying of PEG-6000 through three concentration gradients.
(2) ELISA titration
Anti-BVDV recombinates E2 protein-specific IgY antibody, uses indirect ELISA to measure, after antibody titer is exempted from two, starts to raise, after one week, slowly reduce, three exempt from booster immunization after, tire and continue to raise, four exempt from after, tire and reach 1: 128000.
(3) Western blot identifies
Recombinant protein, after SDS-PAGE electrophoresis, carries out transferring film, uses anti-E2-IgY antibody as primary antibodie, detects antibodies specific.Result shows, the anti-E2-IgY antibody of extraction can preferably in conjunction with E2 albumen (43ku), and with degradation fragment no cross reaction.
The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (8)
1. a preparation method of anti-bovine viral diarrhea virus E2 protein-specific IgY, is characterized in that: comprise the following steps:
(1) cloned and sequenced of bovine viral diarrhea virus raq gene: according to BVDV-E2 gene order, utilize software design pair of primers, pcr amplification BVDV-E2 gene, be connected to pMD18T carrier, transform DH5 α competent cell, after blue hickie screening, extract plasmid, restriction analysis, positive plasmid checks order, and compares analysis to sequencing result;
(2) expression of E2 albumen in intestinal bacteria, purifying: pMD18T and pET-32a carrier uses BamH I and Xho I double digestion, object fragment connects, build pET-32a-E2 expression vector, transform Bal21 (DE3) pLysS competence bacterium, after enzyme is cut and is identified with PCR, optimize IPTG induced concentration and time, carry out a large amount of abduction delivering, use Ni+ affinity column to carry out purifying to recombinant protein;
(3) preparation of anti-E2-IgY antibody: the E2 protein immunization white of purifying carrys out Hangzhoupro laying hen, uses PEG6000 to extract specific IgY antibody, and carries out SDS-PAGE analysis.
2. the preparation method of a kind of anti-bovine viral diarrhea virus albumen E2 specific IgY according to claim 1, it is characterized in that: according to the relevant BVDV-E2 gene order of GenBank report in step (1), utilize primer prime 5.0 software, design pair of primers, pcr amplification BVDV-E2 gene, for 1040bp, be connected to pMD18T carrier, transform DH5 α competent cell, after blue hickie screening, extract plasmid, restriction analysis, positive plasmid checks order.
3. the preparation method of a kind of anti-bovine viral diarrhea virus albumen E2 specific IgY according to claim 1, is characterized in that: in step (2), expression product uses Ni+ affinity column to carry out purifying to recombinant protein after SDS-PAGE and Western blot analyzes.
4. the preparation method of a kind of anti-bovine viral diarrhea virus E2 protein-specific IgY according to claim 1, it is characterized in that: in step (3), SDS-PAGE analysis is carried out to extraction specific IgY antibody, indirect ELISA measures anti-E2-IgY antibody titer after immunity, Western blotting identify obtain the specificity of anti-E2-IgY.
5. the preparation method of a kind of anti-bovine viral diarrhea virus albumen E2 specific IgY according to claim 1, it is characterized in that: described preparation immune egg method is as follows: carry out initial immunity injection to bird in 20 week age, immunizing dose is 200 ~ 800 μ g/, after initial immunity 21 days, carry out 4 booster immunizations, interval time is 21 days, and immunizing dose is 200 ~ 800 μ g/.
6. the preparation method of a kind of anti-bovine viral diarrhea virus E2 protein-specific IgY according to claim 5, is characterized in that: described bird is chicken.
7. the preparation method of a kind of anti-bovine viral diarrhea virus E2 protein-specific IgY according to claim 1, is characterized in that: the method extracting IgY in described step (3) is as follows;
(1) egg of described collection is carried out yolk to be separated with egg white, collect yolk, used by yolk PBS to dilute according to volume ratio 1: 1.5 ~ 2.5;
(2) add to diluent the PEG-6000 that mass volume ratio is 2.5 ~ 5%, fully stir, room temperature effect 20 ~ 30 minutes on shaking table;
(3) mixed solution is centrifugal, collect supernatant, filter, filtrate adds the PEG-6000 that mass volume ratio is 6 ~ 9%, fully stirs, room temperature effect 20 ~ 30 minutes on shaking table;
(4) by centrifugal for the mixed solution after stirring, abandoning supernatant, the PBS of precipitation use 10 milliliters suspends, add the PEG-6000 that mass volume ratio is 10 ~ 13%, fully stir, room temperature effect 20 ~ 30 minutes on shaking table, carry out centrifugal again, obtain throw out;
(5), after using 1.2 ~ 1.5 milliliters of PBS to dissolve described throw out, use dialysis band to dialyse, use PBS dialysed overnight, obtain IgY, and be kept at-20 DEG C for subsequent use.
8. the preparation method of a kind of anti-bovine viral diarrhea virus albumen E2 specific IgY according to claim 7, is characterized in that: the pH value of described PBS is 7.2 ~ 7.5.
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